Labeta M O, Margni R A, Leoni J, Binaghi R A
Immunology. 1986 Feb;57(2):311-7.
The reactions between purified precipitating and non-precipitating anti-DNP sheep and rabbit antibodies and the antigens DNP-BSA and DNP-GABA-BSA have been studied by immunodiffusion, complement fixation and an inhibition test. Both antigens reacted identically with precipitating antibodies. On the contrary, non-precipitating antibodies did not precipitate and did not fix complement with DNP-BSA but were able to do so with DNP-GABA-BSA. A different behaviour with both antigens was also demonstrated by an inhibition test. The properties of these antibodies were also studied after treatment with endo-beta-N-acetylglucosaminidase H. Non-precipitating antibody was able to give precipitin bands in gel diffusion and to fix complement with DNP-BSA after treatment with the enzyme. The treated antibody was able to agglutinate sensitized erythrocytes. Studies by fluorescence quenching showed that the affinity for the ligand DNP-GABA was significantly increased after hydrolysis of the carbohydrate residue. The properties of precipitating antibody were not modified by the endoglycosidase. Affinity chromatography of the F(ab')2 and Fab fragments obtained from precipitating and non-precipitating antibodies was made with Con A-Sepharose. The Con A retained all the F(ab')2 and 50% of the Fab from non-precipitating antibody, which were subsequently eluted with alpha-methyl-D-mannoside. The fragments from precipitating antibody were not retained at all. It is concluded that the asymmetry of the non-precipitating antibody molecule is due to a carbohydrate moiety which is present in only one of the Fab regions. This carbohydrate affects the reaction between the combining site and the antigen, and renders the molecule functionally univalent.
通过免疫扩散、补体结合和抑制试验，研究了纯化的沉淀性和非沉淀性抗二硝基苯（DNP）绵羊及兔抗体与抗原DNP-牛血清白蛋白（BSA）和DNP-γ-氨基丁酸-BSA之间的反应。两种抗原与沉淀性抗体的反应相同。相反，非沉淀性抗体不能与DNP-BSA形成沉淀，也不能结合补体，但能与DNP-γ-氨基丁酸-BSA形成沉淀并结合补体。抑制试验也证明了两种抗原与非沉淀性抗体的反应不同。用内切β-N-乙酰葡糖胺酶H处理后，对这些抗体的性质也进行了研究。处理后的非沉淀性抗体在凝胶扩散中能够形成沉淀带，并能与DNP-BSA结合补体。处理后的抗体能够凝集致敏红细胞。荧光猝灭研究表明，碳水化合物残基水解后，对配体DNP-γ-氨基丁酸的亲和力显著增加。沉淀性抗体的性质未被糖苷内切酶改变。用伴刀豆球蛋白A-琼脂糖对从沉淀性和非沉淀性抗体获得的F(ab')2和Fab片段进行亲和层析。伴刀豆球蛋白A保留了所有非沉淀性抗体的F(ab')2和50%的Fab，随后用α-甲基-D-甘露糖苷洗脱。沉淀性抗体的片段根本不被保留。结论是非沉淀性抗体分子的不对称性是由于仅存在于一个Fab区域的碳水化合物部分。这种碳水化合物影响结合位点与抗原之间的反应，并使分子在功能上呈单价。
