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HIV抗体、抗原检测

2020-09-04 14:18
一、HIV抗体、抗原检测
A, HIV antibody and antigen detection
  HIV抗体一般在人感染后几周逐渐出现,可延续至终生,血清学试验分为初筛和确认试验。很常用的初筛试验和确认试验分别是酶联免疫吸附试验和免疫印迹试验(WB)。常规实验方法:酶联免疫吸附试验、免疫印迹试验(WestBlot)、间接免疫荧光法(IFA)。快速检测方法:明胶颗粒凝集试验、斑点免疫结合试验、P24抗原的检测、分子生物学方法、RTPCR检测法、荧光实时PCR检测技术、支链DNA(bDNA)、连接酶酶促链式反应(LCR)、核酸序列依赖性扩增(NASBA)、转录介导的扩增(TMA)
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)
  (1) 酶联免疫吸附试验
Enzyme-linked immunosorbent assay (1)
  ELISA法的基本原理是免疫反应物通过化学或免疫学的方法形成酶结合物,酶结合物能与待检样品中相应的抗原或抗体结合成为免疫复合物,然后加入酶底物,经酶的催化或水解作用,无色底物产生颜色,用肉眼、分光光度计观察结果。初筛用的HIV ELISA试剂目前已经发展到第四代检测试剂。第一代试剂主要以病毒裂解物或部分纯化的病毒抗原包被反应板,以检测血清中的抗体。由于包被的抗原不很纯,假阳性率较高。第二代试剂使用基因工程方法得到的重组抗原和合成肽包被反应板,由于纯化抗原的使用,特异性有了很大提高。第三代试剂使用双抗原夹心法检测抗体,进一步提高了敏感性。第四代试剂则在第三代的基础上进一步增加了P24抗原的检测,把HIV抗原和抗P24的抗体同时包被反应板,可同时检测血清中的HIV抗体和P24抗原。
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)免疫印迹试验
(2) the western blot test
  免疫印迹试验主要用于确认试验,基本原理是HIV全病毒抗原经过SDSPAGE电泳,将分子量大小不等的蛋白带分离开来,然后再把这些已经分离的不同蛋白带电转移到硝酸纤维素膜上。将此膜切割成条状,每一条硝酸纤维素膜上均含有经电泳分离过的HIV病毒抗原。将待检血清样品用稀释液稀释成1/100,再把它直接加到硝酸纤维素膜上,恒温震荡,使其充分接触反应,血清中若含有抗HIV抗体,就会与膜条上的抗原带相结合。加入抗人IgG酶结合物和底物后,即可使有反应的抗原抗体结合带呈现紫褐色,根据出现条带情况判定结果。有报告说,免疫印迹试验的特异性不是很好, 有大约2%的假阳性率,但免疫印迹试验依然是目前很常用的HIV确认试验。
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)免疫荧光试验(IFA)
(3) immunofluorescence test (IFA)
  基本原理为应用H9或HUT78培养细胞作为载体,用HIV感染细胞,该细胞内就会含有HIV抗原,将HIV感染的淋巴细胞涂于玻片上,固定,制备为抗原片,加入待检血清,待检血清中的抗 HIV抗体与抗原结合后,再与荧光素标记的抗人Ig结合,在荧光下可见到细胞内有黄绿色荧光。
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)明胶颗粒凝集试验(PA)
(4) gelatin particle agglutination test (PA)
  PA的基本过程是先将样品稀释,然后分别加入经抗原致敏的和未致敏的明胶颗粒,混匀后保温(一般为室温)。当血清中有HIV抗体存在时,经抗原致敏的明胶颗粒与抗体发生抗原抗体反应,根据明胶颗粒在孔中的凝集情况判读结果。PA操作简便,无需特殊设备,适合对少量标本的检测。
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)斑点印迹试验(或免疫层析/或渗滤)
(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.