BACKGROUND: The standard assay for the clinical detection of contact allergy against nickel is the patch test. For research purposes, the in vitro lymphocyte transformation test (LTT) is mostly applied. However, the in vivo application of allergens includes the potential of sensitization, whereas the LTT demands 6 days of cell culture and radioactive labelling procedures. OBJECTIVE: The purpose of this study was to validate an enzyme-linked immunospot (ELISpot) assay to specifically detect cellular response against nickel sulphate in correlation with patch test, anamnestic data, and LTT results. METHODS: In probands with positive (n=60) and negative patch test results (n=19), cytokine ELISpot and LTT were performed in parallel using various concentrations of nickel sulphate, various numbers of responding peripheral blood mononuclear cells (PBMC), and various durations of cell cultures. RESULTS: Concentrations of 50 and 75 microM nickel sulphate were found to be optimal to stimulate in the ELISpot 4x10(5) and in the LTT 2x10(5) PBMC, respectively. An increase in ELISpot sensitivity was reached by pre-incubation with nickel sulphate for 24 h prior to transfer to ELISpot plates. In nickel-sensitive probands, an average precursor cell frequency of 19x10(5), 1.7x10(5), and 0.7x10(5) could be defined for IFN-gamma, IL-2, and IL-4 producing PBMC, respectively. In non-sensitive probands IFN-gamma producing cells were detectable, but with significantly lower frequency (2x10(5); P=0.004). The prior performance of patch tests had no significant effect on cytokine production or lymphocyte proliferation. Overall, the parameters patch test, anamnestic nickel allergy, ELISpot, and LTT results were positively correlated (P<0.05). CONCLUSIONS: Based on these experiments, the quantitative in vitro detection of cell-mediated reactions towards nickel as well as other heavy metal ions should be possible utilizing the above-described ELISpot assay.
BACKGROUND: The standard assay for the clinical detection of contact allergy against nickel is the patch test. For research purposes, the in vitro lymphocyte transformation test (LTT) is mostly applied. However, the in vivo application of allergens includes the potential of sensitization, whereas the LTT demands 6 days of cell culture and radioactive labelling procedures. OBJECTIVE: The purpose of this study was to validate an enzyme-linked immunospot (ELISpot) assay to specifically detect cellular response against nickel sulphate in correlation with patch test, anamnestic data, and LTT results. METHODS: In probands with positive (n=60) and negative patch test results (n=19), cytokine ELISpot and LTT were performed in parallel using various concentrations of nickel sulphate, various numbers of responding peripheral blood mononuclear cells (PBMC), and various durations of cell cultures. RESULTS: Concentrations of 50 and 75 microM nickel sulphate were found to be optimal to stimulate in the ELISpot 4x10(5) and in the LTT 2x10(5) PBMC, respectively. An increase in ELISpot sensitivity was reached by pre-incubation with nickel sulphate for 24 h prior to transfer to ELISpot plates. In nickel-sensitive probands, an average precursor cell frequency of 19x10(5), 1.7x10(5), and 0.7x10(5) could be defined for IFN-gamma, IL-2, and IL-4 producing PBMC, respectively. In non-sensitive probands IFN-gamma producing cells were detectable, but with significantly lower frequency (2x10(5); P=0.004). The prior performance of patch tests had no significant effect on cytokine production or lymphocyte proliferation. Overall, the parameters patch test, anamnestic nickelallergy, ELISpot, and LTT results were positively correlated (P<0.05). CONCLUSIONS: Based on these experiments, the quantitative in vitro detection of cell-mediated reactions towards nickel as well as other heavy metal ions should be possible utilizing the above-described ELISpot assay.