Noureldin M S, El-Naggar H M, El-Shinnawy H, Abou Elenin A, El Sherif E A
Department of Parasitology, Faculty of Medicine, Mansoura University, Mansoura, Egypt.
J Egypt Soc Parasitol. 1999;29(3):927-37.
Usually mouse monoclonal antibodies are used in inhibition assays for antibody determination. Interference may occur in these inhibition assays due to presence of naturally occurring anti-mouse antibodies in some human serum samples. To avoid such interference, human IgG isolated from a pool of serum samples of S. mansoni patients and highly positive for IgG against S. mansoni soluble egg antigen (SEA) was used in inhibition ELISA for diagnosis of S. mansoni infection. The assay was based on inhibition of binding of human IgG labeled with fluorescein to S. mansoni SEA coating microtitration plates by tested serum samples. Plates were washed and labeled human IgG reacted with SEA was linked to peroxidase enzyme by incubation with anti-fluorescein/peroxidase conjugate. The assay showed 90% sensitivity and 96.3% specificity. The level of inhibition in ELISA showed highly significant positive correlation with stool egg output (Kandall's tau b = 0.512, P < 0.001). To make the assay quantitative, serial dilutions of the highly positive human serum pool, used for preparation of human IgG, were applied in each plate and concentration of anti-SEA antibodies in serum samples tested was calculated from a 4-parameters logistic curve equation. The highly positive serum pool used as a standard was considered to contain one million arbitrary units of immunoglobulins against S. mansoni SEA. Human IgG is expected to be more practical in inhibition assays than mouse monoclonal antibodies due to elimination of interference caused by naturally occurring human anti-mouse antibodies. Also, large amount of human IgG could be purfied from remnants of serum samples highly positive for the proposed antibodies. A higher specificity and sensitivity could be obtained if IgG is isolated by affinity purification instead of ammonium sulphate precipitation. In conclusion, human IgG isolated from highly positive serum samples could be used in sensitive and specific diagnostic antibody determination inhibition assays for diagnosis of infectious and autoimmune diseases.
通常，小鼠单克隆抗体用于抗体测定的抑制试验。由于某些人血清样本中存在天然抗小鼠抗体，这些抑制试验可能会出现干扰。为避免此类干扰，从曼氏血吸虫患者血清样本库中分离出的、对曼氏血吸虫可溶性虫卵抗原（SEA）的IgG呈高度阳性的人IgG，被用于抑制ELISA法诊断曼氏血吸虫感染。该试验基于受检血清样本对用荧光素标记的人IgG与包被在微量滴定板上的曼氏血吸虫SEA结合的抑制作用。洗涤平板后，将与SEA反应的标记人IgG与抗荧光素/过氧化物酶缀合物孵育，使其与过氧化物酶相连。该试验显示出90%的灵敏度和96.3%的特异性。ELISA中的抑制水平与粪便虫卵排出量呈高度显著正相关（肯德尔氏tau b = 0.512，P < 0.001）。为使试验定量，将用于制备人IgG的高度阳性人血清库进行系列稀释，应用于每块平板，并根据四参数逻辑曲线方程计算受检血清样本中抗SEA抗体的浓度。用作标准的高度阳性血清库被认为含有针对曼氏血吸虫SEA的100万个任意单位的免疫球蛋白。由于消除了天然存在的人抗小鼠抗体引起的干扰，预计人IgG在抑制试验中比小鼠单克隆抗体更实用。此外，可以从对所提议抗体高度阳性的血清样本残余物中纯化出大量人IgG。如果通过亲和纯化而非硫酸铵沉淀来分离IgG，则可获得更高的特异性和灵敏度。总之，从高度阳性血清样本中分离出的人IgG可用于敏感且特异的诊断抗体测定抑制试验，以诊断感染性和自身免疫性疾病。
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