[Preparation and identification of mouse monoclonal antibody against human adenovirus type 55 Fiber protein]

Objective To prepare mouse-derived specific monoclonal antibody against human adenovirus type 55 fiber protein (HAdV55 Fiber). Methods Gene synthesis technology was used to successfully construct the prokaryotic expression vector pET28a-HAdV55 Fiber and the eukaryotic expression vector pCAGGS-HAdV55 Fiber. After transformation of the recombinant plasmid pET28a-HAdV55 Fiber into competent cells BL21(DE3), the target protein was purified using the AKTA pure system after IPTG induction. BALB/c mice were first immunized with pCAGGS-HAdV55 Fiber plasmid by the cupping method, with three basic immunizations at intervals of 21 days; followed by two booster immunizations with purified Fiber protein via the intraperitoneal injection. Three days post-final immunization, splenocytes from mice with high antibody titer were fused to SP2/0 myeloma cells, and hybridomas were screened via limiting dilution to yield mAbs targeting Fiber protein, whose subtypes and ascites titers were subsequently characterized. The immunofluorescence method was used to detect the reactivity of the monoclonal antibodies against Fiber protein expressed in BHK-21 cells transfected with pCAGGS-HAdV55 Fiber. To identify antibody pairs capable of effectively detecting HAdV55, we employed the anti-HAdV55 Hexon monoclonal antibody previously generated by our group as the capture antibody and the aforementioned HRP-labeled Fiber antibody as the detection antibody. Results The pET28a-HAdV55 Fiber and pCAGGS-HAdV55 Fiber expression plasmids were successfully constructed; the Fiber protein was inducibly expressed via IPTG from BL21 (DE3) E. coli cells transformed with the pET28a-HAdV55 Fiber plasmid. Five hybridoma cells are successfully obtained, which can continuously secrete anti-fibritin monoclonal antibodies. Among them, 2A4F3 could recognize the Fiber protein expressed in eukaryotic cells. From a panel of candidate antibodies, an optimal antibody pair with anti-Hexon mAb 2D3 (capture) and anti-Fiber mAb 2A4F3 (detection) was identified for sensitive detection of HAdV55 via double-antibody sandwich ELISA. Conclusion In this study, specific antibodies against the HAdV55 Fiber protein were successfully prepared, and a specific detection method for HAdV55 was initially established.