Development and verification of a crystal violet-methylcellulose plaque reduction neutralization test for detection of neutralizing antibody titer of tick-borne encephalitis virus
10.13200/j.cnki.cjb.004473
- VernacularTitle:蜱传脑炎病毒中和抗体滴度结晶紫-甲基纤维素噬斑减少中和试验检测方法的建立及验证
- Author:
ZHAO Dongmei
- Publication Type:Journal Article
- Keywords:
Crystal violet-methylcellulose;
Plaque reduction neutralization test;
Tick-borne encephalitis virus(TBEV);
Neutralizing antibody titer
- From:
Chinese Journal of Biologicals
2025;38(05):574-580
- CountryChina
- Language:Chinese
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Abstract:
Objective To develop a crystal violet-methylcellulose plaque reduction neutralization test for detecting the titerof neutralizing antibody against tick-borne encephalitis virus(TBEV), and to verify and preliminarily apply the method, so asto provide a reliable method for TBEV antibody products in the detection of live virus neutralizing titer.MethodsOrthogo-nal experimental design was used to determine the important parameters involved in the experiment, namely, the best experi-mental conditions of virus culture time, virus-cell incubation time, neutralization temperature and methylcellulose concentra-tion. According to the experimental parameters determined above, the method was verified for specificity, relative accuracy,linearity, repeatability and intermediate precision. The titer of neutralizing antibody in serum of population immunized withinactivated tick-borne encephalitis vaccine was detected by the developed method.ResultsThe optimum conditions were asfollows: virus culture time of 5 d, virus-cell incubation time of 1. 5 h, neutralization temperature of 37 ℃ and methylcelluloseconcentration of 3%. Under these conditions, clear round plaques were formed, which was convenient for counting. Neutrali-zing antibodies were detected in both diluted test group and accelerated destruction test group in specificity verification withthe detection results not less than 10, while the results of impurity group were less than 10. In the relative accuracy verifica-tion, the relative bias of each titer was 0-10%, all in the range of ± 12%, and the linear regression correlation coefficient(r)was 0. 999 3, which was greater than 0. 98, In the verification of repeatability and intermediate precision, the geometric coef-ficients of variation(GCVs) of each group of samples were between 1. 5% and 4. 1%, not more than 20%. The serum neutrali-zing antibody titers of the population immunized with inactivated tick-borne encephalitis vaccine were less than 10 beforeinitial immunization, and not less than 10 after initial immunization and before and after booster immunization, and theserum neutralizing antibody increased after booster immunization.ConclusionThe developed TBEV neutralizing antibodytiter detection method has good durability, specificity, relative accuracy, linearity and precision, and the results are easy todetermine, which can be used for TBEV neutralizing antibody titer detection.
