A quantitative human monoclonal antibody immunoassay using anti-idiotypic antibody as a membrane antigen surrogate with surface-plasmon-resonance detection

• Published in: Biotechnology and applied biochemistry Vol. 53; no. 1; pp. 51 - 61
• Main Authors: Wong, Rosie B., Lee, An-Horng, Rangan, Vangipuram S., Cheng, Kuang-Chuan
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 01-05-2009; Wiley
• Subjects: Animals; anti-idiotypic antibody (anti-ID); Antibodies, Anti-Idiotypic - analysis; Antibodies, Anti-Idiotypic - immunology; Antibodies, Monoclonal - analysis; Antibodies, Monoclonal - immunology; Biological and medical sciences; Biotechnology; Calibration; CHO Cells; Cricetinae; Cricetulus; Dose-Response Relationship, Immunologic; ELISA; Flow Cytometry; Fundamental and applied biological sciences. Psychology; human monoclonal antibody (hmAb); Humans; Immobilized Proteins - immunology; immunoassay; Immunoassay - methods; Immunoglobulin G - immunology; membrane antigen; Membrane Proteins - analysis; Membrane Proteins - immunology; Sensitivity and Specificity; Surface Plasmon Resonance; surface plasmon resonance (SPR); Human; membrane antigen; anti-idiotypic antibody (anti-ID); surface plasmon resonance (SPR); Monoclonal antibody; Surface plasmon; Immunological method; immunoassay; Antigen; Membrane; Resonance; Detection; ELISA assay; human monoclonal antibody (hmAb); Quantitative analysis; ELISA
• ISSN: 0885-4513; 1470-8744
• DOI: 10.1042/BA20080072

Biologically active membrane proteins are difficult to isolate. Very often the isolated membrane proteins have low binding affinity or no biological integrity at all. Despite some success in isolation, one has to overcome the hurdles of obtaining sufficient quantity of the proteins and maintaining biological activity upon coating them on surfaces for developing an ELISA. Thus an alternative approach may be useful. The present study describes a quantification assay method for a therapeutic hmAb‐1 (human monoclonal antibody‐1) that recognizes a cell‐surface protein employing an anti‐ID (anti‐idiotypic antibody) to hmAb‐1 as a surrogate antigen in an immunoassay format using surface‐ plasmon‐resonance technology. This assay is applicable for quantification of hmAb‐1 in process streams, final drug‐product quality control, as well as low‐concentration drug substances in intravenous‐solution bags. The surrogate nature of the anti‐ID was confirmed by demonstrating that the anti‐ID displaced the interaction between the hmAb‐1 and its membrane antigen in a FACS titration test. The assay format involves first capturing hmAb‐1 on the flow‐cell surface, which then binds quantitatively to anti‐ID in a mixture of increasing quantity of hmAb‐1 in solution. An inverse dose–response relation between this anti‐ID bound signal (or resonance units) and hmAb‐1 concentration was established. The dose–response range of the calibration curve for hmAb‐1 was between 20 and 300 ng/ml. The precision, accuracy and specificity of the assay are reported in the present paper.