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HIV抗體、抗原檢測(cè)
2020-09-04 14:18
一、HIV抗體、抗原檢測(cè)
A, HIV antibody and antigen detection
HIV抗體一般在人感染后幾周逐漸出現(xiàn),可延續(xù)至終生，血清學(xué)試驗(yàn)分為初篩和確認(rèn)試驗(yàn)。很常用的初篩試驗(yàn)和確認(rèn)試驗(yàn)分別是酶聯(lián)免疫吸附試驗(yàn)和免疫印跡試驗(yàn)(WB)。常規(guī)實(shí)驗(yàn)方法：酶聯(lián)免疫吸附試驗(yàn)、免疫印跡試驗(yàn)(WestBlot)、間接免疫熒光法(IFA)。快速檢測(cè)方法：明膠顆粒凝集試驗(yàn)、斑點(diǎn)免疫結(jié)合試驗(yàn)、Ｐ24抗原的檢測(cè)、分子生物學(xué)方法、RTPCR檢測(cè)法、熒光實(shí)時(shí)PCR檢測(cè)技術(shù)、支鏈DNA（bDNA）、連接酶酶促鏈?zhǔn)椒磻?yīng)(LCR)、核酸序列依賴性擴(kuò)增(NASBA)、轉(zhuǎn)錄介導(dǎo)的擴(kuò)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)
?。?） 酶聯(lián)免疫吸附試驗(yàn)
Enzyme-linked immunosorbent assay (1)
ELISA法的基本原理是免疫反應(yīng)物通過(guò)化學(xué)或免疫學(xué)的方法形成酶結(jié)合物，酶結(jié)合物能與待檢樣品中相應(yīng)的抗原或抗體結(jié)合成為免疫復(fù)合物,然后加入酶底物,經(jīng)酶的催化或水解作用,無(wú)色底物產(chǎn)生顏色,用肉眼、分光光度計(jì)觀察結(jié)果。初篩用的HIV ELISA試劑目前已經(jīng)發(fā)展到第四代檢測(cè)試劑。第一代試劑主要以病毒裂解物或部分純化的病毒抗原包被反應(yīng)板,以檢測(cè)血清中的抗體。由于包被的抗原不很純,假陽(yáng)性率較高。第二代試劑使用基因工程方法得到的重組抗原和合成肽包被反應(yīng)板，由于純化抗原的使用,特異性有了很大提高。第三代試劑使用雙抗原夾心法檢測(cè)抗體,進(jìn)一步提高了敏感性。第四代試劑則在第三代的基礎(chǔ)上進(jìn)一步增加了P24抗原的檢測(cè),把HIV抗原和抗P24的抗體同時(shí)包被反應(yīng)板,可同時(shí)檢測(cè)血清中的HIV抗體和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.
?。?）免疫印跡試驗(yàn)
(2) the western blot test
免疫印跡試驗(yàn)主要用于確認(rèn)試驗(yàn)，基本原理是HIV全病毒抗原經(jīng)過(guò)SDSPAGE電泳,將分子量大小不等的蛋白帶分離開(kāi)來(lái),然后再把這些已經(jīng)分離的不同蛋白帶電轉(zhuǎn)移到硝酸纖維素膜上。將此膜切割成條狀,每一條硝酸纖維素膜上均含有經(jīng)電泳分離過(guò)的HIV病毒抗原。將待檢血清樣品用稀釋液稀釋成1/100,再把它直接加到硝酸纖維素膜上,恒溫震蕩,使其充分接觸反應(yīng),血清中若含有抗HIV抗體,就會(huì)與膜條上的抗原帶相結(jié)合。加入抗人IgG酶結(jié)合物和底物后,即可使有反應(yīng)的抗原抗體結(jié)合帶呈現(xiàn)紫褐色,根據(jù)出現(xiàn)條帶情況判定結(jié)果。有報(bào)告說(shuō)，免疫印跡試驗(yàn)的特異性不是很好, 有大約2％的假陽(yáng)性率，但免疫印跡試驗(yàn)依然是目前很常用的HIV確認(rèn)試驗(yàn)。
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.
?。?）免疫熒光試驗(yàn)(IFA)
(3) immunofluorescence test (IFA)
基本原理為應(yīng)用Ｈ9或HUT78培養(yǎng)細(xì)胞作為載體,用HIV感染細(xì)胞,該細(xì)胞內(nèi)就會(huì)含有HIV抗原,將HIV感染的淋巴細(xì)胞涂于玻片上,固定，制備為抗原片,加入待檢血清，待檢血清中的抗 HIV抗體與抗原結(jié)合后,再與熒光素標(biāo)記的抗人Ig結(jié)合,在熒光下可見(jiàn)到細(xì)胞內(nèi)有黃綠色熒光。
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.
?。?）明膠顆粒凝集試驗(yàn)(PA)
(4) gelatin particle agglutination test (PA)
PA的基本過(guò)程是先將樣品稀釋,然后分別加入經(jīng)抗原致敏的和未致敏的明膠顆粒,混勻后保溫(一般為室溫)。當(dāng)血清中有HIV抗體存在時(shí),經(jīng)抗原致敏的明膠顆粒與抗體發(fā)生抗原抗體反應(yīng),根據(jù)明膠顆粒在孔中的凝集情況判讀結(jié)果。PA操作簡(jiǎn)便,無(wú)需特殊設(shè)備,適合對(duì)少量標(biāo)本的檢測(cè)。
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.
?。?）斑點(diǎn)印跡試驗(yàn)(或免疫層析/或滲濾)
(5) spot mark test (or immune chromatography/or percolation)
斑點(diǎn)印跡試驗(yàn)一般采用硝酸纖維素膜作固相載體, 用HIV抗原包被于固相載體,加入待測(cè)標(biāo)本(可為血清、血漿、尿液和其他),一定溫度反應(yīng)后洗去未結(jié)合于固相載體上的標(biāo)本,包被抗原和被檢血清中抗體結(jié)合,用膠體金(或膠體硒)代替底物連接在葡萄球菌蛋白Ａ上,金標(biāo)記蛋白Ａ具有與人Ig結(jié)合的能力,因而可與被捕獲的HIV抗體結(jié)合。若樣品中有HIV抗體,則薄膜上就會(huì)產(chǎn)生一桔紅色斑點(diǎn)(或線條),一般3～10 min出結(jié)果,特異性較好, 較為適宜于偏遠(yuǎn)地區(qū)臨床用血檢測(cè),但不適于城市地區(qū)的獻(xiàn)血員篩查。
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.