Teresa M, Ribela C P, Camargo I M, Oliveira J E, Bartolini P
Department of Application of Nuclear Techniques in Biological Sciences, IPEN-CNEN, São Paulo, 05508, Brazil.
Protein Expr Purif. 2000 Mar;18(2):115-20. doi: 10.1006/prep.1999.1184.
A good quality tracer, to be used in the radioimmunoassay of human growth hormone, was prepared by applying the chloramine-T iodination technique to the recombinant product obtained after a single-step high-performance size-exclusion chromatography purification of a bacterial osmotic shock fluid. The labeling reaction presented a yield of about 65% and the purified tracer exhibited an antibody binding of approximately 50% (NIDDK reference antiserum diluted 1:600,000). These values are very similar to those obtained by radioiodinating highly purified clinical-grade recombinant human growth hormone obtained from the same periplasmic extract after the regular six-step purification process. Both tracers provided the same accuracy, when evaluated with the use of commercial-quality control samples in a classical radioimmunoassay methodology, their stability being practically identical: about 18% decrease in antibody binding after 2 months of storage at -20 degrees C. The novel approach permits the utilization of transformed Escherichia coli strains as a source of freshly prepared, radioiodination-grade recombinant proteins, capable of providing better reproducibility and reagent continuity.
通过将氯胺-T 碘化技术应用于细菌渗透休克液经单步高效尺寸排阻色谱纯化后获得的重组产物，制备了一种用于人生长激素放射免疫测定的优质示踪剂。标记反应的产率约为65%，纯化后的示踪剂显示出约50%的抗体结合率（NIDDK 参考抗血清稀释1:600,000）。这些值与通过对经常规六步纯化过程后从相同周质提取物中获得的高度纯化的临床级重组人生长激素进行放射性碘化所得到的值非常相似。在经典放射免疫测定方法中使用商业质量控制样品进行评估时，两种示踪剂提供了相同的准确性，它们的稳定性几乎相同：在-20℃储存2个月后，抗体结合率下降约18%。这种新方法允许利用转化的大肠杆菌菌株作为新鲜制备的放射性碘化级重组蛋白的来源，能够提供更好的重现性和试剂连续性。
