Development and verification of a method for measuring adenovirus neutralizing antibody titer in immune serum of adenovirus vector vaccines
10.13200/j.cnki.cjb.004648
- VernacularTitle:腺病毒载体疫苗免疫后血清中腺病毒中和抗体滴度检测方法的建立及验证
- Author:
Yan HU
1
Author Information
1. Yunnan Vaccine Laboratory, Kunming 650500, Yunnan Province, China
- Publication Type:Journal Article
- Keywords:
Recombinant fluorescent adenovirus;
Chimpanzee adenovirus;
Adenovirus vector vaccine;
Immune serum;
Vector-specific neutralizing antibodies
- From:
Chinese Journal of Biologicals
2026;39(02):201-207
- CountryChina
- Language:Chinese
-
Abstract:
Objective To develop and verify a method for detecting adenovirus antibodies with neutralizing activity in serum after immunization with recombinant adenovirus vaccines, so as to quantify adenovirus neutralizing antibodies in clinical serum.Methods After neutralizing the recombinant fluorescent adenovirus AdC68 XY4-GFP with continuously serially diluted immune serum, the cells were infected, and the dilution of the test serum corresponding to the number of fluorescent plaques reduced to 10% of the pre-neutralization level was defined as the adenovirus neutralizing antibody titer of the test serum[recorded as inhibitory concentration 90(IC_(90))]. Three different cell lines(293A, HeLa and A549 cells) were inoculated with the same amount of virus to determine the cells with the highest sensitivity for detection. The proliferation curve of the recombinant adenovirus was analyzed and the data interpretation time was determined. The titer range of the recombinant fluorescent adenovirus was determined through neutralization tests. In addition, the specificity, accuracy, repeatability,intermediate precision and robustness of the established method were verified.Results The 293A cells were found to be the most sensitive to adenovirus, and thus were selected for subsequent experiments. The result interpretation time was set at 48 hours post-infection. The mean fluorescent plaque numbers corresponding to different adenovirus titers showed a linear relationship(R~2= 0. 970 3). The recombinant fluorescent adenovirus was found to produce stable results within the infection range of 62. 5 to 500 IFU/50 ??L. The midpoint of the linear range, 250 IFU/50 ??L was chosen for the experiments.The adenovirus neutralizing antibody IC_(90)of fetal bovine serum(FBS) was lower than 1∶20, and the IC_(90)of positive control serum was 1∶96. The recovery rates of neutralizing antibody titers of 003 adenovirus in positive control serum and clinical serum were between 50% and 150%. The RSD of the six test results of positive control serum was 8. 27%, < 50%. The RSD of the two test results of the same sample detected by three experimenters at different times was 4. 52%, < 50%. The effects of virus proliferation culture time and plated cell volume on adenovirus neutralizing antibody titer met the acceptance criteria.Conclusion A method for the detection of adenovirus antibodies with neutralizing activity in serum after immunization with recombinant adenovirus vaccines has been developed. The level of adenovirus-specific neutralizing antibodies can be quickly detected by infecting sensitive cells after neutralization of recombinant fluorescent virus and immune serum.The specificity, accuracy, repeatability, intermediate precision and robustness of this method all meet the verification requirements.
