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

Evelyn McGown, Ph.D. and Michael Su, M.S.
Molecular Devices Corporation, 8/00


INTRODUCTION
Immunoassays have traditionally employed colorimetric indicators. As such, multiple dilutions of samples were necessary to ensure that they fell within the dynamic range of the assay. Recently, chemiluminescent immunoassays, with greatly improved sensitivity and dynamic range, have become commercially available1. The QuantiGlo TNF-α Immunoassay is a chemiluminescent ELISA designed to measure human TNF-α in cell culture supernate, serum and plasma. The assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for TNF-α is pre-coated onto a microplate. Standards and samples are pipetted into the wells, and any TNF-α present is bound by the immobilized antibody. After washing away any unbound substances, horseradish peroxidase (HRP)-linked polyclonal antibody specific for TNF-α is added to the wells. Unbound antibody-enzyme reagent is removed by washing. An enhanced luminol/peroxide substrate solution is added to the wells to produce light. The reaction involves the oxidation of luminol to an excited 3aminophthalate anion (not shown) which decays to the product shown and releases light (Figure 1). Over a wide dynamic range, the amount of light produced is a function of the amount of TNF-α bound in the initial reaction and is easily measured in the LMax microplate luminometer. A brief description of the procedure and results is presented below.


MATERIALS
1. LMax microplate luminometer with SoftMax Pro for LMax (Molecular Devices Corp.)

2. QuantiGlo Human TNF-α Immunoassay kit (R&D Systems1, Cat #QTA50). The kit contains all reagents and standards needed for the assay, including the polystyrene microplate (12 strips of 8 wells@) pre-treated with antibody against TNF-α.


PREPARATION OF REAGENTS AND STANDARDS
1. The kit instructions were followed for preparation of the reagents and substrate solution.

2. The TNF-α stock standard was reconstituted with 0.5 mL deionized water, gently vortexed and allowed to stand for 15 min at ambient temperature. The working standards were prepared by making a serial 1-to10 dilution of the stock standard in calibrator diluent (QDP5). The concentrations of the working standards ranged from 7000 pg/mL to 0.07 pg/mL.


ASSAY PROCEDURE
1. After first putting 50 L assay diluent (QD1-27) into wells of the pre-treated microplate (supplied with the kit), working standards of TNF-α and the blank (QDP5) were pipetted in triplicate (200 L each).

2. The kit instructions were followed for the subsequent aspiration, washing, incubation with conjugate, aspiration and washing steps.

3. After the final step, the addition of substrate solution, the wells were covered with an adhesive strip and incubated with shaking at ambient temperature for 30 minutes.

4. The adhesive cover was removed and the microplate was placed into the LMax. To ascertain the optimal integration time, the plate was read at 0.1, 1, 5 and 10 seconds per well.


DYNAMIC RANGE AND SENSITIVITY OF CHEMILUMINESCENT IMMUNOASSAY
Figure 2 shows the TNF-α standard curves obtained with 1, 5 and 10-second integration times. (The 0.1 sec plot is not shown because it gave significantly poorer sensitivity.) The dynamic range spans approximately 5 logs. As predicted, the longer the integration time, the higher the RLUs. (Recall that the detector is a photon counter which reports the cumulative number of photons detected during the specified integration time.


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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