Greenwalt T J, Swierk E M, Steane E A
J Immunol Methods. 1975 Oct;8(4):351-61. doi: 10.1016/0022-1759(75)90057-5.
Horseradish peroxidase (HRP)-labelled antiglobulin has been used extensively as a histochemical marker. In the method described the stable reaction product formed by using 3,3'-diaminobenzidine (DAB) as the hydrogen donor in the peroxidase reaction was dissolved in a fluoro alcohol, 1,1,1,3,3,3-hexafluoroisopropanol (HPF). Reproducible standardisation curves resulted with HRP. Based on these observations a colorimetric assay procedure was developed to quantitate the amount of antibody coating red cells. This was achieved by treating the coated red blood cells with HRP-labelled rabbit antihuman IgG followed by hypotonic lysis separating the ghosts for colour development with the DAB reagent, solubilisation in HFP, and reading at 450 nm. Preliminary estimates of the number of anti-D molecules on Rhesus positive red cells were found to approximate the results reported using radioiodinated antibodies. The smallest number of anti-D molecules detected was 350 per red cell. Qualitative studies indicate that this procedure can be applied to other blood group systems.
辣根过氧化物酶（HRP）标记的抗球蛋白已被广泛用作组织化学标记物。在所描述的方法中，在过氧化物酶反应中使用3,3'-二氨基联苯胺（DAB）作为氢供体形成的稳定反应产物溶解于氟代醇1,1,1,3,3,3-六氟异丙醇（HPF）中。用HRP得到了可重复的标准化曲线。基于这些观察结果，开发了一种比色测定程序来定量包被红细胞的抗体量。这是通过用HRP标记的兔抗人IgG处理包被的红细胞，然后进行低渗裂解，分离出空泡，用DAB试剂显色，在HFP中溶解，并在450nm处读取来实现的。发现恒河猴阳性红细胞上抗-D分子的数量初步估计与使用放射性碘化抗体报道的结果相近。检测到的抗-D分子的最小数量为每个红细胞350个。定性研究表明，该程序可应用于其他血型系统。
