Objective: To detect anti-Scl-70 antibody with enzyme-linked immunosorbent assay (ELISA) using a recombinant fusion peptide which comprises 207 - 765 amino acid fragment of Scl-70 as antigen.
Methods: cDNA encoding Scl-70 antigen fragment from aa207 to aa765 from human placenta cDNA first strand was amplified with PCR. The obtained cDNA was inserted into expression vector Pet-42b and transferred into E.coli BL21 (DE3) for expression of his-tagged fusion peptide. After Ni(2+)-NTA affinity purification, the antigenicity was identified with Western blotting. Serum samples from 36 systemic sclerosis (SSc) patients, 20 patients with other connective tissue disorder (CTD) and 30 normal controls were retrospectively tested with ELISA.
Results: A soluble fusion peptide was expressed and purified. The antigenicity was confirmed with Western blotting using standard positive anti-Scl-70 antibody serum. Of the 36 serum samples from SSc patients, 5 of 6 samples showing positive reaction with natural Scl-70 antigen in double immunodiffusion (DID) assay also recognized the recombinant fusion peptide with ELISA, only one serum sample which showed positive anti-Scl-70 in DID displayed a negative result in ELISA assay. On the contrary, 3 patients with negative anti-Scl-70 in DID showed positive results in ELISA assay using this recombinant peptide. All the serum samples from patients with other CTD showed negative results in ELISA assay.
Conclusion: The recombinant antigen fragment contains major epitope regions in natural Scl-70 antigen. Detection of anti-Scl-70 antibody with ELISA using the recombinant peptide can improve the sensitivity and has a potential role in determining its clinical association.
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