Development Of A Recombinant Antigen-based Capture Enzyme Immunoassay Kit For Detection Of Recent HIV-1 Infection

ObjectiveDetection of recent HIV-1 infection is an important and difficult issue in the control and prevention of HIV/AIDS.In this study,we developed a recombinant antigen-based capture enzyme immunoassay(RCEIA)kit for detection of recent HIV-1 infection by optimizing the recombinant antigen and other key components,and then preliminarily evaluate its stability,repeatability,reproducibility and test performance.Methods1.Preparation of an HIV-1 subtypes panel.Total RNAs were extracted from HIV-1 positive plasma specimens stored in our specimens bank.The viral pol,gag and env gene region fragments were amplified by nested polymerase chain reaction(PCR)after reverse transcription,sequenced and then analyzed for HIV-1 subtypes.Combining with the other specimens with different subtypes,an HIV-1 subtypes panel covering the popular HIV-1 subtypes in China were prepared.2.Preparation and screening of recombinant antigens.Besides the recombinant protein HIV-Ag-R03,another six HIV-1 recombinant proteins were constructed and expressed by changing the core structures,the expression vectors,and the protein tags,and connecting hydrophilic amino acid sequences.The reactogenicity of each recombinant protein to specimens with different subtypes was detected,respectively.And then the reactogenicity of each recombinant protein labeled with horseradish peroxidase(HRP)to specimens with different subtypes was detected as well,respectively.Finally,a recombinant protein labeled with HRP targeting the specimens with various HIV-1 subtypes in China was selected for use.3.Construction and optimization of RCEIA kit.To assemble the kit after screening the core components of the kit,optimizing the working concentration of recombinant antigen and preparing calibrator and controls.4.Preliminarily evaluation of the RCEIA kit.To predict the storing temperature and time for the kit by thermal stability test.To evaluate the repeatability by precision test and comparing the correlation between initial and confirmatory testing.To evaluate the reproducibility and detection performance through a recent HIV-1 infection detection reagent evaluating panel(BEDn81)prepared by our research group.To compare the consistency and correlation between the results of RCEIA kits and those of BED CEIA or LAg-Avidity EIA.Results1.An HIV-1 subtypes panel named as STn28 was prepared.It was composed of 28 plasma specimens involved in 11 HIV-1 subtypes.2.Six HIV-1 recombinant antigens with high purity and well reactogenicity were obtained.A recombinant antigen labeled with HRP,HIV-Ag-07-HRP,was selected for use.It was shown the best reactogenicity to various HIV-1 subtypes in China.3.An HIV-1 incidence RCEIA kit was developed.The working concentration of HIV-Ag-R07-HRP is 1:2000.All of the high positive control(HPC),calibrator(CAL)and low positive control(LPC)were prepared by diluting a same HIV-1 positive plasma specimen with subtype CRF07-BC.The diluent and the negative control(NC)were HIV negative plasma specimen.4.The results of thermal stability test showed that the enzyme conjugate,controls and calibrator,and entire kit were stable at 37? for at least 7 days,suggesting that they can be storede at 4? for more than 1 year.5.Both of the repeatability and reproducibility of the RCEIA kit were excellent.All of the coefficient of variations(CVs)of OD values for HPC,CAL and LPC in a same microplate were less than 10%,while those for HPC,CAL and LPC in varoius microplate were less than 15%.The correlation coefficients of OD values and ODn values between initial and confirmatory test were 0.989 and 0.997,respectively,while those between two technicians were 0.988 and 0.990,respectively.6.To detect a same panel BEDn81 respectively,the agreement of RCEIA kit with BED CEIA and LAg-Avidity EIA for distinguishing recent infections from long-term HIV-1 infetions were 87.65%(Kappa=0.75,P=0.000)and 85.19%(Kappa=0.68,P=0.000),respectively.The correlation coefficient of ODn values by RCEIA kit and those by BED CEIA was 0.904,while that of ODn values by RCEIA kit and those by LAg-Avidity EIA was 0.892.Conclusions1.An HIV-1 recombinant antigen labeled with HRP(named as HIV-Ag-R07-HRP)targeting the specimens with various HIV-1 subtypes in China was prepared and selected.And then a recombinant antigen-based capture enzyme immunoassay(RCEIA)kit for detection of recent HIV-1 infection was developed.2.The stability,repeatability and reproducibility of the RCEIA kit were excellent.The results of this kit showed well agreement and correlation with those of BED CEIA and LAg-Avidity EIA,respectively.The key technical parameters of the RCEIA kit,such as the mean duration of recent infection(MDRI)and fasle recent rate(FRR),need to be further studied.