Elsevier

Clinica Chimica Acta

Volume 364, Issues 1–2, February 2006, Pages 349-353
Clinica Chimica Acta

Short communication
Comparison of two enzyme immunometric assays to measure tumor necrosis factor-alpha in human serum

https://doi.org/10.1016/j.cca.2005.07.018Get rights and content

Abstract

Background

Tumor necrosis factor-alpha is a 17.5 kDa, 157 amino acid protein that is a potent lymphoid factor, which exerts cytotoxic effects on a wide range of tumor cells and other target cells. TNF-alpha has been suggested to play a pro-inflammatory role by influencing transendothelial migration of monocytes and elicits the expression of proteolytic enzymes by macrophages and smooth muscle cells within the atherosclerotic plaque.

Methods

We compared two methods for the quantitative determination of human tumor necrosis factor-alpha in serum samples. Either kit follows the same assay procedure. Serum samples do not need to be diluted before sampling. Standard is provided lyophilized and serial dilutions after reconstitution generate the standard point curves. The tests are enzyme immunometric assays based on a standard 96-well microtiter plate. The wells are coated with anti-human TNF-alpha antibody.

Results

The range of the standard curve is similar in both kits. It spans from 1000 through 15.6 pg/mL. The median TNF-alpha concentration in samples measured by Pierce assay (n = 368) was 4.23 pg/mL, (range, 1.34–77.2 pg/mL). A very different median was obtained for the same specimen measured with the Titerzyme EIA (median, 176.96 pg/mL; range, 54.7–283.9 pg/mL; n = 364). Substantial significant differences were observed between the two methods.

Conclusion

Our results show that the two kits are unmatchable for results they can give when TNF-alpha concentrations are measured in serum samples. One reason of this disagreement could be the matrix effect or a cross-reactivity of one of the two methods. This study shows that the determination of human serum TNF-alpha needs to be standardized, especially when a comparison of results is required.

Introduction

Tumor necrosis factor-alpha (TNF-alpha) is a 17.5 kDa, 157 amino acid protein that is a potent lymphoid factor, which exerts cytotoxic effects on a wide range of tumor cells and other target cells [1]. TNF-alpha has been suggested to play a pro-inflammatory role by influencing transendothelial migration of monocytes and elicits the expression of proteolytic enzymes by macrophages and smooth muscle cells within the atherosclerotic plaque [2], [3], [4]. This cytokine was originally identified as an endogenous factor that influences energy balance and is associated with weight loss, hypermetabolism and increased resting energy expenditure in infectious or malignant diseases [5], [6], [7]. TNF-alpha is also closely related to metabolic disorders and diabetes [8]. Evidence for a function of TNF-alpha in the state of insulin resistance was provided by the observation that adipocytes in obese animals overexpress TNF-alpha [9]. Because of its interaction with the insulin signalling cascade a major role in the pathogenesis of obesity-linked insulin resistance and type 2 diabetes was postulated for TNF-alpha [10], [11], [12], [13]. In type 1 diabetes, TNF-alpha was found to be involved in the autoimmune process leading to beta cell damage [14], [15], which can be prevented in nonobese mice by sustained antibody-mediated inhibition of TNF-alpha activity [16].

This study was undertaken to evaluate the true TNF-alpha concentrations during pregnancy because TNF-alpha is synthesized and secreted also from placenta [17]. To achieve this goal, we evaluated the analytical properties of two assays based on a standard ELISA platform by comparing the results of one method with those of the other. One method is the human TNF-alpha ELISA kit (catalog number EH3TNFA3 or EH3TNFA5) manufactured by Pierce Endogen (Rockford, IL), the other is the Titerzyme EIA kit (catalog number 900-099) sold by Assay Designs, Inc. (Ann Arbor, Michigan). Since TNF-alpha is involved in several physiological processes, an accurate quantitative determination of TNF-alpha in biological fluids is necessary. Determination of TNF-alpha in human serum is full of risks, since there are a great number of TNF-alpha-related cytokines. On the market, there are scores of available commercial kits, but unfortunately, some of them show cross-reactivities with molecules in human serum. For these reasons we performed a simultaneous determination of serum TNF-alpha with two different commercial kits.

Section snippets

Samples

Pregnant women gave their informed consent to be enrolled in the study and all procedures were according to the Helsinki Declaration of 1975. After an overnight fast, a venous blood sample was drawn from the antecubital vein of each participant. Blood samples were drawn at the beginning of the first and third trimester of gestation. A sample of blood drawn from the umbilical vein was collected at the delivery. Blood samples were centrifuged at 3000 rpm, 4 °C, 10 min to collect serum. The serum

Statistical methods

Each sample was analyzed by Methods I and II and the results are reported as means and standard deviations (S.D.). We compared the serum TNF-α content obtained with the two techniques. A Bland–Altman [18] plot was used to evaluate the agreement between the two techniques. Differences between the two methods were also evaluated by a Deming regression analyses.

The level of statistical significance was set at P < 0.05.

Results and discussion

Either kit follows the same assay procedure. Serum samples do not need to be diluted before sampling. Standard is provided lyophilized and serial dilutions after reconstitution generate the standard point curves. The range of the standard curve is similar in both kits. It spans from 1000 through 15.6 pg/mL and sample buffer is used as the zero calibrators. All the measurements were done with further dilution of the standards. We added two points in the calibration curve: 7.81 and 1 pg/mL. So

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