Rapid and specific screening of monoclonal antibodies in hybridoma supernatants by an enzyme-based dipstick immunoassay

Hybridoma technology remains a cornerstone of monoclonal antibody (mAb) discovery. Classical screening practices such as ELISA, western blot, and dot blot require laborious, time-consuming procedures, rendering them inefficient in time-restricted decision-making. Additionally, due to these assays’ practical and technical limitations, specificity testing of the mAbs is usually omitted during the primary screening of hybridoma libraries. Herein, we present a rapid, dipstick immunoassay (DIA) designed for mAbs screening in cell culture supernatant. The integrated proprietary setup is based on antibody capture and comprises accessible materials such as conjugate pad, nitrocellulose membrane, and absorbent pad. The critical element of this technology is the design of the assay. During the assay run, dipsticks are inserted into supernatant-containing microtiter wells, initiating capillary flow of mAbs towards the conjugate pad where a pre-dried HRP-labeled anti-host species antibody is embedded. This interaction forms an immunocomplex, which migrates to the nitrocellulose membrane and binds to the pre-immobilized target antigen while simultaneously encountering immobilized putative cross-reacting proteins in a multiplex-line fashion. Upon addition of HRP-oxidizable substrate, signal acquisition is performed using a simple camera (colorimetry) or a CCD sensor (chemiluminescence). The system was quantitative up to 2500 ng mL−1 and showed a minimum detectable concentration of 0.61 ng mL−1. Compared to the gold-standard ELISA, the sensitivity was 8-fold higher, while the dynamic range was similar. Critically, the assay allows for the concurrent assessment of antibody specificity in one run. This immunoassay’s unique configuration, simplicity, and rapidity can facilitate antibody discovery. [Display omitted] •A novel dipstick immunoassay for direct selection of mAb-secreting hybridoma was developed.•The enzyme-based method accurately quantified mAb in supernatants.•Supernatants containing mAb can be tested for their cross-reactivity to non-target antigens on the same test run.•Pretreatment-free testing of supernatants provides results in less than 30 min.