Detection of equine antiplatelet and antineutrophil antibodies by enzyme-linked immunosorbent assay.
An enzyme-linked immunosorbent assay (ELISA) was standardised and applied for the detection of antiplatelet and antineutrophil antibodies using a heterologous system consisting of equine platelets or neutrophils and antisera raised in rabbits. The standardised technique consisted of using Immulon type 3 plate, 1 per cent gelatine as a blocking solution, poly-L-lysine buffer as a coating solution, unfixed antigen, 90 microliters test serum, horseradish peroxidase conjugated antibody and o-phenylenediamine dihydrochloride as a substrate. The number of unfixed platelets or neutrophils required for optimum detection of antibodies was 250,000 per well. Unfixed cellular antigens were as good as their extracts and superior to paraformaldehyde-fixed antigens in detecting specific antibodies. Microtitre plates coated with platelet or neutrophil antigens could be stored at 4 degrees and -70 degrees C for four to five weeks without significant loss of antigenicity. The ELISA was very sensitive in that antiplatelet antibody was detected up to a titre of 1:204,800 and antineutrophil antibody to a titre of 1:51,200. Some cross-reactivity (1:1600) was detected in antiplatelet and antineutrophil sera for neutrophil and platelet antigens, respectively. Platelet-associated antibody was also detected in extracts from platelets pretreated with 1:2 and 1:8 dilutions of antiplatelet serum. Standardised ELISA detected antiplatelet antibodies in nine and antineutrophil antibodies in three of 100 isologous equine blood typing sera.
Dhawedkar
R G
RG
Department of Clinical Pathology, School of Veterinary Medicine, University of California, Davis 95616.
Jain
N C
NC
Mount
M E
ME
Bowling
A T
AT
Vegad
J L
JL
D000818:Animals; D001323:Autoantibodies / Q000097:blood*; D001792:Blood Platelets / Q000276:immunology*; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay; D006736:Horses / Q000097:blood / Q000276:immunology*; D007106:Immune Sera / Q000276:immunology; D009504:Neutrophils / Q000276:immunology*; D011817:Rabbits
D001323:Autoantibodies; D007106:Immune Sera