How To Perfect Your ELISA Standards

At Boster, one common question we get from researchers is, “How do I prepare the ELISA standard?” We’re glad you asked because proper construction of the standard curve is the very first step for every ELISA experiment. The standard curve can help confirm that the quality of the kit and the operation procedures are acceptable for further steps.

For ELISA assays, in order to determine a quantity of something, you need to compare your sample results to a set of standards with known quantities. The standards in your assay should be tested at a range of concentrations that yields data from essentially undetectable to maximum signal. This set of results for the standards allows you to “fit” a statistical model and generate a predicted standard curve. You can think of the standard curve as the ideal data for your assay. Once the standard curve is generated, it is relatively easy to see where on the curve your sample lies and interpolate a value.

So how do you prepare this standard curve? The standard curve is prepared through serial dilutions of the standard analyte with known concentrations that should span the standard curve range. This range is expected to be close to the target protein concentrations in the unknown samples. Here is an example of a serial dilution series from a stock solution to generate a standard curve of 3.9 –2000 pg/ml:


Here are some guidelines for making proper dilutions:

  • Make two- or three-fold dilutions.
  • Do not make dilutions that require pipetting a small amount of volume (2 ul or less).
  • Avoid making large, single step dilutions. If the dilution is < 1:1000, use two steps.
  • Prepare enough of each dilution to run in duplicate or triplicate for accuracy.
  • Always include a background, negative control sample containing sample diluent.
  • Make dilutions fresh just before use. Standard solutions are best used within 2 hours.
  • Make all dilutions in tubes that do not absorb protein (polypropylene or glass; i.e. do not make dilutions in microplate).
  • Always use a fresh pipette tip after each dilution.

Once the intensity of each well has been measured on the ELISA plate reader, you can calculate the average absorbance values for each duplicate/triplicate sample. Then generate a standard curve by graphing the mean absorbance for each sample (x-axis) vs. the standard concentration (y-axis). Typically, a standard curve will have a sigmoidal shape in which the higher concentrations of standard dilutions will reach a plateau in absorbance.


In the example graph above, it is the relatively long linear region of the curve that makes the ELISA results accurate and reproducible. Find the portion of the curve that is linear and draw the best-fit trendline for the data. Many ELISA plate readers have built-in programs for generating and analyzing standard curves. Alternatively, you can use Microsoft Excel, or other graphing software to generate the equation of the line (i.e. y=mx + b; m= slope of the line and b= y intercept) and the R2 value. The R2 value is an indication of how closely the data fit the trendline. An R2 value of 1 would be perfect. Use the equation to calculate the concentration of each sample by using the average of the duplicate/triplicate samples for x in the equation. If the concentration of the sample exceeds the highest point of the curve or does not lie within the linear range of the curve, then dilute the sample prior to measurement. If a diluted sample is used, remember to multiply by the dilution factor to obtain the final value.

It is important to understand that a standard curve run at different times will not have the same OD values for each dilution. This is due to operator differences and slight differences in pipetting, incubation times and temperature. So in order to yield reliable data, a new standard curve should be prepared for every experiment and for every new plate along with your unknown samples.

Sometimes after your standard curve is generated, things still don’t seem right. If you’re thinking, “The results of my standards do not look correct. What could be the problem?” Let us offer some advice. Typically, we recommend double checking your protocol for the plate washing step. It should be examined from three aspects:

  1. Did you make the washing buffer correctly?
  2. How long did you let the buffer stay in wells?
  3. What was the volume of the washing buffer added to each well?

The washing steps are necessary to reduce background signal related to unbound, conjugated antibody and thereby increase the assay’s signal-to-noise ratio. Washing between steps ensures that only specific (high-affinity) binding events are maintained for generating signal at the final step. Insufficient washing can result in variation and high background, and thus poor results. Remember to always follow the protocols provided by the manufacturer. Boster provides ELISA protocols both online and on our PicoKine ELISA kit’s datasheets.

If you have any further questions, don’t hesitate to contact [email protected] for more information.

Popular ELISA kits

Below are the 212 most popular ELISA kits.


TNF Alpha elisaIL6 ELISACortisol ELISAVEGF elisa
BDNF elisaIFN Gamma elisaAdiponectin elisaIL-1 Beta elisa
IL10 ELISAIL-8 elisaLeptin elisaIL2 ELISA
IL-12 elisaGranzyme B elisaMPO elisaADA elisa
APP elisaTGF Beta 1 elisaMAG elisaIL4 ELISA
MMP-9 elisaPLAT elisaCystatin C elisaCCL2 ELISA
IL-17 elisaPD-L1 elisaAPOE elisaNGF elisa
CXCL10 elisaPAI-1 elisaS100B elisaGalectin-3 elisa
EGF elisaFibronectin elisaGM-CSF elisaMMP-3 elisa
Cortisol elisa KitInsulin elisa KitIL33 ELISAGDF-15 elisa
Resistin elisaFGF21 elisaAFP elisaAngiopoietin-2 elisa
Clusterin elisaP53 elisaIDS elisaFerritin elisa Kit
MMP-1 elisaOPN elisaEndothelin 1 elisaPCSK9 elisa
HGF elisaG-CSF elisaVWF ELISACXCL1 elisa
PD-1 elisaCaspase 3 elisaTIMP1 ELISAP-Selectin elisa
Tissue Factor elisaTRAIL elisaFetuin A elisaChemerin elisa
IL-15 elisaCOMP elisaIL-22 elisaANG ELISA
CEA elisaPeriostin elisaGalectin-9 elisaMMP2 ELISA
TEK ELISACathepsin B elisaCXCL5 elisaCXCL9 elisa
VEGFC ELISACCL17 elisaCXCL13 elisaIL-27 elisa
PEDF elisaADAMTS13 elisaAPOA1 elisaEotaxin elisa
M-CSF elisaPLGF elisaRANK elisaThrombomodulin elisa
MIA elisaHE4 elisaIL7 ELISAPDGF-AB elisa
C-MET elisaIL-1RA elisaRenin elisaFABP2 elisa
Fractalkine elisaCCL19 elisaCCL21 elisaAngiopoietin-1 elisa
Growth Hormone elisaCCL18 elisaTHBS1 elisaTSLP elisa
SHBG elisaHemopexin elisaTIM-3 elisaMMP7 ELISA
FAS elisaTREM2 elisaMyoglobin elisa KitFGF2 elisa
GDNF elisaPTX3 elisaTGF-Beta 2 elisaMesothelin elisa
Transthyretin elisaDKK1 ELISAFASL elisaCCL4 elisa
MMP-8 elisaOPG elisaRantes elisaACE elisa
CD40 elisaCXCL11 elisaProstate Specific Antigen elisa KitFGF7 elisa
Midkine elisaUromodulin elisaPROC elisaIL11 ELISA
IL31 ELISAIL-3 elisaCCL8 elisaMIF elisa
RBP4 elisaTLR2 elisaFABP4 elisaB2M elisa
CD163 elisaMICA elisaProgranulin elisaFGF19 elisa
FOLR1 elisaSyndecan-1 elisaCEACAM1 elisaMUC1 ELISA
Amphiregulin elisaIL-5 elisaDecorin elisaS100A12 elisa
Tenascin-C elisaIL-32 elisaTFPI ELISAIL18BP ELISA
AXL elisaAggrecan elisaALCAM elisaICAM1 ELISA

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