ELISA Standard Curve Calculator

Figran

Paste standard and sample ODs to fit the curve and get sample concentrations.

  • 4PL, 5PL and linear fits
  • Dilution factors and QC flags
  • Free SVG and 300 DPI PNG
Input

Standards

Samples

X axis
Standard points
Color
Standard curveStandard curve01210¹10²10³10⁴Concentration (pg/mL)Absorbance (OD 450 nm)StandardsFitSamplesOutside standard rangey = d + (a − d) / (1 + (x/c)ᵇ) · R² = 0.9987
Samples (pg/mL)
  • S1< 31.3 pg/mL (below range)
  • S246.8
  • S378.0
  • S4160.4
  • S5249.6
  • S6374.2
  • S7697.3
  • S81191.4

and 2 more · see Results

Outside range: 2

Samples

NameMean ODOD %CVInterpolated (pg/mL)DilutionConcentration (pg/mL)Flag
S10.1922.3—2< 31.3 pg/mL (below range)Below standard range
S20.3535.223.4246.8
S30.5381.439.0278.0
S40.92424.680.22160.4CV > 20%
S51.2103.8124.82249.6
S61.4736.1187.12374.2
S71.8212.9348.62697.3
S82.0354.4595.721191.4
S92.1311.8840.821681.6
S102.1947.2—2> 2000.0 pg/mL (above range)Above standard range

Standards

Nominal (pg/mL)Mean ODOD %CVBack-calculated (pg/mL)Recovery %
0.00.0602.90.02—
15.60.2536.215.63100.0
31.30.4484.131.2299.9
62.50.7761.762.54100.1
125.01.2115.3124.9199.9
250.01.6482.2250.41100.2
500.01.9733.4498.4299.7
1000.02.1701.31002.70100.3

Curve

ParameterEstimateSE
Equationy = d + (a − d) / (1 + (x/c)ᵇ)
a−8.998×10⁻⁵0.02038
b1.1490.04883
c125.15.756
d2.3030.04314
R²0.9987—
Sy.x0.03095—
n16—
df12—

ELISA standard curve examples

Explore common assays and see how the model and results change.

Sandwich ELISA with diluted serum samples01210¹10²10³10⁴Concentration (pg/mL)Absorbance (OD 450 nm)StandardsFitSamplesOutside standard rangey = d + (a − d) / (1 + (x/c)ᵇ) · R² = 0.9987

Sandwich ELISA with diluted serum samples

Estimate cytokine concentrations in duplicate serum samples.

8 standards · 10 samples · duplicates

DNase ELISA from the R datasets00.511.510⁻²10⁻¹10⁰10¹10²Concentration (ng/mL)Absorbance (OD 450 nm)StandardsFitSamplesy = d + (a − d) / (1 + (x/c)ᵇ) · R² = 0.9991

DNase ELISA from the R datasets

Fit a real DNase assay and compare measurements from a second run.

Source: R datasets (DNase; Davidian & Giltinan 1995)

Competitive ELISA standard curve01210¹10²10³Concentration (pg/mL)Absorbance (OD 450 nm)StandardsFitSamplesOutside standard rangey = d + (a − d) / (1 + (x/c)ᵇ) · R² = 0.9998

Competitive ELISA standard curve

Analyze a descending response curve with diluted samples.

6 standards + zero · 8 samples · duplicates

BCA protein assay standard curve00.510500100015002000Concentration (µg/mL)Absorbance (OD 562 nm)StandardsFitSamplesOutside standard rangey = 3.306×10⁻⁵ + 0.0008003x − 9.979×10⁻⁸x² · R² = 0.9998

BCA protein assay standard curve

Fit a quadratic curve to BSA standards and diluted lysates.

Source: Pierce BCA kit; example ODs

Bradford protein assay standard curve00.20.40.601020Concentration (µg/mL)Absorbance (OD 595 nm)StandardsFitSamplesOutside standard rangey = 1.201×10⁻⁶ + 0.02503x · R² = 0.9998

Bradford protein assay standard curve

Use a linear fit for microassay protein measurements.

Source: Bio-Rad; example ODs

Full 96-well plate with 570 nm correction00.511.5210¹10²10³Concentration (pg/mL)Absorbance (OD 450 nm)StandardsFitSamplesy = d + (a − d) / (1 + (x/c)ᵇ) · R² = 1.0000

Full 96-well plate with 570 nm correction

Analyze a complete plate after wavelength correction.

96 wells · 7 standards + zero · 40 samples

How to analyze ELISA data

Fit an ELISA standard curve, check replicate precision, and export dilution-corrected concentrations.

  1. 1Enter standards

    Paste concentrations and replicate absorbances or enter a plate grid.

  2. 2Enter samples

    Add sample ODs and a dilution factor for each sample.

  3. 3Choose a model

    Select the model and weighting recommended for your assay.

  4. 4Download graph

    Review the QC flags, then download the graph and results.

Choosing a standard curve model

Use the assay instructions and the shape of your standards to select a fit.

A 4PL standard curve describes a symmetric sigmoidal response. A 5PL curve adds an asymmetry parameter g. Findlay & Dillard (2007)

4PL model

y=d+a−d1+(xc)b

x: concentration; y: response; a/d: plateaus; b: slope; c: midpoint.

5PL model

increasingy=a+d−a(1+(cx)b)g
decreasingy=d+a−d(1+(xc)b)g

x: concentration; y: response; a/d: plateaus; b: slope; c: location; g: asymmetry.

Inverse calculation

increasingx=c[(d−ay−a)1g−1]1b
decreasingx=c[(a−dy−d)1g−1]1b

y: measured response; x: concentration; a, b, c, d and g: fitted parameters (g = 1 for 4PL).

Sandwich ELISA with diluted serum samples01210¹10²10³10⁴Concentration (pg/mL)Absorbance (OD 450 nm)StandardsFitSamplesOutside standard rangey = d + (a − d) / (1 + (x/c)ᵇ) · R² = 0.99871234
  1. a: response at zero concentration
  2. b: curve steepness near the middle
  3. c: curve location on the concentration axis
  4. d: response at high concentration

Linear and quadratic standard curves

A linear calibration curve is useful over a limited response range; a quadratic fit can capture curvature. Check for a turning point before interpreting inverse values.

BCA and Bradford protein assays

BCA and Bradford assays can use protein standards to estimate concentrations. Select the fit and range recommended by the kit.

Reading the QC flags

Review recovery, replicate CV and the standard range before interpreting sample concentrations.

QC checkWhat to review
RecoveryCompare back-calculated and nominal concentrations.
Replicate CVInspect large variability between replicate wells.
Standard rangeRetest samples outside the calibration range when appropriate.

ELISA calculator FAQ

How do I calculate concentration from an ELISA standard curve?

Average replicate ODs, subtract the zero standard if required, fit the standards, and interpolate the sample OD. Multiply the interpolated concentration by the sample dilution factor.

Which curve fit should I use for ELISA: 4PL, 5PL or linear?

Follow the kit instructions, which often recommend 4PL. A 5PL curve can describe asymmetry; a linear fit is generally suitable only over a limited range.

How do I apply the dilution factor to ELISA results?

Multiply the interpolated concentration by the dilution factor. The dilution factor is the final volume divided by the original sample volume.

Should I subtract the blank or zero standard?

Subtract the mean zero-standard OD when the assay instructions call for it. A constant shift does not change the inverse concentrations of an unconstrained 4PL or linear fit, but it affects a log–log fit.

What should I do with samples outside the standard range?

Retest high samples at a suitable dilution and report low samples according to the assay instructions. This calculator does not calculate a limit of detection.

What %CV is acceptable for ELISA duplicates?

A CV of 20% or less is a common criterion, although individual assays may set a tighter limit. CV is the standard deviation divided by the mean, multiplied by 100.

Is R² enough to judge an ELISA standard curve?

No. Also inspect standard recovery, replicate precision and residual patterns across the concentration range.

Can I make an ELISA standard curve in Excel, and will it match GraphPad Prism?

Excel can fit a 4PL model with Solver. Fits in GraphPad Prism should agree when the equation, data and weighting match.

Should I weight the curve fit (1/Y or 1/Y²)?

Weighting can help when response variability changes across the range. Use the weighting specified by the assay validation; the default here is unweighted.

Ready to analyze your results?

Turn your absorbance readings into a standard curve and sample concentrations.

Calculate your ELISA results