ELISA | GENERAL PROTOCOL FOR SANDWICH ELISA 2021/03/10

GENERAL PROTOCOL FOR SANDWICH ELISA

1. Prepare Reagents
2. Coat Capture Antibody on plate
3. Block
4. Prepare and Load Standards and Samples 
5. Load Biotin Labeled Detector Antibody
6. Load HRP Conjugated Streptavidin
7. Add TMB Substrate
8. Stop Reaction
9. Read Result at 450 nm

Reagents and consumables

1. Biotinylation Kit or HRP Antibody Labeling Kit (for labeling Detector Antibody)
2. Optional: Streptavidin (peroxidase/HRP conjugated)
3. 96-well ELISA plates (for example: Nunc™ MaxiSorp™)
4. Coating buffer: 35 mM NaHCO₃, 15 mM Na₂CO₃, pH 9.6 without NaOH
5. Blocking Buffer for WB, ELISA and IHC (ACE#A1010) or 2% protease-free BSA in wash buffer, pH 7.2 -7.4, 0.2 μm filtered
6. 10X Wash Buffer (10X PBST ACE#C1023).  Dilute in water to 1X: 0.05% Tween® 20 in 1X PBS
7. TMB substrate (ACE#C1013)
8. Stop solution (ACE#C1068): 2N Sulfuric Acid
9. 10X Phosphate Buffered Saline (PBS) (ACE#C1015). Dilute in water to 1X.

Detail Steps:

1. Add 50 µL of 0.5 µg/mL Capture antibody (diluted in Coating Buffer) to each well of a 96-well of a high bind microplate.
2. Seal the plate with a plate seal. Incubate the plate either overnight at 4°C or for 2 hours at room temperature on a plate rocker or shaker.
3. Aspirate each well and wash with at least 300 μl wash buffer, repeating the process two times for a total of three washes. Complete removal of liquid at each step is essential for good performance. After the last wash, remove any remaining wash buffer by inverting the plate and blotting it against clean paper towels.
4. Reduce non-specific binding by adding 300 μL of 1X Blocking Buffer (ACE#A1009) to each well, seal the plate and incubate either overnight at 4°C or for 2 hours at room temperature.
5. Repeat the wash procedure in step 3.
6. Prepare serially diluted standards immediately prior to use. Prepare enough of the standard solutions for duplicate measurements of each concentration. Label eight tubes, Standards #1– 8. To prepare Standard #1, dilute the Protein Standard in 1X Blocking Buffer as the highest concentration. A seven-point standard curve using 2-fold serial dilutions in 1X Blocking Buffer is recommended. Standard #8 is the Blank control (buffer only) and contains no standard protein.
7. Dilute the experimental sample with 1X Blocking Buffer if needed. 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.
8. Add the Standard working solution to the first two columns: Each concentration of the solution is added in duplicate, to one well each, side by side (100 μL for each well). Add the samples to the other wells (100 μL for each well). Cover the plate with the sealer provided in the kit. Incubate for 90 min at 37°C. Note: solutions should be added to the bottom of the micro ELISA plate well, avoid touching the inside wall and causing foaming as much as possible.
9. Repeat the wash procedure in step 3.
10. Dilute Biotinylated Detector Antibody to the working concentration of ~0.5 μg/mL in 1X Blocking Buffer (ACE#A1009) or other appropriate diluent. Add 100 μL of Biotinylated Detection Ab working solution to each well. Cover with the Plate sealer. Gently mix up. Incubate for 1 hour at 37°C.
11. Repeat wash step as described as in step 3.
12. Dilute Streptavidin (peroxidase/HRP conjugated) to the working concentration of ~0.2 μg/mL in 1X Blocking Buffer (ACE#A1009) or other appropriate diluent. Add 100 μL of Streptavidin (peroxidase/HRP conjugated) to each well. Cover with the Plate sealer. Incubate for 30 min at 37°C.
13. Repeat wash step as described as in step 3.
14. Add 90 μL of TMB Substrate (ACE#C1013) to each well. Cover with a new plate sealer. Incubate for about 15 min at 37°C. Protect the plate from light. Note: the reaction time can be shortened or extended according to the actual color change, but not more than 30 min.
15. Add 50 μL of Stop Solution (ACE#C1068) to each well. Note: Adding the stop solution should be done in the same order as the substrate solution.
16. Determine the optical density (OD value) of each well at once with a micro-plate reader set to 450 nm.

Calculation of results:

Average the duplicate readings for each standard and sample and subtract the average optical density of Blank.

Create a standard curve by reducing the data using computer software capable of generating a four parameter logistic (4-PL) curve-fit. Average the duplicate readings for each standard and samples, then subtract the average zero standard optical density. Plot a four-parameter logistic curve on log-log graph paper, with standard concentration on the x-axis and OD values on the y-axis.

If the samples have been diluted, the concentration calculated from the standard curve must be multiplied by the dilution factor. If the OD of the sample surpasses the upper limit of the standard curve, you should re-test it with an appropriate dilution. The actual concentration is the calculated concentration multiplied by the dilution factor.

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