Homologous ELISA for detection of oligomeric human TNF: properties of the assay.
In order to quantify oligomeric human tumor necrosis factor-alpha (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.
Petyovka
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N
Laboratory of Molecular and Cellular Immunology, Foundation for Fundamental Research of the Republic of Belarus, Minsk.
Lyach
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L
Voitenok
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NN
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D000937:Antigen-Antibody Reactions; D001681:Biological Assay; D002478:Cells, Cultured; D017077:Culture Media, Conditioned / Q000032:analysis; D003902:Detergents / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D005189:False Positive Reactions; D006801:Humans; D016209:Interleukin-8 / Q000032:analysis; D007700:Kinetics; D007739:L Cells; D008233:Lymphotoxin-alpha / Q000032:analysis; D051379:Mice; D009000:Monocytes / Q000378:metabolism; D009500:Neutralization Tests; D011136:Polysorbates / Q000494:pharmacology; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000032:analysis / Q000276:immunology; D012680:Sensitivity and Specificity; D014409:Tumor Necrosis Factor-alpha / Q000032:analysis* / Q000737:chemistry
D000911:Antibodies, Monoclonal; D017077:Culture Media, Conditioned; D003902:Detergents; D016209:Interleukin-8; D008233:Lymphotoxin-alpha; D011136:Polysorbates; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha