Table 1.
Conditions used in ELISA assays.
| Assay | Capture antibody | Detection antibody | Standard | Working range (ng/ml) | Sample dilution |
|---|---|---|---|---|---|
| Components | |||||
| C1q | Monoclonal anti-C1q (9H10) | Rabbit anti-C1q | C1q | 16–1000 | 1 in 16 000 |
| FB | Monoclonal anti-FB (JC1) | Monoclonal anti-FB (MBI5) | FB | 16–1000 | 1 in 2000 |
| FD | Rabbit anti-FD | monoclonal anti-FD (D10/4; Hycult)-HRP | FD | 1–1000 | 1 in 10 |
| C5 | Monoclonal anti-C5 (4G2) | Rabbit anti-C5 | C5 | 4–500 | 1 in 2000 |
| Regulators | |||||
| FH | Monoclonal anti-FH (OX24) | Monoclonal anti-FH (35H9) | FH | 16–1000 | 1 in 4000 |
| FHR125 | Monoclonal anti-FHR125 (MBI125) | Monoclonal anti-FH (35H9) | FHR125 | 4–250 | 1 in 4000 |
| FHR4 | Monoclonal anti-FHR4 (4E9) | Monoclonal anti-FHR4 (clone 150)-HRP | FHR4 | 16–1000 | 1 in 20 |
| FI | Monoclonal anti-FI (7B5) | Rabbit anti-FI | FI | 32–2000 | 1 in 200 |
| C1inh | Monoclonal anti-C1inh | Rabbit anti-C1inh | C1 inh | 2–100 | 1 in 6000 |
| Properdin | Monoclonal anti-properdin (clone 1.1.1) | Monoclonal anti-properdin (clone 12.14.2)-HRP | Properdin (CompTech) | 78–5000 | 1 in 200 |
| Clusterin | Monoclonal anti-clusterin (2D5) | Monoclonal anti-clusterin (4E7) | Clusterin | 32–2000 | 1 in 500 |
| Activation products | |||||
| iC3b | Monoclonal anti-iC3b (Clone 9; Hycult) | Monoclonal anti-iC3b (bH6; Hycult)-HRP | iC3b (CompTech) | 16–1000 | 1 in 300 |
| TCC | Monoclonal anti-TCC (aE11; Hycult) | Monoclonal anti-C8 (E2)-biotin | TCC | 10–5000 | 1 in 40 |
All antibodies and standard proteins were prepared in house unless otherwise stated. Commercial sources were Hycult (Hycult Biotech; https://www.hycultbiotech.com/) or CompTech (Complement Technology, Inc.; https://www.complementtech.com/). Anti-properdin mAb clone 1.1.1. and clone 12.14.2 were gifted by Prof Santiago Rodriguez de Cordoba, CISC, Madrid. Antibodies shown to be conjugated to HRP or biotin were labelled in-house using commercial kits (Thermo Fisher, UK).