Heightened sensitivity of quantitative ELISA for IgM rheumatoid factor with the use of the biotin-streptavidin system.
Heightened sensitivity of quantitative ELISA for IgM rheumatoid factor with the use of the biotin-streptavidin system.
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DOI：
10.1093/ajcp/92.5.630
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发表时间：
1989-11
影响因子：
3.5
通讯作者：
G. Moxley
中科院分区：
文献类型：
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作者：
G. Moxley
传统上，通过IgG包被颗粒（如乳胶珠或红细胞）的凝集来检测风湿因子（RF）。这些凝集试验仅为半定量，不能检测五聚体IgM RF以外的物质。为了优化IgM RF的定量酶联免疫测定（ELISA），其可用于检测来自大量生物流体的材料中的少量IgM RF，研究了几个步骤。与使用过氧化物酶缀合抗体的ELISA相比，生物素化抗体随后被过氧化物酶缀合的链霉亲和素取代导致吸光度相对于RF U/mL的斜率更陡，而不增加背景。用四甲基联苯胺（TMB）代替邻苯二胺（OPD）作为显色底物，可获得更陡的斜率和更低的背景。生物素-链霉亲和素ELISA证明比商业乳胶凝集试验更灵敏，并且与使用IgG包被的人红细胞的筛选玻片凝集试验的灵敏度相似。当将在致敏人红细胞和致敏绵羊红细胞的试管稀释试验中均具有阳性结果的血清与仅致敏人细胞凝集而非绵羊凝集具有阳性结果的其他血清进行比较时，通过ELISA，两种试管稀释试验均具有阳性结果的组的IgM RF水平高于仅致敏人细胞阳性试验的组。
Rheumatoid factors (RFs) have been traditionally detected by the agglutination of IgG-coated particles such as latex beads or erythrocytes. These agglutination assays are only semiquantitative and do not detect materials other than pentameric IgM RF. In order to optimize a quantitative enzyme-linked immunoassay (ELISA) for IgM RF that could be used for detecting small amounts of IgM RF in materials derived from large numbers of biologic fluids, several steps were investigated. In comparison with an ELISA using a peroxidase-conjugated antibody, substitution of a biotinylated antibody followed by peroxidase-conjugated streptavidin led to a steeper slope of absorbance versus U/mL of RF without increasing the background. By using as a chromogenic substrate tetramethylbenzidine (TMB) instead of o-phenylenediamine (OPD), an even steeper slope and a lower background were achieved. The biotin-streptavidin ELISA proved to be more sensitive than a commercial latex agglutination test and similar in sensitivity to a screening slide-agglutination test using IgG-coated human erythrocytes. When sera that had had positive results in tube-dilution assays both with sensitized human erythrocytes and sensitized sheep erythrocytes were compared with other sera with positive results for only agglutination of sensitized human cells but not sheep, the group with positive results for both tube dilution assays had higher IgM RF levels by ELISA than those in the group with only the positive assay for sensitized human cells.