Singh K V, Kaur Jasdeep, Varshney Grish C, Raje Manoj, Suri C Raman
Institute of Microbial Technology, Sector 39-A, Chandigarh 160 036, India.
Bioconjug Chem. 2004 Jan-Feb;15(1):168-73. doi: 10.1021/bc034158v.
For the generation of antibodies against small hapten molecules, the hapten is cross-linked with some carrier protein to make it immunogenic. However, the formation of such conjugates is not always reproducible. This may lead to inconsistent hapten-protein stoichiometries, resulting in large variations in the generation of the desired antibodies. In the study described here the hapten (mercaptopropionic acid derivative of atrazine) was coupled to carrier protein at five different molar ratios. The hapten-protein conjugates prepared were characterized thoroughly by spectrophotometric absorption, fluorescence, matrix-assisted laser desorption ionization (MALDI), and gel electrophoresis methods, before being used for the immunization and assay purposes. Electrophoresis and fluorescence methods were very useful in detecting hapten-protein cross-linking while MALDI-MS and spectrophotometric detection provided qualitatively comparable hapten density. The production of specific antibodies was sought following the generation of appropriate hapten-protein conjugates. A high antibody titer with moderate antibody specificity was obtained with hapten density around 15 molecules per carrier protein. The study proved useful for monitoring the course of hapten-protein conjugation for the production of specific antibodies against small molecules.
为了产生针对小分子半抗原的抗体，需将半抗原与某种载体蛋白交联，使其具有免疫原性。然而，此类偶联物的形成并非总能重现。这可能导致半抗原与蛋白质的化学计量比不一致，从而使所需抗体的产生存在很大差异。在本文所述的研究中，半抗原（阿特拉津的巯基丙酸衍生物）以五种不同的摩尔比与载体蛋白偶联。制备的半抗原 - 蛋白质偶联物在用于免疫和检测目的之前，通过分光光度吸收、荧光、基质辅助激光解吸电离（MALDI）和凝胶电泳方法进行了全面表征。电泳和荧光方法在检测半抗原 - 蛋白质交联方面非常有用，而MALDI - MS和分光光度检测提供了定性可比的半抗原密度。在生成合适的半抗原 - 蛋白质偶联物后，寻求产生特异性抗体。当每个载体蛋白的半抗原密度约为15个分子时，获得了高抗体滴度和中等抗体特异性。该研究证明有助于监测半抗原 - 蛋白质偶联过程，以生产针对小分子的特异性抗体。
