To establish a rapid immunological detection method for MPT64 protein based on red microspheres and select highly-sensitive and highly-specific antibody pairs.
A His-tagged prokaryotic expression vector was constructed for expression of MPT64 protein that was used to immunize New Zealand white rabbits after purification and validation. Peripheral blood mononuclear cells (PBMCs) were isolated from the rabbits, and antigen-specific B cells expressing antibodies were sorted using single B-cell flow cytometry. mRNA in B cells was reverse-transcribed into cDNA, and paired antibody heavy-and light-chain sequences were amplified via nested PCR. Expression vectors were constructed, and recombinant antibodies were produced in Expi293F cells. Fluorescent immunochromatography was employed to screen for matched antibody pairs. The selected antibodies were used to establish a rapid detection method based on red microsphere immunochromatography.
Ten high-affinity monoclonal antibodies against MPT64 were generated. Two antibody pairs were selected for MPT64 immunodetection that reached a sensitivity of 0.0125 ng/mL.
High-affinity rabbit monoclonal antibodies against MPT64 are obtained via single B-cell technology, and a rapid red microspheres-based immunodetection method is established, enabling highly sensitive detection of Mycobacterium tuberculosis MPT64 protein.
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