Study on the functional modiffication of Monoclonal Antibodies
Grant-in-Aid for General Scientific Research (C)
|Allocation Type||Single-year Grants|
|Research Institution||National Institute of Health Sciences|
SAWADA Jun-ichi National Institute of Health Sciences, Division of Biochemistry and Immunochemistry, Director, 機能生化学部, 部長 (10111551)
沢田 純一 国立衛生試験所, 研究員
|Project Period (FY)
1992 – 1993
Completed(Fiscal Year 1993)
|Budget Amount *help
¥2,100,000 (Direct Cost : ¥2,100,000)
Fiscal Year 1993 : ¥1,000,000 (Direct Cost : ¥1,000,000)
Fiscal Year 1992 : ¥1,100,000 (Direct Cost : ¥1,100,000)
|Keywords||Monoclonal Antibody / Antibody Engineering / Genetic Recombination / Morphine / UV-photoaffinity Labelling / 発現ベクター|
This project aimed to investigate the basic techniques for the modification of antigen specifficity and affinity of monoclonal antibodies. The project plan included the following antibodies.
1.Determination of the primary structure of the variable regions of anti-hapten monoclonal antibodies.
2.Analysis of the orientation of antigen(hapten)in the combining sites of antibodies by affinity labelling.
3.Modeling of the hypervariable regions of antibodies.
4.Establishment of the expression system of antibodies in E.coli, and modification of antigen specificity by site-directed mutagenesis.
The variabl region uncleotide sequences were determined by mRNA sequencing of 11 monoclonal anti-morphine antibodies with slightly different specificities for morphine-related opiates. The antibodies were classified into four groups in terms of VH/VL combinations : groups 1 and 2, VH-1 and Vlambda-1 ; group 3, VH-1 and Vkappa-10 ; group 4, VH-5 and Vkappa-21d.
Direct UV-photoaffinity labelling with ^3H-morphine revealed the orientation of morphine in the combining sites. These antibodies were classified into two types in terms of the reactivity of the hapten with H and L chains. The morphine phenolic ring appeared to be located near the H chain loops in the combining sites in the VH-5 type group. On the other hand, in the VH-1 type groups, the phenolic ring appeared to be located near the l chain loops.
The plasmids were constructed to express single-chain Fv and Fab fragments with modified amino acid residues in the hypervariable regions in E.coli. The cDNAs for V and C regions of two anti-morphine antibodies were prepared by RT-PCR.The DNAs coding for secretory signal peptide(pelB leader), VH-VL linker, histidine oligomer and ribosome binding site were chemically synthesized. They were ligated and inserted into the vector with the tac promoter. The conditions for the expression are under investigation.
Research Products (3results)