The Study Of Proteins Associated With The Sa Antigen

The Sa antigen/antibody system is a novel serum marker that discovered in the past decade for the early diagnosis of rheumatoid arthritis(RA), and the antigen is highly specific and ecorrelated with the severity of the disease. The study of the Sa antigen/antibody system will promote its clinical application and help to understand the pathogenesis of RA.In this research, the Sa antigen was extraced from fresh human placenta(HPE) by anion exchange chromatography. The anti-Sa antibodies show band(s) with apparent molecular weight of 50 000(and) 55 000 detected by western blotting. The anti-Sa antibodies profile was all negative in 30 health control and 6 reference connective tissue diseaes sera. Test result of sera from 155 patients with connective tissue diseases(71 rheumatoid arthritis,11 ankylosing spondylitis, 7 psoriatic arthrits, 7 reactive arthritis, 4 juvenile idiopathic arthritis, 5 osteoarthritis, 6 polymyalgia rheumatica, 6 gout, 7 systemic lupus erythematous, 10 sjogren's syndorme, 3 adult onset still's disease) yeild a sensitivity and specificity of 47.9% and 95.2%, respectivley. The positive and negative predictive value is 89.5% and 68.4%, correspondingly.Mass spectrometry was then employed in this research to elucidate the character of the Sa antigen. Briefly, the gel bands corresponding to the stained bands on immunoblotting were excised, trypsin-digested in gel and analysised by HPLC-ESI-MS/MS. The data was searched against nr.fasta database(NCBI) and the target protein was identified as vimentin.To verify the target protein, two-dimensional electrophoresis and western blotting was combined to detect the anti-Sa antibodies. The result revealed that the molecular weight and isoelectronic point of Sa antigen is about 50 000 and 5.0, respectively, which were compatible with vimentin.Vimentin gene was cloned and expressed in E.coli. But the antibody against vimentin detected by immunoblotting showed lower sensitivity(36.6%) and specificity(83.3%), with little diagnostic value. And no obvious correlation between anti-vimentin andibody and anti-Sa antibodies was observed(Kappa=0.316), which indicated the great difference betweenthe Sa antigen and vimentin.citrullinated and showed significant change in the antigenic property. The anti-citullinated vimentin antibody yeilded a sensitivity and specificity of 49.3% and 86.9%, respectively, which is highly correlated with anti-Sa antibodies(Kappa=0.746). So, the citrullinated vimentin is closely associated with the Sa antigen. But the relatively high false positive rate of anti-citrullinated vimentin antibody revealed that futher work should be done to clearify the relation between the Sa antigen and vimentin.In this research, the power of the proteomics approaches including mass spectrometry was demonstrated, which will greatly promote the study of the antigens related to autoimmune diseases.