Munari Leonardo, Charchat Sheila, Rodrigues Luiz, von Muhlen Carlos Alberto, Baú Anne Rose, Lavinsky Luiz, Bonorino Cristina
Faculdade de Biociências and Instituto de Pesquisas Biomédicas, Hospital São Lucas, Pontifícia Universidade Católica do Rio Grande do Sul, Av. Ipiranga, 6690 2o andar 90610-000 RS Porto Alegre, Brazil.
J Immunol Methods. 2003 Dec;283(1-2):155-61. doi: 10.1016/j.jim.2003.09.008.
A Western blot to detect anti-HSP70 autoantibodies has been reported to be of diagnostic value for immune-mediated hearing loss patients. While setting up this Western blot in our lab, we detected two main problems. First, some patients were positive for antibodies to a 70-kDa protein when tested against a whole cell lysate, but negative if the antigen used was purified HSP70. Second, if high amounts of purified HSP70 were loaded on the gel, both patients and healthy controls were positive. We have developed and optimized an ELISA as an alternative to the Western blot. This assay is more appropriate to identify positive and negative individuals because it is semi-quantitative. The ELISA is also more sensitive, requiring very low concentrations of the antigen and thus minimizing false positives. Finally, we demonstrated that immune-mediated hearing loss patients recognize mainly the native form of HSP70, a fact that potentially leads to false negatives when a denaturing Western blot assay is used for diagnosis. To test the diagnostic value of the ELISA, we performed a blind test with 70 hearing loss patients, as well as 30 healthy controls. A sensitivity of 84% and a specificity of 93% were obtained, superior to what has been reported so far for the Western blot.
据报道，检测抗HSP70自身抗体的蛋白质印迹法对免疫介导性听力损失患者具有诊断价值。在我们实验室建立这种蛋白质印迹法时，我们发现了两个主要问题。首先，一些患者在针对全细胞裂解物进行检测时，对一种70 kDa蛋白的抗体呈阳性，但如果使用的抗原是纯化的HSP70，则呈阴性。其次，如果在凝胶上加载大量纯化的HSP70，患者和健康对照均呈阳性。我们开发并优化了一种酶联免疫吸附测定（ELISA）作为蛋白质印迹法的替代方法。该检测方法更适合于识别阳性和阴性个体，因为它是半定量的。ELISA也更敏感，需要非常低浓度的抗原，从而将假阳性降至最低。最后，我们证明免疫介导性听力损失患者主要识别HSP70的天然形式，这一事实在使用变性蛋白质印迹法进行诊断时可能导致假阴性。为了测试ELISA的诊断价值，我们对70名听力损失患者以及30名健康对照进行了盲法检测。获得了84%的灵敏度和93%的特异性，优于迄今为止报道的蛋白质印迹法。
