WHOLE CELL ELISA FOR COELIAC DISEASE ANTIBODIES USING HUMAN UMBILICAL CORD-DERIVED FIBROBLASTS AS ANTIGEN

Abstract

Background: Previous studies have shown that human fibroblast-produced soluble molecules are targets for reticulin (ARA) and endomysium (EmA) autoantibodies. We have now studied coeliac disease (CD) antibodies with a quantitative laboratory non-dependent whole cell ELISA using human umbilical cord-derived fibroblasts (HUC-Fb) as antigen. Methods: Wharton's jelly-derived fibroblasts (McElreavey et al., Biochem Soc Trans 1991;1:29S) were allowed to attach onto ELISA plate wells and the cells were fixed. The serum samples were diluted 1:100 and peroxidase-conjugated anti-human IgA was used as second antibody. HUC-Fb autoantibody ELISA results were expressed as relative distance {RD=(sampleabs -negative poolabs)/(positive poolabs-negative poolabs)} from zero. ARA and human umbilical cord antibodies (HUC-ab) were measured with standard indirect immunofluorescence method using composite block of rat kidney, liver, stomach and heart for ARA and human umbilical cord for HUC-ab, as antigen. Gliadin antibodies (AGA) were determined with ELISA. Results: The mean fibroblast whole cell ELISA RD values for 27 children with untreated CD were 1.02 (range 0.55-1.44) and for 15 children on gluten-free diet 0.29 (range -0.01-0.92). The mean value for 66 disease control patients was 0.29 (range 0.05-2.89). Sensitivity of HUC-Fb autoantibody ELISA was 100% (27/27) and specificity 82% (54/66), when the cut-off level for positivity was 0.40. The sensitivity and specificity for ARA and HUC-ab were 100%, and for IgA-class AGA test 85% (23/27) and 67% (44/66), respectively. Conclusions: We conclude that human umbilical cord Wharton's jelly-derived fibroblasts do express antigens detectable by CD patient sera IgA. Fibroblast whole cell ELISA may be used in screening for CD.