Development of an enzyme immunoassay for the measurement of human tumour necrosis factor-alpha (hTNF-alpha) using bispecific antibodies to hTNF-alpha and horseradish peroxidase.
The cytokine tumour necrosis factor-alpha (TNF-alpha) is involved in several pathological processes, and human recombinant TNF-alpha (hrTNF-alpha) is available for testing in preclinical and clinical research. The purpose of this work was the creation of tetradomas producing bispecific anti-hTNF-alpha/anti-HRP (horseradish peroxidase) antibodies and the development of a rapid and sensitive solid-phase enzyme immunoassay. Monoclonal antibodies obtained against hrTNF-alpha could recognize both natural and recombinant hTNF-alpha. The four chosen hybridomas produced IgG1 with an affinity constant of the order of 10(-9) M. Three of them recognized different epitopes. The clone selected for fusion with the hybridoma producing anti-HRP antibodies secreted antibodies against portion 30-50 of the hTNF-alpha N-terminal amino acid residues as found by Western-blot analysis with mutant and chimaeric proteins. The tetradoma producing bispecific anti-hTNF-alpha/anti-HRP antibodies was identified using a fluorescence-activated cell sorter. Bispecific antibodies were isolated by hydroxyapatite chromatography. A sandwich ELISA was developed: one of the monoclonal anti-TNF-alpha antibodies was absorbed to the solid phase as the catcher and was detected by a bispecific anti-hTNF-alpha/anti-HRP antibody. The detection limit of the assay was 1 ng/ml. With such ELISA, the level of hTNF-alpha could be conveniently estimated in different samples containing either natural or recombinant hTNF-alpha in an experimental environment or in clinical trials.
Berkova
N
N
Laboratoire d'endocrinologie de la reproduction, Hôpital Saint-François d'Assise, Québec, Canada.
Karawajew
L
L
Korobko
V
V
Behrsing
O
O
Micheel
B
B
Shamborant
O
O
Stukatcheva
E
E
Shingarova
L
L
D018033:Antibodies, Bispecific; D001483:Base Sequence; D004797:Enzyme-Linked Immunosorbent Assay; D006735:Horseradish Peroxidase / Q000276:immunology*; D006801:Humans; D006825:Hybridomas; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000032:analysis; D012680:Sensitivity and Specificity; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000032:analysis*
D018033:Antibodies, Bispecific; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D006735:Horseradish Peroxidase