Table 2 |.
ELISA estimates of antibodies conjugated per ICGJ-PS under various treatment conditions of ICGJ-PS with aEGFR and aFRα
| Samplea,b | Antibody oxidation time (min)c | Number of washes with 500 μl of 1× PBSd | 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 |
a
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.
b
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.
c
The antibodies were oxidized for different time durations in Step 2.
d
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).