2004 Fiscal Year Final Research Report Summary
Generation of mutant antibodies with ultrahigh affinity based on a novel and universal selection method and their application to trace analysis
| Project/Area Number |
15390180
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Laboratory medicine
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| Research Institution | Kobe Pharmaceutical University |
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Principal Investigator |
KOBAYASHI Norihiro Kobe Pharmaceutical University, Professor, 薬学部, 教授 (90205477)
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| Co-Investigator(Kenkyū-buntansha) |
KOYAMA Junko Kobe Pharmaceutical University, Lecturer, 薬学部, 講師 (60102109)
MORITA Izumi Kobe Pharmaceutical University, Assistant Professor, 薬学部, 助手 (20299085)
GOTO Junichi Tohoku University Hospital, Professor, 病院・教授 (80006337)
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| Project Period (FY) |
2003 – 2004
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| Keywords | antibody / single-chain Fv fragment / phage display / phage antibody / affinity / genetic engineering / cleavable biotin / selection |
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| Research Abstract |
The engineering of hapten-specific antibodies with a much higher affinity than those of conventional antibodies promises hapten immunoassays exhibiting sub-femtomole range sensitivity. From these points of view, we developed a novel and universal method to select phage particles displaying anti-hapten antibody fragments with exceptionally high affinity. 11-Deoxycortisol (11-DC), 17α-hydroxyprogesterone, and estradiol were selected as model target haptens, and were covalently conjugated to biotin via a spacer including a reductively cleavable disulfide bond. These cleavable biotin(CB)-conjugated steroids exhibited desirable biochemical and chemical properties : the conjugates were bound simultaneously with relevant anti-steroid antibody and streptavidin, and the disulfide bond was easily cleaved by the treatment with 50 mM dithiothreitol(DTT). Phage particles displaying high affinity single-chain Fv fragments(scFvs) specific for 11-DC (Ka=1.3×10^<10> M^<-1) were incubated with the CB-conjugated 11-DC and the resulting complexes was captured on immobilized NeutrAvidin. The DTT treatment allowed the recovery of target phages ; this process is fully independent of the dissociation of the antigen-antibody interaction. Five serial rounds of selection enabled the isolation and enrichment of the anti-11-DC-phage from among a 100,000-fold excess of nonspecific phage particles. This method will be universally applicable for selection of extra-high affinity phage antibodies against a wide variety of haptens.
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