Gillenius P, Jäätmaa E, Askelöf P, Granström M, Tiru M
J Biol Stand. 1985 Jan;13(1):61-6. doi: 10.1016/s0092-1157(85)80034-2.
A microplate assay, based on the clustering effect induced by pertussis toxin (PT) in Chinese hamster ovary (CHO) cells, has been developed and standardized. Toxin titration is done directly in the culture microplate by twofold dilutions of 25 microliters of test material to which are added 10 000 freshly trypsinized cells in 200 microliters of culture medium per well. The dilution causing the clustering effect is determined by direct microscopic observation after 48 h of incubation. The method allows detection of 50-100 pg toxin per millilitre. For determination of neutralizing antibodies (antitoxin), twofold dilutions of 25 microliters of antiserum are first made directly in the culture microplate. Thereafter 25 microliters of toxin, containing four times the minimal clustering concentration, is added to each well. After three hours for neutralization at +37 degrees C, cells are added, incubated and examined as above. The assay has been found to be simple and reproducible for measuring the antibody response to PT in human and different animal sera. For titration of bacteria associated toxin, the CHO cells are seeded and incubated for 24 h before the addition of bacteria. Incubation and examination are done as described for toxin titration.
基于百日咳毒素（PT）在中国仓鼠卵巢（CHO）细胞中诱导的聚集效应，已开发并标准化了一种微孔板检测方法。毒素滴定直接在培养微孔板中进行，将25微升测试材料进行两倍稀释，每孔加入200微升培养基中10000个新鲜胰蛋白酶消化的细胞。孵育48小时后，通过直接显微镜观察确定引起聚集效应的稀释度。该方法可检测每毫升50 - 100皮克毒素。为了测定中和抗体（抗毒素），首先在培养微孔板中直接对25微升抗血清进行两倍稀释。此后，向每孔中加入25微升含有四倍最小聚集浓度的毒素。在37℃下中和3小时后，加入细胞，如上述进行孵育和检测。已发现该检测方法在测量人和不同动物血清中对PT的抗体反应方面简单且可重复。对于与细菌相关毒素的滴定，在加入细菌之前先接种CHO细胞并孵育24小时。孵育和检测按照毒素滴定的描述进行。
