Application of In-Cell ELISA assay for hybridoma screening and selection of promising producers of monoclonal antibodies

Abstract

This study was motivated by the pressing need to obtain monoclonal antibodies (mAb) for research and medical purposes. Screening for mAb-producing hybridomas and evaluating various cell cultures requires a technique that combines the advantages of immunohistochemistry (IHC), heterogeneous enzyme immunoassay (ELISA), and Western blotting. Cellular ELISA (In-Cell ELISA, ICE) is valid for in situ measurement of target analytes in both attached and suspended cells with minimal time, specificity, and reproducibility of ELISA. This method can be successfully used to detect surface and intracellular antigens, as well as to assess phosphorylation, methylation, and acetylation. ICE is a simple, fast, inexpensive, and highly sensitive alternative to various common cyto- and histochemical methods. Here, we explored the potential of ICE for screening and selecting the most promising hybridomas and mAbs for IHC staining. The experiments were carried out with the help of the cultural method, the method of heterogeneous and cellular ELISA, and IHC staining. Comparison of ICE and IHC showed a significant agreement in the intensity and localization of staining with the studied mAbs. The most suitable antibodies for subsequent IHC were selected, saving us much time of selection and the number of expensive mAbs. In heterogeneous ELISA, a negative reaction was noted in some cases. In ICE of whole cells, a pronounced response was detected microscopically. With ICE, it is possible to characterize cell cultures by markers that distinguish one cell line from another, show the stages of cell differentiation, and visualize their structures at different stages of cultivation. In some cases, ICE may be more informative than ELISA for selecting monoclones that produce specific mAbs. A preliminary ICE run can be used to determine the optimal concentration of working dilutions of primary antibodies in a subsequent IHC run.

Keywords

• in-cell elisa
• hybridoma
• monoclonal antibodies
• immunohistochemistry