Preparation Of Monoclonal Antibody Against CppB And Establishment Of Fluorescent-microsphere Immunochromatographic Assay For Neisseria Gonorrhoeae

Objective Neisseria gonorrhoeae infection has become a global public health issue due to its high morbidity and high infection rate,which also can cause serious complications.The aim of the present study was to prepare a monoclonal antibody of Cryptic plasmid protein B(CppB)and establish a fluorescent microsphere-based immunochromatographic test strip for rapid detection of gonorrhea.Methods A total of 115N.gonorrhoeae and 93 common bacteria of the genital tract were collected from Guangzhou in 2017,and the broad spectrum and specificity of the cppB gene of N.gonorrhoeae were verified by qRT-PCR.The expression plasmid pGEX-6P-l-cppB was constructed and confirmed by PCR and DNA sequencing.The GST-CppB fusion protein was expressed in E.coli prokaryotic expression system,and the purified GST-CppB fusion protein was obtained using GST-SefinoseTM kit,which was verified by SDS-PAGE and Western Blot.After immunized BALb/c mice,the subtypes and titers of monoclonal antibodies were determined by indirect ELISA,and the reactivity and specificity of monoclonal antibodies were identified by Western Blot.Finally,the immunochromatographic test strip was established based on fluorescent microsphere as a signal,and the sensitivity,specificity and stability of the test strip were verified.Results The positive rate of qRT-PCR was 96.52%(111/115),and the specificity was 100%,proving that the cppB gene is ubiquitous in N.gonorrhoeae,while another bacterium not(0/93).PCR confirmed that the recombinant plasmid pGEX-6P-1-cppB was successfully constructed.The DNA sequencing showed that the cppB gene inserted into the pGEX-6P-1 plasmid was identical to the cppB gene sequence published by GeneBank(FMSZO1000040.1).SDS-PAGE showed that the recombinant host was able to express GST-CppB fusion protein with a large Mnount,and the results of Western Blot were consistent with the results of SDS-PAGE.The purified GST-CppB fusion protein was stained to confirm that a higher purity GST-CppB protein was obtained.Four monoclonal cell-lines were obtained by hybridoma technique,and their subtypes were IgGl(MAb-F11,MAb?G3),IgG2a(MAb-C11),and IgG2b(MAb-E1).The titer of all monoclonal antibodies is greater than 1:64000,of which MAb-El has the highest titer.Western Blot results showed that MAb-F11 had the best response to N.gonorrhoeae,with a positive rate of 87.83%(101/115)and a specificity of 100%,which was consistent with the results of qRT-PCR verification of cppB gene.In addition,we found a mutant of CppB,named CppB-2.Compared with the normal sequence,the CppB-2 gene lacks 54bp that located at 429-482,resulting in a CppB-2 protein of only 21.93kd,slightly lower than CppB(23.94kd).The sensitivity of the immunochromatographic test strip for detecting CppB protein was 20 ng/ml,the sensitivity for detecting Kgonorrhoeae was 1x105 CFU/ml,and the positive rate of the test strip was 82.61%(95/115).Consistent with the results of Western Blot,common genital pathogens were negative on immunochromatographic strips.indicating that the strips were also highly specific.Conclusion CppB protein is ubiquitous in Neisseria gonorrhoeae and can be used as a target for distinguishing common pathogens of Neisseria gonorrhoeae and other reproductive tracts.Immunochromatographic test strips prepared based on monoclonal antibodies against CppB protein are expected to become an important tool for screening for gonorrhea.