Application of a recombinant Fab fragment from a phage display library for sensitive detection of a target antigen by an inhibition ELISA system.
We have found that the recombinant Fab (rFab) produced by phage display system was detectable for a target antigen more sensitive than the parental monoclonal antibody (MoAb). The Fab phage display library was constructed from hybridoma cells producing APU-6 MoAb specific for a modified nucleoside, pseudouridine that have been studied as a urinary marker for malignancy. Fab-displayed phage clones were screened by a direct ELISA, and the single positive clone was finally obtained. Although the reaction pattern of rFab against pseudouridine and uridine was almost identical to that of MoAb, detection sensitivity of rFab was approximately 30 times higher than that of MoAb. Since the sensitivity of rFab was almost identical to that of Fab fragment prepared by papain digestion of MoAb, the increased sensitivity is considered to be the nature of Fab fragment. The sensitivity of established assay system was sufficient for quantitative determination of serum pseudouridine levels in healthy individuals and cancer patients. This procedure may be applicable for improvement of detection sensitivity of a MoAb-based inhibition ELISA system for drugs or low molecular weight compounds.
Itoh
K
K
Department of Pharmaceutical Science, Akita University Hospital, Japan.
Suzuki
K
K
Ishiwata
S
S
Tezuka
T
T
Mizugaki
M
M
Suzuki
T
T
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000235:genetics; D000941:Antigens / Q000276:immunology*; D001435:Bacteriophages / Q000235:genetics / Q000276:immunology*; D001483:Base Sequence; D001667:Binding, Competitive / Q000276:immunology; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis / Q000235:genetics*; D007143:Immunoglobulin Heavy Chains / Q000096:biosynthesis / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000096:biosynthesis / Q000235:genetics; D007938:Leukemia / Q000097:blood / Q000276:immunology; D008223:Lymphoma / Q000097:blood / Q000276:immunology; D008969:Molecular Sequence Data; D019151:Peptide Library; D011560:Pseudouridine / Q000097:blood / Q000276:immunology / Q000378:metabolism; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism*; D012680:Sensitivity and Specificity
D000911:Antibodies, Monoclonal; D000941:Antigens; D007140:Immunoglobulin Fab Fragments; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D019151:Peptide Library; D011994:Recombinant Proteins; D011560:Pseudouridine