Homologous ELISA for detection of oligomeric human TNF: properties of the assay

• Published in: Journal of immunological methods Vol. 186; no. 2; pp. 161 - 170
• Main Authors: Petyovka, Natalia, Lyach, Lyudmila, Voitenok, Nikolai N.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 26.10.1995; Elsevier
• Subjects: Analysis of the immune response. Humoral and cellular immunity; Animals; Antibodies, Monoclonal - immunology; Antibody Specificity; Antigen-Antibody Reactions; Biological and medical sciences; Biological Assay; Cells, Cultured; Culture Media, Conditioned - analysis; Detergents - pharmacology; ELISA; Enzyme-Linked Immunosorbent Assay; False Positive Reactions; Fundamental and applied biological sciences. Psychology; Fundamental immunology; Humans; IL-8; Immunobiology; Interleukin-8 - analysis; Kinetics; L Cells (Cell Line); Lymphokines, interleukins ( function, expression); Lymphotoxin-alpha - analysis; Mice; Monoclonal antibody; Monocytes - metabolism; Neutralization Tests; Oligomeric form; Polysorbates - pharmacology; Protein Conformation; Quaternary structure; Recombinant Proteins - analysis; Recombinant Proteins - immunology; Regulatory factors and their cellular receptors; Sensitivity and Specificity; Tumor necrosis factor; Tumor Necrosis Factor-alpha - analysis; Tumor Necrosis Factor-alpha - chemistry; PBS; Tumor necrosis factor; Oligomeric form; Quaternary structure; MoAb; Monoclonal antibody; IL-8; TNF; Hm-ELISA; Ht-ELISA; ELISA; Human; Investigation method; Cytokine; Detection; Oligomer; ELISA assay; Interleukin 8
• ISSN: 0022-1759; 1872-7905
• DOI: 10.1016/0022-1759(95)00183-B

In order to quantify oligomeric human tumor necrosis factor-α (TNF), we have developed a sensitive homologous enzyme-linked immunosorbent assay (Hm-ELISA) using the same monoclonal antibody (MoAb) for both solid and liquid phase. Different anti-TNF MoAb have been compared in terms of their efficacy in the Hm-ELISA, affinity, neutralization capacity and epitope specificity. The data suggest, that effectiveness in the Hm-ELISA may represent a novel characteristic of MoAb. Of the MoAbs tested, 5 N was capable of recognizing oligomeric TNF in the Hm-ELISA with a detection limit of 15 pg/ml. Furthermore, using Hm-ELISA against human TNF, interleukin-8 (IL-8) and lymphotoxin, we have demonstrated that these cytokines are oligomeric in physiological solutions, but are converted into monomeric forms in the presence of the non-ionic detergent Tween 20. High salt buffer was employed to abrogate a nonspecific false positive reaction in the Hm-ELISA found in nearly half of the plasma samples obtained from healthy subjects. Finally, a good correlation between the Hm-ELISA and the L929 bioassay was observed for natural and recombinant TNF measured in human plasma.