|Budget Amount *help
¥13,900,000 (Direct Cost: ¥13,900,000)
Fiscal Year 2002: ¥4,300,000 (Direct Cost: ¥4,300,000)
Fiscal Year 2001: ¥9,600,000 (Direct Cost: ¥9,600,000)
Antibodies are widely used as a key reagent in various immunoassays or immunoaffinity purification systems, which are essential in the biomedical analyses. However, conventional antibodies have some limitations for their applicability to immunochemical techniques due to their large molecular weight (MW ca. 150,000). In this study, we attempted to generate novel antibody species of low molecular weight (mini-antibodies : MW<30,000), which may allow us to develop excellent analytical systems for various bioactive small molecules.
First, we cloned the V_H- and V_L-genes of various mouse monoclonal antibodies against steroids (corticosteroids, bile acids, and digoxin) or a drug (bufuralol) by RT-PCR using RNA extracted from the corresponding hybridoma cells. Single-chain Fv fragments (scFvs) against these haptens were then prepared by the bacterial expression of scFv genes encoding 5'-V_H-(Gly_4Ser)_3-V_L-3', which had been constructed by combining the cloned V_H- and V_L-genes. The scFvs against 11-deoxycortisol, deoxycholic acid, lithocholic acid sulfate, and bufuralol showed as high affinity and specificity to relevant hapten as the corresponding mouse antibody does. The scFvs could easily be prepared as fusion proteins with marker enzymes or fluorescent proteins, and thus will be useful tools in various immunochemical techniques in biomedical sciences.