| Anti-SSA autobodies are considered as the most prevalent specificity among manyautoimmune diseases, such as systemic lupus erythematosus(SLE), primay orsecondary sjogren's syndrome(SS), subacute cutaneous LE (SCLE) and neonatallupus erythematosus. It was associated with manifestations as rash, exocrine salivarygland dysfunction, neutropenia and so on. The SSA antigen is an heterogeneousantigenic complex, constituted by two different proteins (52 kDa,60 kDa) and four human cytoplasmic RNAs (hYRNA) particles. While theantigenicity lies in the protein component, cDNA encoded the 60 kDa SSA antigenhas been revealed. Harley found that the antigen consists of a serial of discontinuousepitopes that autoantibody responses to SSA antigen can preferentially target.Autoantibodies responsed to different epitopes were found in different diseases andhold different clinical and laboratory associations. For example, our previous workshowed that serum antibodies against multiple antigenic peptide (MAP) 310～323 and482～495 of 60-kDa SSA antigen associated the salivary gland dysfunction. Kurien BTreported, due to the identitial sequence EKVK between 60-kDa Ro and 64-kDa D1antigen predominantly found in the thyroid, anti-SSA antibodies mediate neutropeniain patients with SLE. Aberrant redistribution of intracellular SSA antigen onto thesurface of epithelial cells of SLE, SS and animal models has been evidented in vitro.Immunication with certain epitopes can facilitates epitope spreading and develops thepathological impairment of the salivary gland as involvement of SS. All impled thatanti-SSA may be pathogenic antibodies that affect different organs dependent on thedifferent epitopes on it. Wether organ-special dominant epitopes has its role in thepoint were still unanswered.Objection To investgate difference of the SSA antigen epitopes on the involved organs or cells, following the panning and soluable expression of the Scfv antibodiesagainst corresponding epitopes from the constucted pHEN2 phagmid library. Furtherto study the association of the extent lied between epitopes and impairment.Methods1. Three Octapeptide (residues 482-493 termed as P1 epitopes, residues 310-323termed as P2 epitopes and residues 230-241 termed as P3 epitopes) from thedefined 60 kDa SSA epitopes were synthesized on the Lysine frame.2. Panning and then soluable expression of the ScFv antibodies against P1-P3epitopes from the previously constructed pHEN2 phagmid library.3. With the ScFv McAbs against P1-P3 peptide, expression of epitopes on thesalivary glands of patients with SS and neutrophils of patients with SLE and/or SScomplicated with neutropenia.4. Analysis the association of the extent between epitopes expression and organimpairment.Results1. The repertoire size of the pHEN2 phagmid ScFv antibodies library freezed at-70℃were analysized to be 1.08×1010 cfu. PCR analysis andfingerprinting(digestion with MSP-1 of PCR amplified fragment) showed thatpercentage of phagemid clones with full-length scFv DNA was 55.56% andbacterial clones contained scFv coding regions with great diversity.2. With modified selection method, we successfully obtained reactive scFv clonescontained full-length scFv coding regions (3 for P1,6 for P2,4 for P3 respectively).3 clones were further soluble expreessed induced by IPTG. after reinfection ofselected pHEN2 phagmid into to E coli HB2151. Monoclone Scfv antibodies werepurified from whole cell supernatant fractions by immobilized metal chelateaffinity chromatography. All of them showed sufficient binding and specificity forrespective antigenic peptides in ELISA. Activity in vivo were proved byimmunofluorescence on Hep-2 cells.3. The nucleotide sequences of VH and VL genes were determined and comparedwith the germline sequences in the NCBI Immunoglobin BLAST (IgBlast). VH sequence analysis of the P1-P3 peptides binders showed that half of anti-P2were derived from VH3 families while 75 %of anti-P4 were derived from VH4families.VL of all McAb came from VLκfamily, mostly from A families(P1:100%, P2:66.67%, P3:50%). An average to high level of mutation was identified inall clones, indicative of an antigen-driven respondense.4. Immunohistochemical assay of lip salivary gland biopsy samples, with McAbagainst P1-P3 epitopes showed that epithelial cells of glandular tubes and striatedduct were stained in membrane and cytoplasm. The expression of P1 epitope werestronger than the other two in grade score, while a positive correlation was foundbetween the extent of glandular infiltration and the grade of P1 epitopesexpression.5. Flow cytometry assay showed the positive percentage of P3 epitope was higher onthe neutrophilic granulocyte membrane in both patient with SLE complicated withneutropenia and the normal compared with that of P1-P2 epitopes. But theexpression of P3 epitope were up-regulated when compared the positivepercentage between patient with SLE together with granulocytopenia and the normal.There was significant negative correlation between the positive percentage of P3epitope and neutrophilic granulocyte count.6. The positive percentage of serum anti-P3 antibody was significantly higher inpatient with SLE and/or SS complicated with neutropenia than without. Nodifference of positive percentage of anti-P3 serum antibody was found between thepatients with SLE or pSS complicated with neutropenia. Theretofore, theneutropenia co-morbidities may be better to be incriminated to the presention of theantibodies but rather to SLE.Conclusion1. Aberrant redistribution of intracellular SSA antigen epitopes onto the cellmembrance of the involved organ and cells may break the immune tolerance andthereforth induce the pathogensic autoantibodies.2. There lies the difference between the expression of eptitopes on the menbrancedependent on disease and/or organ involved. It has been evidented that P1 eptitope on the labial gland in patients with SS and P3 eptitope on the neutrophilicgranulocyte in patients with SLE complicated with granulocytopenia.
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