Solid phase enzyme-linked immunosorbent assay (ELISA) for anti-sheep erythrocyte antibody in mouse serum.
The solid phase enzyme-linked immunosorbent assay (ELISA) was developed to measure IgM and IgG anti-SRBC antibody titers in mouse serum. Sheep erythrocytes, which have a surface negative charge, were attached directly to the bottom of an aminoplate well (96-well flat bottom, Sumitomo Bakelite Co.) which is charged positively to provide a solid phase for the ELISA antigen. Serum samples titrated were obtained from mice 5 and 10 days after SRBC immunization. Alkaline phosphatase-labeled goat anti-mouse IgM and/or IgG preparations were used as second antibody. Alkaline phosphatase activity in a well was measured by the Kind and King method [Kind, P. R. N. & King, E. J. (1954). J. clin. Path., 7, 322-326]; the optical density (OD) value of quinone as a final product was measured at 492 nm. (1) A linear relationship was observed between the antiserum concentration and OD value over a wide enough dilution range to assay antibody titers of serum samples; (2) IgM and IgG fractions from antiserum showed almost the same antibody titer as did the reconstituted samples; (3) a good correlation was observed between the serum IgM titer and the number of IgM hemolytic plaque-forming cells (PFCs) in spleen 5 days after the immunization, and was also observed between serum IgG antibody titer and the number of IgG PFCs 5 and 10 days after. Therefore, the ELISA described here requires no fixative for preparation of cell-coated plates, and serum IgM and IgG anti-SRBC antibody titers can be measured without any fractionation technique.
Mori
H
H
Department of Pharmacology, Gifu Pharmaceutical University, Japan.
Sakamoto
O
O
Xu
Q A
QA
Daikoku
M
M
Koda
A
A
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D000906:Antibodies / Q000097:blood*; D000918:Antibody Specificity; D001667:Binding, Competitive; D002021:Buffers; D003720:Densitometry; D004797:Enzyme-Linked Immunosorbent Assay; D004912:Erythrocytes / Q000276:immunology*; D006462:Hemolytic Plaque Technique; D007074:Immunoglobulin G / Q000032:analysis / Q000276:immunology; D007075:Immunoglobulin M / Q000032:analysis / Q000276:immunology; D051379:Mice; D012756:Sheep / Q000276:immunology
D000906:Antibodies; D002021:Buffers; D007074:Immunoglobulin G; D007075:Immunoglobulin M; D000469:Alkaline Phosphatase