Immunological application of the dihydrofolate reductase handle carrying a small peptide, leucine enkephalin.
The "dihydrofolate reductase (DHFR) handle" [Iwakura, M. et al. (1992) J. Biochem. 111, 37-45; Iwakura, M. & Tanaka, T. (1992) J. Biochem. 111, 638-642] fused with a pentapeptide, leucine enkephalin (LEK), has been applied in immunoassays for LEK and for the preparation of anti-LEK monoclonal antibody. DHFR fused with LEK (DHFR-LEK) was first utilized as an immobilized antigen in an enzyme-linked immunosorbent assay for LEK. By using a commercially available anti-LEK and peroxidase-linked anti-IgG, LEK could be quantified in the range between 0.1 ng/ml and 10 micrograms/ml. By using a commercially available anti-LEK and the DHFR-LEK as an enzyme-labeled antigen, LEK was quantified in the range between 0.1 ng/ml and 1 microgram/ml by monitoring the recovery of the DHFR activity from the immuno-precipitates. By using the DHFR-LEK as an immunogen, three mouse monoclonal antibodies against LEK, but not DHFR, were isolated. All three monoclonal antibodies were of IgG1 kappa type. The large-scale preparation of two of these monoclonal antibodies, designated as anti-LEK-36 and anti-LEK-74, was carried out and their recognition specificities were studied by competitive binding assays. The IC50 values of LEK for the anti-LEK-36 and anti-LEK-74 were 3.74 x 10(-6) and 4.66 x 10(-6) M, respectively. The competitive binding assays showed that recognition specificities of the two monoclonal antibodies were high and restricted to LEK and leucine-enkephalin (sulfated form). These results strongly suggest that the DHFR handle is useful in several immunological applications.
Iwakura
M
M
National Institute of Bioscience and Human-technology, Ibaraki.
Kokubu
T
T
Ohashi
S
S
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000737:chemistry; D001412:Bacillus subtilis / Q000201:enzymology; D004743:Enkephalin, Leucine / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000201:enzymology; D005260:Female; D006825:Hybridomas / Q000276:immunology / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D013762:Tetrahydrofolate Dehydrogenase / Q000276:immunology*
D000911:Antibodies, Monoclonal; D004743:Enkephalin, Leucine; D013762:Tetrahydrofolate Dehydrogenase