DEVELOPMENT OF AN ENZYME-IMMUNOASSAY FOR THE MEASUREMENT OF HUMAN TUMOR-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 s everal 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 bisp ecific anti-hTNF-alpha/anti-HRP (horseradish peroxidase) antibodies an d the development of a rapid and sensitive solid-phase enzyme immunoas say, Monoclonal antibodies obtained against hrTNF-alpha could recogniz e both natural and recombinant hTNF-alpha, The four chosen hybridomas produced IgGI with an affinity constant of the order of 10(-9) M, Thre e 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 hi-terminal amino acid residue s as found by Western-blot analysis with mutant and chimaeric proteins , The tetradoma producing bispecific anti-hTNF-alpha/anti-HRP antibodi es was identified using a fluorescence-activated cell sorter, Bispecif ic antibodies were isolated by hydroxyapatite chromatography. A sandwi ch ELISA was developed: one of the monoclonal anti-TNF-alpha antibodie s 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 I ng/ml. With such ELISA, the level of hTNF-alpha could be conveniently estimated in different samples containing either natur al or recombinant hTNF-alpha in an experimental environment or in clin ical trials.