Two-site immunoassay of recombinant hirudin based on two monoclonal antibodies.
Two monoclonal antibodies (mAbs), 10-2 and 10-5, both directed against recombinant hirudin variant 2-Lys47 (rHV2), were selected for their high affinity and epitopic specificities to develop a two-site immunoassay of rHV2. The mAb concentrations, incubation time, and temperature were optimized. The immunoassay has a detection limit for rHV2 of 45 ng/L in plasma and 30 ng/L in urine. The reactivity of the mAbs was tested against rHV2 and several forms of this protein truncated in the carboxyl terminus. The capture mAb 10-2 was found to be mainly directed against rHV2, whereas tracer mAb 10-5 was independent of the carboxyl-terminal region of the protein. This explains the high specificity of the immunoassay for the 65-amino acid form of hirudin.
Mille
B
B
Immunoanalyse et Innovation en Biologie Clinique, CNRS-UMR 9921, Faculté de Pharmacie, Montpellier, France.
Condamines
O
O
Herbert
J M
JM
Maftouh
M
M
Picard
C
C
Dussossoy
D
D
Pau
B
B
D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods* / Q000706:statistics & numerical data; D000939:Epitopes / Q000276:immunology; D006629:Hirudins / Q000032:analysis* / Q000097:blood / Q000652:urine; D006801:Humans; D011863:Radioimmunoassay; D011994:Recombinant Proteins / Q000097:blood / Q000652:urine; D012680:Sensitivity and Specificity; D013917:Thrombin / Q000037:antagonists & inhibitors
D000911:Antibodies, Monoclonal; D000939:Epitopes; D006629:Hirudins; D011994:Recombinant Proteins; D013917:Thrombin