R. Deodato, D. Santos, H. M. Cruz, C. Lima, Carolina Soares van der Meer, L. Villar
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引用次数: 0
摘要
a (ELISA)用于亲和层析。采用聚丙烯酰胺凝胶电泳(SDS-PAGE)、斑点免疫印迹和酶联免疫吸附试验(ELISA)检测IgY抗hbs。用分光光度计测定总蛋白浓度。总蛋白浓度较高的样品被评价为捕获蛋白,用于室内ELISA检测HBsAg。结果:通过SDS Page对IgY进行鉴定,发现了分子量在75 ~ 50kDa和37 ~ 25kDa之间的染色肽带。在斑点斑点试验中,观察到整个样品周期存在抗原-抗体反应。亲和层析纯化后总蛋白的浓度均值±标准差为0.386 mg/mL±0.440 mg/mL。在对不同稀释度的IgY作为ELISA捕获蛋白进行评估后,可以区分阳性和阴性对照,其光密度(OD)的平均值±SD分别为1.99±0.18和0.45±0.06。结论:HBV可在鸡体内大量产生特异性免疫球蛋白,这些蛋白可作为ELISA的捕获蛋白。进一步的研究将用于开发用于乙型肝炎诊断的ELISA试剂盒。
Usefulness of IgY technology for hepatitis B virus diagnosis
an (ELISA) for of affinity chromatography. IgY anti-HBs using polyacrylamide gel electrophoresis (SDS-PAGE), dot blot and enzyme-linked immunosorbent assay (ELISA). Total protein concentrations were measured using spectrophotometer. Samples presenting higher total protein concentration were evaluated as capture protein for in house ELISA to detect HBsAg. Results: The characterization of IgY revealed bands of stained peptides with molecular weight between 75 and 50kDa and 37 and 25kDa using SDS Page. In the dot blot test was observed that there was antigen-antibody reaction throughout the sample period. Mean ± standard deviation (SD) of concentration of total protein obtained after purification by affinity chromatography was 0.386 mg / mL ± 0.440 mg/mL. After evaluation of different dilutions of IgY as capture protein for ELISA, it was possible to distinguish positive from negative controls that presented mean ±SD of optical density (OD) values of 1.99 ± 0.18 and 0.45 ± 0.06, respectively. Conclusion : The results indicated that HBV is able to generate a high production of specific immunoglobulins in chickens and these proteins could be used as capture protein for ELISA. Further studies will be conducting to develop in house ELISA for hepatitis B diagnosis.