ELISA (cont'd)

Introduction to Antibodies - Enzyme-Linked Immunosorbent Assay (ELISA)

ELISA Procedure

Enzyme-linked Immunosorbent Assays (ELISAs) combine the specificity of antibodies with the sensitivity of simple enzyme assays, by using antibodies or antigens coupled to an easily assayed enzyme that possesses a high turnover number. ELISAs can provide a useful measurement of antigen or antibody concentration.

Sandwich ELISA Assays
One of the most useful of the immunoassays is the two antibody “sandwich” ELISA. This assay is used to determine the antigen concentration in unknown samples. This ELISA is fast and accurate, and if a purified antigen standard is available, the assay can determine the absolute amount of antigen in an unknown sample. The sandwich ELISA requires two antibodies that bind to epitopes that do not overlap on the antigen. This can be accomplished with either two monoclonal antibodies that recognize discrete sites or one batch of affinity-purified polyclonal antibodies.

To utilize this assay, one antibody (the “capture” antibody) is purified and bound to a solid phase typically attached to the bottom of a plate well. Antigen is then added and allowed to complex with the bound antibody. Unbound products are then removed with a wash, and a labeled second antibody (the “detection” antibody) is allowed to bind to the antigen, thus completing the “sandwich”. The assay is then quantitated by measuring the amount of labeled second antibody bound to the matrix, through the use of a colorimetric substrate. Major advantages of this technique are that the antigen does not need to be purified prior to use, and that these assays are very specific. However, one disadvantage is that not all antibodies can be used. Monoclonal antibody combinations must be qualified as “matched pairs”, meaning that they can recognize separate epitopes on the antigen so they do not hinder each other’s binding.

Unlike Western blots, which use precipitating substrates, ELISA procedures utilize substrates that produce soluble products. Ideally the enzyme substrates should be stable, safe and inexpensive. Popular enzymes are those that convert a colorless substrate to a colored product, e.g., pnitrophenylphosphate (pNPP), which is converted to the yellow p-nitrophenol by alkaline phosphatase. Substrates used with peroxidase include 2,2’-azo-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), o-phenylenediamine (OPD) and 3,3’5,5’- tetramethylbenzidine base (TMB), which yield green, orange and blue colors, respectively. A table of commonly used enzyme-substrate combinations is included in Appendix D.

The Sensitivity of the Sandwich ELISA is Dependent on Four Factors:

1. The number of molecules of the first antibody that are bound to the solid phase.
2. The avidity of the first antibody for the antigen.
3. The avidity of the second antibody for the antigen.
4. The specific activity of the second antibody.

The amount of the capture antibody that is bound to the solid phase can be adjusted easily by dilution or concentration of the antibody solution. The avidity of the antibodies for the antigen can only be altered by substitution with other antibodies. The specific activity of the second antibody is determined by the number and type of labeled moieties it contains.

General Protocol for the Sandwich ELISA method:

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 100ng/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 hrs. 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 hrs. 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 hrs. 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.

For quantitative results, compare signal of unknown samples against those of a standard curve. Standards must be run with each assay to ensure accuracy.

Competitive ELISA Assays
When two “matched pair” antibodies are not available for your target, another option is the competitive ELISA. Another advantage to the competitive ELISA is that non-purified primary antibodies may be used. Although there are several different configurations for competitive ELISAs, below is an example for one such configuration. In order to utilize a competitive ELISA, one reagent must be conjugated to a detection enzyme, such as horseradish peroxidase. The enzyme may be linked to either the immunogen or the primary antibody. The protocol below uses a labeled immunogen as the competitor. For other configurations of competitive ELISAs, see Appendix F, Harlow and Lane (1996).

Briefly, an unlabeled purified primary antibody is coated onto the wells of a 96 well microtiter plate. This primary antibody is then incubated with unlabeled standards and unknowns. After this reaction is allowed to go to equilibrium, conjugated immunogen is added. This conjugate will bind to the primary antibody wherever its binding sites are not already occupied by unlabeled immunogen. Thus, the more immunogen in the sample or standard, the lower the amount of conjugated immunogen bound. The plate is then developed with substrate and color change is measured.

General Protocol for the Competitive ELISA Method:

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 a 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.

Troubleshooting ELISA Assays:

• Interpret the control results.
  
• If the negative controls are giving positive results, there may be contamination of the substrate solution, or contamination of the enzyme-labeled antibody, or of the controls themselves.
  
• If no color has developed for the positive controls or for the samples, check all reagents for dating, concentration, and storage conditions. Check the integrity of the antibody reagent.
  
• If very little color has developed for the positive controls and the test samples, check the dilution of the enzyme labeled antibody, and the concentration of the substrate.
  
• If color has developed for the test samples but not the positive or negative controls, check the source of the positive controls, their expiration date and their storage. Have they been stored in a dilute form, so that the antigen may have adhered to the surface of the storage vessel?
  
• If color can be seen, but the absorbance is not as high as expected, check the wavelength setting.
  
• When rerunning an assay while troubleshooting, change only one factor at a time.