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High Sensitivity, Wide Dynamic Range, Horseradish Peroxidase (HRP) ELISA Using the LMax Microplate Luminometer (MaxLine Application Note #41)

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The calculated sensitivity of the assay (based on amount giving a signal 3 positive SD of the blank) was approximately 0.3 pg/mL regardless of whether the integration time was 1, 5 or 10 seconds. These results support R&D Systems claims for the kit of a sensitivity of 0.5 pg/mL and an upper limit of 7000 pg/mL.

We have also evaluated R&D Systems Quantikine human TNF-α Colorimetric Sandwich ELISA. Our results essentially confirmed their stated sensitivity of 4.4 pg/mL and upper range of 1000 pg/mL (data not presented here). Thus the luminescent ELISA is an order of magnitude more sensitive with a much greater dynamic range compared to the colorimetric ELISA. R&D Systems also sells a High Sensitivity version of their human TNF-α Colorimetric Sandwich ELISA. Their stated sensitivity (0.18 pg/mL) is similar to that of the luminescent ELISA, but it unfortunately has a very limited dynamic range (0.5 to 32 pg/mL). In contrast, the luminescent ELISA has the advantages of both high sensitivity and wide dynamic range.


SUMMARY
Molecular Devices LMax microplate luminometer provides an easy and sensitive means of measuring chemiluminescent ELISAs in a microplate format. This particular work was done with a commercial kit for human TNF-α; however, excellent results would also be expected with other optimized HRP-based ELISA assays using luminol/peroxide substrate. In addition, SoftMax Pro for LMax provides a powerful and convenient instrument control, data analysis and presentation package.


REFERENCES
1. www.rndsystems.com


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