An immuno-polymerase chain reaction assay for human interleukin-18.

ABSTRACT

Conventional enzyme-linked immunosorbent assays (ELISA) are sufficient to measure normal and elevated serum interleukin (IL)-18 concentrations, but have limited sensitivity when measuring low concentrations of IL-18 such as in patients with the acquired immunodeficiency syndrome. We have developed a highly sensitive method for detecting human (h) IL-18 using an immuno-polymerase chain reaction (PCR). A mouse monoclonal anti-hIL-18 antibody and rabbit polyclonal anti-hIL-18 antibody was used for an indirect sandwich ELISA with a detection limit of 40 ng/l and a very low background. For immuno-PCR, biotinylated DNA was produced from the plasmid Bluescript by PCR amplification with biotinylated M13-20 primer and nonbiotinylated M13 reverse primer. Immuno-PCR for hIL-18 was performed for 40 cycles using 1 ng/l of biotinylated DNA. This immuno-PCR has a detection limit of 2.5 pg/l, 1.6x10(4) times lower than that of the ELISA. In addition, our system avoids sampling error caused by heat transfer from the ELISA plate to the PCR tube because all procedures from immobilization of the antibody to PCR amplification can be performed in the same tube. This immuno-PCR for hIL-18 is the most sensitive method for detecting hIL-18 reported to date.