真菌(1,3)-β-D-葡聚糖多克隆抗体制备及ELISA竞争法检测体系的建立

Preparation of polyclonal antibody and development of a competitive ELISA for fungal( 1,3 )-beta-Dglucan

目的：建立高特异性和敏感性的（1，3）-β-D-葡聚糖酶联免疫吸附试验（ ELISA）检测体系。方法经蛋白修饰的（1，3）-β-D-葡聚糖和真菌提取物作为免疫原制备兔多克隆抗体，并对高效价交叉反应弱的抗体进行纯化和辣根过氧化物酶（HRP）标记，最后建立真菌（1，3）-β-D-葡聚糖ELISA竞争法检测体系。结果建立的ELISA竞争法检测体系线性范围为3．125～200 pg／mL。添加100、25和6．25 pg／mL抗原的血清回收率为97．8％～113．6％，重复试验的变异系数＜15％。该检测体系能有效地从血清样本中检出低浓度的烟曲霉、白念珠菌和高浓度的隐球菌，并且对结核分枝杆菌、大肠埃希菌、沙门氏菌、克雷伯菌和金黄色葡萄球菌5种细菌抗干扰能力强。结论利用（1，3）-β-D-葡聚糖作为包被抗原，酶标抗体HRP-Ab3B作为检测抗体，成功建立了（1，3）-β-D-葡聚糖ELISA竞争法检测体系。

Objective To develop a competitive enzyme-linked immunosorbent assay （ ELISA ） with high specificity and sensitivity for fungal （1,3）-beta-D-glucan.Methods Rabbit polyclonal antibody was produced by immunization with protein-conjugated （ 1, 3 ）-beta-D-glucan and fungal extract.The highest titer and little cross-reactivity polyclonal antibody was labeled with horseradish peroxidase （ HRP ） and purified.Finally, a competitive ELISA was established for fungal （ 1, 3 ）-beta-D-glucan.Results The linear range was 3.125-200 pg/mL.The recovery range for 100, 25 and 6.25 pg/mL serum antigen was 97.8%-113.6%, and the coefficient of variation for repeated test was 〈15%.The detection system could effectively detect low concentrations of Aspergillus fumigatus and Candida albicans and high concentration of Cryptococcus neoformans from serum samples.Meanwhile, the detection system demonstrated little interference against 5 kinds of pathogenic bacteria, including Mycobacterium tuberculosis, Escherichia coli, Salmonella, Klebsiella and Staphylococcus aureus.Conclusions A competitive ELISA is developed successfully for the detection of invasive fungal disease with （1,3）-beta-D-glucan used as a coated antigen and enzyme-labeled antibody HRP-Ab3B used as a detection antibody.