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

Abstract

Background: Tumor necrosis factor-α (TNFα) is an important mediator of inflammatory and autoimmune diseases. Analysis of its pathophysiologic roles has been difficult because low concentrations of TNFα, including those in healthy controls, cannot be measured by existing methods. Methods: We developed a sensitive immuno-PCR assay for the detection of TNFα in human serum. The DNA label was generated by PCR amplification using biotinylated primer and was bound with streptavidin to the biotinylated third antibody. TNFα 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 × 104-fold improvement compared with a conventional ELISA. The mean serum TNFα 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 TNFα concentration in various diseases.

Detection of norovirus capsid proteins in faecal and food samples by a real time immuno-PCR method

“…Depending on the type of antigen captured, different formats of the assay have been developed. Antigen can be coated directly on the plate, or antigen is captured by a sandwich-style dual antibody method (Saito et al 1999). Biotin can be directly conjugated with the second antigen detection antibody or conjugated to a third anti-(second antibody)-antibody (Sanna et al 1995).…”

“…Avidin is used as a bridge to connect the biotinconjugated antibody and biotinylated reporter DNA. The increased sensitivity of I-PCR compared to traditional ELISA results from orders of magnitude increased amplification of the reporter DNA by PCR compared to traditional enzyme amplification (Mweene et al 1996;Saito et al 1999;Liang et al 2003;Adler et al 2003b;Barletta et al 2004;Chao et al 2004).…”

“…I-PCR methods are recognized as one of the most sensitive detection methods that exist (Barletta et al 2004). Compared to ELISA, the sensitivity of I-PCR is increased three to five logs (Mweene et al 1996;Saito et al 1999;Liang et al 2003;Adler et al 2003b;Barletta et al 2004;Chao et al 2004). However, most I-PCR studies reported have tested clinical samples such as blood, body fluid, or purified proteins.…”

Detection of Clostridium botulinum neurotoxin type A using immuno-PCR

“…The major difference in the immuno-PCR assay described here was the use of covalent attachment of the DNA label to reporter antibody. The DNA-labelled reporter reagents used in the earlier reports were assembled by non-covalent attachment such as biotin±avidin or biotin±streptavidin (Sano et al 1992;Sanna et al 1995;Zhang et al 1998;Saito et al 1999;Ren et al 2000). These methods involve numerous steps for the addition of reporter reagents, and need more than 20 washing steps to remove excess reagents.…”

“…In comparison with ELISA, an enhancement in detection sensitivity, from 10 2 to 10 5 -fold (Sano et al 1992), was obtained by immuno-PCR. This methodology had been claimed to have the potential to detect very low concentrations of antigens, such as tumour markers (Zhang et al 1998;Ren et al 2000), cytokines (Sanna et al 1995;Saito et al 1999), hormones Joerger et al 1995) and viral antigens (Maia et al 1995;Mweene et al 1996).…”