Open Access

ARTICLE

Development of a sensitive ELISA for the quantification of human tumour necrosis factor-a using 4 polyclonal antibodies

Nicolai Grebenchtchikov¹, Johanna van der Ven-Jongekrijg², Gerard J. Pesman¹, Anneke Geurts-Moespot¹, Jos W. M. van der Meer², Fred C. G. J. Sweep¹

1 Department of Chemical Endocrinology
2 Department of General Internal Medicine 514 and Nijmegen University Centre for Infectious Diseases, Radboud University Nijmegen Medical Centre, Geert Grooteplein 8, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands

* Corresponding Author: J.W.M. van der Meer,

European Cytokine Network 2005, 16(3), 215-222.

Accepted 23 May 2005;

Abstract

Despite the availability of many assays to measure concentrations of tumour necrosis factor alpha (TNF-α) in body fluids, these assays often lack specificity or sensitivity and are often of questionable reliability, resulting in inconsistent results. Therefore, we have developed an ELISA that is sensitive, reliable and not susceptible to disturbances by interfering substances such as heterophilic antibodies. The assay involves a combination of four polyclonal antibodies. The antibodies, which capture the analyte, were raised in chicken and the trapping anti-analyte antibodies were raised in rabbit. The immobilization of capture antibodies was achieved via a coating antibody raised in a duck against chicken IgY and the recognition of trapping antibodies was achieved by a detection antibody raised in a goat against rabbit IgG and labelled with HRP. The analytical and functional sensitivities of the ELISA are 8 pg/mL and 13 pg/mL, respectively. The assay showed good precision and, in contrast to our in-house RIA, excellent parallelism in serial dilutions. The recovery of TNF-α spiked to plasma samples ranged from 97% to 119%. Comparison of the newly developed, sensitive ELISA with our in-house RIA showed that the median TNF-α value obtained by RIA (range: 0.095-10.0, median 0.578 ng/mL) was found to be 1.5-2 times higher than that obtained with the ELISA (range 0.008-5.84, median 0.213 ng/mL). Spearman correlation was 0.755 (p < 0.0001). In addition, analysis of the TNF-α concentrations in blood from healthy individuals and from patients suffering from tuberculosis, with RIA and ELISA, showed the same differences although TNF-α levels obtained with ELISA were lower. We feel that this ELISA is a major improvement compared to the currently available assays for TNF.

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