PRODUCTION OF ANTIBODIES AGAINST ADP-RIBOSE AND 5'-AMP WITH AID OF N-6CARBOXYMETHYLATED ADP-RIBOSE CONJUGATES
PRODUCTION OF ANTIBODIES AGAINST ADP-RIBOSE AND 5'-AMP WITH AID OF N-6CARBOXYMETHYLATED ADP-RIBOSE CONJUGATES
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DOI：
10.1111/j.1432-1033.1978.tb12001.x
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发表时间：
1978-01-01
期刊：
影响因子：
--
通讯作者：
HILZ, H
中科院分区：
文献类型：
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作者：
BREDEHORST, R;FERRO, AM;HILZ, H
通过碳二亚胺法将ADP-核糖直接偶联到牛血清白蛋白上，导致对5“-AMP具有优先亲和力的抗体的低产量。为了允许ADP-核糖残基以更特异性的方式缩合，以2种方式合成N6-羧甲基化ADP-核糖（cm 6 ADP-核糖）和血清白蛋白的缀合物。首先，腺嘌呤-N6-羧甲基化NAD（cm 6 NAD）通过碳二亚胺几乎完全通过羧基与血清白蛋白偶联，这是从缀合物的表位密度（半抗原/白蛋白的数目）与由未修饰的NAD制备的抗原的比较来判断的。推测由于空间位阻，只能实现NAD半抗原至ADP-核糖半抗原的不完全酶促转化。第二，通过用NAD糖水解酶处理，从cm 6 NAD合成cm 6ADP-核糖。该化合物在碳二亚胺的帮助下与血清白蛋白或甲基化血清白蛋白偶联。只有用最后提到的蛋白质受体获得的缀合物引起ADP-核糖抗体的显着滴度，除了类似的5“-AMP抗体的高产量之外。用cm 6ADP-核糖偶联到非甲基化的血清白蛋白，仅产生5“-AMP抗体，其显示出5”-AMP和3“-AMP或核酸中的AMP残基之间的优异区分。5“-AMP抗体的产生可能是由受体动物中ADP-核糖抗原的水解分解引起的。
ADP-ribose coupled directly to bovine serum albumin by the carbodiimide method resulted in a low production of antibodies with a preferential affinity for 5''-AMP. To allow condensation of the ADP-ribose residue in a more specific manner, conjugates of N6-carboxymethylated ADP-ribose (cm6 ADP-ribose) and serum albumin were synthesized in 2 ways. First, adenine-N6-carboxymethylated NAD (cm6NAD) was coupled by carbodiimide to serum albumin nearly exclusively through the carboxyl group as judged from a comparison of the epitope density (number of haptens/albumin) of the conjugate with an antigen prepared from unmodified NAD. Only incomplete enzymic transformation of the NAD hapten to an ADP-ribose hapten could be achieved due presumably to steric hindrance. Second, cm6 ADP-ribose was synthesized from cm6NAD by treatment with NAD glycohydrolase. This compound was coupled to serum albumin or to methylated serum albumin with the aid of carbodiimide. Only conjugates obtained with the last-mentioned protein acceptor elicited significant titers of ADP-ribose antibodies besides a similarly high production of 5''-AMP antibodies. With cm6ADP-ribose coupled to non-methylated serum albumin, 5''-AMP antibodies were produced exclusively, which showed an excellent discrimination between 5''-AMP and 3''-AMP, or AMP residues in nucleic acids. Production of 5''-AMP antibodies was presumably caused by hydrolytic breakdown of the ADP-ribose antigen in the recipient animals.