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Chapter 39 : Multiplex Cytokine Assays
This chapter explores both traditional enzyme-linked immunosorbent assay (ELISA) and the newer multiplexed assays. For each of the methods, the author examines the technology, instrumentation, and data analysis. The sequential ELISA still has several pitfalls. First, the sample volume necessary is still greater than that used in most multiplex assays. Second, the amount of time required to measure each one of the cytokines is not reduced in the sequential ELISA, so there is no cost savings with regards to time. Third, if there is potential cross-reactivity of the antibodies, then measurement of the first cytokine will result in an overestimate of the amount of that cytokine present and measurement of the next cytokine in the sequence will result in an underestimate of the amount of that cytokine present. A recent paper described a novel method for rapidly detecting cytokines by capillary electrophoresis. Bead array assays are rapidly becoming the rage in cytokine measurement. There are several commercial vendors marketing the bead array assays The major limitation is obtaining antibody with sufficient specificity in order to prevent cross-reactivity. Finally, the volume of sample required to measure multiple cytokines is extremely small. Multiplex cytokine assays for measuring cytokines are rapidly becoming standard across the world. A clear indicator of the enthusiasm for these assays may be found in the large number of companies which are presently producing and manufacturing multiplex cytokine kits.
Direct ELISA. In the direct ELISA, the analyte is first bound to the bottom of the microtiter well. In this example, the analyte is IL-6. Unbound IL-6 is washed away, and excess protein binding sites are blocked in order to reduce background. In the next step, biotin-labeled antibody (biotin-Ab) directed against IL-6 is added; the small circle represents the biotin moiety directly attached to the antibody. After washing, streptavidin (SA) conjugated to horseradish peroxidase (HRP) is added and a final wash is performed. Following addition of a colorimetric substrate, the color develops. The intensity of the color development is directly proportional to the amount of IL-6 in the first step.
Indirect or sandwich ELISA. This ELISA shares many features with the assay described in the legend to Fig. 1 . The major difference is in the first step, in which an antibody (Ab) directed against the analyte is bound to the bottom of the microtiter well. This antibody is termed the capture or coating antibody. Following addition of the analyte, the biotin-labeled detection antibody (biotin-Ab) is applied, followed by horseradish peroxidase (HRP)-conjugated streptavidin (SA-HRP). Color development proceeds, and the intensity of the color is directly related to the amount of the analyte. This is termed a sandwich ELISA because the analyte is sandwiched between two antibodies.
Sequential ELISA. A sample containing a mixture of cytokines is applied to an IL-6 ELISA. IL-6 binds to the anti-IL-6 antibody attached to the bottom of the well. When the sample is removed, cytokines which are not IL-6, such as tumor necrosis factor (TNF), may be used in a subsequent ELISA. Ab, antibody; B, biotin moiety; biotin-Ab, biotin-labeled antibody; HRP, horseradish peroxidase; SA-HRP, horseradish peroxidase-conjugated streptavidin.
Increase in signal intensity with smaller spot size. On the left, each black dot represents the analyte bound to an antibody, which spreads across the entire well. On the right, the same number of antibodies are bound to the analyte, but they now occupy a smaller space, with a resulting increase in signal intensity.
Example of a microarray. Panel A shows six individual nitrocellulose pads arranged on a glass slide. Each pad has been spotted in an 8-by-12 format. The black area between the pads is the place where a silicon gasket was adhered to the surface to allow each of the wells to function individually, similar to the way in which an individual well in a 96-well plate is independent from its neighbors. The antibodies have been spotted in an identical manner onto each of the nitrocellulose pads. Panel B is an enlarged picture of one of the individual pads and highlights the detail of the spots. The antibodies have been spotted in quadruplicate on the pad in a vertical fashion. Each individual spot has a diameter of 150 μl, and the distance between the spot is 300 μm. The total volume delivered to each spot was 350 to 367 pl. In the far-right column of spots, those in the top eight positions are extremely bright and the lower four have virtually no signal. This line of eight plus four spots may be used for alignment of the protein chip. The intensity of the individual spots may be quantified and used to determine the cytokine concentration in the sample. This image shows excellent reproducibility of the quadruple spots.
Specificity of the microarray. For this microarray, 18 different cytokines were tested. A cocktail containing recombinant cytokines was prepared, but IL-12 was not added to the cocktail. There is a strong signal from all the other cytokines, but no fluorescence was observed on the array for IL-12. This demonstrates the specificity for the array.
Mathematical modeling of the standard curve. Data from a microarray standard curve were used to generate a mathematical model for calculations for unknown samples. In panel A, the number of relative light units (RLU) and the concentration of the cytokine (IL-8) were plotted on a linear-linear scale. The correlation coefficient (r2) was not very precise. In panel B, the concentration of the IL-8 was plotted on a log scale and the number of RLU was plotted on a linear scale. This resulted in even worse correlation. In panel C, both the number of RLU and the IL-8 concentration were plotted on a log scale and a very good linear regression could be fitted. However, the optimal modeling of the curve was obtained when the number of RLU was plotted on a linear scale, the IL-8 concentration was plotted on a log scale, and a fourth-order polynomial regression line was used. The results for all four panels were obtained using the same data from the IL-8 standard curve, but similar results are observed with most other cytokines.
Schematic representation of the principle of a flow cytometric bead-based assay. The capture antibody specific for the analyte, in this example IL-6, is attached to a bead. The IL-6 binds to this antibody, which is then followed by a detection antibody which is biotinylated. Streptavidin conjugated to phycoerythrin is then added, and the entire complex is analyzed with the flow cytometer.
Example of the readout from a bead-based flow cytometry assay. In this example, the individual beads have different concentrations of two fluorescent dyes. The groups of beads are detected on the basis of the fluorescence intensities. The individual dots represent individual beads, and the darker-colored dots are doublets. The clear areas around collections of beads indicate those areas used for analysis. In this example, 22 individual cytokines have been detected. The intensity of the fluorescence of each individual bead is captured in a third fluorescent channel and compared to the standard curve in order to determine the concentration of the cytokine.
Example of a standard curve generated from the bead-based flow cytometry assay. In this example, a standard curve using recombinant mouse IL-1β is displayed. MFI, mean fluorescence intensity.
Cost analysis for cytokine measurements a
Sliding-scale cost of measuring cytokines a
Sample volumes required for performing assays a