Peroxidase and fluorescein isothiocyanate as antibody markers. A quantitative comparison of two peroxidase conjugates prepared with glutaraldehyde or periodate anda fluorescein conjugate.
Peroxidase and fluorescein isothiocyanate as antibody markers. A quantitative comparison of two peroxidase conjugates prepared with glutaraldehyde or periodate anda fluorescein conjugate.
复制标题
过氧化物酶和异硫氰酸荧光素作为抗体标记。
DOI：
10.1177/24.9.184204
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发表时间：
1976
期刊：
影响因子：
--
通讯作者：
N. Kors
中科院分区：
文献类型：
--
作者：
D. M. Broorsma;J. G. Steefkerk;N. Kors
用辣根过氧化物酶标记兔抗人免疫球蛋白G抗体，用二步法、高碘酸法或异硫氰酸荧光素(FITC)标记。用两种不同的凝胶类型进行层析分离过氧化物酶结合物。将得到的5种结合物与等量的兔免疫球蛋白G进行标化，用单径向免疫扩散法和被动血凝法测定抗体活性，并与用邻二苯甲胺法测定的酶活性进行比较。使用皮肤冰冻切片和琼脂糖珠模型系统，在直接和间接免疫组织化学试验中确定了5种阳性反应结合物的最终稀释度和绝对量。在过氧化物酶标记过程中，抗体和酶的活性似乎有相当大的损失，而在FITC标记过程中，抗体活性保持不变。然而，用戊二醛制备的过氧化物酶结合物的阳性染色反应仍与荧光素结合物相同或略高。用高碘酸盐制备的过氧化物酶结合物不能被同样程度的稀释。对于间接免疫组织化学方法检测抗体，用戊二醛制备的过氧化物酶结合物与FITC结合物相当。用高碘酸盐制备的过氧化物酶结合物效果较差。
Batches of rabbit anti-human immunoglobulin G antibodies were labeled either with horseradish peroxidase, using the two-step glutaraldehyde method or the periodate method, or with fluorescein isothiocyanate (FITC). The peroxidase conjugates were isolated by chromatography using two different gel types. The five types of conjugates thus obtained were standardized to the same amount of rabbit immunoglobulin G. The antibody activity, as estimated by means of single radial immunodiffusion and passive hemagglutination, and the enzyme activity, determined with orthodianisidine, were compared. The ultimate dilutions and absolute amounts of the five conjugates giving positive reactions were determined in direct and indirect immunohistochemical tests, using both cryostat sections of skin and the agarose bead model system. It appeared that during the peroxidase conjugation procedures there was a considerable loss of abtibody and enzyme activity, whereas in the FITC conjugation procedure the antibody activity remained intact. Neverthe less, peroxidase conjugates prepared with glutaraldehyde still gave positive staining reactions in equal or somewhat higher dilutions than the fluorescin conjugate did. The peroxidase conjugates prepared with periodate could not be diluted to the same extent. For the detection of antibodies by indirect immunohistochemical methods, the peroxidase conjugate, prepared with glutaraldehyde, was comparable to the FITC conjugate. The peroxidase conjugate, prepared with periodate, was less effective.