TABLE 3.
Representative assays used to measure Abs against human therapeutic proteinsa
| Antigen | Antibodies | Assay method | SL of assay | Results |
|---|---|---|---|---|
| HuIFNγR (26) | Mouse anti-HuIFNR MAbs | RIP; indirect ELISA and cell binding assay; bioassay for inhibition of IFN antiviral activity | Bioassay and ELISA: 1.0-7.0 μg of anti-IFN Abs (for Abs binding IFNγR+ cells)/ml | Six MAbs bound to IFNR+ cells, inhibited 125I-labeled γ-IFN binding, inhibited antiviral activity of γ-IFN |
| Recombinant HuIFNα-2a (Roferon-A) (39) | Patients' sera (various treatments) | RIP; bioassay for inhibition of IFN antiviral activity; ELISA (bead-based assay) | RIP and ELISA: 5 ng of IgG/ml of serum; bioassay: 100 IFN NU (50% viral inhibition endpoint) | Excellent correlation (93%) between ELISA and RIP; antiviral bioassay least sensitive |
| Recombinant HuIFNα-2a (Roferon-A) (42) | Patients' sera (various treatments) | ELISA (bead-based assay); competitive commercial RIP; bioassay for inhibition of IFN antiviral activity | ELISA: 5 ng of IgG/ml of serum; RIP: 50 NU of Abs (1 NU per 1 IFN U); Bioassay-100 IFN NU | Poor correlation between ELISA and RIP (48-50% false negatives) or bioassay (100% false negatives) |
| Islet cells (type 1 diabetes mellitis) (3) | Patients' sera (diabetes- related auto-Abs) | Two commercial ELISA kits; RIP using PEG-mediated precipitation; indirect immunofluorescence assay | ELISA: 1,500 mU Glutamic acid decarboxylase, none specified for second ELISA test; RIP: 5 mU of bound insulin/ml | No correlation among assays; 14-50% false-negatives with ELISAs; 98% sensitivity with RIP assay |
| Pooled human serum IgG preparations (75) | Natural anti-cytokine Abs (GM-CSF, IL-5, IL-10) | Protein G affinity chromatography | SL, ≥0.1 μmol of cytokine/mol of Ab | Commercial pooled human IgG preparations contain natural anti-GM-CSF Abs at highest concentration (0.24-5.0 μmol/mol of Ab), followed by anti-IL-5 (1.3 μmol/mol of Ab) and anti-IL-10 (0.12-0.4 μmol/mol of Ab) |
| TPO (4) | Serum from cancer patient treated with pegylated TPO | Solid-phase RIP; bioassay for neutralizing Abs with Mpl-transfected 32D murine cells | RIP: twofold increase in binding of radiolabeled TPO over baseline; bioassay: 50% growth inhibition against 250 pg of TPO/ml in culture | Emergence of binding Abs (RIP) after sixth SC administration of pegylated TPO and neutralizing Abs after seventh administration |
| TPO (54) | Sera from 2 previously healthy volunteers and 1 cancer patient treated with pegylated TPO | RIP (protein A-, protein G-agarose beads) | SL, ≥75 ng/ml of Abs | Anti-TPO Abs detected after second or third SC administration of pegylated TPO in healthy subjects and after multiple administrations (>20) in the cancer patient |
| Recombinant streptokinase (58) | Patients' sera (myocardial infarction) | Indirect ELISA | Titer, ≤1,750 for IgG1 and 4,200 for IgG | Similar results for assays measur:ing IgG and IgG1 Abs (54-59% anti-streptokinase positive) |
| Recombinant streptokinase (11) | Plasma from 30 healthy volunteers, 60 patients treated with streptokinase for myocardial infarction (7-10 days and 12 and 24 mo), and 12 patients with raised Abs | Indirect ELISA; fibrin plate lysis assay; clot lysis assay for streptokinase neutralization | Not specified | ELISA and neutralization assay results were positively correlated (P < 0.001); neutralization preceded ELISA Abs (IgG); by day 10, all patients had elevated IgG and neutralizing Ab levels; Abs persisted for 24 mo in 75% of patients |
| Recombinant HuGM-CSF (2 Escherichia coli-derived proteins) (91, 93) | Sera from patients with colorectal carcinoma treated with either of two E. coli-derived rHuGM-CSF products | Indirect ELISA for binding Abs, bioassay for neutralizing Abs (TF-1 cells), and Western blotting | SLs of assays not specified; 500 ng/well (5 μg/ml) of rHuGM-CSF coated onto ELISA plates; bioassay measured vol of sera required to neutralize 1 IU of WHO International Standard GM-CSF (88/646) | Total of 47 of 58 patient sera recognized rHuGM-CSF at 500 ng/well (5 μg/ml) in ELISA; 9 of 58 sera (various vol) contained neutralizing Abs; Western blots showed heterogeneity of GM-CSF proteins from E. coli, yeast, and CHO cells |
| Recombinant HuGM-CSF (E. coli-derived protein) (86) | Sera from patients with colorectal carcinoma, metastatic carcinoma, or myeloma treated with high or low-dose E. coli-derived rHuGM-CSF plus tumor antigens or idiotype Ab (myeloma patients) | Indirect ELISA for binding Abs, bioassay for neutralizing Abs (TF-1 cells), and Western blotting | SLs of assays not specified; 500 ng/well (5 μg/ml) of rHuGM-CSF coated onto ELISA plates; bioassay measured volumes of sera required to neutralize 1 IU of WHO International Standard GM-CSF (88/646) | Administration of high-dose rHuGM-CSF (425-500 μg/day for 10 days) plus tumor antigens resulted in production of anti-GM-CSF Abs in 11 of 20 (55%) metastatic carcinoma patient samples; low-dose rHuGM-CSF (75-80 μg/day for 4 days) plus tumor antigens resulted in Ab production in only 4 of 25 (16%) patients with myeloma or colorectal carcinoma; no neutralizing Abs were detected; binding Abs did not interfere with clinical benefits or rHuGM-CSF therapy |
| Recombinant human IL-2 (41) | Sera from patients with colorectal carcinoma treated with either of two E. coli-derived rHuIL-2 products | Indirect ELISA, bioassay for neutralizing Abs (CTLL-2 cells), Western blotting | SLs of assays not specified; 25 ng/well (250 ng/ml) of HuIL-2 coated onto ELISA plates; bioassay measured volume of sera required to neutralize 0.1 IU of rHuIL-2 (2 IU/ml) | Total of 10 of 19 patients developed anti-IL-2 Abs, 1 patient developed neutralizing Abs; Western blots showed Abs recognized native-form IL-2 and two E. coli-derived IL-2 products; neutralizing Abs also cross-neutralized different IL-2 preparations in the CTLL-2 bioassay |
| bFGF (99) | MAb 48.1 (anti-bFGF) capture Ab | BIAcore detection of bFGF in various diluents | 5.65 ng of bFGF/ml of buffered saline | Human serum, bovine serum albumin, Pluronic F127 surfactant, and carboxymethylcellulose did not interfere with binding |
| Humanized anti-IL-5 mouse Ab (95) | Mouse sera | BIAcore | 1 μg of antihuman/mouse chimeric Abs/ml | Assay permits detection of serum levels of injected chimeric Abs and any Abs produced against chimeric Abs |
| HIV-1 gp160 peptides (88) | Polyclonal human sera | BIAcore and ELISA | SL, 5 ng/ml with both assays | Linear range of Ab binding greater with BIAcore than with conventional ELISA |
a
Abbreviations: MAbs, monoclonal Abs; IL-5, interleukin-5; PEG, polyethylene glycol; SL, sensitivity limit; HIV, human immunodeficiency virus; rHu, recombinant human HuIFNγR, human α-IFN receptor; bFGF, basic fibroblast growth factor; NU, neutralizing units; TPO, thrombopoietin. Numbers in parentheses are references.