Kit S, Otsuka H, Kit M
Division of Biochemical Virology, Baylor College of Medicine, Houston, TX.
J Virol Methods. 1992 Oct;40(1):45-56. doi: 10.1016/0166-0934(92)90006-y.
A sensitive and specific blocking enzyme-linked immunosorbent assay (ELISA) was developed to distinguish infectious bovine rhinotracheitis virus (IBRV)-infected animals from those immunized with a glycoprotein gIII deletion mutant, IBRV(NG)dltkdlgIII. For this ELISA, undiluted test sera are used to block the binding of an anti-IBRV gIII monoclonal antibody (mAbgIII)-horseradish peroxidase (HRPO) conjugate to gIII antigen. TMB substrate is used for color development. Negative S/N values (defined as the absorbance at 650 nm of test sera/absorbance at 650 nm of negative control sera) of > 0.80 were obtained with immune sera from gnotobiotic cattle immunized with several bovine viruses, with bovine antisera to bovine herpesvirus-2, and vesicular stomatitis virus, with porcine antisera to pseudorabies virus and parvovirus, and with normal sera from heterologous species. Negative S/N values were also obtained with sera from rabbits twice vaccinated with IBRV(NG)dltkdlgIII. However, the S/N values became positive (S/N < 0.8) 10 to 17 days after the rabbits were challenge exposed to virulent IBRV(Cooper). Most of 116 sera (84%) from feedlot cattle with virus neutralization (VN) titers of < 1:2 or < 1:4 had negative S/N values > 0.8, but 18 sera with negative VN titers had positive S/N values, consistent with observations indicating that an IBRV outbreak was occurring in one of the feedlot herds. Thirty nine sera (98%) from feedlot cattle with VN titers of 1:2 to 1:128 had positive S/N values (< 0.8). One serum with a VN titer of 1:2 had a borderline (+/-) S/N value of 0.81. After immunization with a commercial gIII-positive IBRV vaccine, 115/116 sera with VN titers of 1:2 to 1:256 had positive S/N values (< 0.8). One serum with a VN titer of 1:2 had a negative S/N value of 0.83. Serum from one vaccinated animal that failed to seroconvert after vaccination (VN < 1:4) showed a strongly positive ELISA S/N of 0.48.
开发了一种灵敏且特异的阻断酶联免疫吸附测定（ELISA），以区分感染牛传染性鼻气管炎病毒（IBRV）的动物与用糖蛋白gIII缺失突变体IBRV(NG)dltkdlgIII免疫的动物。对于该ELISA，使用未稀释的测试血清来阻断抗IBRV gIII单克隆抗体（mAbgIII）-辣根过氧化物酶（HRPO）偶联物与gIII抗原的结合。使用TMB底物进行显色。用几种牛病毒免疫的无菌牛的免疫血清、抗牛疱疹病毒-2的牛抗血清、水疱性口炎病毒、抗伪狂犬病病毒和细小病毒的猪抗血清以及异源物种的正常血清获得的阴性S/N值（定义为测试血清在650nm处的吸光度/阴性对照血清在650nm处的吸光度）>0.80。用IBRV(NG)dltkdlgIII两次免疫的兔子的血清也获得了阴性S/N值。然而，在用强毒IBRV(Cooper)攻击暴露兔子后10至17天，S/N值变为阳性（S/N<0.8）。116份来自饲养场牛的血清中，病毒中和（VN）效价<1:2或<1:4的血清中的大多数（84%）的阴性S/N值>0.8，但18份VN效价为阴性的血清的S/N值为阳性，这与表明其中一个饲养场牛群正在发生IBRV疫情的观察结果一致。116份来自饲养场牛的血清中，VN效价为1:2至1:128的39份血清（98%）的S/N值为阳性（<0.8）。一份VN效价为1:2的血清的临界（±）S/N值为0.81。用市售gIII阳性IBRV疫苗免疫后，116份VN效价为1:2至1:256的血清中有115份的S/N值为阳性（<0.8）。一份VN效价为1:2的血清的阴性S/N值为0.83。一只接种疫苗后未发生血清转化（VN<1:4）的接种动物的血清显示ELISA S/N为强阳性，为0.48。
