Production and characteristics of monoclonal antibodies to the diphtheria toxin

Abstract

Monoclonal antibodies to the diphtheria toxin were produced without cross reactivity with the thermolabile toxin (LT) from Escherichia coli; ricin; choleraic toxin; the SeA, SeB, SeE, SeI, and SeG toxins of staphylococcus; the lethal factor of the anthrax toxin; and the protective antigen of the anthrax toxin. A pair of antibodies for the quantitative determination of the diphtheria toxin in the sandwich variation of enzyme-linked immunosorbent assay (ELISA) was chosen. The determination limit of the toxin was 0.7 ng/ml in plate and 1.6 ng/ml in microchip ELISA. The presence of a secretion from the nasopharynx lavage did not decrease the sensitivity of the toxin determination by sandwich ELISA. The immunization of mice with the diphtheria toxin and with a conjugate of the diphtheria toxin with polystyrene microspheres demonstrated that the conjugate immunization resulted in the formation of hybridoma clones which produced antibodies only to the epitopes of the A fragment of the diphtheria toxin. The immunization with the native toxin caused the production of hybridoma clones which predominantly produced antibodies to the epitopes of the B fragment.

Determination of Diphtheria Toxin in Bacterial Cultures by Enzyme Immunoassay

“…Cells of mAb-producing hybridomas were used to generate mAbs [ 9 ]. Briefly, the producing cells were introduced into BALB/C mice, and preparative amounts of the antibodies were isolated from the ascitic fluids of these mice.…”

“…The purity of the mAbs was defined by SDS-PAGE. The mAbs antigen-binding activity was confirmed by indirect ELISA as described previously [ 9 ].…”

Generation of a human monoclonal antibody to cross-reactive material 197 (CRM197) and development of a sandwich ELISA for CRM197 conjugate vaccines

“…Cross-reactive material 197 (CRM 197 ) is a non-toxic mutant of diphtheria toxin, which is produced in Corynebacterium diphtheriae and commonly causes respiratory disease among children [1]. Diphtheria toxin is a single-chain 58 kDa protein that consists of two fragments (A and B), which are linked by disulfide bridges and separated after mild trypsin treatment and reduction in vitro [2][3][4]. The fragment A (21 kDa) is the catalytic domain that inhibits protein synthesis, and fragment B (37 kDa) binds to the host cell receptor, heparin binding EGF-like growth factor [5,6].…”