Degl'Innocenti D, Berti A, Stefani M, Ramponi G
Department of Biochemical Sciences, University of Florence, Italy.
Biotechnol Appl Biochem. 1990 Jun;12(3):292-300.
Monoclonal antibodies to human acylphosphatase (muscle isoenzyme) were generated by an improved hybridoma technique. Immunization consisted of four antigen administrations in an overall period of 15 weeks. After cell fusion and repeated subcloning of positive lines, seven monoclonal antibodies with good affinity and specificity were selected. These antibodies were characterized for their affinity constant and immunoreactivity. The latter was determined using peptides generated by CNBr cleavage of the antigen. One of the selected antibodies had an affinity constant such that it could be used to develop a competitive enzyme-linked immunosorbent assay. In our test, the antigen that was coated on the matrix, and the free one, competed for the antibody-horseradish peroxidase conjugate. No cross-reactivity with the erythrocyte iso-enzyme was found, and the test showed a limit in sensitivity of 0.32 ng/ml of antigen. We expect that the enzyme immunoassay could be useful for clinical application.
通过改进的杂交瘤技术制备了抗人酰基磷酸酶（肌肉同工酶）的单克隆抗体。免疫过程在15周的总时间内进行了四次抗原注射。细胞融合并对阳性细胞系进行反复亚克隆后，选择了七种具有良好亲和力和特异性的单克隆抗体。对这些抗体的亲和常数和免疫反应性进行了表征。后者是使用通过溴化氰切割抗原产生的肽来测定的。所选抗体之一的亲和常数使其可用于开发竞争性酶联免疫吸附测定。在我们的测试中，包被在基质上的抗原和游离抗原竞争抗体-辣根过氧化物酶缀合物。未发现与红细胞同工酶有交叉反应，该测试显示抗原的灵敏度极限为0.32 ng/ml。我们期望该酶免疫测定可用于临床应用。
