氧化型琼脂糖凝胶基片结合酶免疫反应的蛋白质分子微阵列

Protein microarray based on oxidation agarose-gel and enzyme immunoassay

目的 研制适于常规免疫学检验的蛋白质分子微阵列。方法 利用分子组装技术 ,以琼脂糖凝胶为基质 ,于玻片表面制成自组装膜 ,经过碘酸钠氧化后再与戊二醛分子偶联 ,而成为具有双功能分子层的薄膜。基片表面衍生的醛基可与各类抗原 (如重组多肽、磷脂和DNA)、辣根过氧化物酶及抗体分子中的氨基形成Schiff′s碱共价连接。结果 基于上述原理 ,制作生物分子微阵列 ,优选出IgG分子的最佳点样浓度为 10 0 μg/ml,点样量以 5 0nl为宜。通过不同的实验模式 ,成功地在基片表面建立了酶免疫检测体系 ,可敏感特异地检出抗原、抗体分子及酶与底物之间的相互作用。结论 该法最突出的优点在于减少了样本和试剂用量 ,可对多种自身抗体实施高通量的平行分析 ,以酶 底物 (HRP DAB H2 O2 )作为芯片信号显示系统 ,结果较为直观 。

Objective To investigate protein microarray which are suitable for antigen and antibody immuno detection.Methods Based on molecular assembly technique, the matrix of agarose gel was deposited on the surface of glass plates,followed by drying, oxidation with NaIO 4 and cross linking with glutaraldehyde.The gel matrix membrane provided double functions with molecular layer, and aldehyde group devired from the surface linked with amino groups in various antigens (recombinant peptides, phospholipid and DNA, etc.),antibodies as well as horseradish peroxydase to form steady Shiff base coupled by covalent bond.Results The biomolecules microarrays were prepared on the basis of the above priciples.The optimum spoting concentration of IgG molecular was 100μg/ml and the sample volume was 50nl.Through several experimentai patterns,an enzyme immunoassay system was successfully developed on the surface of gel matrix. The microarrays could be used for detection of antigens, antibodies,and the interaction of enzyme with its substrate.Conclusions The main advantage of this microarray was highly economical in the use of speciments and reagents, and was performing autoantibodies of massive parallel analysis. Since the enzyme substrate (HRP DAB H 2O 2)was served as the indicator system of signals, the results were directly visible and more stable as in comparison with fluorescent immunoassay. This technique was more practical and broad applications.