ELISA Guide

How To
Run an ELISA:
Indirect, Sandwich, and Competitive!
2007 NOVUS BIOLOGICALS
How This Presentation is Designed: This presentation combines theory with practice and is intended to clarify a difficult subject. Protocol details of any part of the illustration can be accessed by clicking the Details button on any slide. For each type of ELISA covered, the Pros and Cons will be addressed.
This Presentation Will Cover:

General ELISA Theory The three (3) major kinds of ELISA: Indirect, Sandwich and Competitive. (You can jump to that section by clicking on the name.) General problems commonly experienced. Detailed protocol and procedure information linked to each explanatory page.
ELISA Theory Overview:

ELISA is actually an acronym: Enzyme Linked ImmunoSorbent Assay. Similar to Western Blots, antibodies are used to detect the presence of proteins or other antibodies, known as antigens. Unlike Western Blots, the protein or antibody is bound to a well, and hundreds of samples can be analyzed quickly.
ELISA Type Overview: There are three major types of ELISAs:

Indirect, Sandwich and Competitive
Indirect: the protein sample is bound through adsorption, directly (and nonspecifically) to the well. Next, an antibody is used to detect the presence of one of the proteins contained in the sample, known as the antigen.
ELISA Type Overview II:

Sandwich: a capture antibody is bound to the well first, and when the sample is added, only proteins the antibody recognizes are captured. Next, a second detection antibody is used to detect the bound protein. The capture and detection antibodies are commonly called matched-pairs. Finally, a third, enzyme-labeled antibody is added to detect the detection antibody.
ELISA Type Overview III: Competitive: a primary antibody is incubated with the sample, which forms a complex. The complex is then adsorbed to the wells. Next, a secondary antibody is added to the wells, which recognizes the primary antibody only if it is not bound to the antigen. Therefore, the secondary antibody competes with the antigen.
Indirect ELISA
Back
Details
Indirect ELISA, I
First, samples are prepared, usually by serial dilution with PBS. The samples can be complex protein mixtures such as cell lysates, or contain antibodies/proteins of interest in other formats, such as blood aliquots from subjects.
1:2 Dilution w/ PBS
1:2 Dilution w/ PBS
1:2 Dilution w/ PBS
Original Sample
Dilution #1 Final Conc: original
Dilution #3 Final Conc: 1/8 original
Details
Indirect ELISA, II
Samples are then added to the wells of a plate suitable for antigen binding and incubated so that the antigen will be thoroughly adsorbed to the well surface.
Sample: Complex Protein Mixture
Adsorption
Details
Indirect ELISA, III

After incubation, the wells must be washed to remove any unbound, or poorly bound antigen. This step is very important and will occur after every other step.
3 Washes
Details
Indirect ELISA, IV
Next, the wells must be filled with blocking solution, which is non-specific protein that binds to the exposed surfaces in the well and keeps the primary antibody from binding non-specifically to the well.
Blocking Protein
Details
Indirect ELISA, V
Following blocking, the plate is washed again and the primary antibody, in dilution, is added to the well. The antibody will only recognize one antigen, ideally. The primary antibody is usually added in a range of dilutions, and each dilution is usually tested in duplicate or triplicate.
Antibody Binding
Primary Antibody
Details
Indirect ELISA, VI
After primary antibody incubation, another series of washes are performed and then conjugated secondary antibody is added to each well, at a constant dilution.
Enzyme
Conjugated Secondary Antibody

Secondary Binding
Details
Indirect ELISA, VII

Finally, the wells are washed again, and the enzymeconjugated secondary antibody is caused to react, giving off a signal, which is read on a plate reader.
The substrate used varies according to the enzyme the secondary antibody is conjugated to.
Signal
Substrate
(Click: NovaLume)
Indirect ELISA Results

The presence of a signal from the secondary antibody means the antigen of interest is present. In order to determine antigen concentration, a standard curve of known antigen concentration must be run on the same plate. Negative controls should also be run to make sure your antibodies are binding specifically to the antigen only.
Indirect ELISA Pros/Cons

This type of ELISA is commonly used to determine the ideal concentration/dilution of primary antibody to use in other experiments.
This is done by running the ELISA against a known concentration of antigen, and performing serial dilutions of the primary antibody. The dilutions that result in a signal, show the range the primary antibody is effective in, according to the concentration of the antigen.

Because the desired antigen may be present in extremely small quantities relative to the presence of other proteins, and because all proteins in a sample will bind to the well, Indirect ELISA may be unable to detect the presence of antigen in a sample.
In this case, the antigen is usually purified out of the sample, so that it can be more readily detected.

The primary and secondary antibodies used in this type of ELISA can also be used in the same dilutions against the same samples in Western Blot, therefore eliminating the need to purchase and test other antibodies.
Sandwich ELISA
Protein
Back
Details
Sandwich ELISA, I
First, the samples are prepared, usually by serial dilution with PBS. The samples can be complex protein mixtures such as cell lysates, or contain antigens of interest in other formats, such as blood aliquots from subjects.
1:2 Dilution w/ PBS
1:2 Dilution w/ PBS
1:2 Dilution w/ PBS
Original Sample
Dilution #3 Final Conc: 1/8 original
Details
Sandwich ELISA, II
The capture antibody, also known as the first primary antibody, is added to each well, and incubated, to allow the antibody to adsorb to the surface of the well.
Capture Antibody
Adsorption
Details
Sandwich ELISA, III

After incubation of the capture antibody, the wells are washed to remove any unbound antibody.
3 Washes
Details
Sandwich ELISA, IV
Next, blocking solution is added. This is nonspecific protein that binds to the open sites in the well and keeps the non-specific proteins in the sample from binding to the well.
Blocking Protein
Details
Sandwich ELISA, V
Next, samples of unknowns are added to each antibody-coated well and again allowed to incubate and bind to the capture antibody.
Unknown #1 Unknown #2
Unknown #3
Antigen Binding
Details
Sandwich ELISA, VI
After another wash cycle, the detection antibody is added to the wells and allowed to incubate and detect the antigen.
The detection antibody must be a matched-pair with the capture antibody, to make sure that the antibodies dont recognize each other, or the same site on the antigen of interest.
Sandwich
Detection Antibody
Details
Sandwich ELISA, VII

After another wash, the enzyme-linked antibody (sometimes referred to as a secondary, even though it is tertiary in this case) is added to each well and it detects the detection antibody.
Conjugated Tertiary Antibody

Details
Sandwich ELISA, VIII

Finally, another wash cycle is performed, and the enzyme on the tertiary antibody is reacted, to give off a signal, which can be read on a plate reader.
Signal
Substrate
(Click: NovaLume)
Sandwich ELISA Results

The presence of signal means the antigen of interest is present. A standard curve must be included on the same plate to make this ELISA quantitative. Negative controls should also be run to make sure only specific binding is occurring among the many antibodies used.
Sandwich ELISA Pros/Cons

Unlike Indirect ELISAs, antigens of very low or unknown concentration in the sample can be detected because the capture antibody only grabs the antigen of interest and all the other proteins in the sample are washed away.

It is not necessary to use a tertiary enzymelinked antibody, if the detection antibody is already enzyme-linked. However, it can be very difficult, if not impossible to find a matched-pair where the detection antibody is already conjugated. The end-user can conjugate the detection antibody themselves, if so inclined.

Only monoclonal antibodies can be used as matched pairs, because only monoclonals recognize one specific site on an antigen (known as the epitope). Monoclonal antibodies can be more expensive than polyclonal antibodies and matched-pair antibodies can be very difficult to find.
Novus Biologicals can help you find matched-pair antibodies, if you cant find what you are looking for! Email: novus@novusbio.com
Competitive ELISA
Protein
Back
Details
Competitive ELISA, I
To begin with, the samples are prepared by incubating the primary antibody with the samples in tubes, where the antigen of interest forms a complex with the antibody.
Antigen
Complex
Antibody
Sample
Antibody
Combined Sample
Details
Competitive ELISA, II
The sample is added to the well, and allowed to incubate so that it adsorbs to the surface of the well.
The complex, any unbound antibody, and other proteins can all adsorb.
Combined Sample
Details
Competitive ELISA, III

After sample incubation, the wells are washed to remove any unbound protein or antibody/antigen complex.
3 Washes
Details
Competitive ELISA, IV
The wells are then coated with blocking solution, which will keep the secondary antibody from non-specifically binding to the wells.
Details
Competitive ELISA, V
After another wash cycle, the conjugated secondary antibody is allowed to incubate and compete with the antigen of interest for continued binding to the primary antibody.
Conjugated Secondary Antibody
COMPETITION
Details
Competitive ELISA, VI
After a final wash cycle, the conjugated secondary enzyme is reacted to produce a signal, which can be read on a plate reader.
Signal
Substrate
(Click: NovaLume)
Competitive ELISA Results

The strength of the signal is inversely related to the quantity of antigen present.
The more antigen present, the more difficult it is for the secondary antibody to bind to the primary antibody and vice versa.
Just as in other ELISAs, known standards can be run to determine concentration, but remember the inverse rule.
Competitive ELISA Pros/Cons

Like Sandwich ELISA, this form of ELISA can detect smaller quantities of antigen present than Indirect ELISAs can. This type of ELISA does not require the use of matched-pair antibodies, as Sandwich ELISAs do.

Instead of a conjugated secondary antibody being used as the competitor, another conjugated antigen can be used that the primary antibody also recognizes.
In other words, use a protein that the primary antibody also recognizes, that is not the same as the antigen of interest. The benefit of using this method is that you do not have to use a secondary antibody. The disadvantage is that you may have difficulty finding another protein your primary antibody recognizes, and you will also likely have to conjugate the protein yourself.

The primary antibody used can be unpurified, and polyclonal. The antigen of interest that is ideal for this type of ELISA contains only one recognizable epitope by the primary antibody.
Common Problems
Common Problems
The negative controls can give positive results when the blocking solution isnt effective, therefore the secondary antibody or antigen of interest can bind to the open sites in the well. If the positive controls or standards give no signal, check your chemicals and be aware that the enzyme reaction is short-term, so the plate should be read as quickly as possible.
Common Problems
Running your samples in duplicate and triplicate will allow for a more accurate determination of concentration. Applying your primary antibody in a dilution range increases the likelihood that you will get a signal that is neither too weak nor too strong. Past a certain limit, the strength of a signal gives useless information. Dilute your sample or primary antibody if this occurs.
The End ~ Thank You!

*Detailed methods and recipes are on the following slides.
Indirect ELISA Protocol, I

1. Dilute the antigen in PBS and coat the wells of a PVC microtiter plate with 50l of the antigen dilution per well.
Usually a serial dilution is made of either the antigen, the antibody, or both. You must have at least one well per dilution and ideally three wells.
Back to the Slide

Indirect ELISA Protocol, II

2. Cover the plate with adhesive plastic and incubate for 2hr at room temperature or 1hr at 37C.
Back to the Slide

Indirect ELISA Protocol, III

3. Remove the antigen coating solution by flicking the plate over the sink then tapping the plate upside down on a thick paper towel several times. 4. Wash three times by filling each well with 300l PBST. Each wash is removed the same way that the antigen solution was removed.
Back to the Slide

Indirect ELISA Protocol, IV

Block the remaining protein-binding sites in the coated wells by adding 300l blocking buffer: 5% NFDM/PBS. 6. Cover the plate with adhesive plastic and incubate for at least 2hr at room temperature, or overnight at 4C. 5.
Back to the Slide

Indirect ELISA Protocol, V

7. Wash three times by filling each well with 300l PBST. Each wash is removed the same way as the antigen solution was removed. 8. Make 10-fold dilutions (1:100, 1:1,000, 1:10,000. 1:100,000) of antibody in blocking buffer. Add 50ul of each dilution to an antigen-coated well in duplicate or triplicate. 9. Cover the plate with adhesive plastic and incubate for 2hr at room temperature.
Back to the Slide
Indirect ELISA Protocol, VI

10. Wash three times by filling each well with 300l PBST. Each wash is removed the same way as the antigen solution was removed. 11. Add 50l of horse-radish peroxidase conjugated secondary antibody to each sample well, diluted according to manufacturer suggestions. 12. Cover the plate with adhesive plastic and incubate for 2hr at room temperature.
Back to the Slide
Indirect ELISA Protocol, VII

Wash three times by filling each well with 300l PBST. Each wash is removed the same way as the antigen solution was removed. 14. For HRP conjugated secondary, use a chemiluminescent substrate, such as NovuLume, according to suggestions. Add 50l of the substrate solution per well with a multichannel pipette. 15. Measure the absorbance at 405 nm, using a microtiter plate spectrophotometer. Perform an endpoint measurement after 1hr.
Back to the Slide
13.
Sandwich ELISA Protocol, I

1. Dilute the antigen in PBS and coat the wells of a PVC microtiter plate with 50ul of the antigen dilution per well.
Usually a serial dilution is made of either the antigen, the antibody, or both. You must have at least one well per dilution and ideally three wells.
Back to the Slide

Sandwich ELISA Protocol, II

2. Add 50l of the capture antibody to each well.
You should use roughly 1 g of antibody/well, which is more than enough.
3. Cover the plate with adhesive plastic and incubate for 2hr at room temperature, or overnight at 4C.
The antibody solution can be carefully removed and reused if the 4C method is used.
Back to the Slide
Sandwich ELISA Protocol, III

4.
Wash three times by filling each well with 300l PBST.
Back to the Slide

Sandwich ELISA Protocol, IV

Block the remaining protein-binding sites in the coated wells by adding 300l blocking buffer: 5% NFDM/PBS. 6. Cover the plate with adhesive plastic and incubate for at least 2hr at room temperature, or overnight at 4C 5.
Back to the Slide

Sandwich ELISA Protocol, V

7.
Wash three times by filling each well with 300l PBST. Each wash is removed the same way as
the antigen solution was removed. 8. Add 50l of the antigen solution to each well, in serial dilution, with at least duplicate wells for each dilution. 9. Cover the plate with adhesive plastic and incubate for at least 2hr at room temperature, or overnight at 4C.
Back to the Slide
Sandwich ELISA Protocol, VI

10. Wash three times by filling each well with 300l PBST. Each wash is removed the same way as the antigen solution was removed. 11. Add 50l of the detection antibody to each well. This antibody may be conjugated to an enzyme to avoid adding an additional antibody step. You can also use this antibody in serial dilution, but you will need to have duplicate wells per detection antibody dilution per antigen dilution!
Continue
Sandwich ELISA Protocol, VI, Continued

12. Cover the plate with adhesive plastic and incubate for at least 2hr at room temperature, or overnight at 4C.
Back to the Slide

Sandwich ELISA Protocol, VII

This step may be skipped if your detection antibody is already conjugated. 13. Wash three times by filling each well with 300l PBST. Each wash is removed the same way as the antigen solution was removed. 14. Add 50l of enzyme-conjugated antibody, which will recognize your detection antibody, at the manufacturers suggested dilution. 15. Cover the plate with adhesive plastic and incubate for at least 2hr at room temperature, or overnight at 4C.
Back to the Slide
Sandwich ELISA Protocol, VIII

16.
the antigen solution was removed. 17. For HRP conjugated antibody, use a
chemiluminescent substrate, such as NovuLume, according to suggestions. Add 50l of the substrate solution per well with a multichannel pipette.
18. Read on a plate spectrophotometer as quickly as possible, at the recommended wavelength. Take an end-point reading.
Back to the Slide
Competitive ELISA Protocol, I

Setup a competition complex:
1. Create serial dilutions of your antigen in microcentrifuge tubes, ideally with two tubes per dilution. (Volume: 50l) 2. Add 25l of primary antibody at a final concentration of 2X manufacturers suggested dilution, or at a serial dilution. Remember, if you do serial dilutions of both the antigen and the antibody, then you need every dilution of antibody for every dilution of antigen. 3. Incubate the tubes containing the antibody/antigen complex overnight at room temperature. Back to the Slide
Competitive ELISA Protocol, II

4. Add 50l from each competition vial created, to a well. Cover the plate with adhesive plastic and incubate for at least 2hr at room temperature, or overnight at 4C.
5.
Back to the Slide

Competitive ELISA Protocol, III

6. Remove the antigen coating solution by flicking the plate over the sink and then tapping the plate upside down on a thick piece of paper towel several times.
7.
the antigen solution was removed.
Back to the Slide

Competitive ELISA Protocol, IV

Block the remaining protein-binding sites in the coated wells by adding 300l blocking buffer: 5% NFDM/PBS. 9. Cover the plate with adhesive plastic and incubate for at least 2hr at room temperature, or overnight at 4C. 8.
Back to the Slide

Competitive ELISA Protocol, V

10. Wash three times by filling each well with 300l PBST. Each wash is removed the same way as the antigen solution was removed. 11. Add 50ml of the enzyme-conjugated secondary antibody to each well, at the manufacturers suggested dilution. 12. Cover the plate with adhesive plastic and incubate for at least 2hr at room temperature, or overnight at 4C.
Back to the Slide
Competitive ELISA Protocol, VI

13. Wash three times by filling each well with 300l PBST. Each wash is removed the same way as the antigen solution was removed. 14. For HRP conjugated secondary, use a chemiluminescent substrate, such as NovuLume, according to suggestions. Add 50l of the substrate solution per well with a multichannel pipette. 15 15. Read on a plate spectrophotometer as quickly as possible, at the recommended wavelength. Take an end-point reading.
Back to the Slide

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How To

2007 NOVUS BIOLOGICALS

This Presentation Will Cover:

2007 NOVUS BIOLOGICALS

ELISA Theory Overview:

ELISA Type Overview: There are three major types of ELISAs:

ELISA Type Overview II:

2007 NOVUS BIOLOGICALS

1:2 Dilution w/ PBS

1:2 Dilution w/ PBS

1:2 Dilution w/ PBS

Dilution #1 Final Conc: original

Dilution #2 Final Conc: original

Dilution #3 Final Conc: 1/8 original

2007 NOVUS BIOLOGICALS

Indirect ELISA, III

2007 NOVUS BIOLOGICALS

Conjugated Secondary Antibody

Indirect ELISA, VII

Indirect ELISA Results

Indirect ELISA Pros/Cons

Indirect ELISA Pros/Cons

2007 NOVUS BIOLOGICALS

Indirect ELISA Pros/Cons

2007 NOVUS BIOLOGICALS

1:2 Dilution w/ PBS

1:2 Dilution w/ PBS

1:2 Dilution w/ PBS

Dilution #1 Final Conc: original

Dilution #2 Final Conc: original

Dilution #3 Final Conc: 1/8 original

2007 NOVUS BIOLOGICALS

2007 NOVUS BIOLOGICALS

Sandwich ELISA, III

2007 NOVUS BIOLOGICALS

2007 NOVUS BIOLOGICALS

2007 NOVUS BIOLOGICALS

Sandwich ELISA, VII

Conjugated Tertiary Antibody

Sandwich ELISA, VIII

Sandwich ELISA Results

2007 NOVUS BIOLOGICALS

Sandwich ELISA Pros/Cons

2007 NOVUS BIOLOGICALS

Sandwich ELISA Pros/Cons

Sandwich ELISA Pros/Cons

2007 NOVUS BIOLOGICALS

2007 NOVUS BIOLOGICALS

Competitive ELISA, III

2007 NOVUS BIOLOGICALS

2007 NOVUS BIOLOGICALS

Conjugated Secondary Antibody

2007 NOVUS BIOLOGICALS

Competitive ELISA Results

Competitive ELISA Pros/Cons

Competitive ELISA Pros/Cons

Competitive ELISA Pros/Cons

The End ~ Thank You!

2007 NOVUS BIOLOGICALS

Indirect ELISA Protocol, I

Back to the Slide

Indirect ELISA Protocol, II

Back to the Slide