Scott M A, Davis J M, Schwartz K A
Department of Pathology, Michigan State University, East Lansing, USA.
Vet Immunol Immunopathol. 1997 Nov;59(3-4):205-12. doi: 10.1016/s0165-2427(97)00073-1.
Staphylococcal protein A (SpA) binds with high affinity to immunoglobulins from many species, making it a useful reagent for immunoassays and immunoglobulin purification procedures. However, its use is limited by poor reactivity with some immunoglobulin subclasses including human IgG3 and murine IgG1. Reports of SpA's reactivity with canine immunoglobulins have been inconsistent. Because the most recent reports indicated a much greater reactivity of purified SpA with canine immunoglobulins than was suggested by our observations, we quantitatively reevaluated the binding of canine IgG and IgM to Cowan I strain SpA under the conditions of use in our laboratory. IgG and IgM were purified from pooled normal canine plasma by affinity chromatography with heavy chain specific polyclonal anti-IgG and anti-IgM antibodies. The purified IgG and IgM were assessed for SpA reactivity by affinity chromatography using a SpA-agarose column. The relative proportions of total chromatographed IgG or IgM in the flow-through (SpA-nonbindable) and eluate (SpA-bindable) fractions were determined by absorbance at 280 nm. The IgG and IgM in each immunoglobulin fraction were also nonspecifically adsorbed to microtitration plates and tested for reactivity with 125I-SpA using a solid phase immunoradiometric assay (IRMA). Approximately 18% of the affinity purified canine IgG and 33% of the affinity purified canine IgM did not bind to the SpA affinity column and were also unreactive with 125I-SpA using the IRMA. A second approach using a different polyclonal antibody to canine IgG yielded similar results: about 21% of the purified IgG was unreactive with SpA. Incomplete reactivity of SpA with canine IgG and IgM limits the usefulness of SpA in canine immunologic procedures.
葡萄球菌蛋白A（SpA）能与许多物种的免疫球蛋白高亲和力结合，使其成为免疫测定和免疫球蛋白纯化程序中的一种有用试剂。然而，它与包括人IgG3和鼠IgG1在内的一些免疫球蛋白亚类的反应性较差，限制了其应用。关于SpA与犬免疫球蛋白反应性的报道并不一致。由于最近的报道表明纯化的SpA与犬免疫球蛋白的反应性比我们观察到的要强得多，我们在本实验室的使用条件下对犬IgG和IgM与考恩I株SpA的结合进行了定量重新评估。通过使用重链特异性多克隆抗IgG和抗IgM抗体的亲和色谱法从混合的正常犬血浆中纯化IgG和IgM。使用SpA-琼脂糖柱通过亲和色谱法评估纯化的IgG和IgM的SpA反应性。通过在280nm处的吸光度测定流过部分（SpA不可结合）和洗脱部分（SpA可结合）中总色谱IgG或IgM的相对比例。每个免疫球蛋白组分中的IgG和IgM也非特异性吸附到微量滴定板上，并使用固相免疫放射测定法（IRMA）检测与125I-SpA的反应性。大约18%的亲和纯化犬IgG和33%的亲和纯化犬IgM不与SpA亲和柱结合，并且使用IRMA也不与125I-SpA反应。使用针对犬IgG的不同多克隆抗体的第二种方法产生了类似的结果：约21%的纯化IgG与SpA无反应。SpA与犬IgG和IgM的不完全反应性限制了SpA在犬免疫程序中的实用性。
