Characterization of the F(ab')2 and Fab' fragments prepared from a mouse monoclonal insulin antibody.
In this report, optimum conditions were determined for the production of immunoreactive fragments from a mouse monoclonal insulin antibody, and their immunochemical characterization is described. Stable fragments can be obtained in good yield from the purified IgG 1, first by cleavage with pepsin and then by reducing the disulfide bonds with cysteine and subsequent alkylation with iodoacetamide. F(ab')2 and Fab' fragments having molecular weights (Mr) of about 108,000 (108 K) and 55 K, respectively, were produced. Ligand binding assay as well as indirect immunofluorescence on mouse pancreas section demonstrated their reactivity with free and tissue-bound insulin antigen. These results provide methods for the production and characterization of defined fragments of insulin antibody useful in experiments where non-specific interactions mediated by the Fc portion of the whole immunoglobulin may occur.
Diaz-Alonso
J M
JM
Central Institute of Diabetes, Gerhardt Katsch, Karlsburg.
Kohnert
K D
KD
Witt
S
S
Ziegler
M
M
D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis* / Q000276:immunology; D001666:Binding Sites, Antibody; D003545:Cysteine; D005455:Fluorescent Antibody Technique; D007140:Immunoglobulin Fab Fragments / Q000032:analysis* / Q000276:immunology; D007328:Insulin / Q000276:immunology*; D051379:Mice; D010434:Pepsin A / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D007328:Insulin; D010434:Pepsin A; D003545:Cysteine