ELISA Protocols

These protocols are for general guidance only. For specific protocols, please refer to the product description page of the corresponding ELISA kit.

Sandwich assay procedure

1. An appropriate amount of trapping antibody (typically 100 μL) is added to each well of the 96-well plate at a concentration that follows product instructions or optimization results (typically 1-10 μg/mL). Incubate at 4℃ overnight or at room temperature for 2 hours.
2. Remove the trapping antibody solution from the well and flush the plate 3 times with the wash buffer. 200 μL sealing buffer was added to each well and incubated at room temperature for 1-2 hours to block the unbound binding sites.
3. Remove the sealing solution and rinse the plate 3 times with the wash buffer. Add 100μL of test sample or standard product per well (diluted to the required concentration in advance) and incubate at room temperature for 1-2 hours or at 4℃ overnight.
4. Remove the sample and standard solution and rinse the board 3-5 times with the wash buffer. Add 100 μL of enzyme-bound detection antibody per well (diluted at optimal concentration) and incubate at room temperature for 1-2 hours. 

5. Rinse the plate 3-5 times with a wash buffer to thoroughly remove unbound detection antibodies. Add 100 μL enzyme substrate solution to each well and react away from light (generally incubate at room temperature for 10-30 minutes, the specific time according to the substrate reaction rate). Observe color changes (usually the darker the color, the higher the antigen concentration).

6. Add 50-100 μL stopping solution per well to stop the enzyme reaction. After termination of the reaction, the color will change from blue to yellow (TMB substrate for example).

7. The light absorption value (OD value) of each hole is read using a microplate reader at 450 nm wavelength. The standard curve is drawn from the standard product, and the concentration of the target antigen is calculated according to the absorption value of the sample.

Chemicals used in sandwich assay

classification	Name	CAS
Phosphate buffer (PBS)	Sodium chloride	7647-14-5
	Potassium chloride	7447-40-7
	Sodium dihydrogen phosphate	7558-80-7
	Disodium hydrogen phosphate	7558-79-4
Washing buffer (PBS-T)	Tween-20	9005-64-5
Blocker	BSA(Bovine serum albumin)	9048-46-8
Enzyme	Horseradish peroxidase	9003-99-0
Substrate	3,3′,5,5′-Tetramethylbenzidine	54827-17-7
	3,3′,5,5′-Tetramethylbenzidine dihydrochloride	64285-73-0
	ABTS Chromophore Diammonium Salt	30931-67-0
	MTT	298-93-1
	CSPD	142456-88-0
Stop solution	1M H2SO4	7664-93-9

Enzyme immunoassay (EIA) procedure

1. The 96-well microtitration plate was removed, 100 µl was added to each well to capture the antibody, and the antibody was diluted to the appropriate concentration using the coated buffer.

Incubate at 4℃ overnight or at room temperature for 2 hours.

• Wash the well three times with a wash buffer (PBST) of 350 µl each time. Shock well to ensure no residual liquid.
• Add 200 µl sealing buffer to each well. Incubate at room temperature for 1 hour to reduce nonspecific binding. Wash the microtitration plate 3 times to remove excess sealer.
• Add 100 µl sample or standard to each well and dilute the sample to the appropriate concentration. Incubate at room temperature for 2 hours.
• Wash the board 5 times with a wash buffer (PBST) of 350 µl each time. 
• Add 100 µl to each well to detect the antibody and dilute the antibody to the appropriate concentration using a blocking buffer. Incubate at room temperature for 1 hour.
• Discard the solution. Repeat the wash procedure as in step 5.
• Add 100 µl substrate solution (TMB color developer) to each well. Incubate away from light at room temperature for 15-30 minutes until color develops.
• Add 50 µl stop solution (2N sulfuric acid) to each well. Incubate for a few minutes until all reactions are completely stopped.
• The optical density values of each well are read using an enzyme-labeler at a wavelength of 450 nm (or at a wavelength suitable for the substrate used). Calculate the concentration of antigen in the sample according to the standard curve.

Phosphorylation assay procedure

1. Dilute the capture antibody in coating buffer (usually carbonate-bicarbonate buffer, pH 9.6). Add 100 µL of the capture antibody solution to each well of the ELISA plate. Incubate the 96-well plate overnight at 4°C or for 2 hours at room temperature to allow the antibody to adsorb to the plate.
2. Wash the wells three times with washing buffer to remove unbound antibodies. Block non-specific binding sites by adding 200 µL of blocking buffer to each well. Incubate the plate for 1 hour at room temperature.
3. Remove the blocking buffer and wash the plate three times with washing buffer. Add 100 µL of cell lysates or purified protein samples to each well. Incubate the plate for 2 hours at room temperature or overnight at 4°C, allowing the target proteins to bind to the capture antibody.
4. Wash the wells three times with washing buffer to remove unbound proteins. Add 100 µL of the specific anti-phosphorylation antibody to each well. Incubate the plate for 1 hour at room temperature.
5. Wash the plate thoroughly with washing buffer to remove unbound primary antibodies. Add 100 µL of HRP-conjugated secondary antibody to each well. Incubate the plate for 1 hour at room temperature in the dark.
6. Wash the plate again, ensuring thorough removal of any unbound secondary antibody. Add 100 µL of TMB substrate solution to each well. Incubate the plate at room temperature for 15-30 minutes, monitoring the development of the blue color indicating enzyme activity.
7. Add 50 µL of stop solution to each well to halt the enzyme-substrate reaction. The addition of the acid will turn the color from blue to yellow. 
8. Measure the absorbance at 450 nm using a plate reader within 30 minutes of adding the stop solution. Perform data analysis comparing the absorbance values of sample wells to that of standard or control wells.

Cell-based assay procedure

1. Preparation work

Reagents and consumables:

The liquid nitrogen preserved cells must be resuscitated in advance and cultured to a suitable state.

Gather all necessary reagents, including media, fixatives, blockers, primary and secondary antibodies, substrate solutions, and washing buffers. 

96-well plates, cell types suitable for culture conditions.

Cell culture:

Appropriate amount of specific cells were taken and cultured in culture bottles or petri dishes according to conventional cell culture methods.

Cells were collected, cell density was counted, cell concentration was adjusted, and appropriate amount of cells were implanted into the 96-well plate. The number of cells per well should be consistent, usually adding 5,000 to 10,000 cells per well.

The cells were incubated in an incubator at 37°C, 5% CO2, until the cells were overgrown with a moderate density (typically 70-90% confluent).

2. Fixation and blocking

Cell fixation:

Discard the medium and gently wash the cells with PBS per well. Add a fixative (e.g., 4% paraformaldehyde) to each hole and keep it fixed at room temperature or suitable temperature for 10-20 minutes. Wash with PBS several times to remove fixative. Care should be taken to prevent excessive washing resulting in cell shedding.

Cell penetration (if required):

Add an appropriate amount of 0.1% Triton X-100 or other suitable penetrant to make the cell membrane semi-permeable so that the antibody can enter the cell interior. The osmotic treatment is usually carried out at room temperature for 5-10 minutes and washed again with PBS.

Block:

A blocker (5% BSA or 10% normal serum is commonly selected) is added to each well for 1 hour at room temperature to reduce nonspecific binding.

3. Antibody incubation

Primary antibody incubation:

Select the specific primary antibody for the target protein and prepare it according to the concentration recommended in the instructions. An appropriate amount of primary antibody solution is added to each well and is usually left overnight at 4°C or incubated at room temperature for 1-2 hours.

Washing:

The cells on the 96-well plate were washed multiple times with PBST (PBS + 0.05% Tween-20) or an appropriate washing buffer, usually for 3-5 minutes each time, repeated 3-5 times.

Secondary antibody incubation:

Select a suitable enzyme-labeled secondary antibody (usually HRP or AP labeled antibody) and formulate it according to the recommended concentration in the instructions. Add an appropriate amount of secondary antibody solution to each well and incubate at room temperature for 1-2 hours.

Wash again:

Repeat the above washing steps to remove non-specific binding after incubation of secondary antibody.

4. Substrate reaction and detection

Substrate solution:

Add appropriate substrate solution (such as TMB for HRP, PNPP for AP) to protect the incubation from light. The reaction time is usually 10-30 minutes until the color changes significantly.

Stopping the reaction:

After the substrate reaction to a suitable degree, add a termination solution (such as 1M H2SO4) to immediately stop the color reaction.

5. Reading and analysis

Photometric measurement:

Using an ELISA reader, readings are usually taken at a suitable wavelength, such as 450nm. The optical density (OD value) per hole reflects the relative level of the target protein content.

Data analysis:

The standard curve is applied to the experimental sample and the OD value is converted to the protein concentration. Statistical analysis of repeated experimental data, such as calculating the mean value and standard error.

Reference

Wheat, L. J., et al. Diagnosis of invasive aspergillosis by galactomannan antigenemia detection using an enzyme immunoassay. European Journal of Clinical Microbiology & Infectious Diseases. 2008, 27, 245-251.