Development and verification of a detection method for human serum IgG antibodies against nine-valent human papillomavirus vaccine based on domestic multi-channel flow cytometry platform
Objective To develop a detection method for human serum IgG antibodies against the human papillomavirus(HPV)vaccine based on a domestic multi-channel flow cytometry platform,and optimize and verify the method to replace Luminex technology and reduce the testing costs.Methods Nine domestically validated magnetic fluorescent microspheres and nine Luminex magnetic fluorescent microspheres were utilized,each conjugated with specific HPV antigens(HPV6/11/16/18/31/33/45/52/58),respectively.The nine antigen-conjugated microspheres were added into the same well and incubated with serum or monoclonal antibody to be tested,followed by the addition of a biotin-labeled antibody and streptomycin-labeled phycoerythrin.The antibody titers were calculated using the median fluorescent intensity(MFI)tested by the domestic multiple fluorescence bioanalysis system.The compatibility with the domestic flow fluorescence platform,detection range,performance and thermal stability of domestic microspheres and Luminex microspheres were compared.Checkerboard method and Design of Experiment(DoE)were used to optimize the detection conditions,and the methodology was verified.Results The selected domestic fluorescent microspheres and Luminex microspheres were correctly identified by the domestic multiple fluorescence bioanalysis system and microspheres of each type were specifically aggregated.The calibra-tion curve fit R2 values for the nine HPV-type specific monoclonal antibodies all exceeded 0.99.In the thermal acceleration stability study of the microspheres,the domestic microspheres exhibited greater stability and lower detection deviation compared to the Luminex microspheres.Moreover,the method demonstrated high specificity,with concentration deviation of±20％observed in both single and multiple microsphere tests for 9 type-specific standards and clinical serum samples.The results demonstrated that the method had a quantification range exceeding 4 000-fold,with quantification ranges of 0.015 3-62.5 ng/mL for HPV6/16/18/31/58,0.061 0-250 ng/mL for HPV11,0.030 5-125 ng/mL for HPV33/52,and 0.007 6-31.25 ng/mL for HPV45.Moreover,the parallelism observed between the standard curve and clinical serum samples was excellent,thus meeting the requirements for the quantitative detection of clinical serum samples.The accuracy and recovery deviation of the detection method was less than 15％,the precision deviation was less than 10％,and the total error of the method was within 25％.The dilution linearity,selectivity(interference of hyperlipidemia samples)and freeze-thaw stability of serum samples all complied with the requirements specified in Chinese Pharmacopoeia(Volume Ⅳ,2020 edition).Conclusion A quantitative detection method for HPV clinical serum IgG antibody using the domestic flow fluores-cence bioanalysis system and fluorescent microspheres was established,which overcomes the technical barriers of the Luminex platform,resulting in lower detection costs and improved accessibility.It can be utilized for immunogenicity evalua-tion of multivalent vaccines.
Human papillomavirus(HPV)vaccineIgG antibodyQuantitative detectionMultiplex antibody detectionLumi-nex platform
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