Levieux D
Ann Rech Vet. 1978;9(3):523-30.
A solid-phase radioimmunoassay (RIA) has been developed for quantitation of class-specific antibodies against bacteria. Brucella abortus cells were used to sensitize glass tubes after sedimentation, drying and methanol fixation. Bovine antibodies that attached to the bacteria were detected by binding of a rabbit antiserum specific for each class or subclass of bovine immunoglobulins followed by binding of 125I sheep anti-rabbit immunoglobulin reagent. This three-step method was adapted as it was very sensitive (less than 0.3 picogram antibody/ml), gave the best discrimination between sample and background counts and elminated the need for several labeled rabbit anti-bovine immunoglobulin class-specific antisera. Parameters affecting quantitation of the assay were investigated: nature and preparation of the test tube, quantity and fixation of Brucella, temperature and incubation time, incubation volume, concentration of reagents. The RIA and the complement fixation test (CFT) were applied to sera obtained at repeated intervals from seven heifers experimentally infected with Brucella abortus during gestation. Rabbit antisera specific for bovine IgG1 were used in the RIA because of antibodies of this subclass are the predominant type implicated in the CFT. The kinetic of appearance of Brucella specific antibodies were very significantly correlated for the two tests (r = 0.056, P less than 0.01) demonstrating the validity of the proposed RIA. This test was 10,000 times more sensitive than CFT.
已开发出一种固相放射免疫测定法（RIA）用于定量检测针对细菌的类特异性抗体。流产布鲁氏菌细胞经沉降、干燥和甲醇固定后用于包被玻璃管。通过结合针对牛免疫球蛋白每个类或亚类的兔抗血清，随后结合125I标记的羊抗兔免疫球蛋白试剂，检测附着在细菌上的牛抗体。采用这种三步法是因为它非常灵敏（低于0.3皮克抗体/毫升），能最好地区分样品和背景计数，并且无需几种标记的兔抗牛免疫球蛋白类特异性抗血清。研究了影响该测定法定量的参数：试管的性质和制备、布鲁氏菌的数量和固定、温度和孵育时间、孵育体积、试剂浓度。将RIA和补体结合试验（CFT）应用于从7头在妊娠期实验性感染流产布鲁氏菌的小母牛身上反复采集的血清。在RIA中使用针对牛IgG1的兔抗血清，因为该亚类抗体是CFT中涉及的主要抗体类型。两种检测方法中布鲁氏菌特异性抗体出现的动力学具有非常显著的相关性（r = 0.056，P < 0.01），证明了所提出的RIA的有效性。该检测方法比CFT灵敏10000倍。
