Cunningham M W, Russell S M
Infect Immun. 1983 Nov;42(2):531-8. doi: 10.1128/iai.42.2.531-538.1983.
Heart-reactive antibody is known to be produced in animals immunized with group A streptococci. However, the detection and quantitation of these antibodies and their respective antigens have been limited to immunofluorescence or immunoprecipitation tests. In this study a more sensitive technique was used to detect heart-reactive antibody, i.e., the enzyme-linked immunosorbent assay (ELISA). Countercurrent immunoelectrophoresis and agar gel immunodiffusion were also used to confirm the reactivity of rabbit antisera to group A streptococci and human heart tissue. Human heart tissue antigen was prepared by Triton X-100 extraction, and checkerboard titrations of heart tissue antigen versus streptococcal antisera revealed optimal concentrations of each for the ELISA. When immune rabbit serum was reacted with heart tissue antigen, a 5- to 10-fold increase was observed over the reactions of preimmune sera controls. Streptococcal antiserum was found to have a six- to eightfold greater reactivity with heart tissue antigen than with similar concentrations of kidney and skeletal muscle antigens. Heart tissue antigen was partially purified by DEAE-cellulose chromatography, and quantities of greater than 10 to 100 ng (dry weight) were shown to react with streptococcal antisera in the ELISA. Heart, kidney, and skeletal muscle antigens were subjected to electrophoresis in polyacrylamide gel slabs and blotted onto a nitrocellulose filter. These filter blots reacted with streptococcal antisera and confirmed the tissue antigen specificity observed with the ELISA. In addition, the ELISA was found to be an effective method for the detection of heart-reactive antibodies produced by murine hybridomas that were producing antibody to group A streptococci.
已知在用A组链球菌免疫的动物中会产生心脏反应性抗体。然而，这些抗体及其各自抗原的检测和定量仅限于免疫荧光或免疫沉淀试验。在本研究中，使用了一种更灵敏的技术来检测心脏反应性抗体，即酶联免疫吸附测定（ELISA）。还使用了对流免疫电泳和琼脂凝胶免疫扩散来证实兔抗血清对A组链球菌和人心脏组织的反应性。通过Triton X - 100提取制备人心脏组织抗原，心脏组织抗原与链球菌抗血清的棋盘滴定法揭示了ELISA中每种抗原的最佳浓度。当免疫兔血清与心脏组织抗原反应时，与免疫前血清对照的反应相比，观察到增加了5至10倍。发现链球菌抗血清与心脏组织抗原的反应性比与相似浓度的肾脏和骨骼肌抗原高6至8倍。心脏组织抗原通过DEAE - 纤维素色谱法进行部分纯化，并且显示大于10至100 ng（干重）的量在ELISA中与链球菌抗血清反应。将心脏、肾脏和骨骼肌抗原在聚丙烯酰胺凝胶板中进行电泳，然后印迹到硝酸纤维素滤膜上。这些滤膜印迹与链球菌抗血清反应，并证实了ELISA中观察到的组织抗原特异性。此外，发现ELISA是检测由产生抗A组链球菌抗体的鼠杂交瘤产生的心脏反应性抗体的有效方法。
