Competitive ELISA protocol

This competitive ELISA process follows a general protocol in which samples or standards along with a biotinylated antibody are applied to a microplate which has been precoated with the target antigen. The coated antigen and antigen contained within the added sample then compete for sites on the biotinylated detection antibody. Excess conjugate and unbound sample or standard are then washed away, and Avidin-Horseradish Peroxidase (HRP) conjugate are added to each micro plate well and incubated. The addition of a substrate solution to the wells initiates an enzyme-substrate reaction between the HRP-bound antibody and TMB substrate. Following the addition of stop solution, which halts the reaction and turns the colour turns from blue to yellow, the Optical Density (OD) can be measured spectrophotometrically, and the concentration of the target antigen can be calculated by comparing the OD of the samples to the values of the standard curve.

Materials

For this assay the following components are generally included with each kit:

ELISA pre-coated plate
Protein standard
Detection antibody
Concentrated HRP-conjugate

Sample diluent
Biotinylated detection antibody diluent
HRP diluent
Wash buffer
Ready to use substrate solution
Stop solution

The following components are required, but not typically included with a kit:

Microplate reader
Pipettes and tips
Microplate washer
Micro-oscillator
Deionized or double distilled water graduated cylinder
Microvials for serial dilution

Sample collection and storage

Serum
Using a serum separator tube, allow samples to clot for 2 hours at room temperature or overnight at 4°C before centrifugation for 20 minutes at approximately 1000xg. Newly prepared serum should be used immediately or be stored in aliquots at -20°C or -80°C for use at a later time. Avoid repeated freeze-thaw cycles.

Plasma
Plasma can be collected using EDTA or heparin as an anticoagulant. Centrifuge the samples for 15 minutes at 1000xg and keep at 2-8°C. Use within 30 minutes of collection. The plasma should be removed and assayed immediately. Alternatively store the samples in aliquots at -20°C or -80°C for later use. Avoid repeated freeze-thaw cycles.

Urine
Urine samples should be collected with a sterile container. Particulates should be removed with a 15 minute centrifugation step at 1000xg at 2-8°C. Collect the supernatant and process.

Reagent preparation

Bring all reagents to room temperature before use. If crystals have formed in the concentrate bring the reagent to room temperature and mix gently until the crystals have completely dissolved. We recommend making preparations to test out each sample in duplicates.

Add standard-sample diluent to the lyophilised standard to reach the desired kit concentration, and reset for a minimum of 15 minutes with gentle agitation prior to making dilutions. Prepare tubes containing equal measure of standard-sample diluent and then carry out a serial dilution covering the kit detection range. Any remaining standard solution can be aliquoted and stored at -20°C to -70°C.

Washing

During the assay washing steps make sure to aspirate each well and then apply wash buffer, repeating the process 2 times for a total of 3 washes. The wash step can be carried out manually using a squirt bottle, multichannel pipette, or auto washer. Completely removing all of liquid at each step is essential for precision performance. After the last wash, remove any remaining wash buffer by aspirating or decanting, and invert the plate and blot it against clean paper towels.

Assay procedure

Remove any excess microplate strips from the plate frame and return them to the foil pouch containing the desiccant pack, and reseal for later use.

Add standard-sample diluent to the blank well.

In duplicates, add each concentration of standard solution to the wells of the 2 left hand well columns in either ascending or descending dilution. Ensure to pipette delicately into the base of the well to prevent overspill, and avoid touching the walls.

Immediately add biotinylated detection antibody working solution to each of the wells, and cover with an adhesive strip. Again, ensure to pipette delicately into the base of the well to prevent overspill, and avoid touching the walls.

Incubate the microplate for 45 minutes at 37°C.

Following incubation aspirate all of the liquid from each well and wash 3 times.

Add HRP conjugate working solution to each of the wells, cover with new adhesive strip provided and incubate for 30 min at 37°C.

Aspirate the solution from all of the wells and apply a further 5 washes. (We recommend that you ensure the microplate reader is set up during this incubation stage.)

Add ready to use substrate reagent to each of the wells and incubate for 15-20 minutes at 37°C. Keep the microplate protected from light during this step.

Add stop solution to each of the wells.

Determine the optical density of each well within 5 minutes, using a Microplate reader set to 450 nm. If wavelength correction is available, set to 570 nm or 630 nm. If wavelength correction is not available, subtract readings at 570 nm or 630 nm from the readings at 450 nm. This subtraction will correct for optical imperfections in the plate. Readings made directly at 450 nm without correction may be higher and less accurate.