A, HIV antibody and antigen detection
HIV antibodies generally appear in a few weeks after infection gradually, can continue into lifelong, serologic test is divided into early screening and confirmation test. At the beginning of the most commonly used screening test and validation test are respectively enzyme-linked immunosorbent assay and western blot (WB). Conventional experimental methods: enzyme-linked immunosorbent assay and western blot test (WestBlot), indirect immunofluorescence (IFA). Rapid detection methods: gelatin particle agglutination test, digfa combined test, P24 antigen detection method, molecular biology, RTPCR method, real-time fluorescent PCR detection technology, branched DNA (bDNA), ligase enzymatic chain reaction (LCR) and nucleic acid sequence dependency amplification (NASBA), transcription mediated amplification (TMA)
Enzyme-linked immunosorbent assay (1)
ELISA method is the basic principle of immunoreactants are formed by the method of chemical or immunology enzyme, enzyme combined with influenza virus samples corresponding antigen or antibody combined with become immune complex, then add the enzyme substrate, the enzyme catalysis or hydrolysis, colorless color substrate, with the naked eye, spectrophotometer observations. Early screening of HIV ELISA reagent at present has developed to the fourth generation detection reagent. The first generation of reagent mainly virus cracking thing or part of a package of purified virus antigen was reaction plate, to detect antibody in the serum. Antigens as a result of the package is very pure, high rate of false positives. The second generation of recombinant antigen of reagent to use genetic engineering approach and synthetic peptide package is reaction plate, due to the use of purified antigens, specificity have greatly improved. The third generation of reagent to use double antigen clamp method to detect the antibody, to further improve the sensitivity. Fourth generation reagent is on the basis of the third generation of further increased the P24 antigen detection, the HIV and resistance to P24 antigen antibody package is reaction plate at the same time, can simultaneously detect HIV antibodies in the serum and P24 antigen.
(2) the western blot test
Western blot test is mainly used for validation test, the basic principle is HIV virus antigen by SDSPAGE electrophoresis, the molecular size of protein separation, and then put these have different protein separation charged transferred to nitrocellulose membrane. The film cut into strips, each a nitrocellulose membrane has contained the electrophoresis separation of HIV virus antigen. Influenza virus serum samples with diluent dilution into 1/100, then add it directly to the nitrocellulose membrane, constant temperature oscillation, make its full contact reaction, if contain HIV antibody in serum, the article will with the membrane antigen on the belt. Joined the resistance after IgG enzyme combination and the substrate, can make a reaction of antigen antibody belt show puce, according to the results of strip case judgement. Have reported that the specificity of the western blot test is not very good, about 2% of the false positive rate, but western blot test is still by far the most commonly used HIV confirmation trial.
(3) immunofluorescence test (IFA)
Basic principle for application H9 or HUT78 cultured cells as a carrier, with HIV infected cells, the cells will contain HIV antigen, coated on the glass of lymphocytes to HIV infection, fixed, preparation of antigen, join the influenza virus serum, influenza virus in the serum HIV antibody and antigen, and combine with fluorescein labeled anti human Ig, visible to the cell with yellow-green fluorescence under the fluorescent.
(4) gelatin particle agglutination test (PA)
PA is the basic process of dilute the sample of the first, and then join the antigen sensitization and no sensitization of gelatin particles, heat preservation after blending normally (room temperature). When the presence of HIV antibodies in serum, antigen sensitization in the gelatin particle antigen antibody reaction with antibody, according to the situation of gelatin particle agglutination in the hole interpretation results. PA easy operation, no special equipment and is suitable for the detection of a small amount of samples.
(5) spot mark test (or immune chromatography/or percolation)
斑点印迹试验一般采用硝酸纤维素膜作固相载体, 用HIV抗原包被于固相载体,加入待测标本(可为血清、血浆、尿液和其他),一定温度反应后洗去未结合于固相载体上的标本,包被抗原和被检血清中抗体结合,用胶体金(或胶体硒)代替底物连接在葡萄球菌蛋白Ａ上,金标记蛋白Ａ具有与人Ig结合的能力,因而可与被捕获的HIV抗体结合。若样品中有HIV抗体,则薄膜上就会产生一桔红色斑点(或线条),一般3～10 min出结果,特异性较好, 较为适宜于偏远地区临床用血检测,但不适于城市地区的献血员筛查。
Macular imprinting test generally using cellulose nitrate film as the solid phase carrier, with HIV antigen package is in solid phase carrier, join the specimen under test (for serum, plasma, urine, and other), and A certain temperature reaction after wash not on the solid phase carrier of specimens, envelope antigen and checked antibody in serum, using colloidal gold (or colloid selenium) instead of the substrate connection on staphylococcus protein A, gold marker protein A has the ability to combine Ig with people, and therefore can be combined with captured HIV antibodies. If samples have HIV antibodies, the film is produced on the orange spots (or lines), generally 3 ~ 10 min as a result, the specificity is better, more suitable for clinical use detection in remote areas, but not suitable for urban areas to donate blood screening.