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Clinical Chemistry 45: 665-669, 1999;
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(Clinical Chemistry. 1999;45:665-669.)
© 1999 American Association for Clinical Chemistry, Inc.

Endocrinology and Metabolism

Detection of Human Serum Tumor Necrosis Factor-{alpha} in Healthy Donors, Using a Highly Sensitive Immuno-PCR Assay

Kaori Saito, Daisuke Kobayashi, Masateru Sasaki, Hiroshi Araake, Takashi Kida, Atsuhito Yagihashi, Tomomi Yajima, Hidekazu Kameshima and Naoki Watanabea

Department of Laboratory Diagnosis, Sapporo Medical University, School of Medicine, South-1, West-16, Sapporo 060-0061, Japan.
a Author for correspondence. Fax 81-11-622-7502; e-mail watanabn{at}sapmed.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Tumor necrosis factor-{alpha} (TNF{alpha}) is an important mediator of inflammatory and autoimmune diseases. Analysis of its pathophysiologic roles has been difficult because low concentrations of TNF{alpha}, including those in healthy controls, cannot be measured by existing methods.

Methods: We developed a sensitive immuno-PCR assay for the detection of TNF{alpha} in human serum. The DNA label was generated by PCR amplification using biotinylated primer and was bound with streptavidin to the biotinylated third antibody. TNF{alpha} sandwiched by antibodies was detected by amplification of the DNA label using PCR.

Results: The limit of detection of the assay was 0.001 ng/L, an ~5 x 104-fold improvement compared with a conventional ELISA. The mean serum TNF{alpha} concentration (± SD) in healthy donors was 0.021 ± 0.044 ng/L in men (n = 29) and 0.033 ± 0.065 ng/L in women (n = 25).

Conclusion: This method may be useful for analyzing the significance of TNF{alpha} concentration in various diseases.© 1999 American Association for Clinical Chemistry


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Tumor necrosis factor-{alpha} (TNF{alpha})1 is a multifunctional cytokine identified initially as a monocyte/macrophage-derived serum protein that mediates necrosis of solid tumors in mice (1)(2)(3)(4). TNF{alpha} has various physiologic activities that not only affect tumor cells, but non-tumor cells as well (5)(6)(7)(8). TNF{alpha} plays an important role in inflammation by eliminating foreign substances, e.g., bacteria and grafts, through the activation of chemotaxis and phagocytosis, induction and release of oxygen free radicals, and degranulation of monocytes/macrophages (9)(10)(11)(12)(13). Several inflammatory and autoimmune diseases are thought to caused by excessive TNF{alpha} activity. Previous studies have attempted to measure human serum TNF{alpha} in Crohn disease (14), ulcerative colitis (15), systemic lupus erythematosus (16), rheumatoid arthritis (17), and human immunodeficiency virus type-1 infection (18)(19). However, it is unclear whether measurement of serum TNF{alpha} can be useful in understanding various pathophysiologic processes because low concentrations of TNF{alpha}, including those in healthy donors, could not be measured by the methods used in those studies (20)(21). In this study, therefore, we established a highly sensitive method for measuring human serum TNF{alpha}, using an immuno-PCR assay (22)(23)(24) and determined reference values in healthy donors.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
serum samples
Serum samples were collected from 29 men (mean age, 40.5 years; range, 25–61 years) and 25 women (mean age, 36.6 years; range, 21–58 years) and used for immuno-PCR assays. These donors were selected randomly from workers in our hospital who had been determined to be healthy by clinical examination. Serum samples were stored at -70 °C until being assayed.

human recombinant tnf{alpha} and antibodies
Human recombinant TNF{alpha}, mouse anti-human recombinant TNF{alpha} monoclonal antibody (mAb), and rabbit anti-human recombinant TNF{alpha} polyclonal antibody (pAb) were kindly provided by ASAHI Chemical Industry Co., Tokyo, Japan (25).

elisa
Mouse anti-human recombinant TNF{alpha} mAb (100 mg/L in 0.05 mol/L borate buffer, pH 9.6) was immobilized at 4 °C overnight on TopYieldTM Strips (Nalgenunc). After the plate (TopYield Strips) containing immobilized mAb was blocked with a 1:4 dilution of Blockace (Dainihon Pharmaceutics) at 4 °C overnight, human recombinant TNF{alpha} or serum samples diluted 1:1 with 1 g/L gelatin in phosphate-buffered saline were added, and incubation was continued at 4 °C overnight. To examine the effect of the serum matrix on the lower limit of detection for TNF{alpha}, we also used recombinant human TNF{alpha} diluted 1:1 with the serum from a healthy donor in the experiment. The plate was washed with buffer A (2 g/L Triton X-100 in a 1:10 dilution of Blockace) five times, after which rabbit anti-human TNF{alpha} pAb (3 mg/L in buffer A) was added, and incubation was continued at 25 °C for 2 h. After the plate was washed five times, 0.5 mg/L horseradish peroxidase-labeled goat anti-rabbit mAb (Biosource) in buffer A was added and incubated at 25 °C for 1.5 h. o-Phenylenediamine (0.7 g/L in citrate buffer) with 300 mL/L H2O2 was then added and incubated at 25 °C for 15 min. The reaction was stopped by the addition of 2.25 mol/L sulfuric acid. The absorbance of the sample was determined at 492 nm in an ELISA reader EAR400 (SLT-Labinstruments).

dna label
Biotinylated double-stranded DNA for the DNA label was generated by PCR amplification of plasmid Bluescript (pBluescript; Toyobo) with a 5'-biotinylated M13-20 primer (biotin-5'-GTAAAACGACGGCCAGT-3') and a nonbiotinylated M13 reverse primer (5'-GGAAACAGCTATGACCATG-3') (26). PCR was performed in a Gene Amp PCR System 9600-R (Perkin-Elmer Cetus) under the following reaction conditions: 10 mmol/L Tris-HCl buffer (pH 8.3), 50 mmol/L KCl, 3.0 mmol/L MgCl2, 0.2 mmol/L each deoxyribonucleotide, 0.2 µmol/L each primer, 1 U of Taq DNA polymerase (Perkin-Elmer Cetus), and 5 pg of pBluescript. The temperature profile was as follows: initial denaturation at 95 °C for 5 min; 30 cycles of denaturation at 94 °C for 30 s; annealing at 58 °C for 60 s, extension at 72 °C for 30 s; and final extension at 72 °C for 5 min. The 227-bp PCR products were purified on CHOROMA SPIN-200 columns (Toyobo).

immuno-pcr
A schematic representation of the immuno-PCR method is shown in Fig. 1 . Briefly, the procedures up to the addition of the second pAb were identical to those for ELISA except for the addition of 1 g/L salmon sperm DNA to the 1:4 dilution of Blockace used as the blocking agent. The plate was washed to remove unbound second pAb; 0.5 mg/L biotinylated goat anti-rabbit mAb (Biosource) in buffer A was then added, and incubation was continued at 25 °C for 90 min. After the plate was washed five times, 0.1 mg/L streptavidin (Chemicon International) in buffer A was added and incubated at 25 °C for 30 min. The plate was washed with buffer A five times, and then the biotinylated DNA label was bound with streptavidin and incubated at 25 °C for 30 min. The plate was washed five times with buffer A and five times with distilled water, and then was subjected to PCR using a Gene Amp PCR System 9600-R. PCR was carried out under the following reaction conditions: 10 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, 3.0 mmol/L MgCl2, 0.2 mmol/L each deoxyribonucleotide, 0.2 µmol/L each forward primer (5'-AGCGCGCGTAATACGACTC-3') and reverse primer (5'-ACCATGATTACGCCAAGCG-3'), and 1 U of AmpliTaq DNA polymerase in a total volume of 50 µL. The temperature profile was as follows: initial denaturation at 95 °C for 5 min; 40 cycles of denaturation at 94 °C for 15 s, annealing at 56 °C for 15 s, extension at 72 °C for 30 s; and final extension at 72 °C for 5 min. The 196-bp PCR product was electrophoresed on a 1% agarose gel at 100 V for 40 min and then stained with 0.5 mg/L ethidium bromide for 20 min. The stained gel was washed and scanned immediately with a FluorImager SI apparatus (Molecular Dynamics; pixel size, 100 µm; digital resolution, 16 bits; detection sensitivity, high sensitivity). Distilled water and the biotinylated DNA label with reaction mixture added were used as the negative and positive controls, respectively, for the PCR.



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  		Figure 1. Schematic representation of the immuno-PCR assay.

A mouse mAb (1st mAb) immobilized on an ELISA microtiter plate was used to capture antigen sandwiched with rabbit pAb (2nd pAb), after which biotinylated monoclonal anti-rabbit antibody (3rd biotinylated mAb) was reacted with the captured pAb, and biotinylated DNA label was bound using streptavidin. Finally, the reporter DNA was amplified using PCR.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
determination of the detection limit of elisa
We determined the detection limit of the ELISA by using serial dilutions of human recombinant TNF{alpha}. The calibration curve is shown in Fig. 2 . The detection limit was equivalent to ~50 ng/L, as determined by quadruplicate determinations. To examine the effect of the serum matrix on the lower limit of detection for TNF{alpha}, ELISA was performed using human recombinant TNF{alpha} dissolved in serum from a healthy donor in which the TNF{alpha} concentration was below the detection limit of conventional ELISA (Fig. 3 ). The detection limit was 50 ng/L, as defined by the antigen concentration in which the mean ± 2 SD value did not overlap that of the serum without TNF{alpha}. There was no difference in the detection limit between the experiments using phosphate-buffered saline and serum.



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  		Figure 2. Calibration curve obtained with a conventional ELISA (as described in Materials and Methods).

Data are expressed as means of quadruplicate measurements and are represented by the absorbance at 492 nm.



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  		Figure 3. The effect of serum matrix on the lower limit for detection of TNF{alpha} in ELISA.

Two lower concentrations of TNF{alpha} (25 and 50 ng/L) diluted 1:1 with healthy human serum were used; serum without TNF was used as the zero concentration. Data are the means ± 2 SD (error bars) of quadruplicate measurements and are represented by the absorbance at 492 nm.

determination of optimal streptavidin concentration for immuno-pcr
To determine the optimal streptavidin concentration for the immuno-PCR study, the effect of different streptavidin concentrations on the ELISA calibration curve was examined. Horseradish peroxidase-labeled streptavidin (1, 0.1, and 0.01 mg/L) was used instead of free streptavidin, and o-phenylenediamine was used as a chromogenic substrate. A strong nonspecific reaction was seen at a streptavidin concentration of 1 mg/L, and a plateau in o-phenylenediamine reactivity was seen at high antigen concentrations. In contrast, at a streptavidin concentration of 0.01 mg/L, the sensitivity was clearly reduced, although only a weak nonspecific reaction was noted. At a streptavidin concentration of 0.1 mg/L, both a weak nonspecific reaction and the proper slope for the reaction curve were obtained. Therefore, we used a streptavidin concentration of 0.1 mg/L for the immuno-PCR study (Fig. 4 ).



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  		Figure 4. Analysis of optimal streptavidin concentration for immuno-PCR.

Streptavidin concentrations are 1 mg/L ({bullet}), 0.1 mg/L ({circ}), and 0.01 mg/L ().

effect of dna label concentration on nonspecific amplification by immuno-pcr
We next examined the optimal concentration of DNA label because this may have a marked influence on the intensity of false-positive signals. We used 1.0 g/L gelatin in phosphate-buffered saline instead of antigen, and free streptavidin was used to attach the biotinylated reporter DNA to the biotinylated third mAb. Serial 10-fold dilutions (0–5000 ng/L) of DNA label were added, and each resulting complex was subjected to PCR. As shown in Fig. 5 , the intensity of the 196-bp band decreased in a dose-dependent manner. The band disappeared at a DNA label concentration of 0.5 ng/L. Consequently, the optimal reporter DNA concentration was determined to be 0.5 ng/L for immuno-PCR using antigen.



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  		Figure 5. Analysis of nonspecific amplification in immuno-PCR assay. Lane 1, DNA size marker; lane 2, negative control; lanes 3–7, serial 10-fold dilutions of the DNA label ranging from 0.5 to 5000 ng/L; lane 8, positive control (5 pg of reporter DNA only).

comparison of immuno-pcr and elisa detection limits
Serial logarithmic dilutions of recombinant TNF{alpha} were used to compare the limits of detection of immuno-PCR and ELISA. In the conventional ELISA system for the detection of TNF{alpha}, the limit of detection was 50 ng/L. Immuno-PCR was performed using optimal concentrations of streptavidin (0.1 mg/L) and DNA label (0.5 ng/L). With immuno-PCR, the 196-bp band was at TNF{alpha} concentrations down to 0.001 ng/L. This result indicated that the limit of detection of the immuno-PCR was approximately 5 x 104-fold lower than that of the ELISA for TNF{alpha} (Fig. 6 ).



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  		Figure 6. Comparison of limit of detection between immuno-PCR ({circ}) and ELISA ({bullet}) for TNF (as described in Materials and Methods).

The detection values in immuno-PCR and ELISA are represented by the relative fluorescence units (RFU) and absorbance at 492 nm, respectively.

detection of serum tnf{alpha} in healthy donors by immuno-pcr
To examine whether this highly sensitive immuno-PCR method is actually useful, we measured TNF{alpha} concentrations in 54 samples obtained from healthy blood donors in which the TNF{alpha} concentration was below the detection limit of the ELISA. The mean value was 0.021 ± 0.044 ng/L (n = 29) in men and 0.033 ± 0.065 ng/L (n = 25) in women (Fig. 7 ).



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  		Figure 7. Detection of TNF{alpha} in healthy donors using immuno-PCR.

The samples used were sera from 29 healthy men (M) and 25 healthy women (W).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we established a highly sensitive method for the detection of human serum TNF{alpha}, using an immuno-PCR assay. The results show that the limit of detection of the immuno-PCR was 0.001 ng/L, which is ~5 x 104-fold lower than the that of the traditional ELISA, for which the limit of detection was 50 ng/L. The reference values for TNF{alpha} concentrations in healthy donors as determined by this method were more than three orders of magnitude below the detection limit of ELISA, with values of 0.021 ± 0.044 ng/L in men and 0.033 ± 0.065 ng/L in women.

Sanna et al. (27) reported previously that an immuno-PCR assay allowed the detection of TNF{alpha} in cerebrospinal fluid in the early stages after intracerebroventricular administration of lipopolysaccharide in a rat model. However, the detection limit of their immuno-PCR assay was 6.25 ng/L, which was only ~20-fold lower than the detection limit of their ELISA, which was 100 ng/L. The differences between the detection limit in their immuno-PCR system and ours may be attributable to differences in the antigens or antibodies used in the assays, the number of PCR cycles, and the DNA label concentrations. The DNA label concentration in their system was not reported, but they performed PCR for 25 cycles. In contrast, we used 40 PCR cycles to improve detection. Although high sensitivity is obtained with an increased number of cycles, nonspecific reactions also are increased. High DNA label concentrations in the immuno-PCR assay also cause nonspecific reactions, although sensitivity again is increased (28). In contrast, low concentrations of DNA label decrease sensitivity, but the nonspecific reactions are also decreased (29). High sensitivity was obtained in our system through the optimization of cycle number and DNA label concentration. Actual use of immuno-PCR has been problematic because of the prozone phenomenon, given the narrow detection range of immuno-PCR (30). We have shown that this phenomenon is avoidable by dilution and reanalysis of samples in the prozone area.

In our system, PCR products are electrophoresed and the intensity of each band is calculated by densitometry. Recently, Niemeyer et al. (31) compared the detection of immuno-PCR products by three different analytic methods, using fluorometry for detection of recombinant hepatitis B surface antigen. These investigators demonstrated that the enzymatic assay, carried out with either chromogenic or fluorogenic substrates for enzymatic signal amplification, is more sensitive than gel electrophoresis. Therefore, a more sensitive, practical assay suitable for routine laboratories could be developed by improving the analytic method in our system. The TaqMan® PCR method using a fluorogenic probe may be a good application of this strategy (32)(33).

In this study, we examined human serum TNF{alpha} concentrations by an immuno-PCR method, using samples in which TNF{alpha} was undetectable by ELISA. Despite the failure of ELISA to detect the presence of TNF{alpha}, immuno-PCR detected TNF{alpha} in all samples, with no difference between the samples from men and women. Given the important role played by TNF{alpha} in a variety of pathologic conditions, a readily available method for quantifying its presence may generate new insights into some types of pathophysiology and may eventually lead to the development of new treatment strategies for some disease states.


   Footnotes
 
1 Nonstandard abbreviations: TNF{alpha}, tumor necrosis factor-{alpha}; mAb, monoclonal antibody; and pAb, polyclonal antibody.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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