Du Weidong, Xu Zhenshan, Ma Xueling, Song Lihua, Schneider E Marion
Sektion Experimentelle Anaesthesiologie, Universitaetsklinikum Ulm, Steinhoevelstrasse 9, Ulm D-89075, Germany.
J Biotechnol. 2003 Dec 5;106(1):87-100. doi: 10.1016/j.jbiotec.2003.08.001.
The formation of antibodies against cytokines may play a major role in the generation of the immune response and may affect treatment protocols with recombinant cytokines. Interferon (IFN) is one of the effective therapeutic agents with anti-viral and anti-tumor specific effects. The appearance of IFN antibodies in patients may limit the natural and the therapeutic effect by IFNs. In contrast to conventional ELISA techniques, we here report a simple biochip methodology that enables identification of antibodies against cytokines and peptides. The method takes advantage of a functionalized self-assembled monolayer modified by N-hydroxysuccinimide (NHS). To validate this surface, four human proteins: IFNalpha2b, leptin, growth hormone and human IgG, with molecular sizes ranging between 14 and 150 kDa, were used. A number of other parameters for protein assay conditions by array technology were evaluated concomitantly. Finally, 56 serum samples from patients treated with recombinant human IFNalpha2b were simultaneously tested on single chip. In these patients, 16.1% (9 of 56 cases) were positive for IFNalpha2b antibodies. All results were confirmed in an ELISA, specific for the identification of IFNalpha specific antibodies in human samples. The potential application of this protein biochip can be amplified rapidly and reliably to test not only IFNalpha2b, but also other cytokine specific antibodies. The clinical relevance of such assays for investigations in autoimmune disorders is expected.
抗细胞因子抗体的形成可能在免疫反应的产生中起主要作用，并且可能影响重组细胞因子的治疗方案。干扰素（IFN）是具有抗病毒和抗肿瘤特异性作用的有效治疗剂之一。患者体内IFN抗体的出现可能会限制IFN的天然作用和治疗效果。与传统的ELISA技术不同，我们在此报告一种简单的生物芯片方法，该方法能够鉴定抗细胞因子和肽的抗体。该方法利用了由N-羟基琥珀酰亚胺（NHS）修饰的功能化自组装单层。为了验证该表面，使用了四种人类蛋白质：IFNα2b、瘦素、生长激素和人IgG，其分子大小在14至150 kDa之间。同时评估了通过阵列技术进行蛋白质检测条件的许多其他参数。最后，在单个芯片上同时检测了56例接受重组人IFNα2b治疗患者的血清样本。在这些患者中，16.1%（56例中的9例）IFNα2b抗体呈阳性。所有结果均在ELISA中得到证实，该ELISA专门用于鉴定人样本中的IFNα特异性抗体。这种蛋白质生物芯片的潜在应用可以快速可靠地扩展，不仅用于检测IFNα2b，还可用于检测其他细胞因子特异性抗体。预计此类检测在自身免疫性疾病研究中的临床相关性。
