@Article{,
AUTHOR = {Nicolai Grebenchtchikov, Johanna van der Ven-Jongekrijg, Gerard J. Pesman, Anneke Geurts-Moespot, Jos W. M. van der Meer, Fred C. G. J. Sweep},
TITLE = {Development of a sensitive ELISA for the quantification of human tumour necrosis factor-a using 4 polyclonal antibodies},
JOURNAL = {European Cytokine Network},
VOLUME = {16},
YEAR = {2005},
NUMBER = {3},
PAGES = {215--222},
URL = {http://www.techscience.com/ECN/v16n3/66217},
ISSN = {1952-4005},
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.},
DOI = {}
}