PRINCIPLES, PROBLEMS, AND STRATEGIES IN THE USE OF ANTIGENIC MIXTURES FOR THE ENZYME-LINKED IMMUNOSORBENT-ASSAY
PRINCIPLES, PROBLEMS, AND STRATEGIES IN THE USE OF ANTIGENIC MIXTURES FOR THE ENZYME-LINKED IMMUNOSORBENT-ASSAY
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DOI：
10.1128/jcm.17.4.655-665.1983
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发表时间：
1983-01-01
影响因子：
9.4
通讯作者：
DUNSMOOR, CL
中科院分区：
文献类型：
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作者：
KENNY, GE;DUNSMOOR, CL
通过酶联免疫吸附测定 (ELISA) 研究了蛋白质和其他大分子之间对塑料上吸附位点的竞争，以确定使用抗原混合物或微生物提取物对 ELISA 抗体和抗原测定的影响。对以牛白蛋白和人 IgG 作为抗原的许多不同聚苯乙烯微孔板的比较显示出 2 个主要类型的板：那些吸附白蛋白较差的板和那些吸附白蛋白良好的板。 IgG 在所有板上都有很好的吸附，但吸附白蛋白最好的板也给出了显着的非特异性缀合物结合背景水平。当使用IgG和牛血清白蛋白的混合物作为包被抗原时，观察到显着的竞争； .ltoreq 中存在的组件。除非使用过量的缀合物，否则混合物中的 1% 基本上检测不到。重要的因素是竞争剂与抗原的比例，而不是绝对量。其他蛋白质（卵白蛋白、兔白蛋白、人白蛋白和明胶）同样是塑料吸附位点的有效竞争者。非离子去垢剂（Tween 20、Triton X-100）即使在竞争剂与抗原的比例为 10:1 时也是强有力的竞争者。在抗原捕获测定中，正常血清成分会阻断抗原特异性 IgG 的附着；通过使用强结合聚苯乙烯板，这种竞争在一定程度上减轻了。在用于测量血清抗体的间接 ELISA 中，当所需抗原小于包被总蛋白的 1% 时，使用抗原混合物会显着降低抗体滴度。因此，在 ELISA 中使用混合或粗抗原会带来有关测试灵敏度和特异性的重大问题。
Competition between proteins and other macromolecules for adsorption sites on plastic was studied with the enzyme-linked immunosorbent assay (ELISA) to determine effects of the use of antigenic mixtures or extracts of microorganisms on assays of antibodies and antigens by ELISA. A comparison of a number of different polystyrene microplates with bovine albumin and human IgG as antigens showed 2 major classes of plates: those which adsorbed albumin poorly and those which adsorbed albumin well. IgG adsorbed well on all plates, but plates which adsorbed albumin best also gave significant background levels of nonspecific conjugate binding. When mixtures of IgG and bovine serum albumin were used as coating antigens, significant competition was observed; the component present at .ltoreq. 1% in the mixture was essentially undetectable unless excessive amounts of conjugate were used. The important factor was the ratio of competitor to antigen, not the absolute amount. Other proteins (ovalbumin, rabbit albumin, human albumin and gelatin) were equally effective competitors for adsorption sites on plastic. Nonionic detergents (Tween 20, Triton X-100) were strong competitors even at 10:1 competitor-to-antigen ratios. In antigen capture assays, normal serum components blocked attachment of antigen-specific IgG; this competition was lessened to a degree by the use of strongly binding polystyrene plates. In indirect ELISA for measurements of serum antibody, the use of antigenic mixtues gave significantly lower antibody titers when the desired antigen was < 1% of the total protein coated. Therefore, the use of mixed or crude antigens in ELISA presents significant problems concerning the sensitivity and specificity of tests.