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Our first-to-market chimera with mouse IgG2c backbone, this functional antibody specifically depletes neutrophils in vivo for up to 72h.
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ELISAs can be run on a number of sample types. Here we provide ways to prepare different sample formats.
Prepare the extraction buffer as recommended by the manufacturer.
Keep all buffers, reagents and equipment on ice at 4 °C.
Isolate the cells and suspend them in the extraction buffer.
Some adherent cells may require enzymatic or mechanical detachment.
Lyse the cells.
Spin down the suspension in a centrifuge to pellet insoluble contents.
Determine the concentration of protein in your extract using a Bradford or BCA assay.
Aliquot supernatant into several tubes.
Minimize freeze/thaw cycles.
Now your samples are prepared, the antigen needs to be attached to the wells of the microplate so that it can be recognized by detection antibodies.
Dilute your samples and standards in coating buffer to the desired concentration.
If you don’t know what dilutions to use, consult the literature and optimize by performing serial dilutions down the plate.
Adsorb samples to the wells.
Keep the wells covered to prevent them from drying out.
Wash each well three times with wash buffer.
Perform background blocking.
Keep the plate covered to prevent wells from drying out.
Wash each well three times with wash buffer.
Now that antigens are adsorbed onto the plate, you're ready to add antibodies to detect them. This can be done through indirect or direct methods.
Dilute the primary and secondary antibodies in blocking buffer.
If no suggested dilutions are found, you may need to perform serial dilutions to find the antibody concentration that works best.
Add primary antibody to the wells.
Keep the plate covered to prevent wells from drying out.
Wash each well three times with wash buffer.
Add secondary antibody to the wells
It’s important to keep the plate covered to prevent wells from drying out.
Wash each well three times with wash buffer.
ELISA typically uses antibodies conjugated with enzymes such as horseradish peroxidase (HRP). These react with a substrate in oxidizing conditions to produce either a colored or fluorescent product. The signal generated is proportional to the concentration of the protein of interest. This signal can be measured at several time points throughout the substrate incubation (kinetic mode), or at a defined point in time after the reaction is complete (end-point mode).
Horseradish peroxidase (HRP) and alkaline phosphatase (AP) are the two most widely used enzymes for detection in ELISA.
Consider that some biological materials have high levels of endogenous enzyme activity (such as high AP activity in alveolar cells or high peroxidase activity in red blood cells) that may result in a nonspecific signal. If necessary, perform an additional blocking treatment with levamisol (for AP) or 0.3% H2O2 in methanol (for peroxidase).
Set up your plate reader to observe the color change or fluorescence at the expected wavelength.
Bring all reagents to room temperature.
All wells need to reach an equal temperature to minimize edge effects.
Add the enzyme substrate solution to each well.
HRP substrates | The substrate for HRP is hydrogen peroxide. The cleavage of hydrogen peroxide is coupled to the oxidation of a hydrogen donor, which changes color during the reaction | |
TMB (3,3’,5,5’-tetramethylbenzidine) | Add TMB solution to each well, incubate for 15–30 min, add an equivalent volume of stopping solution (2 M H2SO4), and measure the optical density at 450 nm | |
OPD (o-phenylenediamine dihydrochloride) | Keep and store the substrate in the dark, as it is light-sensitive. Add the OPD solution and incubate for 10-30 min. The reaction can be stopped using sulfuric acid 2.5-3.0 M H2SO4. For stopped reactions, measure the reaction using a plate reader at 450 nm; for reactions that are not stopped, measure the reaction at 490 nm. | |
ABTS (2,2’-azino-di-[3-ethyl-benzothiazoline-6 sulfonic acid] diammonium salt) | ABTS is an HRP substrate. Add ABTS solution to each well and incubate for 20 min on a rocker. If color generation occurs rapidly, then reduce the concentration of the conjugated antibody in the assay. Do not dilute this solution. This highly sensitive substrate produces a green product. The end product is measured at 405 nm. Always handle carefully and wear gloves as some enzyme substrates are considered hazardous (potential carcinogens) | |
AP substrate | P-Nitrophenyl-phosphate (pNPP) is the most widely used substrate for most applications. Measure the yellow color of nitrophenol at 405 nm after a 15–30 min incubation at room temperature and stop the reaction by adding an equivalent volume of 0.75 M NaOH. |
Add the stop solution to each well
Read the signal development in the plate reader.
The reaction time for end-point reading may need some optimization.
Here we have provided step-by-step best-practice guidelines to analyzing data for quantitative ELISAs.
Tip: If the plate reader does not come with software, you can use a general statistical analysis tool such as GraphPad Prism
An in-depth guide on the analysis of data from ELISA can be found here.
Plot the standard curve from your standard controls using curve-plotting software.
A 4 or 5 parameter log plot (4/5-PL) provides the best fit for most ELISA standard curves. However, you can try different models to see which one gives the best fit to your data.
Determine the curve fit and regression coefficient.
If R2 < 0.99, your data cannot be reliably used for quantitative analysis. See our troubleshooting guide for possible causes.
Perform a spike recovery test.
If using an ELISA kit, expected recovery ranges will be listed on the datasheet. At concentrations where the recovery falls outside the acceptable range, the assay cannot be reliably used for quantitative analysis of the given sample type.
Calculate the coefficient of variation.
If using an ELISA kit, expected CV values will be listed on the datasheet. If the CV is > 15, there is too much variation in your data for quantitative analysis. See our troubleshooting tips for possible causes of high CV.
Calculate the sample concentration.