Measurement of anti-cardiolipin antibodies by an enzyme-linked immunosorbent assay (ELISA): standardization and quantitation of results.
Measurement of anti-cardiolipin antibodies by an enzyme-linked immunosorbent assay (ELISA): standardization and quantitation of results.
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DOI：
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发表时间：
1985-12
影响因子：
4.6
通讯作者：
S. Loizou;J. Mccrea;A. Rudge;R. Reynolds;C. Boyle;E. Harris
中科院分区：
文献类型：
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作者：
S. Loizou;J. Mccrea;A. Rudge;R. Reynolds;C. Boyle;E. Harris
我们描述了一种简单和高灵敏度的双抗体夹心酶联免疫吸附试验（ELISA）的发展，用于测量IgG和IgM抗心磷脂抗体（ACA）。用浓度为45微克/毫升的心磷脂在氮气下蒸发包被微滴板。用10%胎牛血清在磷酸盐缓冲盐水（PBS/FCS）中阻断平板2小时，消除稀释后血清的非特异性结合。然后将稀释为1:100的100微升血清在孔中孵育1小时。随后加入浓度为1微克/毫升的亲和纯化山羊抗人IgG或IgM（100微升）孵育1小时；用碱性磷酸酶偶联兔抗山羊IgG试剂检测ACA，与底物孵育后在405 nm处读比色收率。建立参考血清池，研究该方法在整个灵敏度范围内的重现性，并建立标准曲线。IgG和IgM-ACA的定量正常范围分别为0 ~ 9.0抗心磷脂ELISA单位和0 ~ 8.0抗心磷脂ELISA单位。ELISA和放射免疫测定法测量IgG和IgM类ACA之间存在很强的相关性。报告了65例系统性红斑狼疮（SLE）患者和45例血清阳性类风湿性关节炎患者的结果。ELISA法定量测定ACA水平的优势，应使其成为临床和实验监测SLE及相关自身免疫性疾病患者有用和可靠的方法。
We describe the development of a simple and highly sensitive double antibody sandwich enzyme-linked immunosorbent assay (ELISA) for measuring IgG and IgM anticardiolipin antibodies (ACA). Microtitre plates were coated with cardiolipin at a concentration of 45 micrograms/ml by evaporation under nitrogen. Non-specific binding of diluted sera was eliminated by blocking of plates with 10% fetal calf serum in phosphate buffered saline (PBS/FCS) for 2 h. Then sera (100 microliters) at a dilution of 1:100 were incubated in the wells for 1 h. Affinity purified goat anti-human IgG or IgM (100 microliters) at a concentration of 1 microgram/ml was subsequently added and allowed to incubate for 1 h; detection of ACA was achieved using an alkaline phosphatase conjugated rabbit anti-goat IgG reagent by reading the colorimetric yield at 405 nm after incubation with substrate. Reference serum pools were established to study reproducibility of the assay throughout its sensitivity range, and Standard curves were established. The quantitative normal range was 0-9.0 Anticardiolipin ELISA Units (AEU) for IgG and 0-8.0 (AEU) for IgM-ACA. A strong correlation was found between the ELISA and radioimmunoassay methods for measuring ACA of both IgG and IgM classes. Results from 65 patients with systemic lupus erythematosus (SLE) and 45 patients with seropositive rheumatoid arthritis are also reported. The advantages of the ELISA method for quantitative determination of ACA levels, should make it a useful and reliable method for clinical and experimental monitoring of patients with SLE and associated autoimmune disorders.