Detailed Diagram of a Sandwich ELISA protocol

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This weeks topic is a sandwich ELISA protocol to use with our Leinco Technologies ELISA development kits or for general purposes. Leinco Technologies validates many of their products using ELISA (Enzyme-linked immunosorbent assay) methods.  This protocol provides an initial set of conditions; however, further optimization may be required on an individual basis.

STEP 1:

Coating the Plate with Capture Antibody

1.  Dilute unlabeled capture antibody to a final concentration of 1-10 μg/mL using PBS or carbonate/bicarbonate buffer (pH7.4).

2.  Transfer 50-100 µL per well to ELISA microplate (Nunc Maxisorp Prod.#442404).

3.  Seal the plate and incubate overnight at 4°C.

4.  Bring the plate to room temperature and flick off the capture antibody solution.

5.  Wash the plate three times with PBS/Tween by using a squirt bottle, multi-channel pipettor or automatic plate washer.

6.  Tap the plate on paper towels after the last wash to remove residual wash solution.

Step 2:

Blocking the Plate

7.  Make a 1x blocking solution consisting of BSA in PBS azide and add 200 ul/well

8.  Seal the plate and incubate at 37°C for 2 hr or at 4°C overnight.

Step 3:

Add Antigen Coating Sample

Dilute samples and standards to desired concentrations using Blocking buffer.

9.  Add appropriate amount of diluted samples to each well.

10.  Seal plate and incubate for 2-4 hrs at RT or overnight at 4°C.

11.  Wash the plate three times with PBS/Tween and blot on paper towels after last wash.

Step 4:

Adding Detection Antibody

12. Dilute the biotin-labeled detection antibody to 0.25-2 µg/mL in Blocking buffer and add 100 µL to each well.

13.  Seal the plate and incubate for 1-2 hr at RT.

14.  Wash the plate three times with PBS/Tween and blot on paper towels after last wash.

Step 5:

Adding UltraAvidin-Horseradish Peroxidase (Av-HRP)

15.  Dilute the Av-HRP conjugate to its optimal concentration in Blocking buffer (typically 1/500-1/2000).

16.  Add 100 μL per well, seal the plate and incubate for 30 min at RT.

17.  Wash the plate five times with PBS/Tween and blot on paper towels after last wash.

Step 6:

Detection

18.  Add 100 µL of TMB Microwell Substrate (Leinco Prod. #T118) solution to each well (substrate solution should be at RT prior to use).

19.  Once a soluble blue reaction product develops, the plate can be read at 370 nm or in the range of 620 nm to 650 nm. The absorbance values of the sample should be monitored so that a linear curve can be plotted. For increased sensitivity, add 50 μl of a stop solution such as 1.0 N HCl or 1.0 N sulfuric acid to create a soluble bright yellow reaction product. After stopping the enzymatic reaction the plate should be read at 450 nm. Estimated incubation times for substrate range from 20 to 30 minutes.

Solutions You Need:

Note: Do not use sodium azide in any buffers or solutions, as sodium azide inactivates the horseradish-peroxidase enzyme.  All solutions should be at ambient temperature prior to use.

PBS: Dilute 10xPBS to 1xPBS in sterile water.

Wash Buffer: 0.05% Tween-20 in PBS

Block Buffer: 1% BSA in PBS *

Diluent: 0.05% Tween-20, 0.1% BSA in PBS *

* Sterile filter and store at 4°C for up to 1 week.

Thanks and see you again next week