Quantification of antigen loading efficiency in nanoadjuvant-antigen covalent conjugates
CUI Minxuan
SHI Zhuoyue
XI Jiayue
ZHU Yupu
MA Zhengjun
SONG Chaojun
FAN Li
Abstract：Objective To establish an accurate quantification method for antigen loading efficiency in nanoadjuvant-antigen conjugates.Methods PLGA nanoparticles(NPs)were synthesized as nanoadjuvant by emulsification solvent volatilization method using PLGA-PEG-COOH copolymer as raw material,bovine serum albumin(BSA)was used as a model antigen,and crosslinked NPs were prepared by covalently cross-linking nanoadjuvant and antigen using 1-3-(dimethylamino)propyl-3-ethylcarbodiimide methiodide(EDC)/N-hydroxysuccinimide(NHS)as a cross-linking agent.Based on bicinchoninic acid(BCA)and Bradford method,several methods were adopted to determine the A562nm and A595 nm values of BSA antigen in crosslinked NPs,shielding the interference caused by NPs scattering on the determination,and the reliability and stability of the determination results were analyzed to establish an accurate quantitative method of antigen loading efficiency.Results Trace amounts of cross-linker EDC/NHS could react with BCA working solution to produce strong A 562 nm values,making the measurement results of the BCA method inaccurate.In the Bradford method,the source of the A595 nm values produced by the interaction of blank NPs with the Bradford working solution could be split as follows:A595 nm(blank NPs+Bradford working solution)=A595 nm(blank NPs+PBS buffer)+A595 nm(PBS buffer+Bradford working solution).Therefore,the absorbance value of antigens in crosslinked NPs could be calculated based on the following formula:A595 nm(BSA)=A595 nm(crosslinked NPs+Bradford working solution)-A595 nm(crosslinked NPs+PBS buffer)-A595 nm(PBS buffer+Bradford working solution),which achieved accurate determination of antigen crosslink efficiency in conjugates.Conclusion Using the Bradford method to split the A595 nm values generated by the nanoadjuvant-antigen conjugates and Bradford working solution by the source of A595 nm can effectively shield the interference caused by NPs scattering on the determination and realize the accurate determination of antigen loading efficiency in nanoadjuvant-antigen conjugates.
Keywords：nanoadjuvantcovalent crosslinkingantigen loading efficiencyquantification method
Publication Date：2025-11-30
Online Publishing Date：2026-08-26(First online date of this platform, not the publication date of the document)
Pages：8( 1442-1449 )