Chen B X, Szabolcs M J, Matsushima A Y, Erlanger B F
Department of Pathology, Columbia University, New York, NY 10032, USA.
J Histochem Cytochem. 1996 Aug;44(8):819-24. doi: 10.1177/44.8.8756754.
We report a novel strategy, called end-product (EP) amplification, capable of enhancing the sensitivity of immunohistochemical procedures by about an order of magnitude or more. The strategy employs an antibody (anti-EP) to the product generated by the action of horseradish peroxidase on 3,3'-diaminobenzidine (DAB), and can be extended to the products of other enzymes as well, e.g., alkaline phosphatase. Amplification is the consequence of the ability of anti-EP to detect the multiplicity of product moelcules resulting from the turnover of substrate by a single enzyme molecule. The subsequent detection of anti-EP was by biotinylated goat anti-rabbit antibody, followed by avidin-peroxidase and DAB or by avidin-alkaline phosphatase and Vector Red. Further amplification can be accomplished by repeated cycles of the protocol. Anti-EP was produced by immunization with a bovine serum albumin (BSA) conjugate of a soluble polymer of DAB, prepared by a carefully controlled reaction of DAB with horseradish peroxidase and hydrogen peroxide. Coupling to BSA (and to RSA) was accomplished with glutaraldehyde. The titer of anti-EP was established by ELISA. Formalin-fixed, paraffin-embedded sections of five cases of Hodgkin's disease and five tonsils with follicular hyperplasia were immunolabeled for the following lymphoid markers: CD3, CD20, CD30, CD45RA, and CD68. EP amplification with anti-EP was also applied to cases of CMV pneumonia and cerebral toxoplasmosis to determine whether this procedure could improve detection of the infectious agents. Immunolabeling of the primary antibody was performed by the avidin-biotin-peroxidase technique with DAB as the reaction substrate. The specificity of EP amplification was tested by demonstrating binding of anti-EP with Vector Red with the generation of a fluorescence end-point. There was complete congruence in the distribution of the DAB signal and the red immunofluorescence representing EP amplification. The intensity of the DAB signal was increased as much as 16-fold by EP amplification, making possible a reduction in the amount of the primary antibody by as much as 85-90%. Sensitivity also increased with respect to weakly expressed antigens and low concentrations of infectious agents.
我们报告了一种名为终产物（EP）放大的新策略，它能够将免疫组织化学程序的灵敏度提高约一个数量级或更多。该策略使用一种针对辣根过氧化物酶作用于3,3'-二氨基联苯胺（DAB）所产生产物的抗体（抗-EP），并且也可扩展至其他酶的产物，例如碱性磷酸酶。放大是抗-EP能够检测单个酶分子催化底物周转所产生的多种产物分子的结果。随后对抗-EP的检测是通过生物素化的山羊抗兔抗体，接着是抗生物素蛋白-过氧化物酶和DAB，或者是抗生物素蛋白-碱性磷酸酶和Vector Red。通过重复该方案的循环可以实现进一步放大。抗-EP是通过用DAB可溶性聚合物的牛血清白蛋白（BSA）缀合物免疫制备的，该缀合物是通过DAB与辣根过氧化物酶和过氧化氢的精心控制反应制备的。通过戊二醛实现与BSA（以及与RSA）的偶联。抗-EP的效价通过ELISA确定。对5例霍奇金病和5例滤泡性增生扁桃体的福尔马林固定、石蜡包埋切片进行免疫标记，检测以下淋巴标志物：CD3、CD20、CD30、CD45RA和CD68。用抗-EP进行的EP放大也应用于巨细胞病毒肺炎和脑弓形虫病病例，以确定该程序是否能改善对感染因子的检测。一抗的免疫标记通过抗生物素蛋白-生物素-过氧化物酶技术，以DAB作为反应底物进行。通过证明抗-EP与Vector Red结合并产生荧光终点来测试EP放大的特异性。代表EP放大的红色免疫荧光与DAB信号的分布完全一致。通过EP放大，DAB信号强度增加了多达16倍，使得一抗的用量有可能减少多达85 - 90%。对于弱表达抗原和低浓度感染因子，灵敏度也有所提高。
