How to use
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).
1. Add Capture Antibody diluted in coating buffer.
2. Incubate and then wash.
3. Block non-specific protein interactions by incubating with blocking buffer and
then wash.
4. Add standards, controls and samples to the plate in duplicate.
5. Incubate the plate and then wash.
6. Add Detection Antibody. Incubate and then wash.
7. Add HRP Streptavidin solution for detection.
8. Incubate and then wash.
9. Detect by adding TMB substrate.
10. Add Stop solution.
11. Measure plate on a microplate reader at 450 nm and 620 nm.
2
How to use
matched antibody
pair kits for
sandwich ELISA
1. Coat the plate (we recommend Nunc Maxisorb 96-well plate (ab210903)).
2. 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.
3. 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.
4. Plate can be used immediately after coating or up to 2 weeks after if kept
sealed at 4oC.
5. 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.
6. 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.
7. Repeat wash step as described in 5.
8. Prepare serially diluted standards immediately prior to use. Prepare enough of
the standard solutions for duplicate measurements of each concentration.
9. 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.
10. The reconstituted protein standard should be aliquoted for single use and
stored -80oC.
11. Label eight tubes, Standards 1–8.
12. 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.
eg Stock Solution Concentration = 100 pg/µL (as example only – see lot
specific certificate of analysis for the concentration of the protein standard)
3
Required Standard #1 Concentation = 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.
13. 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.
14. 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.)
15. Repeat wash step as described as in step 5.
16. Add 50 µL of 1x HRP-Streptavidin solution to each well. Seal the plate and
incubate for 1 hour at room temperature on a plate shaker set to 400 rpm.
17. Repeat wash step as described in step 5.
18. 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.
19. Add 100 µL of Stop Solution to each well. Shake plate on a plate shaker for 1
minute to mix.
20. Measure the plate at 450 nm.
21. Analyze the data:
a. Calculate the average absorbance value for the blank control (zero)
standards. Subtract the average blank control standard absorbance
value from all other absorbance values.
b. 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.
4
c. 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.
d. 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.
Detecting biomarkers of disease requires techniques that are sensitive and highthroughput. Find out which approach is most suitable for your experiment.
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.
Browse all products related to Matched Antibody Pair Kits and Reagents.
5