title: [Affinity optimization of an anti-hIL17A single-chain Fv antibody through error-prone PCR technique].
abstract: OBJECTIVE
  To optimize antibody affinity of a  humanized  anti-hIL17A single-chain Fv (scFV) antibody through error-prone polymerase chain reaction (PCR) so as to develop a therapeutic humanized antibody for the treatment of rheumatoid arthritis (RA).
  
  
  METHODS
  Variable regions of heavy chain and light chain were subjected to random mutation by error-prone PCR and a scFv library was constructed by overlapping PCR. The library was screened for improved mutants by the bacterial inner membrane display technique and flow cytometry (FCM).  Real-time PCR was used to detect neutralization effects of the purified scFv antibody mutants on mRNA expressions of IL-6 and IL-8 in HeLa cells stimulated by hIL17A.
  
  
  RESULTS
  We obtained five mutants with improved affinity.  FCM revealed that the affinity of three clones was greatly enhanced as compared with the parent clone. All mutants retained binding specificity to hIL17A. Real-time PCR results showed that all five mutants could block hIL17A stimulation of HeLa cells to express IL-6 and IL-8, and the neutralization effects were positively related to mutant affinity.
  
  
  CONCLUSIONS
  Error-prone PCR technique is a feasible method for antibody affinity optimization in vitro, which is able to improve the affinity and neutralization capacity of antibody on the basis of its unchanged antigen binding specificity. This study provides potential drug candidates aimed for the treatment of rheumatoid arthritis and an alternative method of optimizing antibody  affinity in vitro.
authors: Department of Biopharmaceutics, College of Life Science, Northeast Agricultural University, Harbin 150030, China. woxiangxintianhui@yahoo.cn
authors: Hui
authors: H
authors: Tian
authors: Fu-liang
authors: FL
authors: Bai
authors: Li-ming
authors: LM
authors: Xu
authors: Wen-fei
authors: WF
authors: Wang
authors: Ze-shan
authors: ZS
authors: Niu
authors: Gui-ping
authors: GP
authors: Ren
authors: De-shan
authors: DS
authors: Li
mesh_terms: D000915:Antibody Affinity / Q000235:genetics / Q000276:immunology*; D000918:Antibody Specificity / Q000276:immunology; D015870:Gene Expression / Q000187:drug effects; D015723:Gene Library; D006367:HeLa Cells; D006801:Humans; D020381:Interleukin-17 / Q000276:immunology* / Q000494:pharmacology; D015850:Interleukin-6 / Q000235:genetics; D016209:Interleukin-8 / Q000235:genetics; D016296:Mutagenesis; D016133:Polymerase Chain Reaction; D011485:Protein Binding / Q000276:immunology; D057127:Single-Chain Antibodies / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*
chemical_list: D020381:Interleukin-17; D015850:Interleukin-6; D016209:Interleukin-8; D057127:Single-Chain Antibodies