( ated with antibody against TNF-α....)High Sensitivity, Wide Dynamic Range, Horseradish Peroxidase (HRP) ELISA Using the LMax Microplate Luminometer (MaxLine Application Note #41)

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
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