QuantiGlo IL-6 Immunoassay

Background

The quantitative determination of human interleukin 6 (IL-6) in serum, plasma and cell culture supernatant is now possible using a chemiluminescent sandwich immunoassay developed by R&D Systems and the MLX™, a Microtiter luminometer, by DYNEX TECHNOLOGIES.

The QuantiGlo kit for quantifying IL-6 has an anti-IL-6 monoclonal antibody to capture IL-6 and an enzyme-linked anti-IL-6 polyclonal antibody to detect the captured analyte. An enhanced luminol peroxide substrate emits light in the presence of the enzyme (horseradish peroxidase), and the light produced is proportional to the amount of IL-6 captured. This new luminescent technique for assaying cytokines utilizing luminescence offers substantial advantages over previously used methodologies, including increased sensitivity, smaller sample volume, and a broad range (0.3-3000 pg /mL).

The MLX is used to measure the intensity of the light, to plot a standard curve, and to analyze the results. Since the substrate emits light over a long time period, the test can be read any time between 20 and 40 minutes after addition of the substrate.

Use of a QuantiGlo Human IL-6 Immunoassay Kit with an MLX Luminometer to measure light output is described here. Kits for detecting other cytokines are also available from R&D Systems.

Materials

• QuantiGlo Human IL-6 Immunoassay Kit Catalog Number Q6000
• MLX Luminometer Catalog Number ML1000 DYNEX TECHNOLOGIES, Inc.

Methods

All reagents necessary to perform the assays are supplied with the kits. Instructions in the package insert were followed. Light was quantified in the MLX 20-25 minutes after substrate addition and all data reduction was performed using the MLX Revelation™ software.

1. A Glow - Endpoint assay was defined with the following Reader Settings:
   Start Mode: After 1 minute
   Shake and Temperature: Off
   Reader Options:
   • Auto Gain
   • Sum all Readings
   • Read Time/Well 1.00 second
2. A template was defined with the following well definitions:
   Wells containing the zero standard (Calibrator Diluent) were defined as Blanks.
   Wells containing the other standards were defined as S1 - S5.
   Wells containing Controls were defined under "User Definable Types" with the designations on the vial (LL, L, M, H)
   Wells containing samples should be defined as Tests.
3. In the blank mode window, select Average. All Blank (zero standard) wells will be averaged and the average subtracted from all other wells.
4. In the curve fit window, the IL-6 concentrations for Standards 1 - 5 were entered and plotted versus the Relative Light Window using a log/log Cubic Spline-fit.
5. The Test Report included the Data Matrix and the Curve-Fit graph results.

Results

A set of dilutions was prepared for the IL-6 standard curve. The mean MLX relative light unit (RLU) of each standard was calculated and the mean value of the zero standard was subtracted from each (Table 1).

The RLU values of the standards were plotted against the concentration of each standard. A cubic spline-fit was used in order to determine IL-6 concentrations of the kit controls and test samples. The dose-response curve for the standards is shown in figure 1.

Replicates of the four Control specimens were placed in various wells at the top and bottom of the plate. IL-6 concentrations were determined from the standard curve (Table 2). The IL-6 concentration of each Control specimen was within 10% of the midpoint value given in the package insert.

References

1. QuantiGlo™ Human IL-6 Immunoassay Package Insert.