Development of a sensitive radioimmunoassay for Fab fragments: application to Fab pharmacokinetics in humans

• Published in: Pharmaceutical research Vol. 10; no. 5; p. 692
• Main Authors: Thanh-Barthet, C V, Urtizberea, M, Sabouraud, A E, Cano, N J, Scherrmann, J M
• Format: Journal Article
• Language: English
• Published: United States 01.05.1993
• Subjects: Animals; Digitoxin - poisoning; Digoxin - immunology; Electrophoresis, Polyacrylamide Gel; Enzyme-Linked Immunosorbent Assay; Female; Half-Life; Humans; Immune Sera - immunology; Immunoglobulin Fab Fragments - blood; Immunoglobulin Fab Fragments - urine; Middle Aged; Radioimmunoassay; Sensitivity and Specificity
• ISSN: 0724-8741
• DOI: 10.1023/A:1018903614997

Anti-sheep Fab fragment antisera were produced in rabbits using sheep digoxin-specific Fab fragments (Digidot) as immunogen. These antisera were used for the development of a radioimmunoassay (RIA) of sheep Fab fragments in human plasma and urine using 125I-labeled Fab fragments. Interference in the assays by digoxin, human proteins, and antibodies from different species was insignificant, but cross-reactivity between anti-sheep Fab antisera and goat IgG or Fab fragments was 22 to 67%. The limit of detection was 0.1 microgram/mL and the assay was linear over a 0.6-28 micrograms/mL range of Fab fragments. Intra- and interassay coefficients of variation were less than 6.9 and 10.5%, respectively. Accuracy of plasma and urine assays at various Fab fragment levels ranged from 96 to 106%. RIA was applied to the pharmacokinetic study of sheep digoxin-specific Fab fragments in one patient acutely intoxicated by digitoxin and treated with Digidot. The Fab elimination half-life was 12.1 hr. Steady-state volume of distribution and total-body clearance were 10.8 L and 23.4 mL/min, respectively. Unchanged Fab fragments (50 kD) and degradation products (25 kD) isolated by gel filtration chromatography of a urine sample cross-reacted with the anti-Fab antiserum.