Improved sensitivity in the antibody-forming cell assay for Japanese encephalitis virus in mice by optimal fixation of cells and avidin-biotin complex (ABC) immunocytochemistry.
A sensitive modified method for the detection in vitro of antibody-forming cells (AFCs) in Japanese encephalitis (JE) virus infected mice is described. The procedure involves the selection of the most appropriate incubation period, after the infection of monolayer cells, for expression of viral antigens to be recognized by AFCs. Adequate preservation of the expressed antigens was achieved after fixation with paraformaldehyde-lysine-periodate buffer (PLP). The development of spots which corresponded to AFCs was successfully obtained with the use of the avidin-biotin complex (ABC) amplification reaction. The number of the spots developing after completion of the assay was greater after PLP fixation compared with other fixation solutions such as methanol, formalin and acetone-water. Optimum antigen expression on the infected cells for recognition by lymphoid cells was reached after 24 h of infection. However, it was possible to detect viral antigens on the infected cells 8 h after infection. The ABC reaction was shown to be the best procedure for developing spots compared with other immunocytochemical methods. A linear increment was observed in the number of spots that developed at different spleen cell densities. This assay is simple to perform and could detect virus-specific Ig producing cells from the spleens of mice infected with JE virus. It also makes it possible to enumerate accurately the kinetics of the AFC response in different lymphoid organs during infection or after vaccination.
García-García
J
J
Department of Veterinary Public Health, Faculty of Veterinary Medicine, Hokkaido University, Sapporo, Japan.
Takashima
I
I
Kariwa
H
H
Hashimoto
N
N
D000818:Animals; D000921:Antibody-Producing Cells; D000956:Antigens, Viral / Q000032:analysis; D001360:Avidin; D001710:Biotin; D002452:Cell Count; D002522:Chlorocebus aethiops; D003513:Cycloheximide / Q000494:pharmacology; D004664:Encephalitis Virus, Japanese / Q000276:immunology*; D005260:Female; D007150:Immunohistochemistry; D051379:Mice; D008813:Mice, Inbred ICR; D013154:Spleen / Q000166:cytology
D000956:Antigens, Viral; D001360:Avidin; D001710:Biotin; D003513:Cycloheximide