Real-time Immuno-RT-PCR For Antigen Detection With Armored RNA As Reporter

Objective To develop an expression system producing the virus-likeparticles (VLPs) which contain the HCV recombinant RNA (reRNA).The reRNA within VLPs is ribonuclease resistant due to theencapsidation by MS₂ bacteriophage coat proteins. The aim of the designis to improve the sensitivity of immunoassay because of the power ofRT-PCR.Methods We described a reverse transcription polymerase chainreaction (RT-PCR) reporter system for the detection of protein antigen,termed "immuno-RT-PCR". First, develop an expression systemproducing,the virus-like particles (VLPs) which contain the HCVrecombinant RNA (reRNA). According to the 5'UTR sequence of HCV,we design one pair primers to amplify HCV 5'UTR seqences by RT-PCR.A Hindâ…¢restriction sequence was incorporated into the primers. ThepNCCL1 previously digested with Hindâ…¢restriction enzyme wascombined with the digested fragment to treat a new expression plasmidpNCCL1-HCV.Under the induction of IPTG, MS₂ coat proteins wereexpressed into the cytoplasma of E.coli strain BL21-DE3 harboringpNCCL1-HCV, and then assembled with HCV reRNA into VLPs. Inimmuno-RT-PCR, a recombinant MS₂ phage particle encapsulatedspecific RNA (armored RNA) which is similar to the repoters inconventional ELISA is covalently attached to an specific Ab; thus, in the presence of solid antibody, antigen to be detected and coupled antibodywith armored RNA, sandwich complexes would be formed. The armoredRNA bond to the solid phase would be relased and detected by RT-PCR.Results The expression plasmid vector pET-MS₂-HCV can be usedas a platform to produce noninfectious and RNase-resistant HCV-1bRNA quantitative standards and controls. The detection sensitivity couldbe improved through the power of RT-PCR. The idea has beendemonstrated by applying HBsAg as detection target in sandwichprotocol and real-time immuno-RT-PCR. The detection sensitivity wasincreased 10000-100000-folds compared with conventionalenzyme-linked immunosorbent assays (ELISAs). With differentarmored-RNA for different targets antigens and multiple real-timeRT-PCR, multiple targets could be detected at same time. This idea mayserve as a new model to develop ultrasensitive immunoassay method forresearch and clinical diagnosis application.Conclusions The plasmid packaging system for VLPs containingHCV RNA was successfully constructed, couple of monoclonal antibodyto armored RNA was successfully carried out by two-step glutaraldehydeprocedure. The detection sensitivity for HBsAg was increased 10000folds compared with conventional enzyme-linked immunosorbent assays(ELISAs).