Monath T P, Wands J R, Hill L J, Gentry M K, Gubler D J
J Gen Virol. 1986 Apr;67 ( Pt 4):639-50. doi: 10.1099/0022-1317-67-4-639.
A monoclonal radioimmunoassay (RIA) was developed for detection of dengue virus in infected cell culture fluids and blood samples from dengue patients. Antibodies used to construct the RIA were selected on the basis of high binding avidity, the demonstration of synergism in competitive binding assays and empirical trials with different antibody combinations. Optimal binding of all four dengue virus serotypes was achieved by use of a flavivirus group-reactive and a dengue virus complex-reactive antibody as radiolabelled probe. A 'simultaneous sandwich' format and prolonged (18 h) incubation at 37 degrees C yielded optimal results. The limit of sensitivity of the RIA for detection of dengue type 2 virus was 2.7 log10 mosquito 50% infectious doses (MID50). The assay was tenfold more sensitive for dengue type 2 than for dengue types 1 and 3 viruses and 100-fold more sensitive than for dengue type 4 virus. Specificity, assessed using over 500 disease control human sera, was increased by addition of monoclonal anti-tetanus blocking antibodies, resulting in a false positive rate of only 0.2%. Heterologous dengue virus antibodies were shown to inhibit the RIA in assays performed with artificial immune complexes. Acute phase human sera containing 10(4.2) to 10(7.6) MID50 but no detectable antigen by RIA, were also shown to inhibit binding of the homologous dengue virus serotype; this effect was attributed to heterologous antibody from a prior infection. Among 116 viraemic sera from dengue patients, the RIA was positive in 43 to 47% of patients with dengue type 1, 2 or 3 infections but in only 10% of the dengue type 4 cases. Virus was more frequently detected in cases of primary infection (54%) than in cases of superinfection (16%). Despite the limitations imposed by immunological interference, the antigen capture RIA appears useful as a rapid diagnostic technique for dengue surveillance.
开发了一种单克隆放射免疫测定法（RIA），用于检测感染细胞培养液和登革热患者血样中的登革热病毒。用于构建RIA的抗体是根据高结合亲和力、竞争性结合试验中的协同作用证明以及不同抗体组合的经验性试验来选择的。通过使用黄病毒属反应性和登革热病毒复合物反应性抗体作为放射性标记探针，实现了对所有四种登革热病毒血清型的最佳结合。“同时夹心”形式和在37℃下延长（18小时）孵育产生了最佳结果。RIA检测登革热2型病毒的灵敏度极限为2.7 log10蚊虫50%感染剂量（MID50）。该测定法对登革热2型病毒的敏感性比对登革热1型和3型病毒高10倍，比对登革热4型病毒高100倍。使用500多份疾病对照人血清评估的特异性通过添加单克隆抗破伤风阻断抗体而提高，导致假阳性率仅为0.2%。在用人造免疫复合物进行的试验中，异源登革热病毒抗体显示会抑制RIA。急性期人血清含有10(4.2)至10(7.6) MID50，但通过RIA未检测到可检测抗原，也显示会抑制同源登革热病毒血清型的结合；这种效应归因于先前感染产生的异源抗体。在116份登革热患者的病毒血症血清中，RIA在登革热1型、2型或3型感染患者中的阳性率为43%至47%，但在登革热4型病例中仅为10%。病毒在原发性感染病例（54%）中比在重叠感染病例（16%）中更频繁地被检测到。尽管存在免疫干扰带来的局限性，但抗原捕获RIA似乎作为一种用于登革热监测的快速诊断技术是有用的。
