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    Types of ELISA protocols

    ELISA protocols vary by subtypes, but share basis

    elisaThere are five types of ELISA, thus, about ELISA protocol, a few differences exist amid indirect ELISA protocol, direct ELISA protocol, sandwich ELISA protocol, competitive ELISA protocol and ELISPOT protocol. However, the main ELISA principle and lots of procedures are the same. General ELISA protocol includes plate preparation, assay procedure and calculation of results.

    (ELISA Protocol) Plate Preparation

    1.Dilute the capture antibody to the working concentration in CBS. Immediately coat a 96-well microplate with 100μL per well of the diluted capture antibody. Seal the plate and incubate overnight at 4℃.

    2.Aspirate each well and wash with at least 300μl 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.

    3.Block plates by adding 300 μL of blocking buffer to each well. Incubate at room temperature for a minimum of 1 hour.

    4.Repeat the aspiration/wash as in step 2. The plates are now ready for sample addition.

    (ELISA Protocol) Assay Procedure

    1.Add 100 μL of sample or standards in sample dilution buffer per well. Seal the plate and incubate 2 hours at room temperature.

    2.Repeat the aspiration/wash as in step 2 of plate preparation.

    3.Add 100 μL of the detection antibody, diluted in antibody dilution buffer, to each well. Seal the plate and incubate 1 hour at room temperature.

    4.Repeat the aspiration/wash as in step 2 of plate preparation.

    5.Add 200 μL of substrate solution to each well. Incubate for 20 minutes at room temperature ( if substrate solution is not as requested, the incubation time should be optimized ). Avoid placing the plate in direct light.

    6.Add 50 μL of stop solution to each well. Gently tap the plate to ensure thorough mixing.

    7.Determine the optical density of each well immediately, using a microplate reader set to 450 nm.

    (ELISA Protocol) Calculation of Results

    1.Calculate the mean absorbance for each set of duplicate standards, controls and samples. Subtract the mean zero standard absorbance from each.

    2.Construct a standard curve by plotting the mean absorbance for each standard on the y-axis against the concentration on the x-axis and draw a best fit curve through the points on the graph.

    3.To determine the concentration of the unknowns, find the unknowns’ mean absorbance value on the y-axis and draw a horizontal line to the standard curve. At the point of intersection, draw a vertical line to the x-axis and read the concentration. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.

    4.Alternatively, computer-based curve-fitting statistical software may also be employed to calculate the concentration of the sample.

    Indirect ELISA Protocol


    From the sample to the reading, Indirect ELISA Protocol

    Buffer Preparation

    1. Bicarbonate/carbonate coating buffer (100 mM)

    Antigen or antibody should be diluted in coating buffer to immobilize them to the wells:

    • 3.03 g Na2CO3
    • 6.0 g NaHCO3
    • 1000 ml distilled water,
    • pH 9.6

    2. PBS:

    • 1.16 g Na2HPO4
    • 0.1 g KCl
    • 0.1 g K3PO4
    • 4.0 g NaCl (500 ml distilled water)
    • pH 7.4

    3. Blocking solution:

    • Commonly used blocking agents are 1% BSA , serum, non-fat dry milk, casein, gelatin in PBS.

    4. Wash solution:

    • Usually PBS or Tris -buffered saline (pH 7.4) with detergent such as 0.05% (v/v) Tween20 (TBST).

    5. Antibody dilution buffer:

    • Primary and secondary antibody should be diluted in 1x blocking solution to reduce non-specific binding.

    Indirect ELISA Protocol

    1. Dilute antigen to a final concentration of 1-20 μg/ml using PBS or Bicarbonate/carbonate coating buffer. Coat the wells of a PVC microtiter plate with the antigen by pipeting 50μl of the antigen dilution in the top wells of the plate. Dilute down the plate as required. Seal the plate and incubate overnight at 4°C or 2 h at room temperature.

    2. Wash plate 3 times with PBS.

    3. Block the remaining protein-binding sites in the coated wells by adding 200 μl blocking buffer, 5% non fat dry milk/PBS, per well. Alternative blocking reagents include BlockACE or BSA.

    4. Cover the plate with an adhesive plastic and incubate for at least 2 h at room temperature or, if more convenient, overnight at 4°C.

    5. Wash the plate 3 times with PBS.

    6. Add 100 μl of diluted primary antibody to each well.

    7. Cover the plate with an adhesive plastic and incubate for 2 h at room temperature.

    8. Wash the plate 4 times with PBS.

    9. Add 100 μl of conjugated secondary antibody, diluted at the optimal concentration (according to the manufacturer) in blocking buffer immediately before use.

    10. Cover the plate with an adhesive plastic and incubate for 1-2 h at room temperature.

    11. Wash the plate 5 times with PBS.

    12. Dispense 100 μl (or 50 μl) of the substrate solution per well with a multichannel pipet or a multipipet. 13. After sufficient color development (if it is necessary) add 50-100μl of stop solution to the wells. 14. Record the absorbance at 450 nm on a plate reader within 30 minutes of stopping the reaction.


    Direct ELISA Protocol


    From the sample to the reading, Direct ELISA Protocol

    1. Coat ELISA plate (96 well plate) with testing antigen (10 μg/ml to 0.01 ng/ml in 50 mM Na2C03, pH 9.6, adjust based on the reactivity of antibody, 100 μl/well. Seal the plate and incubate overnight at 4°C.

    2. Wash plate 3 times with PBS-T (0.05 % Tween-20 in PBS).

    3. Block plate with 0.2% non-fat dry milk in PBS at room temperature for 1 hour at 4°C

    Note: Milk should be thoroughly dissolved. It’s recommended to dissolve 0.5 g milk in 50 ml PBS (1%) for at least 30 minutes at room temperature with rotation or stirring, then dilute to 0.2 %, and keep rotating or stirring for another 10-15 minutes at room temperature. If high background is experienced, 1% milk in PBS could be applied for both blocking and antibody dilution.

    4. Wash plate 3 times with PBS-T.

    5. (a) Incubate with biotinylated, affinity-purified rabbit IgG (0.1-0.5 μg/ml in PBS, 100 μl/well) at room temperature for 1 hour, followed by washing 6 times with PBS-T, then go to 8a.

    (b) Alternatively, incubate with affinity purified rabbit IgG (0.2-1 μg/ml in PBS, 100 μl/well) at room temperature for 1 hour, followed by washing 6 times with PBS-T, then go to 8b.

    6. (a) Incubate with HRP-Streptavidin (1:4000-10,000 dilutions) in 0.2 % milk-PBS, 100 μl/well, at room temperature for 1 hour.

    (b) Incubate with HRP-anti-rabbit IgG (1:3,000-10,000 dilutions of 1 mg/ml or 0.25 μg/ml) in PBS, 100 μl/well, at room temperature for 1 hour.

    7. Wash plate 8 times with PBS-T.

    8. Develop color using TMB as a substrate (100 μl/well) and incubate at room temperature for 15-30 minutes without shaking.

    9. Stop reaction by addition of 2N H2S04 (100 μl/well). Record the absorbance at 450 nm on a plate reader within 30 minutes of stopping the reaction.


    Sandwich ELISA Protocol


    1. Before the assay, both antibody preparations should be purified and one must be labeled.

    2. For most applications, a polyvinylchloride (PVC) microtiter plate is best; however, consult manufacturer guidelines to determine the most appropriate type of plate for protein binding.

    3. Bind the unlabeled antibody to the bottom of each well by adding approximately 50 μL of antibody solution to each well (20 μg/ml in PBS). PVC will bind approximately 100 ng/well (300 ng/cm2). The amount of antibody used will depend on the individual assay, but if maximal binding is required, use at least 1 μg/well. This is well above the capacity of the well, but the binding will occur more rapidly, and the binding solution can be saved and used again.

    4. Incubate the plate overnight at 4°C to allow complete binding.

    5. Wash the wells twice with PBS. A 500 ml squirt bottle is convenient. The antibody solution washes can be removed by flicking the plate over a suitable container.

    6. The remaining sites for protein binding on the microtiter plate must be saturated by incubating with blocking buffer. Fill the wells to the top with 3% BSA/PBS with 0.02% sodium azide. Incubate for 2 h to overnight in a humid atmosphere at room temperature.

    Note: Sodium azide is an inhibitor or horseradish peroxidase. Do not include sodium azide in buffers or wash solutions if an HRP-labeled antibody will be used for detection.

    7. Wash wells twice with PBS.

    8. Add 50 μL of the antigen solution to the wells (the antigen solution should be titrated). All dilutions should be done in the blocking buffer (3% BSA/PBS). Incubate for at least 2 h at room temperature in a humid atmosphere.

    9. Wash the plate four times with PBS.

    10.Add the labeled second antibody. The amount to be added can be determined in preliminary experiments. For accurate quantitation, the second antibody should be used in excess. All dilutions should be done in the blocking buffer.

    11.Incubate for 2 h or more at room temperature in a humid atmosphere.

    12.Wash with several changes of PBS.

    13.Add substrate as indicated by manufacturer. After suggested incubation time has elapsed, optical densities at target wavelengths can be measured on an ELISA plate reader.

    Note: Some enzyme substrates are considered hazardous, due to potential carcinogenicity. Handle with care and refer to Material Safety Data Sheets for proper handling precautions.


    Competitive ELISA Protocol


    1. For most applications, a polyvinylchloride (PVC) microtiter plate is best; however, consult manufacturer guidelines to determine the most appropriate type of plate for protein binding.

    2. Add 50 μL of diluted primary antibody (capture) to each well. The appropriate dilution should be determined using an checkerboard titration prior to testing samples. PVC will bind approximately 100 ng/well (300 ng/cm2). The amount of antibody used will depend on the individual assay, but if maximal binding is required, use at least 1 μg/well. this is well above the capacity of the well, but the binding will occur more rapidly, and the binding solution can be saved and used again. Allow to incubate for 4 hrs. at room temperature or 4°C overnight.

    Note: If a purified capture antibody is not available, the plate should first be coated with a purified secondary antibody directed against the host of the capture antibody according to the following procedure:

    a. Bind the unlabeled secondary antibody to the bottom of each well by adding approximately 50 μL of antibody solution to each well (20 μg/mL in PBS).

    b. Incubate the plate overnight at 4°C to allow complete binding.

    c. Add primary capture antibody (as above).

    3. Wash the wells twice with PBS. A 500 mL squirt bottle is convenient. The antibody solution washes can be removed by flicking the plate over a suitable container.

    4. The remaining sites for protein binding on the microtiter plate must be saturated by incubating with blocking buffer. Fill the wells to the top with 3% BSA/PBS with 0.02% sodium azide. Incubate for 2 hrs. to overnight in a humid atmosphere at room temperature

    5. Wash wells twice with PBS.

    6. Add 50 μL of the standards or sample solution to the wells. All dilutions should be done in the blocking buffer (3% BSA/PBS with 0.05% Tween-20)

    Note: Sodium azide is an inhibitor or horseradish peroxidase. Do not include sodium azide in buffers or wash solutions, if an HRP-labeled conjugate will be used for detection.

    7. Add 50 μL of the antigen-conjugate solution to the wells (the antigen solution should be titrated). All dilutions should be done in the blocking buffer (3% BSA/PBS with 0.05% Tween-20). Incubate for at least 2 hrs. at room temperature in a humid atmosphere.

    8. Wash the plate four times with PBS.

    9. Add substrate as indicated by manufacturer. After suggested incubation time has elapsed, optical densities at target wavelengths can be measured on an ELISA reader.

    Note: Competitive ELISAs yield an inverse curve, where higher values of antigen in the samples or standards yield a lower amount of color change.


    ELISpot Protocol


     

    ELISpot Four Parts of Protocol

    Note: Use ELISPOT plates and reagents under aseptic conditions (e.g., in laminar flow hood) for Steps 1 – 3. Plates are pre-coated with antibody and ready for the addition of cells. Blocking and wetting are not required.

    Cell Activation (under aseptic conditions)

    1. Prepare mitogen or antigen, diluted in complete medium (eg, RPMI 1640 with FBS, Pen/Strep, and L-glutamine).

    2. Add 100 μl/well to ELISPOT plate.

    3. Prepare cell suspensions at different densities, (e.g., 1 × 105 to 2 × 106 cells/ml). Add 100 μl/well of each cell suspension to ELISPOT plate microwells.

    4. Replace lid and incubate at 37ºC in a 5% CO2 humidified incubator. The duration of the incubation time will vary (eg, 2h – 24h). Specific activation conditions will vary, depending on cell type, kinetics, and analyte of interest.

    Note: Cells may be diluted in a regular tissue culture plate starting at 105/well in triplicate wells with 1:3 or 1:4 serial dilutions down the plate, then transferred to the ELISPOT plate.

    Detection Antibody

    5. Aspirate cell suspension. Wash wells 2 times with deionized (DI) water. Allow wells to soak for 3 – 5 min at each wash step.

    6. Wash wells 3 times with prepared Wash Buffer, 200 μl/well. Discard Wash Buffer.

    7. Add prepared Detection Antibody Solution at 100 μl per well.

    8. Replace lid and incubate for 2 h at room temperature.

    Enzyme

    9. Discard Detection Antibody solution. Wash wells 3 times with 200 μl/well prepared Wash Buffer. Allow wells to soak 1 – 2 min at each wash step.

    10. Add prepared Streptavidin-HRP Solution at 100 μl/well

    11. Replace lid; incubate for 1 h at room temperature.

    Substrate

    12. Discard Streptavidin-HRP solution. Wash wells 4 times with 200 μl/well prepared Wash Buffer. Allow wells to soak 1 – 2 min at each wash step.

    13. Wash wells 2 times, 200 μl/well, with prepared PBS.

    14. Add 100 μl of prepared AEC Substrate Solution to each well. Monitor spot development from 5 – 60 min. Do not let color overdevelop as this will lead to high background.

    15. Stop substrate reaction by washing wells with DI water.

    16. Air-dry plate at room temperature for 2 h to overnight until it is completely dry. Removal of plastic tray supporting the 96-well plate will facilitate drying. Store plate in a sealed plastic bag, in the dark, until it is analyzed.

    Analysis

    17. Enumerate spots manually by inspection under a dissecting microscope (or magnifying glass), or automatically using an ELISPOT plate reader.