Development of a method for detecting AVA-antigen specific antibody secreting cells

Objective To establish a method for detecting AVA-antigen specific antibody-secreting cells(ASCs) with enzyme linked immunospot essay,which can be used to detect and evaluate the function of AVA-antigen specific ASCs,and to observe the humoral immune responses in mice after being inoculated with AVA.Methods The level of Ig and its subtype in serum was detected.Mice were sacrificed and spleen cells were isolated.A series of parameters for detecting AVA specific ASCs was established: including the optimal concentration of coated antigen,optimal incubation time of spleen cells,and the performance concentration of Biotin-Igs.The sensitivity and specificity of the method was checked by detecting IgG levels in the culture supernatant and coating with irrelevant antigen.Results When antigen concentration was 7.5 μg/mL,the reaction curve was the most stable compared with the others.The amount of spots in cell with concentration of 5×106 mL-1 were in moderate level and easy to be recognized.Besides,the detected spots were the clearest and the background was the lightest when the incubation time was 3-6 h comparing with the others under the same circumstance.Lastly,the amount of spots coated with AVA-antigen was much higher than that coated with irrelevant antigen(P0.01).Antigen-specific ASC was undetected in the culture supernatant.Conclusion The ELISPOT essays represent a sensitive,specific,and reliable tool for tracking humoral immune responses after AVA vaccination.