Human fibroblast-derived molecules as antigens in enzyme-linked immunosorbent assay for coeliac disease-specific IgA.
We have recently shown that cultured human fibroblasts synthesize and secrete protein molecules that bind to IgA-class anti-reticulin and anti-endomysium antibodies but not to anti-gliadin antibodies in coeliac disease patient sera. In the present report, we describe a reproducible method for purification of these antigen molecules from fibroblast culture medium. Using reversed-phase chromatography as the final purification step, four different protein molecules reacting with coeliac disease patient sera IgA were obtained. In enzyme-linked immunosorbent assay (ELISA) for coeliac disease-specific IgA, a mixture of 0.5 microgram of the four reversed-phase-separated molecules was used as antigen. The optical density values in ELISA of the sera from newly diagnosed coeliac disease patients (n = 34) were 0.740-3.400 (mean 1.830) and in control patients (n = 66) 0.090-0.850 (mean 0.320). Using an arbitrary cut-off level of 0.700, the sensitivity of the present autoantibody test was 100%, specificity 91% and positive predictive value 85%. Our identified autoantigens may generate the production of the classical tissue antibodies, known as anti-reticulin and anti-endomysium antibodies, and may be used as antigen in an immunoassay for the antibodies.
Marttinen
A
A
Medical School, University of Tampere, Finland.
Sulkanen
S
S
Mäki
M
M
D000293:Adolescent; D000918:Antibody Specificity; D001323:Autoantibodies / Q000737:chemistry / Q000378:metabolism; D001324:Autoantigens / Q000276:immunology* / Q000378:metabolism; D002446:Celiac Disease / Q000178:diet therapy / Q000276:immunology* / Q000378:metabolism; D002648:Child; D002675:Child, Preschool; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005347:Fibroblasts / Q000276:immunology / Q000378:metabolism; D005526:Food, Formulated; D005903:Gliadin / Q000276:immunology; D006801:Humans; D007070:Immunoglobulin A / Q000097:blood* / Q000378:metabolism; D007223:Infant; D015203:Reproducibility of Results; D012155:Reticulin / Q000276:immunology / Q000378:metabolism; D012680:Sensitivity and Specificity
D001323:Autoantibodies; D001324:Autoantigens; D007070:Immunoglobulin A; D012155:Reticulin; D005903:Gliadin