Preprint Article Version 1 This version is not peer-reviewed

Antibody Screening by Microarray Technology – Direct Identification of Selective High-Affinity Clones

Version 1 : Received: 1 November 2019 / Approved: 3 November 2019 / Online: 3 November 2019 (17:00:59 CET)

How to cite: Paul, M.; Weller, M.G. Antibody Screening by Microarray Technology – Direct Identification of Selective High-Affinity Clones. Preprints 2019, 2019110023 (doi: 10.20944/preprints201911.0023.v1). Paul, M.; Weller, M.G. Antibody Screening by Microarray Technology – Direct Identification of Selective High-Affinity Clones. Preprints 2019, 2019110023 (doi: 10.20944/preprints201911.0023.v1).

Abstract

The primary screening of hybridoma cells is a time-critical and laborious step during the development of monoclonal antibodies. Often critical errors occur in this phase, which supports the notion that the generation of monoclonal antibodies with hybridoma technology is difficult to control and hence a risky venture. We think that it is crucial to improve the screening process to eliminate most of the immanent deficits of the conventional approach. With this new microarray-based procedure, several advances could be achieved: Selectivity for excellent binders, high throughput, reproducible signals, avoidance of misleading avidity (multivalency) effects, and simultaneous performance of competition experiments. The latter can directly be used to select clones of desired cross-reactivity properties. In this paper, a model system with two excellent clones against carbamazepine, two weak clones and blank supernatant has been designed to examine the effectiveness of the new system. The excellent clones could be detected largely independent of the IgG concentration, which is unknown during the clone screening since the determination and subsequent adjustment of the antibody concentration is not possible in most cases. Furthermore, in this approach, the enrichment, isolation, and purification of IgG for characterization is not necessary. Raw cell culture supernatant can be used directly, even when fetal calf serum (FCS) or other complex media had been used. In addition, an improved method for the oriented antibody-immobilization on epoxy-silanized slides is presented. Based on the results of this model system, we conclude that this approach should be preferable to most other protocols leading to many of false positives, causing expensive and lengthy confirmation steps to weed out the poor clones.

Subject Areas

monoclonal antibodies; Mabs; fusion; false positives; hapten immunoassays; competitive immunoassays; ELISA; antibody validation; antibody quality; microarray; hybridoma technology; linker recognition; high-throughput screening; HTS; heterology concept

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