Based on our cutting-edge technology, Creative Biolabs offers anti-drug antibody (ADA) neutralization activity evaluation services through cell-based assays for our worldwide customers. With experienced scientists, our immunoanalytics department will provide the most suitable assays to assess your biologic. Please refer to Non-Cell Based Competitive Ligand Binding Assay for non-cell-based assays.
It is widely known that neutralizing antibodies (NAbs) block the biological activity of the therapeutic molecule by either binding directly to the epitope or by blocking its active site by steric hindrance. Besides immunoassay methods such as colorimetric enzyme-linked immunosorbent assay (ELISA), immunofluorescence-based receptor binding assays, competitive ligand binding immunoassay (CLB), NAbs could also be detected using cell-based bioassays in which the presence of NAbs can be detected by their ability to inhibit the biological activity of the biotherapeutic agent.
During the past years, cell-based neutralizing antibody assays have been the preferred method to detect NAbs because they could best mimic the mechanism of NAbs in a living biological system. Evidence regarding NAbs and clinical impact has mainly been amassed from data derived from cell-based NAb assays.
Fig.1 Schematic outline of the experimental procedure neutralizing antibody assay.1, 2
In a cell-based assay, the sample is pre-incubated with the therapeutic and if NAbs are present, the therapeutic is unable to bind to its target resulting in an inhibition of assay response. Various assay types can be used for cell-based NAb assays, such as cell proliferation, phosphorylation of intracellular substrates, apoptosis, chemokine/cytokine release, and gene expression. However, cell-based assays have inherent technical challenges. They are often influenced by the sample matrix and require significant validation. For instance, the assay should differentiate the antibody from other sample components such as complement, coagulation factors, soluble receptors, lipids, concomitant medications, and an administered product. The use of novel approaches such as quantitative PCR assays or reporter gene assays using chemically treated division-arrested cells can overcome some of the problems seen with cell-based assays. Depending on the mechanism of action (MOA) of the therapeutic and how it interacts with cells, the assay format can vary.
1. Direct cell-based NAb assay format
This assay format uses the cell line expressing the target receptor to measure the assay response induced by the direct interaction between the drug molecule and its cognate receptor. NAb blocks the drug function and reduces the assay response. A direct NAb assay format is used for drug products that directly exert a biological effect on the cell, such as drug product-induced proliferation.
2. Indirect cell-based NAb assay format
In addition to drug and an appropriate cell line, this assay format usually requires an additional component such as a ligand that binds to the cognate cellular receptor. Interaction of the ligand with the receptor generates an assay response. The drug neutralizes the ligand, reducing the assay response. NAb inhibits the drug function and restores the assay response. NAb assays for drug products including mAb therapeutics, soluble receptors, receptor antagonists, often fall into the indirect NAb assay format.
Table 1. Types of NAb Assay Formats
| Assay Format | Drug Product's MOA | Drug Examples |
| Direct | Acts directly on the cells to induce a biological response | Therapeutic cytokines, growth factors, peptides, agonistic monoclonal antibodies (mAbs) |
| Indirect | Acts by interfering or enhancing the binding of a ligand to a receptor on the cell surface | Therapeutic mAbs, soluble receptors, receptor antagonists |
It is important to develop reliable NAb assays of appropriate sensitivity, specificity, and robustness that can detect and distinguish NAbs from non-neutralizing antibodies in non-clinical or clinical samples collected for antibody testing. Creative Biolabs provides high-quality neutralization activity evaluation services by cell-based bioassay. If you are interested in our cell-based assay, please don’t hesitate to contact us for more information.
References
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