Thraenhart O, Ramakrishnan K
Institute for Medical Virology and Immunology, University of Essen--Medical School, F.R.G.
J Biol Stand. 1989 Oct;17(4):291-309. doi: 10.1016/s0092-1157(89)80001-0.
A non-competitive enzyme-linked immunoassay (ELISA) has been standardized to supplement the in vivo potency test used for the quality control of inactivated tissue culture vaccines against rabies. The essentials of the ELISA were: fixation of the virus in different dilutions of vaccine on the surface of microtitre plates; testing of the reference and up to six test vaccines on one plate; incubation with polyclonal antisera to rabies virus glycoprotein containing an excess of antibody; further incubation with a species-specific anti-IgG coupled to peroxidase; a final incubation with a substrate. The incubation periods were 1 h, 1 h and 30 min both at +37 degrees C. The relative potency determinations were made graphically or by a computer using a parallel line bioassay in which the potencies of the vaccines of unknown potency were tested against the reference preparation on a single microtitre plate. Under these conditions inactivated rabies vaccines of different types (virus strains, cell substrates, inactivation and concentration procedures) were tested for potency. Furthermore, it was possible with this in vitro method to assay adjuvanted vaccines, in process samples such as tissue culture supernatants with live or inactivated rabies virus, concentrates, and vaccines undergoing thermal stability tests. The rabies glycoprotein antigen-antibody reaction was highly specific according to the results and the glycoprotein content was measured quantitatively. The potency determined by the in vitro ELISA correlated with the in vivo NIH protection potency test. The lower limit of detection of the ELISA was 0.015 IU/ml. Quantitative antigen determination was possible with both homologous and heterologous antisera to rabies virus glycoprotein when vaccines of the same virus strain were tested. When the potencies of vaccines of different virus strain specificity were calculated, it was necessary to take into account the strain-specific antigenicity. Even so vaccines of high potency were found to give a stronger reaction with a heterologous serum than did weak vaccines with a homologous antiserum. Stability tests made on inactivated tissue culture vaccines such as vaccine from the human diploid cell strain (HDCS), from purified chicken embryo cell (PCEC) or from purified Vero cell rabies vaccine (PVRV), showed high stability of the glycoprotein antigen even after four months of storage at +37 degrees C or 24 h at +56 degrees C, provided that the vaccines were stored in a lyophilized state. The antigenicity of liquid vaccines was inactivated after a few hours at +56 degrees C. For tropical areas, therefore, only lyophilized vaccines should be considered.
一种非竞争性酶联免疫吸附测定法（ELISA）已标准化，以补充用于狂犬病灭活组织培养疫苗质量控制的体内效价试验。ELISA的要点如下：将不同稀释度疫苗中的病毒固定在微量滴定板表面；在一块板上检测参考疫苗和多达六种试验疫苗；与含有过量抗体的狂犬病病毒糖蛋白多克隆抗血清孵育；再与与过氧化物酶偶联的种属特异性抗IgG孵育；最后与底物孵育。在37℃下，孵育时间均为1小时、1小时和30分钟。相对效价测定通过图形法或使用平行线生物测定法的计算机进行，其中在单个微量滴定板上针对参考制剂测试未知效价疫苗的效价。在这些条件下，对不同类型（病毒株、细胞底物、灭活和浓缩程序）的灭活狂犬病疫苗进行了效价测试。此外，使用这种体外方法还可以检测佐剂疫苗、过程样品，如含有活的或灭活狂犬病病毒的组织培养上清液、浓缩物以及正在进行热稳定性测试的疫苗。根据结果，狂犬病糖蛋白抗原-抗体反应具有高度特异性，并且可以定量测量糖蛋白含量。通过体外ELISA测定的效价与体内NIH保护效价试验相关。ELISA的检测下限为0.015 IU/ml。当测试相同病毒株的疫苗时，使用狂犬病病毒糖蛋白的同源和异源抗血清都可以进行定量抗原测定。当计算不同病毒株特异性疫苗的效价时，有必要考虑毒株特异性抗原性。即便如此，高效价疫苗与异源血清的反应仍比低效价疫苗与同源抗血清的反应更强。对灭活组织培养疫苗，如人二倍体细胞株（HDCS）疫苗、纯化鸡胚细胞（PCEC）疫苗或纯化Vero细胞狂犬病疫苗（PVRV）进行的稳定性测试表明，只要疫苗以冻干状态储存，即使在37℃下储存四个月或在56℃下储存24小时后，糖蛋白抗原仍具有高稳定性。液体疫苗在56℃下放置数小时后抗原性即被灭活。因此，对于热带地区，应仅考虑冻干疫苗。
