| Objective:At present,the most commonly used method of HIV-1 blood screening is ELISA.Domestic mainstream method for detection of HIV-1 is the third generation HIV ELISA Kit,the third generation of reagents used double antigen sandwich method,detection of HIV antibodies of different subtype of HIV-1/2/O IgG/M/A,improved the reactive of O group specimens,Enzyme markers by specific antigen HIV-1 replace the anti human IgG.The detection the sensitivity and specificity is higher than the first,the two generation of reagents,the window period is shortened to 22 days.However,some domestic and foreign study found that due to HIV-1 ELISA reagent detect several antibodies in the same reaction hole at the same time.Produced the interaction of steric,caused by the interference of immune response,Effect of the detection sensitivity and specificity.HIV-1 positive samples of Initial test and review by ELISA must pass through the Western blot confirmation experiment to report HIV-1 antibody positive.In this paper,using liquid chip detection technology for independent and simultaneous determination HIV-1 gp41,gp120,p24 antibody at the same time,increase accuracy of HIV-1 p17,P31,p66 antibody to verify this method,and compare between the experimental results and Western blot results.Liquid phase chip detection technology has high sensitivity and specificity of detection,has been widely used in research and clinical detection in the domestic and foreign.The sensitivity and specificity of HIV-1 detected by liquid phase chip was significantly higher than that of HIV-1 at home and abroad conventional detection reagent.Method:This study through the use of gene cloning technology,Express HIV-1 Chinese e-pidemic strains of CRP01-AE p17,p24,p31,p66 antigen by PET prokaryotic vect or,and purify it by affinity chromatography.HIV-1 gp41,gp120,p17,p24,P31,p6 6 antigen respectively combined with different fluorescence microspheres(50#,56#,65#,71#,77#,83#,)the biotinylated two antibody as indicator molecules,form indepe-ndent and Simultaneous detection of HIV-1 gp41,gp120,p24 three kinds of an-tibody,at the same time,increase accuracy of HIV-1 p17,P31,p66 antibody to v-e rify this method,Six sets of fluorescence immune reaction formed a liquid phasec hip multipleantibody immunofluorescence reaction system.The mean fluorescenc-e intensity detected each microspheres by Luminex-200 liquid phase chip,The Wes-t ern blot confirmed HIV-1 positive samples,HIV-1 antibody indeterminate speci-m ens and HIV-1 negative specimens were qualitative and quantitative analysis.Result:1.Successfully amplified HIV-1 p17,p24,P31,p66 gene fragment,Restriction enzyme digestion,BL21 Escherichia coli expression SDS-PAGE results showed thatmolecular weight of expressed protein with the expected size protein molecular weight.2.Experimental conditions of the best reaction time of package microspheres and serum specimens,optimum concentration of Biotinylated two anti,the optimum concentration of SA-PE,the best reaction method and so on are optimized.The experiment about 2.5 hours,Compared with the WB test,greatly shorten the detection time,Simpler and quicker,and It has the advantages of high throughput.3.Luminex-200 test results show:1)Confirm the cut-off values of six kinds of microspheres(50#,56#,65#,71#,77#,83#)based on negative specimens of MFI,they are 897.67,788.32,1055.52,909.77,798.55,989.44.2)Find that 50 samples by WB confirmed HIV-1 antibody are positive,Luminex test showed that 50 samples were also positive.3)Among the 5 copies of WB HIV-1 antibody indeterminate samples(No.13,No.14,No.25,No.31 and No.51),Luminex HIV-1 antibody positive samples are 3 copies(No.13,No.31 and No.51),HIV-1 antibody indeterminate samples are 2 copies(No.14 and No.25).4)1 copies of the ELISA initial and review HIV-1 positive specimens(No.42),WB test was negative(undetected),but the results of Luminex HIV-1 were antibody indeterminate.5)At the same time,increased HIV-1 RN A nucleic acid detection for 5 copies of WB detection of HIV-1 antibody indeterminate samples and 1 copies of the ELISA initial and review HIV-1 positive but WB HIV-1 test negative specimen,These 6 specimens'HIV-1 RNA nucleic acid were positive.6)Among the 92 copies of ELISA positive and WB negative samples,85 copies of the Luminex results determination is negative;3 copies of the Luminex results determination is positive;4 copies of the results determination is indeterminate.7)49 copies of HIV negative samples in ELISA detection,the Luminex test results were all negative.Conclusion:Successfully amplified HIV-1 p17,p24,p31,p66 gene fragment,expressed and purified four the HIV-1 high purified antigen.Set up HIV-1 antibody liquid phase chip reaction system,optimization of the optimal experimental conditions.Solve the problem that the third generation of HIV-1 ELISA kit for detection of low specificity.Test specificity is better than WB test.At the same time,Liquid phase chip reaction system has the advantage of less amount of sample,short reaction time,cost saving,simple operation,has the strong value of clinical detection. |
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