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

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 critical 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 performance of simultaneous competition experiments. The latter can also 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 containing fetal bovine serum was designed to examine the effectiveness of the new system. The excellent clones could be detected largely independent of the immunoglobulin G (IgG) concentration, which is usually unknown during the clone screening since the determination and subsequent adjustment of the antibody concentration are not feasible 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 is 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 with simulated hybridoma supernatants, we conclude that this approach should be preferable to most other protocols leading to many false positives, causing expensive and lengthy elimination steps to weed out the poor clones.

Cocaine Detection by a Laser-Induced Immunofluorometric Biosensor

“…The dye is based on a cyanine backbone with four sulfonic acid groups (see Figure S6 , which results in highly hydrophilic behavior. The dye works well with the used excitation source of 638 nm and has proven to display negligible non-specific binding to epoxy-functionalized glass substrates [ 51 ] or trinitrophenyl-BSA affinity columns [ 46 ]. The degree of labeling of the IP3G2-Dy-654 conjugate was determined with MALDI-TOF MS to be approx.…”

Fast Confirmation of Antibody Identity by MALDI-TOF MS Fingerprints

“…We propose to attach such an antibody fingerprint to any scientific publication or other studies critically relying on antibodies, irrespective of in-house or commercial origin. In projects for hybridoma or other antibody development, it could be a component of good laboratory practice (GLP), in order to characterize all positive clones [56] also with their respective peptide fingerprints. In the future, the determination of the species and the antibody subclasses might also be included, which would provide an extra benefit of the technique.…”

Hybridoma technology a versatile method for isolation of monoclonal antibodies, its applicability across species, limitations, advancement and future perspectives

“…Additionally, this approach eliminates the enrichment, isolation, and purification of IgG for the characterization process. The crude culture supernatants can be directly used and thus avoids expensive and lengthy screening steps [100]. The screening process has advanced through flow cytometry-based methodology where single cells could be sorted from a bulk mixture of fused hybridoma cells.…”