A detailed protocol on how to use our matched antibody pair kits for sandwich ELISA.
Matched antibody pair kits are a great choice for scientists who need flexibility to scale up their ELISA-based assays but still require capture and detector antibody pairs that perform reliably in biological samples. Matched antibody pair kits can be used for a broad range of assays or for a standard sandwich ELISA. Here we provide a detailed protocol on how to use our matched antibody pair kits for sandwich ELISA.
Titrated capture and biotinylated detector antibody pair
Calibrated protein standard
Enough reagents for 10 x 96-well plates using in a sandwich ELISA
Protocol to use reagents in a sandwich ELISA
Additional buffers and plates are required for the assay. An accessory pack can be purchased which includes all the reagents required to perform 10 x 96-well plates sandwich ELISA (ab210905).
Coat the plate (we recommend NuncMaxisorb 96-well plate (ab210903)).
Dilute Capture Antibody from 1 mg/mL to the suggested working concentration of 2 µg/mL in the Coating Buffer. Add 50 µL per well.
eg, for one plate: 10 µL of 1mg/mL Capture Antibody 4,990 µL of Coating Buffer (ab210899)
Note: For best results, optimization of the concentration of capture antibody in the coating buffer may be required.
Seal the plate with a plate seal. Place plate on a plate rocker or shaker. Incubate plate overnight at 4°C or 2 hours at room temperature.
Plate can be used immediately after coating or up to 2 weeks after if kept sealed at 4oC.
Wash plate 3 times with 350 µL of 1 x Wash Buffer (ab206977). Remove liquid completely from last wash by tapping the plate vigorously against a pad of absorbent towels.
Reduce non-specific binding of proteins in the sample or the Detector Antibody by incubating plates with 300 μL of 1 x Blocking Buffer (ab210904) to per well. Cover plate with plate seal or plate cover. Incubate plate either overnight at 4°C or for 2 hours at room temperature.
Repeat wash step as described in 5.
Prepare serially diluted standards immediately prior to use. Prepare enough of the standard solutions for duplicate measurements of each concentration.
Reconstitute the standard protein by adding 100 µL of ddH2O water. Gently mix the solution at room temperature for 10 minutes to ensure that the protein has completely dissolved. See the certificate of analysis for the concentration of the protein standard.
The reconstituted protein standard should be aliquoted for single use and stored -80oC.
Label eight tubes, Standards 1–8.
Use the Stock Standard to prepare a seven-point standard curve using 2-fold dilution series. Standard #8 contains no protein and is the Blank control. Each dilution should contain enough volume of standard to provide duplicate readings.
egStock Solution Concentration = 100 pg/µL (as example only – see lot specific certificate of analysis for the concentration of the protein standard) Required Standard #1Concentation = 4,000 pg/mL(as an example only – see the linear range of the assay in 12 Typical Data)
4,000 pg/mL x 300 µL total volume required = 1,200 pg Stock Standard required. 1,200 pg/100 pg/µL = 12 µL of Stock Solution required.
To prepare Standard #1, combine 12 µL Stock Solution with 288 µL assay buffer.
Prepare the sample by dilute with blocking buffer. Dilute the sample so that the resulting concentration is within the dynamic range of the assay. Multiple sample dilutions using 1:2 dilution series is advised if the concentration of the target protein is unknown.
Dilute Detector Antibody from stock concentration of 0.25 mg/mL to the suggested working concertation of 0.5 µg/mL in Blocking Buffer (ab210904) or other appropriate diluent. Add 50 µL per well. e.g. for one plate: 10 µL Detector Antibody and 4,990 µL of Blocking Buffer
Note: For best results, the concentration of detector antibody in the working solution may require optimization. (See ELISA troubleshooting tips for more information.)
Repeat wash step as described as in step 5.
Add 50 µL of 1xHRP-Streptavidin solution to each well. Seal the plate and incubate for 1 hour at room temperature on a plate shaker set to 400 rpm.
Repeat wash step as described in step 5.
Add 100 µL of TMB Substrate to each well and incubate for up to 20 minutes in the dark on a plate shaker set to 400 rpm.
Add 100 µL of Stop Solution to each well. Shake plate on a plate shaker for 1 minute to mix.
Measure the plate at 450 nm.
Analyze the data:
Calculate the average absorbance value for the blank control (zero) standards. Subtract the average blank control standard absorbance value from all other absorbance values.
Create a standard curve by plotting the average blank control subtracted absorbance value for each standard concentration (y‑axis) against the target protein concentration (x‑axis) of the standard. Use graphing software to draw the best smooth curve through these points to construct the standard curve.
Note: Most microplate reader software or graphing software will plot these values and fit a curve to the data. A four parameter curve fit (4PL) is often the best choice; however, other algorithms (eg linear, semi-log, log/log, 5 parameter logistic) can also be tested to determine if it provides a better curve fit to the standard values.
Determine the concentration of the target protein in the sample by interpolating the blank control subtracted absorbance values against the standard curve. Multiply the resulting value by the appropriate sample dilution factor, if used, to obtain the concentration of target protein in the sample.
Samples generating absorbance values greater than that of the highest standard should be further diluted and re-analyzed. Similarly, samples which measure at an absorbance values less than that of the lowest standard should be retested in a less dilute form.
Matched antibody pair kits and reagents
Our Matched Antibody Pair Kits provide a titrated antibody pair composed of an unlabeled capture antibody and a biotinylated detector antibody as well as a calibrated protein standard for quantification.