Prostaglandin E2 FPIA Kit - Red Validation

July 2004

Standard Curve Validation

The antibody and tracer (i.e., rhodamine labeled PGE₂) were titered to optimize dynamic range and limit of detection. Dynamic range is the difference in mP (ΔmP) between the highest and lowest standard. Based on previously published assays, this difference should be ≥100. The assay described here has a ΔmP of 200. Limit of detection is the lowest concentration of analyte reliably quantified (i.e., CV<20%). Currently, the limit of detection is approximately 100 pg/mL.

Standard curves were then constructed by serially diluting PGE₂ in FP buffer (PBS + 0.1 mg/mL BSA) between 200 ng/mL and 91 pg/mL. To determine the %CV, mP values were determined at each concentration from a total of twenty-four curves. For each curve, the mP data was transformed using a logit function, y = ln(x/(1-x)), and plotted versus concentration. The data was then fit using a log-regression algorithm in Microsoft Excel. The concentration of each standard was recalculated using the regression parameters. All values corresponding to a particular standard were averaged and the coefficient-of-variance determined. Figure 1 is a typical standard curve overlaid with the %CV curve.

Figure 1. PGE₂ Standard Curve with %CV of Each Point

Z′-Factor

The Z′-Factor is a statistic designed to reflect both the assay signal dynamic range and the variation associated with the signal measurements. Prior to the determination of this coefficient, the quality of assays were determined based on their signal-to-noise (S/N) or signal-to-background (S/B) ratios. Both of these parameters, however, suffer because they do not take into account the variability in the sample and background measurements and the signal dynamic range. The Z′-Factor accomplishes this, and, because it is dimensionless, can be used to compare similar assays. The equation and the meaning of possible values are summarized in table 1.

To determine the Z′-Factor for the PGE₂-FPIA, four 96 well plates were run. Each plate had 48 negative control wells and 48 positive control wells. The negative control wells contained only FP buffer, and the positive control wells contained FP buffer spiked with 5000 pg/mL PGE₂. The mP values were determined for all wells in every plate, and %B/B₀ was calculated from this data. The Z′-Factor had a value of 0.69, indicating a robust assay. Figure 2 is a plot of the data used to determine the Z′-Factor.

Figure 2. Z′-Factor Data

PGE₂-FPIA in Culture Media

In order to test the effects of cell culture medium on PGE₂ measurements, standard curves were prepared in the presence of various types of culture media. When fetal bovine serum (FBS) and Phenol Red (PR) are excluded from DMEM, the FPIA can be performed directly in the medium without any dilution (Figure 3). Inclusion of 10% FBS and PR in DMEM requires a minimum dilution of 1:20 to produce a standard curve similar to the one obtained in FPIA buffer alone (Figure 4). Further characterization of the effects of FBS and PR in the assay showed that FBS, but not PR, contributes the majority of the interference observed in the FPIA (data not shown).

Figure 3. Comparison of PGE₂ FPIA standard curves in DMEM not containing FBS or Phenol Red

Figure 4. PGE₂ Standard curves in various dilutions of DMEM containing 10% FBS and Phenol Red

PGE₂ FPIA in Plasma

Experiments were performed to characterize the interference associated with measurements of PGE₂ in plasma samples. Direct measurement of PGE₂ in plasma resulted in significant interference thereby making the assay unusable in this matrix (data not shown). To circumvent this problem, plasma proteins were precipitated with 4 volumes of ethanol. After removal of the ethanol from the supernatant, the plasma solution was adjusted to the original volume with water. The data in Figure 5 shows PGE₂ standard curves performed in the presence of the plasma extract. A minimum matrix dilution of 1:5 is required for reasonable measurement of PGE₂ in this sample.

Figure 5. PGE₂ standard curves in an ethanol-extracted plasma sample.

Discussion

The above outlines the validation of a rhodamine-based PGE₂-FPIA. The variance of the standard curve is below similar assays (e.g., EIA) available for the quantitation of PGE₂. The robustness of this assay for high-throughput screening was established by determining the Z′-Factor (0.69). Finally, the utility of this assay is applicable to measurements of PGE2 in cell culture media and plasma samples.