The optimization of ELISA for multiple analytes requires two processes during assay development.
1. Screening for capture and detection antibody pairs with the best performance
2. Optimization of the ELISA experimental conditions to allow simultaneous development of the readout

1. Screening for capture and detection antibody pairs with the best performance

We have screened every possible combination of capture and detection antibody commercially available for each of our cytokine and chemokine antigens. We systematically tested all combinations of capture and detection antibodies using the following criteria:

1. Low Background: The lowest antigen concentration produces a signal less than twice the background.
2. High Sensitivity: The highest antigen concentration produces the maximum signal (OD₄₅₀ ~ 2.0).
3. Good Linearity: The calibration curve is linear with a correlation factor (R) > 0.975.

Figure 1 shows three examples of antibody combinations screened during the ELISArray optimization process. Antibody combination 1 failed due to its high background. Antibody combination 3 failed due to its poor linearity. Antibody combination 2 passed, because it met all three criteria.

	Figure 1: The antigen standard curves from the best performing capture and detection antibody concentrations for three combinations are shown. Only the combination of antibodies B and C passes all three rigorous quality control criteria (Antibody Combo 2). The other two combinations produce too high a background (Antibody Combo 1) or a response that is not linear enough (Antibody Combo 1).

2. Optimization of the ELISA experimental conditions to allow simultaneous development of the readout

Once the optimal capture and detection antibody combination has been identified, we optimized their concentrations under the same experimental ELISA conditions to achieve the same development or incubation time (Figure 2).

	Figure 2: Similar standard curves are achievable for all optimized cytokine and chemokine assays in the Multi-Analyte ELISArray Kits. Shown are twelve different cytokine antigen standard curves generated using the respective pre-optimized capture and detection antibody combinations and concentrations. All standard curves are virtually super-imposable indicating that all twelve assays provide similar linear and sensitive responses under the same standardized conditions and development or incubation time.

The same assay conditions, particularly development time, are essential for the analysis of multiple cytokines or chemokines from the same samples at the same time. The convenience of analyzing multiple analytes allows researchers to quickly survey their cytokines and chemokines of interest. The absolute quantification of each cytokine or chemokine can then by achieved by using the Single Analyte ELISArray Kits.