Optimización de un ELISA celular versátil para la detección de anticuerpos contra la proteína de membrana BCMA

Monoclonal antibodies against membrane proteins are commonly used for diagnostic and therapeutic applications. They are still mostly produced using hybridoma technology or phage display, generating large numbers of antibody candidates that need to be tested in a short period of time. As most membrane proteins do not retain their three-dimensional structure after their isolation from the cell membrane, cell-based high-throughput assays are needed for the primary screening of hundreds of antibody candidates. We designed a simple cell-based ELISA by taking the protocols for indirect cell immunostaining and integrating them into a standard ELISA format. This cell ELISA is conducted in 96-well round bottom plates and consists of two immunostaining and washing steps, and a detection step. We designed the assay on B-cell maturation antigen as a model membrane protein antigen. We used several cell lines and a wide range of antibody concentrations. The assay was validated with imaging flow cytometry. Although we obtained comparable results in specificity with cell ELISA and flow cytometry, screening multiple samples with cell ELISA is substantially faster than with flow cytometry, due to its ability to test several samples simultaneously. Using cell ELISA, a single operator can easily test several hundred antibodies per day. Key advantages of the protocol presented here are that it can be used for suspension or adherent cell lines and for cell lines that express the membrane protein in native or recombinant form. As such, it can potentially be translated to diverse antibody-antigen-cell line systems. This was confirmed by successfully detecting anti-CD3 and anti-CD19 antibodies binding to Jurkat and Raji cells, respectively. Cell ELISA developed in our laboratory is a high-throughput, inexpensive, and easy-to-perform method for screening hybridoma that produce antibodies targeting membrane proteins. It does not require the use of soluble pure protein and it can be performed in any laboratory that specializes in antibody development and ELISA testing.