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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:
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:
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.
Below are the 212 most popular ELISA kits.
|TNF Alpha elisa||IL6 ELISA||Cortisol ELISA||VEGF elisa|
|BDNF elisa||IFN Gamma elisa||Adiponectin elisa||IL-1 Beta elisa|
|IL10 ELISA||IL-8 elisa||Leptin elisa||IL2 ELISA|
|IL-12 elisa||Granzyme B elisa||MPO elisa||ADA elisa|
|APP elisa||TGF Beta 1 elisa||MAG elisa||IL4 ELISA|
|MMP-9 elisa||PLAT elisa||Cystatin C elisa||CCL2 ELISA|
|IL-17 elisa||PD-L1 elisa||APOE elisa||NGF elisa|
|CXCL10 elisa||PAI-1 elisa||S100B elisa||Galectin-3 elisa|
|EGF elisa||Fibronectin elisa||GM-CSF elisa||MMP-3 elisa|
|Cortisol elisa Kit||Insulin elisa Kit||IL33 ELISA||GDF-15 elisa|
|Resistin elisa||FGF21 elisa||AFP elisa||Angiopoietin-2 elisa|
|Clusterin elisa||P53 elisa||IDS elisa||Ferritin elisa Kit|
|MMP-1 elisa||OPN elisa||Endothelin 1 elisa||PCSK9 elisa|
|HGF elisa||G-CSF elisa||VWF ELISA||CXCL1 elisa|
|PD-1 elisa||Caspase 3 elisa||TIMP1 ELISA||P-Selectin elisa|
|Tissue Factor elisa||TRAIL elisa||Fetuin A elisa||Chemerin elisa|
|IL-15 elisa||COMP elisa||IL-22 elisa||ANG ELISA|
|CEA elisa||Periostin elisa||Galectin-9 elisa||MMP2 ELISA|
|TEK ELISA||Cathepsin B elisa||CXCL5 elisa||CXCL9 elisa|
|VEGFC ELISA||CCL17 elisa||CXCL13 elisa||IL-27 elisa|
|PEDF elisa||ADAMTS13 elisa||APOA1 elisa||Eotaxin elisa|
|M-CSF elisa||PLGF elisa||RANK elisa||Thrombomodulin elisa|
|MIA elisa||HE4 elisa||IL7 ELISA||PDGF-AB elisa|
|C-MET elisa||IL-1RA elisa||Renin elisa||FABP2 elisa|
|BMP2 ELISA||IL17C ELISA||LCN2 ELISA||MMP12 ELISA|
|Fractalkine elisa||CCL19 elisa||CCL21 elisa||Angiopoietin-1 elisa|
|Growth Hormone elisa||CCL18 elisa||THBS1 elisa||TSLP elisa|
|SHBG elisa||Hemopexin elisa||TIM-3 elisa||MMP7 ELISA|
|FAS elisa||TREM2 elisa||Myoglobin elisa Kit||FGF2 elisa|
|GDNF elisa||PTX3 elisa||TGF-Beta 2 elisa||Mesothelin elisa|
|Transthyretin elisa||DKK1 ELISA||FASL elisa||CCL4 elisa|
|MMP-8 elisa||OPG elisa||Rantes elisa||ACE elisa|
|CD40 elisa||CXCL11 elisa||Prostate Specific Antigen elisa Kit||FGF7 elisa|
|Midkine elisa||Uromodulin elisa||PROC elisa||IL11 ELISA|
|IL31 ELISA||IL-3 elisa||CCL8 elisa||MIF elisa|
|RBP4 elisa||TLR2 elisa||FABP4 elisa||B2M elisa|
|IL1A ELISA||LTA ELISA||ERBB2 ELISA||FAP ELISA|
|SERPINA1 ELISA||COL1A1 ELISA||IGFBP2 elisa||CCL22 elisa|
|CD163 elisa||MICA elisa||Progranulin elisa||FGF19 elisa|
|FOLR1 elisa||Syndecan-1 elisa||CEACAM1 elisa||MUC1 ELISA|
|Amphiregulin elisa||IL-5 elisa||Decorin elisa||S100A12 elisa|
|Tenascin-C elisa||IL-32 elisa||TFPI ELISA||IL18BP ELISA|
|AXL elisa||Aggrecan elisa||ALCAM elisa||ICAM1 ELISA|
|CCL20 ELISA||TIMP2 ELISA||TIMP4 ELISA||PROK1 ELISA|
|DPP4 ELISA||ANXA1 ELISA|
|Haptoglobin elisa||FGF23 elisa||CRP elisa||EPO elisa|
|SAA elisa||Elastase elisa||IL-13 elisa||S100A8 elisa|
|LBP elisa||PF4 ELISA||Stat3 ELISA||IL-23 elisa|
|CD14 elisa||CTLA4 elisa||LIF elisa||IL-21 elisa|
|CD30 elisa||S100A9 elisa||TREM-1 elisa||TrkB elisa|
|SAP elisa||Thrombopoietin elisa||SCF elisa||MFGE8 elisa|
|GAS6 elisa||CXCL2 ELISA||Sclerostin elisa||Mpl ELISA|
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