Gupta R K, Siber G R
Massachusetts Public Health Biologic Laboratories, Boston, USA.
Dev Biol Stand. 1996;86:207-15.
Current United States (US) regulations for potency testing of vaccines containing adsorbed diphtheria and tetanus toxoids require in vivo toxin neutralization (TN) tests on the pooled sera of immunized guinea pigs. To reduce the number of animals required for testing, two in vitro tests have been evaluated, the Vero cell assay for diphtheria antitoxin and ELISA for IgG antibody to tetanus toxin; these have been correlated with in vivo TN tests. In the Vero cell method, diphtheria antitoxin titres of the guinea pig sera, obtained four weeks after immunization as per US potency requirements, were markedly dependent on the toxin dose level used in the assay. A toxin dose level termed the Lcd/1 dose (limit of cytopathic dose at a IU/ml) for Vero cells gave comparable estimates of antitoxin activity to the in vivo TN test performed at L+/1 dose of toxin. When lower dose levels of toxin were used in the Vero cells (Lcd/10 to Lcd/1000), diphtheria antitoxin levels in four weeks guinea pig sera were two to 11.7 times lower than with the Lcd/1 dose level. The most likely reason for these differences is that guinea pig sera a 4 weeks are of lower avidity than the equine antitoxin standard. Antibodies of low avidity bind antigen less well at low reactant concentrations. Therefore, to obtain similar estimates of diphtheria antitoxin in the Vero cell method and in vivo TN test, the use of toxin dose for the Vero cell method similar to that for the in vivo TN test is suggested. Another alternative, in which any dose of toxin may be used for the Vero cell method, is the use of a reference guinea pig serum (calibrated in IU/ml by the in vivo TN test at L+/1 level of toxin) that has similar avidity or similar immunization status as the test sera (i.e. 4 week serum). IgG antibodies to tetanus toxin in guinea pig sera were found early in the course of immunization when tetanus antitoxin could not be detected by TN test. Tetanus toxin IgG antibody levels of guinea pig sera calculated in IU/ml against an ELISA guinea pig reference serum (calibrated in IU/ml by TN test) depended upon the immunization status of the animals. To obtain similar estimates of tetanus antibodies in IU/ml by TN and ELISA, the ELISA reference guinea pig serum should have similar immunization status (and presumably similar avidity) as the test serum (i.e. six week serum). We propose that the Vero cell method and ELISA deserve further evaluation to determine whether they can replace in vivo TN tests for titration of diphtheria and tetanus antitoxins in the US potency test.
美国目前针对含有吸附白喉和破伤风类毒素疫苗的效力测试规定，要求对免疫豚鼠的混合血清进行体内毒素中和（TN）试验。为减少测试所需的动物数量，已对两种体外试验进行了评估，即用于检测白喉抗毒素的Vero细胞试验和用于检测破伤风毒素IgG抗体的ELISA试验；这些试验已与体内TN试验进行了相关性分析。在Vero细胞法中，按照美国效力要求，免疫四周后获得的豚鼠血清白喉抗毒素效价明显取决于试验中使用的毒素剂量水平。对于Vero细胞，一种称为Lcd/1剂量（每毫升国际单位时细胞病变剂量的极限）的毒素剂量水平，对白喉抗毒素活性的估计与在L+/1剂量毒素下进行的体内TN试验相当。当在Vero细胞中使用较低剂量水平的毒素（Lcd/10至Lcd/1000）时，四周龄豚鼠血清中的白喉抗毒素水平比Lcd/1剂量水平低两至11.7倍。这些差异最可能的原因是四周龄豚鼠血清的亲和力低于马抗毒素标准品。低亲和力抗体在低反应物浓度下与抗原的结合能力较差。因此，为了在Vero细胞法和体内TN试验中获得对白喉抗毒素的相似估计值，建议在Vero细胞法中使用与体内TN试验相似的毒素剂量。另一种选择是，在Vero细胞法中可以使用任何剂量的毒素，即使用与测试血清（即四周龄血清）具有相似亲和力或相似免疫状态的参考豚鼠血清（通过在L+/1毒素水平下的体内TN试验校准为每毫升国际单位）。在免疫过程早期，当通过TN试验无法检测到破伤风抗毒素时，在豚鼠血清中发现了破伤风毒素IgG抗体。以ELISA豚鼠参考血清（通过TN试验校准为每毫升国际单位）计算的豚鼠血清中破伤风毒素IgG抗体水平（以每毫升国际单位计）取决于动物的免疫状态。为了通过TN和ELISA获得以每毫升国际单位计的破伤风抗体的相似估计值，ELISA参考豚鼠血清应与测试血清（即六周龄血清）具有相似的免疫状态（大概具有相似的亲和力）。我们建议对Vero细胞法和ELISA进行进一步评估，以确定它们是否可以在美国效力试验中替代体内TN试验来滴定白喉和破伤风抗毒素。
