Methods in immunochemistry of viruses. 3. Simple techniques for labelling antibodies with 131-I and 35S.
Methods in immunochemistry of viruses. 3. Simple techniques for labelling antibodies with 131-I and 35S.
复制标题
病毒免疫化学方法。
DOI：
10.1038/icb.1962.36
复制
发表时间：
1961
期刊：
影响因子：
--
通讯作者：
S. FAZEKAS DE ST GROWTH
中科院分区：
文献类型：
--
作者：
R. Webster;W. Laver;S. FAZEKAS DE ST GROWTH
本文介绍了用S/SUP35/和I/SUP131/标记抗体的方法。最终产物在血清学上是不变的，因此适合于研究稀释体系或微量的免疫相互作用。所描述的碘化过程包括碘与酪氨酰的结合，以及在较小程度上蛋白质的组氨酸残基，以及伴随而来的碘化氢的释放。载体0.001.5M KI(0.25ml)与无载体放射性碘混合，用蒸馏水将体积提高至3.5ml，并用0.2mlNH/sub2/so/sub4/酸化。加入0.2mlH/sub2/O/sub2/(100体积)释放碘，反应混合物以2.0mlCCl/sub4/间隔摇动30min，将游离碘提取到有机相中。然后移除水相并洗涤CCl/sub4/洗涤。血清球蛋白用0.1M碳酸氢盐缓冲液在pH 8.9时加入洗涤的CCL/SUB4/中，立即混合。将球蛋白-CCl/Sub4/混合物轻轻搅拌10分钟，离心分离两相，水相在自来水中透析12小时，最后在SSL线上透析1小时。由于上述程序的结果，20%的输入放射性结合到蛋白质上。这相当于每11.95毫克蛋白质含0.05微克碘，即每3.96×10/sup-16/mg蛋白质含一个碘原子，或每个抗体分子含0.71个碘原子。将S/sup 35/LABEL掺入兔抗体中，在抗体合成高峰期喂饲S/sup 35/标记的蛋氨酸和胱氨酸，从含有硫酸盐-S/sup 35/的培养基中获得。用5000剂猪流感病毒血凝剂第3次加强免疫后第5天，静脉注射含氨基酸的S/苏普35/-氨基酸水解物。24小时后，兔子放血，血清分离并储存在-15℃下。细菌中存在的约43%的标记在蛋白质水解后的氨基酸部分中得到回收，假设从兔体内回收50ml血清，注射的标记3.5%被结合到血清蛋白中。该抗体的生物学特性与未标记的抗体相同。因此，用S/sup 35/内部标记抗体提供了一种廉价的标记抗体来源，与未标记的抗体在化学上难以区分，具有相当长的半衰期(87.1d)。然而，这种方法有点复杂，与所有体内标记抗体的方法一样，99%以上的活性被结合到其他蛋白质中。使用i/sup 131/作为放射性标记的主要缺点是其半衰期相对较短(8.1天)；但由于该试剂可获得无载体且价格低廉，因此有可能获得几乎任何所需的放射性水平，而不会增加平均每个蛋白质分子的单个放射性原子以上的碘化程度。这种常规标记方法固有的另一个缺点是，标记的分子与原始分子在化学上不相同，并且每个抗体分子中超过1到2个碘原子可以改变免疫反应性。
Methods for labeling antibody with S/sup 35/ and I/sup 131/ are described. The final product is serologically unaltered and thus suited to the study of immunologic interactions in dilute systems or in microquantities. The described iodination procedure involves the combination of iodine with tyrosyl and, to a smaller extent, histidyl residues of the protein, and the concomitant liberation of hydrogen iodide. Carrier 0.001 M KI (0.25 ml) was mixed with carrier-free radioiodide, the volume brought up to 3.5 ml with distilled water and acidified with 0.2 ml N H/sub 2/SO/sub 4/. Iodine was liberated by the addition of 0.2 ml of H/sub 2/O/sub 2/ (100 volumes), and the reaction mixture was shaken with 2.0 ml CCl/sub 4/ at intervals for 30 min to extract the free iodine into the organic phase. The aqueous phase was then removed and the CCl/ sub 4/ washed. Serum globulin, buffered at pH 8.9 with 0.1 M bicarbonate buffer, was added to the washed CCl/sub 4/ and mixed immediately. This globulin-CCl/sub 4/ mixture was gently agitated for 10 min, the 2 phases separated by centrifuging, and the aqueous phase dialyzed against running tap water for 12 hr and finally against ssline for 1 hr. Asmore » a result of the procedure described above, 20% of the input radioactivity was bound to protein. This amounts to 0.05 mu moles of iodine per 11.95 mg protein and hence to one atom of iodine per 3.96 x 10/sup -16/ mg protein, or 0.71 atoms of iodine per antibody molecule. The S/ sup 35/ label was incorporated into rabbit antibody by feeding the animals, during maximum antibody synthesis, S/sup 35/-labeled methionine and cystine obtained from Escherichia culi grown in a medium containing sulfate-S/sup 35/. On the fifth day after boosting rabbits for the third time with 5000 hemagglutinating doses of swine influenza virus a S/sup 35/-amino acid-containing hydrolyzate was injected intravenously. The rabbits were bled 24 hr later and the serum separated and stored at --15 deg C. About 43% of the label present in the bacteria was recovered in the amino acid fraction after hydrolysis of the protein and, assuming a recovery of 50 ml of serum from the rabbit, 3.5% of the injected label was incorporated into serum proteins. The biological properties of the antibody were identical with those of unlabeled antibody. Thus, internal labeling of antibody with S/sup 35/ offers a cheap source of tagged antibody, chemically indistinguishable from unlabeled antibody, which has a reasonably long half-life (87.1 days). However, this method is somewhat more complex and, as with all in vivo methods of labeling antibody, over 99% of the activity is incorporated into other proteins. The major disadvantage of using I/sup 131/ as a radioactive label is its relatively short half-life (8.1 days); but as the reagent is obtainable carrier-free and is inexpensive, it is possible to attain almost any desired level of radioactivity without increasing the degree of iodination beyond a single radioactive atom, on the average, per protein molecule. The other disadvantage inherent in this general method of labeling is that the labeled molecaie is not chemically identical with the original and that more than 1 or 2 atoms of iodine per antibody molecule can alter the immunological reactivity.(BBB)« less