The development of a quantitative assay for the detection of anti-Ro/SS-A and anti-LA/SS-B autoantibodies using purified recombinant proteins.
A characteristic of patients with autoimmune diseases such as Sjögren's syndrome and systemic lupus erythematosus is the presence of anti-Ro/SS-A and anti-La/SS-B autoantibodies in their circulation. In order to investigate specific autoantibody levels in the sera of these patients quantitative assays for the detection of both anti-Ro/SS-A and anti-La/SS-B reactivity were developed. Ro/SS-A (60 kDa) and La-SS-B (50 kDa) cDNAs were cloned and expressed in E. coli as non-fusion proteins. These were purified to homogeneity using two different purification protocols. With these recombinant antigens, specific enzyme-linked immunosorbent assays (ELISAs) were developed. 40 sera positive for anti-Ro/SS-A autoantibodies in counterimmunoelectrophoresis (CIE) were tested in both the Ro/SS-A and La/SS-B ELISA. Activity values reproducibly ranged from 1536 to 120,000 U in the Ro/SS-A ELISA and from 763 to 2,500,000 U in the La/SS-B ELISA. The suitability of these ELISAs as screening assays was further investigated by testing 200 sera sent to our laboratory for routine detection of autoantibodies to extractable nuclear antigen (ENA: anti-Sm, anti-RNP, anti-Ro/SS-A and anti-La/SS-B). Both ELISAs showed a high sensitivity and specificity (Ro/SS-A ELISA 85% and 94%, La/SS-B ELISA 100% and 98% respectively), when compared to the standard assays, the RNA-precipitation assay and the HeLa immunoblotting test. From these data we conclude that a quantitative analysis of both anti-Ro/SS-A and anti-La/SS-B autoantibodies is now possible using purified recombinant non-fusion proteins. For screening purposes the La/SS-B ELISA showed a great improvement in sensitivity for the detection of anti-La/SS-B activity in comparison to the La/SS-B CIE, while the Ro/SS-A ELISA almost equalled the performance of the Ro/SS-A CIE.
Veldhoven
C H
CH
Department of Autoimmune Diseases, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam.
Meilof
J F
JF
Huisman
J G
JG
Smeenk
R J
RJ
D001323:Autoantibodies / Q000276:immunology*; D001324:Autoantigens / Q000235:genetics / Q000276:immunology*; D015153:Blotting, Western; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006801:Humans; D007118:Immunoassay / Q000379:methods; D007123:Immunoelectrophoresis, Two-Dimensional; D008970:Molecular Weight; D020733:RNA, Small Cytoplasmic; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology; D012261:Ribonucleoproteins; D012995:Solubility; D000097585:SS-B Antigen; D000099207:SS-A Antigen
D001323:Autoantibodies; D001324:Autoantigens; D020733:RNA, Small Cytoplasmic; D011994:Recombinant Proteins; D012261:Ribonucleoproteins; D000097585:SS-B Antigen; D000099207:SS-A Antigen; C116006:RO60 protein, human