Martinez D, Mari B, Aumont G, Vidalenc T
IEMVT-CIRAD, Pointe à Pitre Guadeloupe, French West Indies.
Vet Microbiol. 1993 Jan;34(1):47-62. doi: 10.1016/0378-1135(93)90006-s.
A solid phase immunosorbent assay to detect antibodies to Dermatophilus congolensis in ruminant sera was developed to be used as a single dilution ELISA in large epidemiological surveys. Optimal conditions for the test are described. The use of blocking proteins to reduce non specific binding was necessary. Non fat dry cow milk and fetal calf serum were the only two efficient blocking agents out of six tested. Comparison of 4 antigenic fractions obtained after sonication and differential centrifugations of D. congolensis cultures showed that cell-wall (CW) or membrane (M) enriched preparations were more specific than a crude extract (CR) or a soluble (S) antigen. Whole spores and filaments performed poorly as antigens. The best sensitivity and specificity of the ELISA were obtained when the cut-off point of positivity was fixed at mean absorbance of negative sera + 2.58 sd. The specificity was then 97.6% either with M or CR antigen. The sensitivity was improved from 93.4% with CR to 98.2% with M antigen. Threshold values for a positive test varied between the 3 geographical areas tested. CW and M were also the most efficient antigens for discerning between serotypes of D. congolensis. The precision of the test was evaluated with CR antigen and expressed in residual expressed in residual coefficient of variation (CV). The precision was CV = 5.1% when each serum was titrated in duplicate and the antibody levels were expressed in absorbances. The expression of antibody levels in arbitrary standard units estimated from calibration curves reduced the precision (CV = 13.8%). Several methods were tested to decrease between plate variability but these did not greatly improve the reproducibility since it was shown that the main source of variation was within the plate.
开发了一种固相免疫吸附测定法，用于检测反刍动物血清中抗刚果嗜皮菌的抗体，以在大型流行病学调查中用作单稀释酶联免疫吸附测定（ELISA）。描述了该检测的最佳条件。使用封闭蛋白以减少非特异性结合是必要的。脱脂奶粉和胎牛血清是六种测试物中仅有的两种有效封闭剂。对刚果嗜皮菌培养物进行超声处理和差速离心后获得的4种抗原组分的比较表明，富含细胞壁（CW）或膜（M）的制剂比粗提物（CR）或可溶性（S）抗原更具特异性。全孢子和菌丝作为抗原表现不佳。当阳性判定点固定为阴性血清平均吸光度+2.58标准差时，ELISA获得了最佳的敏感性和特异性。然后，使用M或CR抗原时，特异性均为97.6%。敏感性从使用CR抗原时的93.4%提高到使用M抗原时的98.2%。阳性检测的阈值在三个测试地理区域之间有所不同。CW和M也是区分刚果嗜皮菌血清型最有效的抗原。用CR抗原评估了检测的精密度，并以残余变异系数（CV）表示。当每份血清一式两份滴定且抗体水平以吸光度表示时，精密度为CV = 5.1%。根据校准曲线以任意标准单位估计抗体水平会降低精密度（CV = 13.8%）。测试了几种方法以减少板间变异性，但这些方法并没有显著提高重现性，因为已表明主要变异来源在板内。
