. Author manuscript; available in PMC: 2026 Oct 3.

Published in final edited form as: Nat Protoc. 2025 Nov 19;21(5):1841–1868. doi: 10.1038/s41596-025-01272-3

Directional conjugation of monoclonal antibodies to nanoparticles using metal-free click chemistry

Ananthakrishnan S Jeevarathinam ¹, Mohammed R Kawelah ², Sangheon Han ¹, Brian J Grindel ³, Steven W Millward ³, Keith P Johnston ^2,⁴, Konstantin V Sokolov ^1,^5,⁶

PMCID: PMC13633081 NIHMSID: NIHMS2139065 PMID: 41258016

Abstract

Here, we describe a practical step-by-step protocol for directional conjugation of monoclonal antibodies to polymersome nanoparticles via the Fc antibody moiety using metal-free click chemistry. This Protocol Extension details procedures for synthesis, quality control and evaluation of nanoparticle–antibody conjugates. The synthesis includes three main stages: (i) a mild oxidation of the glycosyl moiety that is present only on the Fc region of the antibody to produce functional aldehyde groups; (ii) attachment of a heterobifunctional linker, aminooxy-PEG-dibenzocyclooctyne, to the aldehyde groups in the Fc moiety; and (iii) click conjugation of the PEG-linker-modified antibodies to nanoparticles with azide functional groups. This protocol enables covalent surface conjugation of monoclonal antibodies that inherently does not involve the antigen-binding Fab region, thus minimizing the impact of the conjugation on the avidity of antibodies. In contrast, conventional conjugation methods that use amine groups of antibodies for covalent immobilization result in a random antibody orientation that can lead to a loss of the antibody’s binding efficiency. The entire conjugation protocol requires ~18 h. The nanoparticle–antibody conjugates synthesized using this method are expected to display a high molecular specificity toward epitopes of the conjugated antibodies while maintaining the physicochemical properties of the core nanoparticles. The protocol described here does not require any special expertise other than general laboratory training on equipment such as a centrifuge, ultraviolet–visible spectrophotometers, dynamic light scattering, ELISA plate readers and cell culturing.

Introduction

Targeted nanoparticles are being developed for a wide variety of biomedical applications including screening and diagnostic assays^1–3, molecular imaging⁴, drug delivery^5–7, therapy monitoring⁸, radiosensitization⁹ and theranostics^10,11. Molecule-specific targeting is generally achieved through conjugation of biomolecules such as antibodies, peptides, nucleic acids and small ligands with specific biorecognition properties to the surface of nanoparticles. Monoclonal antibodies and their fragments are one of the most commonly used types of targeting moieties because of their high stability in biological media and well-established manufacturing protocols including existing capabilities to produce antibodies for new biomarkers^12,13. Antibody fragments can provide better tissue extravasation because of their lower molecular weight^14,15 and reduced immunogenicity because of the absence of an Fc portion¹⁶. However, the lack of the Fc (fragment crystallizable) domain could reduce the antibody’s stability and is also associated with an inherent reduction in binding affinity^17,18. Furthermore, the directional conjugation protocol described here sterically hinders nonspecific interactions with the Fc moiety while preserving the high affinity of the whole monoclonal antibody¹⁹.

Development of the protocol

Synthesis of antibody-targeted nanoparticles with high binding avidity requires conjugation strategies that do not interfere with antibody-antigen interactions. To this end, we previously developed a protocol for directional conjugation of antibodies to gold nanoparticles using thiol chemistry¹⁹. This protocol has been successfully implemented by multiple research laboratories in diverse biomedical applications including biosensing^20–23, optogenetic retinal prosthetics²⁴, cancer immunotherapy^25–27, tracking of epigenetic cell modulation^28,29, optoporation³⁰, augmented tumor extravasation^31,32, blocking of metastasis³³ and immunoassays^34–39. However, recent analysis revealed that the translational and clinical research on metal nanoparticles accounts for only 11% of clinical trials, whereas organic nanoparticles including polymeric, liposomal and lipid-based nanoparticles account for 62%⁴⁰. Therefore, here we extend the idea of directional conjugation of antibodies to organic nanoparticles (e.g., polymeric and liposomal nanoparticles) by implementing a copper-free click chemistry approach.

The key feature of our protocol is conjugation through a carbohydrate antibody moiety that is located on the Fc portion of the antibody (Fig. 1). The protocol consists of three main stages: (i) mild oxidation of the carbohydrate antibody moiety to form functional aldehyde groups, (ii) conjugation of heterobifunctional aminooxy-PEG-dibenzocyclooctyne (DBCO) linker to the oxidized antibody’s Fc region and (iii) antibody conjugation via a terminal alkyne to nanoparticles functionalized with azide groups. The directional conjugation (i.e., conjugation via the Fc portion) is achieved because of the intrinsic property of most IgG antibodies to contain a carbohydrate moiety in the Fc region. In contrast to most commonly used antibody conjugation protocols via antibody amine groups, the directional conjugation approach does not hinder specific antibody interactions via the Fab region. Furthermore, it allows for the co-labeling of antibodies with fluorescent dyes or other labeling molecules through the antibody’s amine groups. In addition, the conjugation through the Fc region can minimize interactions with Fc cell receptors that cause non-specific interactions with macrophages and results in a fast in vivo clearance by the reticuloendothelial (RES) system⁴¹.

We used directional copper-free antibody (Ab) conjugation in development of targeted polymersomes (PSs) loaded with J-aggregates of indocyanine green (ICG) dye (ICGJ) (ICGJ-PS-Ab) for molecular specific photoacoustic imaging (PAI)^42,43. We showed that ICGJ-PS-Abs conjugated with cetuximab antibodies that are specific to epidermal growth factor receptor (EGFR) can be used in molecular PAI of breast and ovarian cancer cells with a low level of non-specific binding. The PAI signal was proportional to the known EGFR expression level in different cell lines. Furthermore, the cetuximab-targeted ICGJ-PS-Abs enabled highly sensitive PAI of labeled cancer cells in tissue mimicking phantoms with a detection limit of just ~100 total cells. The protocol presented here is based on these validation studies and also demonstrates conjugation of antibodies for folate receptor-α (FRα), another important cancer biomarker^44,45.

Applications of the method

The directional conjugation approach can be used for any glycosylated antibodies, proteins or peptides. In addition, the directional protocol can be adapted to other conjugation chemistries such as the maleimide-thiol reaction. Recently, we used a heterobifunctional hydrazide–PEG–thiol linker to conjugate monoclonal antibodies to maleimide-functionalized liposomes⁴⁶. In this chemistry, the antibody’s sugar moieties on the Fc portion were first mildly oxidized to produce aldehyde groups as described below (Steps 1–4). The reaction between the aldehydes and the linker’s hydrazide groups resulted in thiol-modified antibodies, which were attached to liposomes through a maleimide-thiol covalent bond.

Similarly, any heterobifunctional PEG linkers with an aldehyde reactive functionality can be used, while the other functional group can be selected on the basis of the surface that needs to be modified. For example, commercially available heterobifunctional PEG linkers such as alkyne-PEG-amine (Nanocs, cat. no. PG2-AKAM-3k), tetrazine-PEG-hydrazide (Nanocs, cat. no. PG2-HZTZ-3k) and azide-PEG-aminoxy (Nanocs, cat. no. PG2-AOAZ-3k) can be adopted for the conjugation approach described here.

Comparison with other conjugation strategies

Antibody conjugation methods have been surveyed in a number of comprehensive review articles^47–49. Here, we provide a brief overview of the available approaches from the standpoint of controlling antibody orientation relative to the surface during attachment.

Methods with limited control of orientation

Perhaps the simplest approach is immobilization of antibodies through non-covalent interactions that include a combination of hydrophobic, electrostatic, dipole and nonpolar interactions and hydrogen bonds⁵⁰. In principle, this method does not require any nanoparticle or antibody modifications, and it was initially developed for synthesis of immunogold stains for tissue and cell sections in transmission electron microscopy (TEM)⁵¹. However, the major drawback is a lack of conjugate stability because the attached molecules can be easily displaced in a biological medium, which largely limits application of this approach to well-controlled environments such as pure aqueous buffers.

Stable conjugates can be produced by covalent binding through an antibody’s carboxyl or amine groups. The reaction with carboxyl groups is activated by carbodiimide and is usually carried out in combination with N-hydroxysuccinimide (NHS), which results in a semi-stable, amine reactive intermediate that is used either for a direct conjugation with amine groups on a nanoparticle’s surface or to react with an amine-containing linker molecule⁵². Conversely, amine groups on an antibody can be used to link to activated carboxyl groups on a nanoparticle surface or to a linker with a carboxyl moiety. Furthermore, an antibody’s primary amine groups have also been used for conjugation reactions through aldehydes, epoxides and cyanogen bromide^53–55. However, because carboxyl and amine groups are located in various antibody regions, these conjugation approaches, in general, result in a random surface orientation of the molecules. Furthermore, it was suggested that the most reactive primary amine groups are located in the antibody’s antigen-recognition region⁴⁸. Therefore, the conjugation through primary amine groups can interfere with the binding affinity of conjugated antibodies.

Methods with an intrinsic control of orientation (i.e., directional conjugation)

In addition to the directional conjugation method through carbohydrate antibody moiety that was introduced by us¹⁹, there are two other approaches that allow control over an antibody’s orientation: (i) conjugation through thiol groups either on half-antibodies^16,56 or Fab antibody fragments⁵⁷ and (ii) conjugation using Fc-binding proteins and antibodies^58,59. The first strategy can be used either with a thiol-reactive cross-linker (e.g., maleimide)⁵⁷ or by a direct interaction of thiol groups with a gold surface⁵⁶. However, the major drawbacks of using antibody fragments are an increase in production costs, loss of stability and a higher propensity to aggregation in comparison with full antibodies, as well as a reduction in avidity^17,60. The use of Fc-binding molecules such as proteins A, G and A/G or Fc-specific antibodies requires conjugation of a binding protein that increases the size of the nanoparticle conjugates and is associated with higher costs¹⁷.

Because boronic acid groups have high affinity to diol (1,2 and 1,3) moieties in the carbohydrates of the Fc region of monoclonal antibodies, the diol-boronic acid interaction was also explored for directional antibody conjugation via an antibody’s carbohydrate moiety⁶¹. However, this reaction is known to be reversible in the aqueous in vivo environment, and, therefore, it cannot be used in the synthesis of stable antibody–nanoparticle conjugates⁶². Special linkers containing a boronic acid and a proximally located functional group have been developed for irreversible antibody binding^63,64. In these approaches, an antibody first interacts with boronic acid through the Fc sugar moiety, followed by a covalent attachment through the secondary functional group. Although this is an interesting approach, it relies on highly specialized linkers that are not commercially available, and it has been evaluated only for conjugation to flat surfaces.

Other methods

Streptavidin–biotin interactions⁶⁵ and nucleic acid hybridization⁶⁶ have also been explored for conjugation of antibodies. Antibodies are first modified with either biotin or a single-stranded DNA molecule, followed by specific interactions with either streptavidin or a complementary DNA strand on nanoparticles, respectively. The antibody modification is carried out using one of the aforementioned chemistries, which determines the antibody’s surface orientation.

Overview of the protocol

Here, we describe a detailed protocol for directional conjugation of two types of monoclonal IgG antibodies, humanized (i.e., cetuximab, an anti-EGFR antibody) and mouse (i.e., anti-FRα antibody), to PSs (Fig. 1). PSs are self-assembled vesicles composed of bilayer assemblies of amphiphilic block copolymers⁶⁷. We use PSs that are formed by azide-poly(ethylene glycol)-b-poly(d,l-lactide-co-glycolide) amphiphilic di-block polymers and are loaded with ICGJ for applications in molecular PAI, synthesized in house as described in refs. 42,43.

The procedure starts with a mild oxidation of glycosyl moieties on the Fc fragment of the antibody to produce aldehyde groups (Steps 1–5). The successful oxidation is confirmed by a qualitative, colorimetric Purpald test⁶⁸. Then, we modify the oxidized antibodies with aminoxy-PEG-DBCO linker to insert DBCO functional groups in the glycosylated Fc antibody’s moiety for a copper-free click chemistry with azide (Steps 6–10). The DBCO-functionalized antibodies are conjugated to azide groups on the surface of polymersomes to produce antibody-targeted nanoparticles (Steps 11–17).

Quality control

Different stages of the protocol require different quality control methods. The ICGJ-loaded PSs (ICGJ-PSs) and ICGJ-PS-antibody conjugates are monitored by visual inspection, ultraviolet (UV)–visible absorption at 780 and 895 nm, dynamic light scattering (DLS) and TEM. Visual inspection ensures that the ICGJ-PSs are evenly dispersed in the storage medium and do not settle. The ratio of absorbance at 895 nm to 780 nm signifies the ratio of ICG monomer to ICG J-aggregates within the ICGJ-PS. DLS and TEM provide information on the potential aggregation state of ICGJ-PSs upon the conjugation of antibodies.

To assess the efficiency of conjugation of antibodies, a sandwich ELISA was developed. For this purpose, the standard response curves from known concentrations of monoclonal antibodies are used to calculate the number of antibodies per particle.

Note that ELISA could underestimate the number of antibodies conjugated to nanoparticles, especially at a high conjugation density, because of a steric hindrance of secondary antibody interactions with conjugated antibodies that are proximal to the capture surface⁶⁹. Alternative approaches include the use of fluorescently labeled antibodies, where the conjugated amount is calculated by measuring the difference in fluorescence of the antibody’s solution used during the conjugation and the total fluorescence recovered during washing of the nanoparticle conjugates⁴². Furthermore, conjugates of metal nanoparticles with fluorescently labeled antibodies were chemically dissolved, and the fluorescence of the released antibodies was used to estimate the number of conjugated molecules⁷⁰. However, a limitation of these methods is the need for fluorescently labeled antibodies, which means that they cannot be used as a standard quality control procedure during the production of antibody–nanoparticle conjugates.

Finally, cancer cell labeling with the ICGJ-PS-antibody conjugates was evaluated in cells with different expression levels of either EGFR or FRα for ICGJ-PSs conjugated with anti-EGFR antibodies (ICGJ-PS-aEGFR) and anti-FRα antibodies (ICGJ-PS-aFRα), respectively. The cell labeling efficiency was studied using flow cytometry and confocal fluorescence microscopy. To further confirm binding specificity, we carried out a blocking cell labeling assay with either free EGFR or FRα antibodies for ICGJ-PS-aEGFR and ICGJ-PS-aFRα, respectively.

Limitations

One of the major limitations of the proposed protocol is the requirement for the antibody to be glycosylated. Commonly used antibodies are produced by hybridomas and generally contain glycosylated regions⁷¹. However, glycosylation-free recombinant antibody fragments and aglycosylated recombinant antibodies^72,73 produced in bacteria are not suitable for this conjugation protocol.

In principle, the proposed conjugation strategy should work for any glycosylated antibody with the glycoside moieties present in the 1,2-dihydroxy groups in a quasi-planar conformation for oxidation by periodate. An unknown antibody can be tested for glycosylation by a glycosylation kit (e.g., Sigma-Aldrich, cat. no. GLYCOPRO-1KT). Then, the presence of the oxidizable glycoside moieties can be evaluated by either the Purpald assay or by a glycan assay test that provides data on the relative amount of different monosaccharide units in the antibody’s glycosylated moiety (e.g., RayBiotech, cat. no. GA-Glycan-300–1).

Furthermore, this protocol requires azide functional groups on the surface of nanoparticles. Here, we used azide-functionalized polymers in the synthesis of PSs. Alternatively, azide groups can be introduced to nanoparticles with maleimide, amine or carboxyl groups by using commercially available, heterobifunctional linkers such as thiol-PEG-azide (Nanocs, cat. no. PG2-AZTH-3k)⁷⁴, NHS-PEG-azide (Nanocs, cat. no. PG2-AZNS-3k)⁷⁵ or amine-PEG-azide (Nanocs, cat. no. PG2-AZCR-5k)⁷⁶, respectively, using well-established protocols⁷⁷. For lipid-coated or liposomal nanoparticles, azide-modified lipids such as 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE)-PEG-azide (Avanti Research, cat. no. 880255P)⁷⁸ can be included in the lipid formulation at a required density.

Experimental design

Determination of nanoparticle concentration

Quantifying nanoparticle concentration for optimization and characterization of antibody conjugation depends on a specific nanoparticle that is used in a study. Noticeable examples include the use of the qNano Gold system (Izon Science Ltd.) to determine the concentration of liposomes⁴⁶, ¹⁹F NMR for perfluorocarbon nanodroplets^79,80, nanoparticle tracking analyzer (NTA) for exosomes⁸¹ and inductively coupled plasma mass spectrometry for gold⁸² and iron oxide⁸³ nanoparticles.

For nanoparticles with a strong absorbance such as ICGJ-loaded PSs used in this protocol, a linear relationship can be established between absorbance and concentration. We previously estimated using NTA that 1 ml of ICGJ-PS suspension with an optical density (OD) of 1.0 contains ~1.5 × 10¹⁰ PSs⁴³. Hence, the OD of ICGJ-PSs can be used to determine the concentration of the PSs. Other types of nanoparticles might require a different method for determining their concentration or a suitable surrogate parameter that is proportional to the nanoparticle’s concentration.

Optimal ligand density on nanoparticle–antibody conjugates

Nanoparticle conjugates are inherently heterogeneous because of the distribution of nanoparticle sizes and variations in the number of conjugated antibodies per nanoparticle. Research evaluating how ligand density influences the properties of nanoparticle conjugates has been summarized in refs. 84,85. The analysis and generalization of these data are complicated by the fact that the reported studies were carried out with nanoparticles that had different sizes, shapes, core materials, surface coatings and conjugation chemistries. Nevertheless, some general trends could be identified. For small molecule–based ligands such as folic acid, the avidity of nanoparticle conjugates was shown to increase to the maximum value at 100% of ligand surface density⁸⁶. However, for bigger ligands such as human transferrin and IgG1 antibodies, the targeting efficiency significantly increased only up to ~25%, with no further changes at higher densities of transferrin and only modest improvement in the case of IgG1 antibodies⁸⁷. Similarly, the optimum cell binding in vitro was achieved at ~25% surface density of prostate-specific membrane antigen (PSMA)-targeted single-chain variable fragment antibodies conjugated with polymeric micelle nanoparticles, with decreased binding at 75% and 100%⁸⁸. The optimum tumor accumulation of the targeted micelles was also observed at ~25% surface density, with a sharp decrease beyond the optimal ligand density because of increased immunogenicity and clearance by the RES⁸⁸. These data indicate that there is an optimum ligand density for effective cell targeting and delivery in vivo that probably needs to be optimized for each nanoparticle conjugate. Increasing the ligand density beyond the optimum value could lead to several drawbacks such as steric hindrance and depletion of cellular receptors at the cellular level and increased immunogenicity that is associated with increased RES clearance and lower target accumulation in vivo⁸⁴.

Emerging tools such as direct stochastic optical reconstruction microscopy that was recently developed to measure the number of binding ligands per each individual nanoparticle conjugate⁸⁹ will allow evaluation of the heterogeneity of nanoparticle conjugation, which can be used to establish acceptable levels of ligand density variance for biomedical applications. Furthermore, a narrow distribution of nanoparticle conjugates with a specific ligand density for optimization studies can be obtained by previously evaluated purification techniques such as gel electrophoresis^90–92, field flow fractionation⁹³ and size exclusion chromatography⁹⁴.

Materials

Reagents

Human epidermal growth factor receptor monoclonal antibody (aEGFR) (ERBITUX; Eli Lilly and Company, ImClone; RRID: AB_2459632)
Folate receptor-α monoclonal antibody (Thermo Fisher Scientific, cat no. MA5–23917; RRID: AB_2609390)
The linker molecule aminoxy-PEG-dibenzocyclooctadiene (NH₂-O-PEG-DBCO; Nanocs, cat. no. PG2-AODB-3K)
The linker molecule used for non-directional control, NHS-PEG-DBCO (Nanocs, cat. no. PG2-DBNS-3k)
Alexa Fluor 594 antibody labeling kit (Thermo Fisher Scientific, cat. no. A20185)
Alexa Fluor 647 antibody labeling kit (Thermo Fisher Scientific, cat. no. A20186)
Alexa Fluor 488 (AF488) antibody labeling kit (Thermo Fisher Scientific, cat. no. A20181)
DAPI (Life Technologies Corporation, cat. no. D3571)
16% (wt/vol) paraformaldehyde (Electron Microscopy Sciences, cat. no. 15710)
Fluoromount aqueous mounting medium (Millipore Sigma, cat. no. F4680–25ML)
OmniPur 10× PBS (Millipore Sigma, cat. no. 6505–4L)
Dulbecco’s PBS (DPBS), 10× (Corning, cat. no. 20–030-CV)
Cell culture–grade water (OmniPur water; Millipore Sigma, cat. no. 4.86505.0500)
0.07541 M sodium phosphate dibasic (NaH₂PO₄) (Sigma-Aldrich, cat. no. RES20908-A702X)
0.02459 M sodium phosphate monobasic (Na₂HPO₄) (Sigma-Aldrich, cat. no. RES20906-A702X)
HEPES (Fisher Scientific, cat. no. BP310–100)
Sodium periodate (Millipore Sigma, cat. no. 311448–100G)
Sodium hydroxide (10 M solution; Fisher Scientific, cat. no. SS255–1)
Hydrochloric acid (6 M; Honeywell Fluka, cat. no. 72033–1L)
Purpald (Millipore Sigma, cat. no. 162892)
BSA (Millipore Sigma, cat. no. A7906)
Tween 20 (Millipore Sigma, cat. no. P1379–500ML)
Promega 3,3′,5,5′-tetramethylbenzidine (TMB) One solution, used as a substrate solution for ELISA (Fisher Scientific, cat. no. G7431)
Pierce Protein L coated 96-well plates, used for ELISA tests in estimation of antibodies (Thermo Fisher Scientific, cat. no. 15190)
Goat anti-human IgG (H+L) antibody, horseradish peroxidase (HRP) conjugate (goat anti-human IgG-HRP) (Thermo Fisher Scientific, cat. no. A18805; RRID: AB_253558)
Pierce goat anti-mouse IgG (H+L) antibody, HRP conjugate (goat anti-mouse IgG-HRP)

(Thermo Fisher Scientific, cat. no. 31430; RRID: AB_228307)
DMEM/F-12 medium 1:1 mixture with l-glutamine and HEPES, sterile filtered, used to culture MDA-MB-468 (American Type Culture Collection (ATCC), cat. no. 30–2006)
EMEM medium with l-glutamine, sterile filtered, used to culture MCF7 cells (ATCC, cat. no. 30–2003)
RPMI-1640 medium with high glucose, l-glutamine and HEPES, sterile filtered, used to culture SKOV3 and A2780 cells (ATCC, cat. no. 30–2001)
MDA-MB-468 cells (RRID: CVCL_0419)
MCF7 cells (RRID: CVCL_0031)
SKOV3 cells (RRID: CVCL_0C84)
A2780 cells (RRID: CVCL_0134)
FBS, triple 0.1 μm sterile filtered (Cytiva, cat. no. SH30910.03)
Trypsin 0.25% (wt/vol) with 2.21 mM EDTA (Corning, cat. no. 25–053-CI)
Penicillin–streptomycin, stabilized, with 10,000 units of penicillin and 10 mg of streptomycin ml⁻¹, 0.1 μm filtered (Sigma-Aldrich, cat. no. P4333–100ML)
Universal Fc blocker, used to block Fc receptors on MDA-MB-468 cells and SKOV3 cells (INNOVEX Biosciences, cat. no. NB309–5S)
Invitrogen human IgG isotype control, used as isotype control for MDA-MB-468 cell labeling in flow cytometry (Thermo Fisher Scientific, cat. no. 02–7102)
Mouse IgG1 isotype control, obtained as a part of the Invitrogen mouse IgG1 ELISA kit

(Thermo Fisher Scientific, cat. no. 02–6100)
ICG (Tokyo Chemical Industry, cat. no. I0535)
Azide-poly(ethylene glycol)-b-poly(l,d lactide-co-glycolide) (molecular weight: 100,000 Da; PEG (10 kDa) and poly(d,l-lactide-co-glycolide) (90 kDa); the lactide (L) to glycolide (G) ratio (L:G) is 90:10) (custom synthesized by Nanosoft Polymers Inc.)
0.01% (wt/vol) sterile filtered poly-l-lysine solution (Sigma-Aldrich, cat. no. P4707–50ML)
Fluoromount aqueous mounting medium (Sigma-Aldrich, cat. no. F4680–25ML)
No. 1.5 glass coverslip, 22 mm (Warner Instruments, cat. no. CS-22S15)

Reagent setup

1× PBS pH 7.4 for antibody processing

Prepare by diluting OmniPur 10× PBS in milli-Q water. This solution need not be freshly prepared. A stock of 1,000 mL of the buffered solution can be stored in a closed glass container for up to one month at 4°C in a refrigerator and must be visually inspected for any precipitation or microbial contamination before use.

Buffer to rinse cell specimens, 1× phosphate buffer with calcium and magnesium

Prepare by diluting DPBS 10× in cell culture–grade water. This solution need not be freshly prepared, and a 500-ml stock can be stored at 4 °C in a refrigerator for ≤1 month. Check the pH and inspect the solution for potential microbial contamination or precipitation before use. If a pH adjustment is required, perform sterile filtration using a 0.2-μm filter after adjusting the pH.

10 mM sodium phosphate buffer solution pH 7.5

Prepare a solution containing 0.075 M sodium phosphate dibasic (NaH₂PO₄) and 0.025 M sodium phosphate monobasic (Na₂HPO₄). Adjust the pH using 1 N hydrochloric acid or 1 N sodium hydroxide solutions with a pH meter. This solution can be prepared in 1,000-ml stocks and stored at 4 °C in a refrigerator in tightly sealed glass containers for ≤1 month.

40 mM HEPES buffer pH 8.5

Prepare by dissolving 1.9 g of HEPES in 200 ml of Millipore water and adjust the pH to 8.5 using 1 N hydrochloric acid or 1 N sodium hydroxide solutions with a pH meter. A 200-ml stock of HEPES buffered solution can be prepared and stored at 4 °C in a refrigerator for ≤1 month in tightly sealed glass containers.

Solution of human aEGFR

Anti-EGFR antibody is supplied as a solution in PBS at a concentration of 2 mg ml⁻¹. Store it as 1-ml aliquots at 4 °C for ≤1 month or at −20 °C for ≤6 months. Before the antibody oxidation step, perform a buffer exchange by washing with 100 mM sodium phosphate buffer (pH 7.0) in a 30,000-Da molecular weight cutoff (MWCO) centrifuge filter, as described in Box 1.

BOX 1. Optional protocol for buffer exchange for removal of cryoprotectant from the antibody solution.

If an antibody is received as a solution, perform a buffer exchange using a 30-kDa MWCO centrifuge filter to transfer the antibody to 100 mM sodium phosphate buffer, as follows:

Pipette the antibody solution into a 30-kDa centrifugal filter, add five to six volumes of 100 mM sodium phosphate buffer and centrifuge at 3,100g for 8 min in a swinging bucket centrifuge at 4 °C.
Again add five to six volumes of 10 mM sodium phosphate buffer to the residual solution that remained (from the previous step) inside the centrifuge filter and centrifuge again at 3,100g for 8 min at 4 °C.
Determine the concentration of antibody in the retentate using UV–visible absorbance at 280 nm and adjust the concentration to 1 mg ml⁻¹ by adding 100 mM sodium phosphate buffer (Supplementary Methods, section I).

The concentration of antibody can be determined using the following equation.

Antibody concentration = A_{280} \times \frac{{MW}_{Ab}}{ε_{Ab}}

(1)

where $A_{280}$ is the absorbance at 280 nm for the path length of 1 cm, ${MW}_{ab}$ is the molecular weight of the antibody (typically 146,000–170,000 g mol⁻¹)¹¹⁰ and $ε_{ab}$ is the absorption coefficient of the antibody (typically 202,500–229,500 L mol⁻¹ cm⁻¹)¹¹¹. The appropriate molecular weight for a specific class or a subclass of an antibody can be found in the literature¹¹² or from the antibody’s manufacturer.

◆ TROUBLESHOOTING

Solution of FRα monoclonal antibody

Folate receptor-α monoclonal antibody (aFRα) is supplied as a lyophilized powder at 100-μg size. Store aFRα as the lyophilized powder in the sealed pack at −20°C. Follow the procedure described in Box 1 for preparation of the antibody solution before use.

Sodium periodate (NaIO₄) solution, 100 mM

Prepare by dissolving 21.3 mg of sodium periodate powder in 1 ml of deionized water. Use this solution within 5 min of preparation. The remainder of the solution must be safely discarded.

Purpald solution for testing oxidation of the antibody

Prepare 10 mg ml⁻¹ of Purpald solution by dissolving 10 mg of Purpald (powder) in 1 ml of freshly prepared 1 N NaOH solution. Use the Purpald solution within 24 h and discard the remainder of the solution after 24 h.

DAPI solution used for cell staining in confocal microscopy

Prepare the stock solution of 4.9 mg ml⁻¹ (10.9 mM) DAPI in deionized water before the experiment and dilute the 10.9 mM DAPI solution in 1× PBS to the final concentration of 1.09 μM before cell staining. Store the stock solution of 10 mM DAPI at −20 °C for ≤6 months. Store the diluted 1.09 μM solution of DAPI in the dark at room temperature (in the range of 20–23 °C) for ≤3 months.

Aminoxy-PEG-DBCO solution, 46.5 mM in dimethyl sulfoxide (DMSO)

NH₂O-PEG-DBCO is supplied as a powder at 20-mg size. Dissolve 20 mg of NH2-PEG-DBCO in 127 μl of DMSO and store the solution as 10-μl aliquots in 300-μl centrifuge tubes at −20 °C for ≤90 d.

Paraformaldehyde 4% in 1× PBS

Prepare 4% (wt/vol) paraformaldehyde solution by mixing 10 ml of 16% (wt/vol) paraformaldehyde with 27 ml of cell culture–grade water and 3 ml of 10× DPBS with calcium and magnesium. Use the solution within 24 h and discard the remainder of the solution.

Blocking buffer for ELISA

Prepare the blocking buffer for ELISA by dissolving 500 mg of BSA in 50 ml of 1× PBS. Filter the solution through a 0.45-μm PVDF syringe filter to remove protein aggregates. Store the blocking buffer at 4 °C and use it within 24 h.

Washing buffer for ELISA

Prepare the washing buffer for ELISA by dissolving 250 μl of 0.05% (vol/vol) Tween 20 in 500 ml of 1× PBS. Store the washing buffer under ambient conditions for ≤30 d.

Reagent buffer for ELISA

Prepare the reagent buffer for diluting sample and standard solutions of free antibodies for ELISA by mixing 5 ml of blocking buffer with 45 ml of washing buffer. Store the reagent buffer at 4 °C and use it within 24 h of preparation.

Goat anti-human IgG-HRP and goat anti-mouse IgG-HRP

These antibodies are received as lyophilized powder made from a PBS solution of the respective antibodies. According to the manufacturer’s instructions, reconstitute the lyophilized secondary antibodies in 2 ml of Milli-Q water to obtain 1× PBS solutions and store in 20-μl aliquots at −20 °C for ≤180 d. Before use in the ELISA experiment, dilute secondary antibodies 2,000 times in the reagent buffer.

DMEM/F-12 medium 1:1 mixture, EMEM and RPMI-1640 high-glucose medium

Supplement (mix) the fresh medium with 10% (vol/vol) FBS and 1% (vol/vol) penicillin–streptomycin solution under sterile conditions and use for cell culture. Store the supplemented medium at 4 °C for ≤30 d or until the time of expiration specified by the manufacturer, whichever is earliest.

Equipment

UV–visible spectrophotometer (BioTek SYNERGEY H1 microplate reader)
Malvern Zetasizer NS instrument (Malvern Panalytical)
Glacios 200-kV cryo-transmission electron microscope (Thermo Fisher Scientific) fitted with a Falcon 4 direct electron detector
Electrical vortex mixer (VWR micro plate shaker, 120 V)
Centrifuge for centrifugal filtration (Eppendorf centrifuge 5810R 15-amp version)
Microcentrifuge to wash nanoparticles (VWR Symphony 2417R)
Leica TCS SP8 confocal microscope or equivalent to perform confocal microscopy
LSRFortessa X-20 analyzer (5-laser, 18-color) or equivalent for flow cytometry analysis

Procedure

Stage 1: modification of antibody

Oxidation of the antibody

● TIMING 40 ± 16 min depending on the need for buffer exchange and concentration determination before the reaction (Box 1)

1
Add 10 μl of freshly prepared 100 mM sodium periodate solution in Milli-Q water to 100 μl of 1 mg ml⁻¹ antibody solution, to achieve 1,700 molar excess of periodate.

▲ CRITICAL STEP This step is important to oxidize diol groups in the carbohydrate moiety of the Fc non-targeting domain of the antibody to produce aldehyde groups (Fig. 2).

◆ TROUBLESHOOTING
2
Incubate antibodies with periodate for 30 min at room temperature in the dark on a shaker at the rate of 350 rpm.

▲ CRITICAL STEP The reaction must be performed under dark conditions at room temperature and must not extend beyond 30 min, to avoid potential adverse effects on the bioactivity of the antibody.
3
Quench the reaction by adding 500 μl of 1× PBS (or ~50× the volume of sodium periodate solution used in a reaction depending on the batch size). Transfer the quenched reaction mixture into a 30-kDa MWCO centrifuge filter and centrifuge at 3,100g for 8 min to reduce the volume to ~200 μl. Add 2 ml of 1× PBS and repeat the process twice to wash the residual oxidizing agent. Finally, adjust the concentration to 1 mg ml⁻¹ in 1× PBS. The oxidized antibody cannot be stored and should be used in the next step within 30 min.

▲ CRITICAL STEP The solution must be mixed well after the addition of 1× PBS to ensure complete quenching.

Fig. 2 | — Glycosyl moieties on the Fc region of the antibody are mildly oxidized by sodium periodate (NaIO₄). In this step, 1,2-dihydroxy groups are converted into aldehyde groups.

Optional fluorescent labeling

4
This step is recommended if the antibody needs to be fluorescently labeled for flow cytometry analysis or fluorescence microscopy. Although manufacturers provide a protocol for fluorescent antibody labeling with commercial kits, we recommend verifying the antibody’s binding activity and optimizing the fluorescence labeling using ELISA (Supplementary Methods, sections II and III and Supplementary Tables 1–3).

Quality control

4
Perform the Purpald assay to verify the oxidation of the carbohydrate moiety and formation of aldehyde groups. Mix 20 μl of the antibody solution with 100 μl of freshly prepared Purpald solution (10 mg ml⁻¹ in 1 N sodium hydroxide). The color of the solution should turn purple (with λ_max = 550 nm) in 15–20 min to indicate the presence of aldehydes. This step is used to confirm the presence of oxidizable diol groups in the glycosyl antibody’s moiety before conjugation. The unmodified antibody in 1× PBS at the same concentration as the oxidized antibody (i.e., 0.5 mg ml⁻¹) can be used as a non-treatment negative control, and 1× PBS buffer can be used as a blank control. We observed an increase in absorbance at 550 nm for the oxidized antibodies of at least threefold, respectively, relative to the unmodified antibody controls (Supplementary Methods, section IV). The Purpald assay can also be performed quantitatively if needed by using formaldehyde as a standard^95,96.

▲ CRITICAL STEP The Purpald solution must be freshly prepared and should be used within 1 h of preparation.

◆ TROUBLESHOOTING

Stage 2: attaching a PEG linker to antibodies

● TIMING 1 h 30 min

4
Add 2.0 μl of 46.5 mM aminoxy-PEG-DBCO linker in DMSO per 100 μl of 1 mg ml⁻¹ oxidized antibody in 1× PBS (from Step 3) and immediately mix the solution using a 200-μl pipette. Incubate the reaction mixture for 1 h in the dark at room temperature on a shaker at 350 rpm (Fig. 3).

▲ CRITICAL STEP PEG linkers can be dissolved in DMSO at 46.5 mM concentration and can be stored at −20 °C in 500-μl centrifuge tubes as 10-μl aliquots. After using a portion of each aliquot, discard the rest of the linker solution if it is not immediately used or needed for other reactions.
5
After 1 h, add 2 ml of 1× PBS and transfer the reaction mixture to a 30-kDa MWCO centrifuge filter and centrifuge at 3,100g for 8 min. Perform this washing step two more times.
6
Add 2 ml of 40 mM HEPES buffer (pH 8.5) to the residual volume of the antibody solution in the centrifuge filter, mix the solution using a pipette and centrifuge at 3,100g for 8 min.
7
Add 500 μl of 40 mM HEPES buffer to the residual volume of antibody solution from Step 8.
8
Measure the concentration of the antibody using absorbance at 280 nm and Eq. 1 in Box 1. Note that DBCO groups of the conjugated linker molecules contribute to the overall absorbance of antibody–linker conjugates at 280 nm. We determined that this contribution accounts for ~5% of the total absorbance of linker–aEGFR conjugates, which corresponds to approximately two linker molecules per modified aEGFR antibody. Similarly, in the case of aFRα antibody, DBCO functional groups account for 8% of the total absorbance of the linker–antibody conjugate, and each modified aFRα antibody had about three linker molecules (Supplementary Methods, section V). The absorbance of attached linker molecules should be subtracted from the overall absorbance of linker–antibody conjugates before using Eq. 1 to calculate the antibody’s concentration. Follow the steps in Box 2 to determine this correction factor for a new antibody.

▲ CRITICAL STEP Mix the antibody solution well using a pipette for homogeneous dispersion of the antibodies in the solution before measuring the concentration. Poor mixing of the solution can result in an error in the antibody’s concentration measurement.

■ PAUSE POINT The antibody–linker solution can be stored at 4 °C for the recommended duration for a specific monoclonal antibody.

Fig. 3 | — In this step, the aldehyde groups on the glycosyl moiety of the oxidized antibody react with the aminoxy group of the aminoxy-PEG-DBCO linker.

BOX 2. Optional protocol to determine the number of linker molecules attached to the antibody.

Make a calibration curve by measuring the absorbance of serial dilutions of NH₂-O-PEG-DBCO linker molecules from 0 to 3.10 × 10⁻⁵ M in 1× PBS.
Make a solution of NH₂-O-PEG-DBCO molecules in 1× PBS at the same concentration as in the antibody conjugation reaction. Measure its concentration using the calibration curve in step 1 of this Box; this is the initial concentration of the linker molecules.
After conjugation of NH₂-O-PEG-DBCO with an antibody, add 2 ml of 1× PBS to the reaction mixture and transfer the entire solution to a centrifuge filter.
Centrifuge at 3,100g for 8 min, collect the filtrate with the unbound NH₂-O-PEG-DBCO linker molecules in a separate container and label it.
Repeat the washing step two more times with 2 ml of 1× PBS for each wash, collect the filtrates in different containers and label them.
Measure the absorbance of all supernatants at 280 nm.
Estimate the total amount of collected NH₂-O-PEG-DBCO linker molecules using the calibration of absorbance of serial dilutions of NH₂-O-PEG-DBCO in 1× PBS from step 1 of this Box.
Determine the total amount of conjugated NH₂-O-PEG-DBCO linker molecules by subtracting the total amount of the collected linkers (determined in the previous step) from the initial amount of the linker molecules (determined in step 2 of this Box).
Calculate the total number of linkers per antibody by dividing the total amount of conjugated linkers in moles from the previous step by the amount of antibodies in moles that was used during the conjugation reaction.

Stage 3: conjugation of antibodies to nanoparticles by metal-free click chemistry

● TIMING 16–24 h

▲ CRITICAL In this stage, the antibody with the linker containing the DBCO group is conjugated to the nanoparticle surface functionalized with azide groups (Fig. 4). Nanoparticles are synthesized and functionalized as described in ref. 43.

Fig. 4 | — Azide groups on the nanoparticle surface and DBCO groups on the antibody undergo a strain-promoted alkyne-azide cycloaddition¹⁰⁷ reaction, also known as a ‘metal-free click reaction’.

Quality control of nanoparticles before antibody conjugation

▲ CRITICAL The absorbance spectra and DLS measurements of ICGJ-PSs are used to determine the quality of the nanoparticles before antibody conjugation.

4
Determine the OD of the stock suspension of ICGJ-PS nanoparticles by measuring their UV–visible–near infrared (NIR) absorbance spectra. The ratio of absorbance at 895 nm (A₈₉₅) to absorbance at 780 nm (A₇₈₀) can be used as a surrogate measure of the integrity of the polymersome’s shell, which prevents dissociation of the encapsulated ICG J-aggregates, and, based on our experience, it should be >4.0 (Fig. 5a).

▲ CRITICAL STEP This step is always performed every time that we take aliquots for antibody conjugation, to ensure that the concentration of the nanoparticles is known at each use.

Fig. 5 | — a, UV–visible–NIR absorbance spectra of ICGJ-PS, ICGJ-PS-aEGFR and ICGJ-PS-aFRα showing almost dentical spectral properties; note that the spectrum of ICGJ-PS overlaps with the spectrum of ICGJ-PS-aEGFR and ICGJ-PS-aFRα. b and c, DLS of ICGJ-PS before and after conjugation with a-EGFR and a-FRα antibodies, respectively. d and e, Corresponding number distribution DLS of ICGJ-PS-aEGFR and ICGJ-PS-aFRα, respectively. After a successful conjugation, the polydispersity index (PDI) of nanoparticle conjugates is expected to be ≤0.3, as shown in b and c. It should be noted that aggregate peaks were visible in the volume distribution of DLS in b and c. However, the number distribution showed only unimodal distribution (d and e). This indicates that the apparent aggregation seen in volume distribution is numerically insignificant relative to the non-aggregated ICGJ-PS conjugates. f, Photographs of ICGJ-PS, ICGJ-PS-aEGFR and ICGJ-PS-aFRα showing examples of uniform nanoparticle suspensions on the left and an aggregated sample on the right. The suspensions should look uniform and be without clumps under a visual examination (f). Source data are provided as Supplementary Information. D_h, hydrodynamic diameter.

4
Use DLS to determine the size and heterogeneity of nanoparticles in solution (Fig. 5b–e). Visually inspecting the suspension of nanoparticles for inhomogeneity due to flocculation or instability during storage is also recommended (Fig. 5f). The quality criteria for DLS of polymersomes that we used were the volume average size of polymersomes between 68 and 77 nm and the polydispersity index (PDI) of nanoparticles ≤0.3 (see Supplementary Methods, sections VI and VII for a detailed description of DLS measurements).

Antibody-nanoparticle conjugation by metal-free click chemistry

4
Mix the required amount of antibody solution with the nanoparticle suspension for conjugation. This step needs to be optimized for a specific type of nanoparticle or antibody experimentally. The calculations for ICGJ-PSs used in our studies is as follows. On the basis of the concentration of nanoparticles (see Experimental design), calculate the amount of antibodies that needs to be added to achieve the desired ratio of antibody molecules per nanoparticle in the reaction mixture. This ratio is determined experimentally as described in Supplementary Methods, section VIII. The amount of antibodies or nanoparticles needed for a conjugation can be calculated as described in Box 3.
5
In a typical reaction with 300 OD (i.e., ~4.5 × 10¹² nanoparticles ml⁻¹) ICGJ-PS stock solution, add 40 μl of 0.46 mg ml⁻¹ antibody solution in 40 mM HEPES buffer (pH 8.5) to 81 μl of the ICGJ-PS stock in water to achieve the ratio of ~169 aEGFR antibodies per nanoparticle in the reaction. Note that this ratio can be adjusted by increasing the concentration or volume of the antibody solution. Mix the suspension using a 200-μl pipette and incubate the reaction mixture at 4 °C overnight (i.e., for a minimum of 16 h to a maximum of 24 h).

▲ CRITICAL STEP Incubating the nanoparticle–antibody reaction mixture beyond 24 h may result in a non-specific attachment of the antibody to the nanoparticle surface.
6
Centrifuge the reaction mixture at 17,200g for 30 min at 4 °C to form a pellet of nanoparticles with unbound free antibodies in the supernatant. Nanoparticles that are not suitable for centrifugation can be purified using alternative methods such as tangential flow filtration, size exclusion chromatography or density gradient centrifugation with sucrose⁹⁷.

▲ CRITICAL STEP The centrifugation conditions depend on the stability, size and density of nanoparticles and, therefore, may need to be optimized for a specific type of nanoparticle.
7
Remove the supernatant using a pipette and wash the nanoparticles twice by resuspending the pellet in 1× PBS using the same volume as the reaction mixture (mix using a 200-μl pipette) and centrifuging the suspension at 17,200g for 30 min at 4 °C.
8
Reconstitute the final pellet in 160 μl of 1× PBS or 5% (wt/vol) glucose in deionized water to achieve the stock concentration of 100 OD or ~1.5 × 10¹² nanoparticles ml⁻¹ and store the nanoparticle–antibody conjugates (i.e., ICGJ-PS-Ab) at 4–8 °C in the dark.

▲ CRITICAL STEP Note that azide and DBCO functional groups are not naturally found in biological systems and do not have cross-reactivity in a biological environment^98,99. Therefore, we did not include a blocking step in this protocol. However, these groups could be quenched using methoxy PEG with either DBCO or azide functionality for blocking azide and DBCO moieties, respectively (see Supplementary Methods, section IX for blocking recommendations for an excess of azide or DBCO functional groups).

■ PAUSE POINT The nanoparticle–antibody conjugates are stable when stored at a high concentration (OD 100) in 1× PBS at 4 °C for ≤24 h.

BOX 3. Calculating the amount of antibodies or nanoparticles needed for a conjugation.

The amount of antibodies or nanoparticles needed for a conjugation can be calculated using the following equations:

R_{opt} = \frac{N_{Ab}}{N_{NP}}

(2)

where $N_{Ab}$ and $N_{NP}$ are the number of antibody molecules and the number of nanoparticles, respectively. $R_{opt}$ is the ratio of antibodies to nanoparticles, which is determined experimentally. This equation can be rewritten as follows:

R_{opt} = \frac{M_{Ab} \times N_{A}}{M W_{Ab} \times C_{NP} \times V_{NP}}

(3)

where $M_{Ab}$ is the total mass of the antibody in grams, $N_{A}$ is the Avogadro number, ${MW}_{Ab}$ is the molecular weight of antibody, $C_{NP}$ is the concentration of nanoparticles and $V_{NP}$ is the volume of the nanoparticle stock suspension that is used for conjugation. This equation can be rewritten as:

M_{Ab} = \frac{C_{NP} \times V_{NP} \times {MW}_{Ab} \times R_{opt}}{N_{A}}

(4)

Use the value of $M_{Ab}$ to calculate the volume of antibody for the conjugation:

Volume (ml) = M_{Ab} (mg) / Antibody concentration (mg / ml)

(5)

For conjugation of aEGFR antibodies, we determined that a ~169:1 antibody per nanoparticle ratio resulted in ligand surface coverage of ~22% (Supplementary Methods, section VIII, Supplementary Table 4 and calculations in “Anticipated results”), which was reported to be near the optimal density for achieving the maximum efficiency of cell labeling in vitro and of tumor delivery of nanoparticle conjugates in vivo^84,87,88,113. We recommend a titration-based approach for other types of nanoparticles and antibodies to arrive at the optimum ratio to achieve the desired antibody surface coverage (Supplementary Methods, section VIII).

Therefore, in the case of aEGFR antibodies, we used Ropt = 169 and MWAb = 170,000 g mol⁻¹. Furthermore, considering the concentration of ICGJ-PS of −1.5 × 10¹⁰ nanoparticles ml⁻¹ per 1 OD (Supplementary Methods, section XI and Supplementary Table 5), we arrive at the following equation for the total amount of the antibodies in milligrams:

M_{Ab} = \frac{{OD}_{NP} \times V_{NP}}{1.4 \times 10^{3}}

(6)

Eq. 5 is then used to calculate the required volume of the antibodies. V_NP is the volume of nanoparticle suspension in milliliters.

Quality control and characterization of the conjugates

4
Use UV–visible–NIR spectroscopy (see Supplementary Methods, section I for details) to evaluate any potential changes in the optical properties of ICGJ-loaded polymersomes that are indicative of destabilization of the polymersome’s shell (Fig. 5a).
5
Use DLS (see Supplementary Methods, section VI for details) to evaluate the size and polydispersity of the conjugates (Fig. 5b,c). The samples with moderately aggregated conjugates exhibited a PDI >0.3 in DLS measurements (Supplementary Methods, section X). The autocorrelation function intercept in DLS measurement of a nanoparticle suspension should be ≥0.8 to obtain an acceptable quality of DLS data.

▲ CRITICAL STEP In case of nanoparticles with a strong absorbance or fluorescence in the wavelength range that is used for DLS measurements, NTA can be used as an alternative method to measure size distribution (see Supplementary Methods, section XI for more details).
6
Use TEM or cryo-TEM (see Supplementary Methods, section XII for details) to assess morphology, which should reveal that the nanoparticles have a similar morphology before and after the antibody’s conjugation (Fig. 6); note that antibodies do not provide sufficient contrast to be visible in TEM or cryo-TEM. The zeta potentials of unmodified ICGJ-PS, ICGJ-PS-aEGFR and ICGJ-PS-aFRα were −14.9, −16.6 and −15 mV, respectively, which is consistent with previously published data⁴².

◆ TROUBLESHOOTING
7
To determine the number of antibodies conjugated to nanoparticles, we recommend a sandwich ELISA using protein-L–coated ELISA plates that are specific to the Fab moiety of antibodies (Fig. 7) and using the protocol described in Box 4.

Fig. 6 | — **a–c**, ICGJ-PS (a), ICGJ-PS-aEGFR (b) and aggregated sample of ICGJ-PS-aEGFR (c). There were no apparent changes in the morphology of nanoparticles between a and b, indicating that the antibody’s conjugation does not have a significant impact on either the structure of the nanoparticles or their agglomeration.

Fig. 7 | — a, An illustration of a sandwich ELISA used for determining the amount of antibodies conjugated to nanoparticles. b and c, ELISA response curves for ICGJ-PS-aEGFR (b) and ICG-PS-aFRα (c). Note differences in the concentration range for ICGJ-PS-aEGFR (OD 0–2 corresponding to 0–2.4 × 10¹⁰ particles ml⁻¹) and ICGJ-PS-aFRα (OD 0–10 corresponding to 0–12.0 × 10¹⁰ particles ml⁻¹) conjugates. The control of ICGJ-PS without conjugated antibodies was used as a reference for baseline correction. ICGJ-PS-aEGFR contained 23 a-EGFR antibodies per nanoparticle after the first wash, and the conjugates retained 21 (91.3%) a-EGFR antibodies per nanoparticle after four consecutive washes. ICGJ-PS-aFRα contained 17 a-FRα antibodies per particle after the first wash and retained 13 (76.5%) a-FRα antibodies per particle after four washes. Each point in b and c represents the mean (n = 2), and the error bars represent the s.d. The illustration in a was Created in BioRender. Source data are provided as Supplementary Information.

BOX 4. ELISA of nanoparticle–antibody conjugates.

Blocking non-specific interactions on the ELISA plate

● TIMING 3–5 h at room temperature or 12 h at 4 °C

1
Add 300 μl of the blocking buffer to each well of a protein-L–coated 96-well ELISA plate (these are 96 well plates that are precoated with bacterial protein-L, which has affinity toward the light chains of IgG antibodies) and cover the plate with Parafilm. Incubate the plate at 4 °C overnight or for 3–5 hours at room temperature. The purpose of this step using blocking buffer is to block potential non-specific binding on the L-protein–coated wells.

▲ CRITICAL STEP Use 10-ml pipettes during preparation of blocking buffers and a 300-μl multichannel pipette during the blocking step to minimize error and contamination at this step.

Incubation of samples and calibration standards on the ELISA plate

● TIMING 12–15 h

2
Remove the blocking buffer by directly inverting the plate on a waste container and then tapping the plate inverted on a folded lint-free wipe to remove residual droplets of the blocking buffer.
3
Next, wash the plate three times with 300 μl of washing buffer. First, use a multichannel pipette to add 300 μl of washing buffer into the wells, wait for 1 min and remove the wash buffer by inverting the plate directly into a waste container followed by tapping the plate in inverted position on a lint-free wipe. Repeat the washing step to complete three repeats.
4
Prepare the reagent buffer for dilution of samples and standards. To this end, mix 5 ml of the blocking buffer with 45 ml of the wash buffer. The final reagent buffer composition is 0.1% BSA (wt/vol) in 1× PBS with 0.045% (vol/vol) Tween 20.
5
Prepare standard serial dilutions of a free antibody in the reagent buffer. A series of eight different concentrations with a dilution factor of 2 need to be prepared. For example, the standard serial dilutions of aEGFR antibodies were prepared in triplicate in the range of 500 to 0 ng ml⁻¹ (typically 500, 250, 125, 62.5, 31.25, 15.62, 7.81 and 0 ng ml⁻¹). Similarly, the standard solutions of aFRα were prepared in triplicate in the range of 10,000 to 0 ng ml⁻¹. For the 0 ng ml⁻¹ point, the equivalent volume of the reagent buffer is used.

▲ CRITICAL STEP The concentrations of standard serial dilutions can vary for different antibodies on the basis of the sensitivity of ELISA to the given antibody and require prior optimization in a wider concentration range.

2
Prepare a series of up to eight serial dilutions of a sample in the reagent buffer with a dilution factor of 2. Note that the concentration range for nanoparticle–antibody conjugates could be different for different antibodies and nanoparticles. For example, the concentration range for ICGJ-PS-aEGFR conjugates used in this study was from OD 2.0 to 0 (corresponding to ~3.0 × 10¹⁰ to 0 nanoparticles ml⁻¹), and the concentration range for ICGJ-PS-aFRα was from OD 10 to 0 (corresponding to ~1.5 × 1011 to 0 nanoparticles ml⁻¹). The concentration range of nanoparticle–antibody conjugates should be optimized to receive a sigmoidal ELISA response curve.
3
Add 100 μl of each dilution for the samples and the standards to the pre-blocked ELISA wells in triplicate. We recommend using at least three replicates for each dilution of samples and standards. Incubate the plates overnight at 4 °C. Parafilm or an ELISA plate cover must be used to avoid evaporation during incubation.

Application of a secondary antibody and reading out of ELISA results

● TIMING 3–6 h

2
Remove the solutions by inverting the ELISA plates and wash the plates three times with 300 μl of the wash buffer as described in steps 2 and 3 of this Box.
3
Dilute the secondary antibody solutions 2,000 times in the reagent buffer (typically 6 μl of a secondary antibody stock solution in 12 ml of the reagent buffer). The secondary antibodies used here were goat anti-human IgG-HRP conjugate (for ICGJ-PS-aEGFR) and goat anti-mouse IgG-HRP conjugate (for ICGJ-PS-aFRα), which are specific for the humanized aEGFR and the mouse aFRα antibodies, respectively.
4
Add 100 μl of a corresponding secondary antibody solution to each well and incubate the ELISA plate at room temperature for 3 h. Cover the ELISA plate with Parafilm during this step.
5
Remove the secondary antibody solution and wash the wells three times with 300 μl of the wash buffer. Use the same procedure as described in steps 2 and 3 of this Box.
6
Add 100 μl of TMB one solution and wait for 5–10 min for a blue color to develop.
7
After the blue color is prominently visible, quench the HRP oxidation of TMB by quickly adding 50 μl of 0.5 N HCl to every well of the ELISA plate. The color should instantly turn yellow upon the addition of 0.5 N HCl.

▲ CRITICAL STEP This step must be performed using a multichannel pipette within a 60–90-s interval.

2
Measure the optical density of each well at 450 nm (A₄₅₀) and 570 nm (A₅₇₀). Calculate the difference between A₄₅₀ and A₅₇₀ (A_450–570). The ELISA standard plot and sample response curves are generated by plotting the values of A_450–570 as a function of the concentration of the serial dilutions of free antibodies and of nanoparticle conjugates, respectively (Fig. 7).

◆ TROUBLESHOOTING

2
Determine the number of antibodies per nanoparticle by fitting a polynomial equation to the standard curve (the corresponding average A_450–570 versus known concentrations of free antibody) as follows. First, plot the A_450–570 values (x axis) of the wells with serial fee antibody dilutions (step 5 of this Box) versus the antibody’s concentration in milligrams per milliliter (y axis). This is the standard curve.
3
Determine the best polynomial or linear fit for the standard curve with y intercept = 0 that results in the best R² value (typically ≥0.9).
4
Determine the linear portion of the ELISA sample response curve (from step 14 of this Box) and calculate the corresponding antibody concentration for each nanoparticle conjugate dilution in the linear region by using the fitting equation from the previous step.
5
Determine the number of antibodies per nanoparticle conjugate by averaging the values obtained in the previous step (see Supplementary Methods, section XIII for additional details).

▲ CRITICAL STEP Use only the linear region of the sample response curve to calculate the amount of antibodies.

▲ CRITICAL STEP The buffers for ELISA should be used within 72 h after preparation and should be stored in a refrigerator at 4 °C before the use.

◆ TROUBLESHOOTING

Evaluation of the antibody–nanoparticle conjugates for selective labeling of cells

● TIMING 12–14 h

▲ CRITICAL To assess the specificity of the nanoparticle–antibody conjugates for selective labeling of cells with overexpression of a specific receptor, nanoparticles conjugated with fluorescently labeled antibodies (see Step 4) are used to enable implementation of flow cytometry and fluorescence confocal microscopy to confirm specificity of cell labeling.

4
To evaluate the molecular specificity of cell labeling with ICGJ-PS-aEGFR conjugates, fix the high-EGFR-expressing (EGFR⁺) MDA-MB-468 cells and low-EGFR-expressing (EGFR⁻) MCF-7 cells by suspending the cells in 4% (wt/vol) paraformaldehyde in 1× PBS solution for 5 min (at concentration of 1 × 10⁶ cells ml⁻¹).
5
Wash the fixed cells twice by suspending the cells in 1 ml of 1× PBS using a micropipette and centrifuging the cells at 300g for 5 min at room temperature to remove excess paraformaldehyde.
6
Incubate 4 × 10⁵ cells (MDA-MB-468 or MCF7 cells) with ICGJ-PS-aEGFR-AF488 conjugates at a concentration of 7.92 × 10¹⁰ (OD = 6.6) particles in 1 ml of cell culture medium for 3 h at room temperature.
7
In addition to EGFR-positive and -negative cells, include Fc blocking control, nanoparticles conjugated with non-targeted IgG1 isotope control and a blocking assay with free aEGFR antibodies to confirm the specificity of the conjugates (Fig. 8a,b). Evaluate the molecular specificity of antibody–nanoparticle conjugates using flow cytometry as described in Box 5.
8
Evaluate the molecular specificity of ICGJ-PS-aFRα conjugates as described in Steps 22–26 using SKOV3 and A2780 cells with high and low FRα expression, respectively¹⁰⁰(Fig. 8c,d).

◆ TROUBLESHOOTING

Fig. 8 | — ICGJ-PS-aEGFR-AF488 and ICGJ-PS-aFRα-AF488 conjugates were synthesized using aEGFR and aFRα antibodies, respectively, and pre-labeled with AF488 dye to assess cell labeling efficiency using flow cytometry. Nanoparticle conjugates with fluorescently labeled nonspecific IgG1 antibodies were used as an antibody isotype control. Flow cytometry of MDA-MB-468 EGFR⁺ cells (a) and MCF-7 EGFR⁻ cells (b) showed EGFR-specific labeling of MDA-MB-468 cells by ICGJ-PS-aEGFR conjugates and no significant labeling of MCF-7 cells. c and d, SKOV3 cells with a high FRα⁺ expression (c) and A2780 cells with a low FRα⁻ expression (d) labeled with ICGJ-PS-aFRα-AF488 conjugates. As expected, A2780 cells exhibited significantly lower labeling efficiency with ICGJ-PS-aFRα-AF488 conjugates than did SKOV3 cells, which correlates well with the expression profile of these cell lines¹⁰⁰. Note the efficient blocking of cell labeling by free aEGFR and aFRα antibodies (green curves in a and c, respectively). Fluorescence of cells labeled with antibody isotype control (a and c, orange curves) overlapped with the free antibody blocking controls, indicating a low non-specific binding of conjugated nanoparticles. Furthermore, the Fc blocker control did not show a substantial decrease in fluorescence labeling with the targeted nanoparticles (a and c, purple curves). Note that the lower fluorescence intensity of labeled SKOV3 cells in comparison with MDA-MB-468 cells was probably associated with the lower expression level of FRα (i.e., ~87,000 receptors per cell) by SKOV3 cells¹⁰⁸ relative to the EGFR expression of ~1.9 × 10⁶ per MDA-MB-468 cell¹⁰⁹ (see Supplementary Table 6 for the precise fluorescence intensity of peaks and Supplementary Figures 8–11 for the gating strategy). Source data are provided as Supplementary Information.

BOX 5. Protocol for evaluation of the molecular specificity of antibody–nanoparticle conjugates using flow cytometry.

Cell preparation

1
Culture ~2 million of MDA-MB-468, MDA-MB-231, SKOV3 or A2780 cells in a recommended cell culture medium to ~90% confluence. We used the cell culture media listed in Reagents.
2
Remove the cell culture medium by pipetting and replace it with 5–7 ml of 0.25% (wt/vol) trypsin solution and incubate at 37 °C for 5–8 min in a cell culture incubator.
3
Add 5–7 ml of fresh medium (i.e., equal to the volume of trypsin solution in the previous step) and mix thoroughly by repeated pipetting to detach the cells, transfer the cells to a 15-ml centrifuge tube, centrifuge at 300g for 5 min at room temperature to obtain the cells as a pellet and carefully remove the supernatant by pipetting without disturbing the pellet.
4
Resuspend the cells in 2 ml of 1× PBS. Count the cells using trypan blue staining on a hemocytometer or cellometer, centrifuge the suspension at room temperature at 300g for 5 min at room temperature, remove the supernatant by pipetting without disturbing the cell pellet and discard it.
5
Resuspend the cell pellet in a freshly prepared solution of 4% (wt/vol) paraformaldehyde in 1× PBS at a concentration of ~1 × 10⁶ cells ml⁻¹ and incubate for 10 min at room temperature.
6
Wash the cells twice by centrifugation at 300g for 5 min in the same volume of 1× PBS as the initial suspension in paraformaldehyde and resuspend the washed fixed cells in fresh medium at 4 × 10⁵ cells ml⁻¹ (see Reagents for the recommended medium).

Cell labeling using antibody–nanoparticle conjugates and controls

7
Transfer 1 ml of the cell suspension from the previous step to 1.5-ml centrifuge tubes.
8
Measure the OD of the stock solution of ICGJ-PS conjugate and calculate the volume of conjugates that is needed to achieve the final concentration of 6.6 OD in cell suspension from the previous step.
9
Add the required volume of ICGJ-PS conjugate to 1 ml of cell suspension (~4 × 10⁵ cells ml⁻¹ from step 7 of this Box) to get the final OD of 6.6 for the ICGJ-PS conjugates. Thoroughly mix the suspension by repeated pipette aspiration and incubate the mixture for 3 h at room temperature on a vortex shaker at 250 rpm.
10
Centrifuge the cells at 300g for 5 min, remove the supernatant by pipetting to remove the excess of nanoparticles without disturbing the cell pellet and discard the supernatant. Wash the cells again by resuspending the cell pellet in 1 ml of 1× PBS using a micropipette and centrifuge to obtain the labeled cells as a pellet.

▲ CRITICAL STEP This washing is necessary to remove excess ICGJ-PS from the cells to avoid artifacts in flow cytometry.

7
Resuspend the washed labeled cells in 500 μl of 1× PBS to get ~8 × 10⁵ cells ml⁻¹. This mixture is used for flow cytometry analysis of labeled cells.
8
Add ICGJ-PS-aEGFR-AF488 and ICGJ-PS-aFRα-AF488 conjugates at 6.6 OD, which corresponds to ~7.92 × 10¹⁰ particles ml⁻¹ to MDA-MB-468 and SKOV3 cells, respectively. Incubate the cells with nanoparticles for 3 h at room temperature with vortex shaking at 250 rpm.
9
For the antigen competitor control, preincubate 4 × 10⁵ fixed MDA-MB-468 and SKOV3 cells with 64 μg ml⁻¹ either free aEGFR or aFRα antibodies, respectively, for 30 min, followed by immediate addition of either ICGJ-PS-aEGFR-AF488 or ICGJ-Ps-aFRα-AF488 conjugates, respectively, at 6.6 OD and 3 h of incubation (i.e., ICGJ-PS-aEGFR + aEGFR Ab block in Fig. 8a and ICGJ-PS-aFRα + aFRα Ab block in Fig. 8c).
10
14. Prepare the Fc blocking controls by adding 60 μl of Fc receptor blocker (see Reagents) to 4 × 10⁵ of either MDA-MB-468 or SKOV3 cells suspended in their respective culture medium for 30 min. Then, wash the cells twice by suspending the cells in 1 ml of 1× PBS followed by centrifugation at 300g for 5 min at room temperature. Resuspend the washed cells in their cell culture medium and incubate with either ICGJ-PS-aEGFR-AF488 for MDA-MB-468 cells (Fig. 8a: ICGJ-PS-aEGFR + Fc block) or ICGJ-PS-aFRα-AF488 for SKOV3 cells (Fig. 8c sample: ICGJ-PS-aFRα + Fc block) at 6.6 OD concentration for 3 h at room temperature.
11
For isotype controls, conjugate either human or mouse IgG isotype controls (see Reagents) to ICGJ-PS using the protocol for preparation of ICGJ-PS-aEGFR or ICGJ-PS-aFRα with AF488 fluorescent labeling as described in Steps 1–17. Incubate MDAMB-468 and SKOV3 cells with ICGJ-PS-human IgG1 isotype and ICGJ-PS-mouse IgG1 isotype conjugates, respectively, using the same conditions as for the targeted nanoparticles described in steps 1–9 of this Box (Fig. 8a,c, orange graphs). Note that the human-IgG1 isotype and mouse-IgG1 isotype controls were fluorescently labeled with AF488 before conjugation by using the labeling conditions that were optimized for the corresponding targeted antibodies (Supplementary Methods, section III and Supplementary Table 3).

◆ TROUBLESHOOTING

7
Evaluate the molecular specificity of ICGJ-PS-aFRα conjugates using the same approach as described in steps 1–15 of this Box by using SKOV3 and A2780 cells with a high and low FRα expression, respectively¹⁰⁰ (Fig. 8c,d).
8
Fluorescence confocal images of cells with high and low expressions of corresponding receptors further confirm molecular specificity of antibody nanoparticle conjugates as shown in Fig. 9. Prepare the samples using conditions described for ICGJ-PS-aEGFR and ICGJ-PS-aFRα conjugates in Steps 11–17, respectively, using aEGFR and aFRα antibodies pre-labeled with AF488.
9
Wash no. 1.5 glass cover slips with 70% (vol/vol) ethanol and sterile water. Dry the coverslips in a biosafety cabinet at room temperature.
10
Place one coverslip per well into a sterile six-well plate and fill the wells with 750 μl of 0.01% (wt/vol) sterile filtered poly-l-lysine solution and incubate for 1 h.
11
Wash the coated coverslips with sterile deionized water and dry them in the biosafety cabinet at room temperature for 12 h.
12
Use the coverslips immediately to plate 200,000 MDA-MB-468, MDA-MB-231, SKOV3 or A2780 cells per coverslip, suspended in 500 μl of their respective culture medium. Apply the cells to the poly-l-lysine–coated side of the coverslips so that the medium with cells is confined within the surface of the coverslip.
13
After 12 h, remove the cell medium by pipetting and add 500 μl of 2.0 OD solution of antibody nanoparticle conjugates in a cell culture medium and incubate in a cell culture incubator at 37 °C for 12 h.
14
Remove the nanoparticle solution using a micropipette, gently wash the coverslips with 500 μl of 1× DPBS and add 500 μl of 4% (wt/vol) paraformaldehyde for 10 min to fix the adhered cells.
15
Wash the cells with 500 μl of 1× DPBS, add 500 μl of DAPI staining solution for 5 min and wash the coverslips with 500 μl of 1× DPBS.
16
Place a drop of fluoromount aqueous mounting medium on a clean microscope slide and gently place the coverslips with the adhered cells facing down on the fluoromount mounting medium.
17
Use the prepared sides for confocal fluorescence microscopy by following the procedure described in ref. 43.

Troubleshooting

Troubleshooting advice can be found in Table 1.

Table 1 |.

Troubleshooting

Step	Problem	Possible reason	Solution
Box 1 and Step 1	Buffer exchange takes too long to complete	Use of centrifuge filters with very low MWCO values	Use centrifuge filters with optimal MWCO for a reasonable filtration rate (0.25 ml min⁻¹). For aEGFR and aFRα antibodies in this study, a 30-kDa centrifuge filter is optimal, whereas filtration rates were too slow with a 10-kDa filter
5	The Purpald test shows no the antibody	The presence of cryoprotectant in the oxidation or very low oxidation of antibody sample	Wash the initial antibody solution with 6× volume excess of sodium phosphate buffer, using a centrifuge filter to remove the cryoprotectant
	Purpald does not dissolve fully at the concentration of 10 mg ml⁻¹ in the 1 N sodium hydroxide solution	The sodium hydroxide solution is not fresh	Prepare fresh sodium hydroxide solution
	The Purpald solution is already pink	The Purpald solution is not fresh	Prepare a fresh solution of Purpald in fresh 1 N sodium hydroxide
	The Purpald solution turned yellow instead of pink upon addition of antibody solution	Sodium peroxide is still present in the antibody solution	After antibody oxidation, wash the antibody solution with 3× volume of 1× PBS
	No observable change in color after the Purpald antibody oxidation assay	The antibody does not have oxidizable glycans	Test antibodies for glycosylation by using commercially available kits (e.g., Sigma-Aldrich, cat. no. GLYCOPRO-1KT), or a detailed analysis of the antibody’s glycosylation moieties can be done using commercial services (e.g., RayBiotech, cat. no. GA-Glycan-300–1)
20	The ICGJ-PS conjugates show severe or moderate aggregation	1× PBS was used to store the conjugates for >24 h	Use 5% (wt/vol) glucose in deionized water to avoid aggregation when storing the suspensions of conjugates for >24 h. If the sample is already aggregated, reverse the aggregation by using a two-step process: first, transfer the conjugates to 5% (wt/vol) glucose solution in deionized water. Next, centrifuge the suspension at 17,200g for 25 min, remove the supernatant and resuspend the conjugates back in 1× PBS or 5% (wt/vol) glucose solution based on the storage duration
Box 4, step 14	No color development is seen in ELISA	A wrong type of protein coating on the ELISA plate	Use the right ELISA plate for antibody capture after determining the type of antibody under study
		Use of the wrong secondary antibody	Use the HRP antibody conjugate that specifically binds to the isotype of the antibody under study
		Improper preparation of the reagent buffer	Cross-check the reagent buffer and the blocking buffer if the BSA is dissolved
	Very high background signal seen in ELISA	The amount of BSA in the blocking buffer is insufficient, or the wrong grade of BSA was used The secondary antibody solution was too concentrated	Verify that the right quantity of the correct grade of BSA in the blocking buffer. The BSA blocks nonspecific interaction on the was used ELISA well plate Consult the manufacturer’s instructions for dilution in ELISA
	The absorbance at 450 nm corresponding to the wells with maximum concentrations is saturated	Longer than optimal wait time between the addition of TMB and quenching the addition of TMB solution and quenching leads to intense color development	Try a shorter duration between with 0.5 N hydrochloric acid
	The A_(450–570) absorbance value or the number of antibodies per nanoparticle value is negative	This can be due to the background absorbance of nanoparticles attached to the ELISA plate, to the absorbance value at 570 nm, or to other variations in the baseline absorbance	Use the A_(450–570) values from the control wells with antibodyconjugated nanoparticles that were not incubated with TMB solution (Step 12 of the ELISA protocol; Box 4) to correct for the intrinsic absorbance of nanoparticles at 570 nm During the curve-fitting process of the standard ELISA response curve, set the intercept with the y-axis to zero
22–26 and Box 5, steps 7–16	Conjugated nanoparticles do not label the cells	Modifications of the antibody (i.e., oxidation and/or fluorescent labeling) before conjugation result in a decreased affinity of antibodies	Use ELISA to optimize the time and amount of fluorescent labeling to ensure that the affinity of the antibody is retained after fluorescent labeling (see Supplementary Methods, section III)
		No nanoparticle conjugation due to interference of sodium azide present in the antibody’s solution	An elution buffer that is provided as part of an antibody’s labeling kit could contain 2.0 mM sodium azide as a preservative, which would interfere with the antibody’s conjugation
		Bleaching of the fluorescent label	Wash the fluorescently labeled antibody after fluorescent labeling in 1× PBS using 30-kDa centrifugal filters Always perform the oxidation step before fluorescent labeling. Oxidation of fluorescently labeled antibodies can lead to bleaching of fluorescent labels and loss of fluorescence signal Always store the fluorescently labeled antibodies in the dark to prevent photobleaching of fluorescent labels Store the ICGJ-PS-antibody conjugates under refrigeration in 1× PBS suspensions at or above the concentration equivalent to an OD of 100 (~1.5 × 10¹² particles ml⁻¹) to avoid deactivation. Avoid frequent thawing of the samples and avoid sonication of ICGJ-PS-antibody conjugates
	High levels of non-specific cell labeling are observed in the case of isotype controls	Use of sub-optimal cell-labeling conditions	Fully supplemented cell culture medium is recommended to minimize non-specific interactions

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Timing

Steps 1–5, oxidation of the antibody: 40 ± 16 min depending on the need for buffer exchange and concentration determination before reaction

Steps 6–10, attaching the PEG linker: 1 h 30 min

Steps 11–17, conjugation of antibodies to the polymer nanoparticles: 16–24 h

Steps 18–19, quality control and characterization by UV-visible-NIR spectroscopy and DLS: 30 min–1 h

Step 20, TEM or cryo-TEM characterization: 12–14 h

Step 21, ELISA to determine the number of antibodies: 18–26 h

Steps 22–26, in vitro evaluation stage: 12–14 h

Box 4, step 1, blocking non-specific interactions in the ELISA plate: 3–5 h at room temperature, 12 h at 4 °C

Box 4, steps 2–7, incubation of samples and standards in the ELISA plate: 12–15 h

Box 4, steps 8–14, adding secondary antibody and reading out the ELISA: 3–6 h

Anticipated results

Here, we present a protocol for the directional conjugation of monoclonal antibodies to polymersome nanoparticles (i.e., ICGJ-PSs), quality control of the nanoparticle–antibody conjugates, estimation of antibody conjugation efficiency using ELISA and evaluation of the molecular specificity of the antibody-targeted nanoparticles. ICGJ-PS-Ab conjugates prepared using this protocol are expected to have the same absorbance spectrum as the initial unconjugated nanoparticles, with the ratio of the absorbance at 895 nm to that at 780 nm (A_{895 nm}/A_{780 nm}) greater than 4.0 (Fig. 5a). This high value of A_{895 nm}/A_{780 nm} indicates that the most ICG molecules in the polymersomes are present as J-aggregates^42,101. Comparison of sizes of the initial ICGJ-PS nanoparticles and of their conjugates with a-EGFR (i.e., ICGJ-PS-aEGFR) and aFRα (i.e., ICGJ-PS-aFRα) antibodies by DLS showed a consistent ~20-nm increase in the hydrodynamic diameter (Fig. 5b,c). The Fc moieties of aEGFR and aFRα antibodies are ~10.55 nm¹⁰² and ~11.7 nm¹⁰³, respectively. In addition, the PEG linker with a molecular weight of 3 kDa has a Flory radius of ~3.5 nm¹⁰⁴. The sum of the Fc moiety and the PEG linker lengths would suggest a larger size increase than the DLS-measured increase of ~10-nm radius. However, the conjugated antibodies are expected to be tilted relative to the nanoparticle’s surface because glycosylated moieties on IgG1 antibodies (i.e., the location where the PEG linkers are conjugated) are generally located in the CH₂ region of the Fc domain¹⁰² rather than at the end of the antibody’s stem. This arrangement can explain the lower increase in the diameter of the conjugated nanoparticles than the one that would be expected for perfectly aligned antibodies with their Fc moieties perpendicular to the surface of the nanoparticles. Furthermore, TEM can be used as an effective tool to confirm that the conjugation does not change the morphology of the underlying ICGJ-PS⁴³ (Fig. 6).

We evaluated the potential cross-linking of nanoparticle conjugates due to the presence of an excess of azide groups on nanoparticles and multiple DBCO functionalities on modified antibodies. To this end, we carried out NTA on a typical batch of ICGJ-PS before and after conjugation of either aEGFR or aFRα antibodies (Supplementary Methods, section XI and Supplementary Fig. 6). NTA did not detect any large particles in the conjugates, confirming that they are present only in a minute quantity. In addition, we did not see any significant differences in particle concentrations between the unconjugated and conjugated polymersomes (Supplementary Table 5), which further confirmed the lack of aggregation or cross-linking that would have been associated with a decrease in particle number concentration.

We developed an ELISA assay to quantify the number of antibody molecules conjugated to nanoparticles. This assay was used to evaluate and optimize the protocol (Table 2). We showed that the 30-min antibody oxidation duration in Step 2 resulted in significantly better conjugation efficiency and antibody retention after four washings than the 5-min oxidation duration. Therefore, the 30-min oxidation was chosen for our protocol. We also evaluated the level of non-specific attachment of oxidized antibodies without linker molecules to the polymersomes (Table 2). When antibodies oxidized for a 30-min duration (without any PEG linker modification) were incubated with ICGJ-PS, only ~27% and ~52% of a-FRα and a-EGFR antibodies, respectively, were retained after four consecutive washings; this is significantly lower than the retention of ~76.5% and ~91.3% for the linker-modified a-FRα and a-EGFR antibodies, respectively. The data indicate that the linker-modified antibodies are predominantly covalently conjugated to the nanoparticles; otherwise, we would have observed significantly lower retention values for these antibodies.

Table 2 |.

ELISA estimates of antibodies conjugated per ICGJ-PS under various treatment conditions of ICGJ-PS with aEGFR and aFRα

Sample^a,b	Antibody oxidation time (min)^c	Number of washes with 500 μl of 1× PBS^d	Number of antibodies per ICGJ-PS particle (rounded off to the nearest whole number) estimated by ELISA
ICGJ-PS-aFRɑ	30	1	17
ICGJ-PS-aFRɑ	30	4	13
ICGJ-PS-aFRɑ	5	1	11
ICGJ-PS-aFRɑ	5	4	9
ICGJ-PS physical mixture with aFRɑ	30	1	11
ICGJ-PS physical mixture with aFRɑ	30	4	3
ICGJ-PS physical mixture with aFRɑ	5	1	15
ICGJ-PS physical mixture with aFRɑ	5	4	6
Unmodified ICGJ-PS	–	–	0
ICGJ-PS-aEGFR	30	1	23
ICGJ-PS-aEGFR	30	4	21
ICGJ-PS-aEGFR	5	1	14
ICGJ-PS-aEGFR	5	4	6
ICGJ-PS physical mixture with aEGFR	30	1	19
ICGJ-PS physical mixture with aEGFR	30	4	10
ICGJ-PS physical mixture with aEGFR	5	1	12
ICGJ-PS physical mixture with aEGFR	5	4	5
Unmodified ICGJ-PS	–	–	0

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ICGJ-PS-aEGFR or ICGJ-PS-aFRα indicates the covalently bonded conjugates of ICGJ-PS via click chemistry (carried out for 12 h), which retain most of the antibodies after repeated washing.

ICGJ-PS physical mixture with aFRɑ or aEGFR antibodies indicates that the oxidized antibodies without a linker were mixed and incubated with ICGJ-PS for the same duration of 12 h as was used during the covalent antibody conjugation.

The antibodies were oxidized for different time durations in Step 2.

The number of washes of unbound antibodies during Steps 15 and 16. The numbers of antibodies presented in this table represent the mean (n = 3).

A typical size of a human IgG1 antibody that closely corresponds to aEGFR antibody is 17.1 nm (the length of the Fab region) by 10.55 nm¹⁰². When assuming a circular footprint of an antibody molecule, the directional orientation with the Fab portion oriented outward and a 2D hexagonal close packing efficiency of 0.9069, the maximum number of aEGFR antibodies that can be accommodated on a single ICGJ-PS nanoparticle is ~107. On the basis of our evaluation of three different batches, the mean ± s.d. number of aEGFR antibodies per ICGJ-PS-aEGFR conjugate was ~23.3 ± 3.5 (see Supplementary Methods, section XVI and Supplementary Tables 7 and 8), which corresponds to ~21.8 ± 3.3% surface coverage. In the case of ICGJ-PS-aFRα conjugates, we calculated a maximum of 263 antibodies per nanoparticle on the basis of the dimensions of a mouse IgG1 antibody with 11.8-nm Fab and 11.7-nm Fc lengths¹⁰³. This corresponds to ~5.6 ± 1.1% surface coverage, considering an average of ~14.7 ± 2.9 conjugated aFRα antibodies per ICGJ-PS-aFRα based on the average of three different batches (see Supplementary Methods, section XVI and Supplementary Tables 7 and 8).

We compared our directional conjugation with a non-directional, commonly used NHS chemistry. To this end, we synthesized non-directional conjugates of aEGFR antibodies with ICGJ-PS by using an NHS-PEG-DBCO linker, which has the same molecular weight (i.e., 3 kDa) as the NH₂-O-PEG-DBCO linker used in the directional conjugation. In this chemistry, the NHS group interacts with primary amine groups on an antibody to introduce the DBCO functionality that is used for conjugation with azide groups on ICGJ-PS, resulting in conjugates with a limited orientational control of the attached antibodies (i.e., non-directional (ND) conjugation) – ICGJ-PS-ND-aEGFR.

The directional and ND conjugates were compared by ELISA with protein L as the capture molecule. Protein L specifically binds to the Fab portion of an IgG1 isotype antibody (e.g., aEGFR antibody used in this study). Therefore, any difference in the ELISA curves of ICGJ-PS-aEGFR and ICGJ-PS-ND-aEGFR is a direct result of Fab availability on these conjugates. We obtained binding curves to determine the K_D values for directionally and non-directionally conjugated nanoparticles of 2.71 × 10⁻² nM and 4.81 × 10⁻² nM, respectively (Supplementary Fig. 12 and Supplementary Table 9). Because K_D values correspond to the concentration of the Fab ligands required to saturate half of the available capture molecules¹⁰⁵, this result indicates that the half-binding point is achieved with ~1.77-fold less concentration of directionally conjugated polymersomes relative to the ND conjugates. Thus, the directional conjugation resulted in ~1.77 times as much Fab availability as the ND NHS-based conjugation. To evaluate if this result was due to differences in the number of conjugated antibodies, we determined that ICGJ-PS-aEGFR had ~1.3-fold more conjugated antibodies than ICGJ-PS-ND-aEGFR conjugates. After correction for this variance, the data indicated that our directional conjugation protocol resulted in ~36% more accessible Fab moieties than with ND carbodiimide/NHS chemistry (Supplementary Methods, section XVII).

Furthermore, we determined the dissociation constants of ICGJ-PS-aEGFR and of free unmodified aEGFR antibodies using ELISA with human EGFR as the capture molecule (Supplementary Methods, section III and Supplementary Table 10). Nanoparticle conjugates had K_D of ~5.3 × 10⁻³ nM (Supplementary Methods, section XVIII and Supplementary Table 10), which was significantly lower (i.e., greater avidity) than the K_D of ~1.5 × 10⁻¹ nM of free antibodies (Supplementary Methods, section III and Supplementary Table 2). This difference is due to a multivalency effect that was previously reported for nanoparticle conjugates^85,106. These data indicate the preservation of the affinity of conjugated antibodies.

Finally, we evaluated the specificity of antibody–nanoparticle conjugates in labeling breast (MDA-MB-468 and MCF-7) and ovarian (SKOV3 and A2780) cancer cells by flow cytometry and confocal fluorescence microscopy (Figs. 8 and 9). To this end, we conjugated the nanoparticles with fluorescently labeled antibodies. The fluorescent labeling did not interfere with antibody conjugation and could be easily implemented in our protocol at Step 4. The results for both antibody conjugates showed a high level of molecular specificity in cell labeling that was confirmed using cells with high and low expression of a respective molecular biomarker (EGFR or FRα) and by a blocking assay with an appropriate free antibody (anti-EGFR or anti-FRα) (Figs. 8 and 9). Conjugates of other types of nanoparticles prepared using our protocol are expected to show a similar specificity in cell binding.

Fig. 9 | — Top row, SKOV3 and A2780 cells with a high and low FRα expression labeled with ICGJ-PS-aFRα-AF488 conjugates. Bottom row, MDA-MB-468 (EGFR⁺) and MCF-7 (EGFR⁻) cells labeled with ICGJ-PS-aEGFR-AF488 conjugates. DAPI staining was used to highlight cell nuclei. The conjugates were detected using a 488-nm excitation laser with a hybrid detector, and DAPI was imaged using a 405-nm excitation laser with a standard photomultiplier tube detector. All images were acquired using a Leica HC PL APO CS2 40×/1.30 oil objective and represent maximum intensity projections.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Supplementary Material

Supplementary information

NIHMS2139065-supplement-Supplementary_information.pdf^{(2.9MB, pdf)}

Additional information

Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41596–025-01272–3.

Key points.

This Protocol Extension provides steps for the directional conjugation of monoclonal antibodies to azide-functionalized nanoparticles via the Fc antibody moiety using metal-free click chemistry, including procedures for synthesis, quality control and evaluation of nanoparticle–antibody conjugates.
The nanoparticle–antibody conjugates synthesized using this method are expected to display high molecular specificity toward epitopes while maintaining the physicochemical properties of the core nanoparticles.

Acknowledgements

This study was supported by National Institutes of Health (NIH) grant R01EB028762 and grants RP240311 and RP190131 from the Cancer Prevention & Research Institute of Texas (CPRIT). This study used MD Anderson’s Advanced Microscopy Core (supported by NIH S10RR029552) and Flow Cytometry and Cellular Imaging Core Facility (supported in part by The University of Texas MD Anderson Cancer Center and NIH grant P30CA016672). The authors gratefully acknowledge the Center for Electrochemistry, University of Texas at Austin, for providing research support for this work through the CEC Instrumentation Facility maintained under grant no. H-F-0037 from the Welch Foundation. The authors gratefully acknowledge Joseph Munch, Sr. Scientific Editor from the MD Anderson editorial service for editing this manuscript.

Footnotes

Competing interests

The authors declare no competing interests.

Peer review information Nature Protocols thanks Srikanth Singamaneni and the other, anonymous reviewer(s) for their contribution to the peer review of this work.

This protocol is an extension to Nat. Protoc. 3, 314–320 (2008): https://doi.org/10.1038/nprot.2008.1

Key references

Kawelah, M. R. et al. ACS Appl. Mater. Interf. 16, 5598–5612 (2024): https://doi.org/10.1021/acsami.3c16584

Changalvaie, B. et al. ACS Appl. Mater. Interf. 11, 46437–46450 (2019): https://doi.org/10.1021/acsami.9b14519

Data availability

Source data for Figs. 5, 7 and 8 are provided with this manuscript. Any additional data are available from the corresponding author upon request. Source data are provided with this paper.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary information

NIHMS2139065-supplement-Supplementary_information.pdf^{(2.9MB, pdf)}

Data Availability Statement

Source data for Figs. 5, 7 and 8 are provided with this manuscript. Any additional data are available from the corresponding author upon request. Source data are provided with this paper.

PERMALINK

Directional conjugation of monoclonal antibodies to nanoparticles using metal-free click chemistry

Ananthakrishnan S Jeevarathinam

Mohammed R Kawelah

Sangheon Han

Brian J Grindel

Steven W Millward

Keith P Johnston

Konstantin V Sokolov

Abstract

Introduction

Development of the protocol

Fig. 1 |. Overview of the proposed protocol for directional conjugation of monoclonal antibodies to nanoparticles.

Applications of the method

Comparison with other conjugation strategies

Methods with limited control of orientation

Methods with an intrinsic control of orientation (i.e., directional conjugation)

Other methods

Overview of the protocol

Quality control

Limitations

Experimental design

Determination of nanoparticle concentration

Optimal ligand density on nanoparticle–antibody conjugates

Materials

Reagents

Reagent setup

1× PBS pH 7.4 for antibody processing

Buffer to rinse cell specimens, 1× phosphate buffer with calcium and magnesium

10 mM sodium phosphate buffer solution pH 7.5

40 mM HEPES buffer pH 8.5

Solution of human aEGFR

BOX 1. Optional protocol for buffer exchange for removal of cryoprotectant from the antibody solution.

Solution of FRα monoclonal antibody

Sodium periodate (NaIO4) solution, 100 mM

Purpald solution for testing oxidation of the antibody

DAPI solution used for cell staining in confocal microscopy

Aminoxy-PEG-DBCO solution, 46.5 mM in dimethyl sulfoxide (DMSO)

Paraformaldehyde 4% in 1× PBS

Blocking buffer for ELISA

Washing buffer for ELISA

Reagent buffer for ELISA

Goat anti-human IgG-HRP and goat anti-mouse IgG-HRP

DMEM/F-12 medium 1:1 mixture, EMEM and RPMI-1640 high-glucose medium

Equipment

Procedure

Stage 1: modification of antibody

Oxidation of the antibody

Fig. 2 |. An illustration of the antibody oxidation step.

Optional fluorescent labeling

Quality control

Stage 2: attaching a PEG linker to antibodies

Fig. 3 |. Schematic representation of conjugation of the aminoxy-PEG-DBCO linker to the oxidized antibody.

BOX 2. Optional protocol to determine the number of linker molecules attached to the antibody.

Stage 3: conjugation of antibodies to nanoparticles by metal-free click chemistry

Fig. 4 |. Illustration of antibody conjugation to polymersomes.

Quality control of nanoparticles before antibody conjugation

Fig. 5 |. Quality control of nanoparticles and nanoparticle–antibody conjugates.

Antibody-nanoparticle conjugation by metal-free click chemistry

BOX 3. Calculating the amount of antibodies or nanoparticles needed for a conjugation.

Quality control and characterization of the conjugates

Fig. 6 |. Cryo-TEM of ICGJ-PS and their antibody conjugates for quality control.

Fig. 7 |. ELISA of ICGJ-PS antibody conjugates.

BOX 4. ELISA of nanoparticle–antibody conjugates.

Blocking non-specific interactions on the ELISA plate

Incubation of samples and calibration standards on the ELISA plate

Application of a secondary antibody and reading out of ELISA results

Evaluation of the antibody–nanoparticle conjugates for selective labeling of cells

Fig. 8 |. Flow cytometry of cancer cells labeled with antibody–nanoparticle conjugates.

BOX 5. Protocol for evaluation of the molecular specificity of antibody–nanoparticle conjugates using flow cytometry.

Cell preparation

Cell labeling using antibody–nanoparticle conjugates and controls

Troubleshooting

Table 1 |.

Timing

Anticipated results

Table 2 |.

Fig. 9 |. Confocal fluorescence microscopy of cells labeled with antibody–nanoparticle conjugates.

Reporting summary

Supplementary Material

Sodium periodate (NaIO₄) solution, 100 mM