Ⅰ型登革病毒NS1抗原捕获ELISA的建立和初步临床诊断应用

目的以登革病毒特异性非结构蛋白1（NS1）单克隆抗体为基础建立Ⅰ型登革病毒（DEN1）抗原检测的酶联免疫吸附（ELISA）法，并探索从病人早期血清样品中检测DEN1-NS1的可行性。方法利用已制备的抗DEN1-NS1单克隆抗体（单抗），进行多种抗体组合配对优化模式的分析，建立双抗体夹心抗原捕获ELISA，以469份健康人血清样品确定cut off值，检测DEN1感染患者急性期血清样品。结果对多种抗体组合反复筛选，最终确立了最佳的包被单抗和酶标测定单抗，建立了抗体夹心捕获DEN1-NS1抗原的酶联免疫测定方法，能特异检测DEN1，与其他血清型登革病毒不发生交叉反应。检测16例临床确诊DEN1感染病人急性期血清样品，15例呈特异的抗原反应阳性。结论成功建立了DEN1-NS1抗原捕获ELISA并应用于临床血清样品的检测，为登革热的早期实验室诊断提供技术方法。

Antigen capture enzyme-linked immunosorbent assay for specific detection of dengue virus serotype 1 nonstructural protein

Objective To develop a monoclonal antibody(McAb)-based capture ELISA for dengue virus serotype 1 nonstructural protein NS1 (DEN1-NS1) and to study the feasibility of NS1 antigen detection in acute phase serum samples from DEN1-infected patients. Methods Among the McAb against DEN1-NS1, antibody pairs were analyzed to choose the optimal coating McAb and detecting McAb by determinating the detection sensitivity and specificity. Four hundred and sixty-nine samples from healthy donors were tested to calculate the cut off value. Results The best coating antibody and HRP-conjugate detection antibody were ultimately selected by antibody pairing method to construct the DEN1-NS1 antigen capture ELISA. This test is specific to dengue virus serotype 1 without cross reaction to the other three serotypes. The cut off value was determined as the average value of negative control added to three standard deviations. 15 out of 16 DEN1-infected serum samples were detected positive to the NS1 antigen. Conclusion A sensitive and specific monoclonal antibody-based capture ELISA for detecting NS1 antigen in sera from dengue virus serotype 1-infected patients was successfully established, which will be helpful in early diagnosis and disease monitoring of dengue virus infection.