[The soluble expression and identification of single-chain fragment V antibodies against SSA antigen epitopes from the pHEN2 phagemid library].
OBJECTIVE
To obtain the soluble single-chain fragment V (ScFv) monoclonal antibodies (McAbs) against the SSA antigen epitopes.


METHODS
Three octapeptides (60000 SSA antigen residues 482-493 termed as P1 epitope, residues 310-323 termed as P2 epitope and residues 230-241 termed as P3 epitope) were synthesized on the lysine frame. The McAbs were panned by coating the corresponding as targets. The specificity, affinity and gene sequences of the positive clones were assessed. Soluble single-chain fragment V antibodies special for SSA antigen epitopes were expressed and then identified.


RESULTS
After 5 rounds of panning, reactive scFv clones contained full-length scFv antibodies coding regions were obtained, with sufficient affinity and specificity for respective antigen peptides. The absorbance values at 410 nm of the fusion protein of anti-P1-P3 activity with the corresponding peptides were 1.43±0.23, 0.82±0.31 and 0.80±0.25, and there was also statistically significant difference in the cross reactions (P<0.01). Three clones were successfully expressed and then purified by His-bind resin. The activity in vivo of soluble ScFv antibodies was identified to be positive by the indirect immune-fluorescence assay on Hep-2 cells.


CONCLUSIONS
Soluble ScFv McAbs against corresponding SSA antigen peptides with high affinity, specificity and activity in vivo were obtained, which are to be competent enough for epitopes expression on the target organs.
Li
Hong-bin
HB
Department of Rheumatology, Peking Union Medical College Hospital, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing 100032, China.
Zhang
Xuan
X
Tang
Fu-lin
FL
Zhang
Feng-chun
FC
D000918:Antibody Specificity / Q000276:immunology; D000939:Epitopes / Q000276:immunology; D019151:Peptide Library; D011994:Recombinant Proteins / Q000276:immunology / Q000302:isolation & purification; D012261:Ribonucleoproteins / Q000276:immunology*; D057127:Single-Chain Antibodies / Q000276:immunology* / Q000302:isolation & purification*; D000099207:SS-A Antigen
D000939:Epitopes; D019151:Peptide Library; D011994:Recombinant Proteins; D012261:Ribonucleoproteins; D057127:Single-Chain Antibodies; D000099207:SS-A Antigen