Anticardiolipin antibodies by an enzyme-linked immunosorbent assay: fundamental studies on the conditions for antigen-application and specificity of the assay.
We have established an enzyme-linked immunosorbent assay (ELISA) for anticardiolipin antibodies (aCL) using standard sera obtained from the Rayne Institute, St. Thomas' Hospital (London, U.K.). In this study, we compared several fundamental requirements for the assay with the standard assay as a reference, such as conditions for antigen application and test samples using our patients' samples. In addition, the specificity of our assay and cross-reactivities of aCL were also evaluated. In the standard assay, the concentration of antigen was optimal in the range of 30-100 micrograms/ml. The antigen-coating temperature was optimal at 4 degrees C for 16 hours. The method based on rapid evaporation of CL-ethanol solution can be used instead of the standard method. On the other hand, there was no significant difference in the results between physical conditions of the antigen (CL-ethanol solution vs. CL-micelles), between washing solutions (saline vs. PBS containing 0.05% Tween-20) and between test samples (sera vs. plasma). The aCL activity in our patients' samples was almost completely inhibited by pre-incubation of sera with either CL or phospholipid reagent for activated partial thromboplastin time (APTT). Interestingly, the aCL activity of the lupus anticoagulant was negative, but aCL-positive samples were also absorbed by the reagent for APTT. No inhibition of the aCL activity, however, was observed when patients' sera were preincubated with ss-DNA.
Ichikawa
Y
Y
Department of Internal Medicine, School of Medicine, Tokai University, Kanagawa, Japan.
Kobayashi
N
N
Kawada
T
T
Morita
K
K
Tokunaga
M
M
Uchiyama
M
M
Shimizu
H
H
Moriuchi
J
J
Takaya
M
M
Watanabe
K
K
D001323:Autoantibodies / Q000032:analysis* / Q000592:standards; D001324:Autoantigens / Q000592:standards; D001327:Autoimmune Diseases / Q000276:immunology; D002308:Cardiolipins / Q000276:immunology* / Q000592:standards; D004797:Enzyme-Linked Immunosorbent Assay / Q000592:standards; D006801:Humans; D007074:Immunoglobulin G / Q000032:analysis / Q000592:standards; D007075:Immunoglobulin M / Q000032:analysis / Q000592:standards; D008180:Lupus Erythematosus, Systemic / Q000276:immunology; D012015:Reference Standards
D001323:Autoantibodies; D001324:Autoantigens; D002308:Cardiolipins; D007074:Immunoglobulin G; D007075:Immunoglobulin M