Sandwich ELISA kit protocol

This Sandwich-ELISA process follows a general protocol in which standards or samples are added to a microplate, which has been precoated with detection antibody. After the incubation period a biotinylated detection antibody specific to the protein target of interest is added, and Avidin-Horseradish Peroxidase (HRP) conjugate is added successively to each well for further incubation. Any free components are then aspirated and washed away. A substrate solution is then added to each well, and only those wells which contain the bound protein-antibody-HRP conjugate will appear blue in colour. This enzyme-substrate reaction is terminated with the addition of stop solution, which turns the colour from blue to yellow, and from which the optical density (OD) is measured spectrophotometrically, at a wavelength of 450 nm ±2 nm. The OD value is directly proportional to the concentration of the target protein, which can be calculated by taking the concentration of the protein of interest in the samples and comparing the OD of the samples to 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

The types of samples which can be used for a sandwich ELISA kit will vary depending on the precise kit being used, and we recommend checking this on the protocol provided.

Cell Culture Supernatant
Centrifuge the sample at 1000xg for 10 min and then immediately move to the kit protocol, or alternatively, aliquot and store the samples between -20°C to -70°C (or 2-8°C if testing within 24 hours). Avoid freeze-thaw cycles. If the cell culture supernatant samples require larger dilutions, perform an intermediate dilution with culture media and the final dilution with the standard/sample diluent.

Serum
Using a serum separator tube, allow the samples to clot for 2 hours at room temperature, or overnight at 4°C, before centrifuging for 20 minutes at approximately 1000xg. Assay the freshly prepared serum immediately, or store samples in aliquot at -20°C or -80°C for later use. Avoid repeated freeze-thaw cycles.

Plasma
Collect the plasma using EDTA or heparin as an anticoagulant. Centrifuge the samples for 15 minutes at 1000xg and keep at 2-8°C until ready to use, which should be within 30 minutes of collection. Remove the plasma and assay immediately, or store the samples at -20°C or -80°C for later use. Avoid repeated freeze-thaw cycles.

Cell Lysates
Cells need to be lysed before assaying according to the following directions. Adherent cells should be washed by cold PBS gently, and then detached with trypsin. Collect the cells following centrifugation at 1000xg for 5 minutes (the cell suspension can be collected by centrifugation directly). Wash 3 times in cold PBS before resuspending the cells in fresh lysis buffer. If it is necessary, the cells may be ultrasonicated until the solution is clarified. A centrifugation step of 1500xg for 10 minutes at 2-8°C should help to remove cellular debris. Assay the cells immediately or aliquot and store below -20°C.

Tissue homogenates
The preparation of tissue homogenates will vary depending upon tissue type. Tissues should be rinsed thoroughly in ice-cold PBS to remove excess blood and weighed before homogenization. Mince the tissues to small pieces and homogenise them in fresh lysis buffer (different lysis buffer needs to be chosen based on subcellular location of the target protein) (E.g., 1mL lysis buffer in 200mg tissue sample) with a glass homogenizer on ice. The resulting suspension should be sonicated with an ultrasonic cell disrupter until the solution is clarified. Centrifuge the homogenates for 5 minutes at 10000×g and collect the supernatant. Assay immediately or aliquot and store below -20°C.

Other biological fluids
Centrifuge the sample for 20 minutes at 1000xg. Collect the supernatant and assay immediately or store the sample at -20°C or -80°C for later use. Avoid repeated freeze-thaw cycles. Avoid haemolytic and hyperlipidaemia samples for serum and plasma. Dilute the samples at the appropriate multiple - for this we recommend carrying out a pre-test to determine the dilution factor.

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.

Then, also in duplicates, add each of the samples to be tested to the columns to the right of the controls. Once this is carried out cover the microplate with the adhesive strip provided and incubate for 90 minutes at 37°C. Again, ensure to pipette delicately into the base of the well to prevent overspill, and avoid touching the walls.

Following incubation remove all the liquid from each well, but DO NOT wash.

Add the biotinylated detection antibody to all the wells and cover with new adhesive strips before incubating for a further 1 hour, at 37°C.

Then aspirate the solution from all of the wells. Add wash buffer, and aspirate after 60-120 seconds, repeating the process 2 times for a total of 3 washes.

Add Streptavidin-HRP working solution to each of the wells. Cover with a new adhesive strip and incubate for 30

minutes at 37°C. We recommend that you ensure the microplate reader is set up during this incubation stage.

Add wash buffer to each well, and aspirate after 60-120 seconds. Repeat the process 4 more times, to total 5 washes.

Add substrate to each well and incubate for 15-20 minutes at 37°C. Protect the plate from light.

Add stop solution to each well. Determine the optical density of each well within 5 minutes with a microplate reader set to 450 nm. If wavelength correction is applicable, set the reader to 570 nm or 630 nm. If wavelength correction is not available, subtract the 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.

Upon completion of the experiment ensure you return unused reagents to their appropriate storage locations.