ELISA Standard Curve

ELISA Standard Curve

If you are using a Competitive Inhibition kit, please view our Competitive Inhibition ELISA Standard Curve Guide.

Enzyme linked immunosorbent assay (ELISA) can be used for the detection of target antigen. There are four types of ELISA: direct ELISA, indirect ELISA, sandwich ELISA and competitive inhibition ELISA. The most widely used ELISA type is the Sandwich ELISA, where the analyte is sandwiched between two antibodies, the capture antibody and the detection antibody. Results can collected by measuring the absorbance, where a high absorbance is indicative of a high concentration, and vice versa.

The standard curve is used to determine the concentration of the samples. In the example below, the standard absorbance values for abx155737, Rat IL6 ELISA Kit, are shown as a reference. As shown in Figure 1, the standard was diluted from 100 pg/ml to 1.56 pg/ml. A negative control is also run. This contains no standard (0 pg/ml). To accurately determine the true absorbance of any well, the background must be subtracted. This will give the corrected absorbance values.

WellConcentration of standard (pg/ml)AbsorbanceCorrected
A00.1150
B1.560.2960.181
C3.120.3720.257
D6.250.4590.344
E12.50.6840.569
F251.141.025
G501.9171.802
H1003.1483.033

Figure 1. Data of standard concentrations, absorbance values and corrected absorbance values

Next, the O.D. value of the standard (X-axis) can be plotted against the known concentration of the standard (Y-axis), as an XY scatter plot. In the example, these values are highlighted in green in Figure 1. As shown in Figure 2, the X-axis is the Optical Density, and the Y-axis is the Standard Concentration in pg/ml.

Data of standard concentrations, absorbance values and corrected absorbance values

Figure 2. Plot of 7-point XY scatter

A polynomial order 2 trendline can be added to the graph. In Excel, this can be achieved by selecting any point in the XY scatter graph, right-clicking and selecting “Add Trendline”. The trendline formatting should then be amended to polynomial order 2 (the default trendline is linear) as shown in Figure 3 below. In the formatting section, further select “Display Equation on chart” and “Display R-squared value on chart”. Excel will then display the corresponding trendline formula and R-squared value on the graph. This is shown in Figure 4 below.

Addition and formatting of the trendline

Figure 3. Addition and formatting of the trendline

Schematic diagram of a typical standard curve with trendline, equation and R-squared

Figure 4. Schematic diagram of a typical standard curve with trendline, equation and R-squared

In general, a good standard curve should have the following characteristics:

  1. R-squared value is greater than 0.95, and as close to 1 as possible.
  2. The OD of the blank well should be lower than 0.25.
  3. The maximum absorbance value should be higher than 0.8.

Troubleshooting

ProblemPossible SourceCorrection
Poor backgroundContaminated negative control wellChange and use new pipette tips, containers and sealers
Poor R-squaredImproper standard curve preparationEnsure accurate dilution procedure
Improper standard curveEnsure accurate dilution of the standard
Inaccurate PipettingCheck and Calibrate pipettes
Reused pipette tips, containers and sealersChange and use new pipette tips, containers and sealers
Low OD ValuesImproper standard curve preparationEnsure accurate operation of the dilution
Improper standard curve dilutionEnsure accurate dilution of the dilution
Inaccurate PipettingCheck and Calibrate pipettes
Incorrect incubation timesEnsure sufficient incubation times
Incorrect incubation temperatureReagents balanced to room temperature
Conjugate or substrate reagent failureMix conjugate and substrate; color should develop immediately
No stop solution addedFollow the assay protocol in the kit manual