ELISA Guide

How ToHow To

Run an ELISA:Run an ELISA: Indirect, ‘Sandwich’, and Indirect, ‘Sandwich’, and

Competitive!Competitive!

© 2007 NOVUS BIOLOGICALS

http://www.novusbio.com/

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

button on any slide.•

For each type of ELISA covered, the Pros and Cons will be addressed.

Details



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 non-

specifically) 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 ELISAIndirect ELISA

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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.

Details


Original Sample Dilution #1Final Conc: ½ original

1:2 Dilution w/ PBS

1:2 Dilution w/ PBS

1:2 Dilution w/ PBS

Dilution #2Final Conc: ¼ original

Dilution #3Final Conc: 1/8 original


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.

Details


Sample: Complex Protein Mixture

Adsorption


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.

Details


3 Washes


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.

Details


Primary Antibody

Antibody Binding


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.

Details


Conjugated Secondary Antibody

Secondary Binding

Enzyme


Indirect ELISA, VII

• Finally, the wells are washed again, and the enzyme- conjugated 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.

Details


Substrate

Signal

(Click: NovaLume)


http://www.novusbio.com/data_sheet/index/LUM-500&prev_module=search

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’ ELISASandwich’ ELISA


Protein


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.

Details


Original Sample Dilution #1Final Conc: ½ original

1:2 Dilution w/ PBS

1:2 Dilution w/ PBS

1:2 Dilution w/ PBS

Dilution #2Final Conc: ¼ original

Dilution #3Final Conc: 1/8 original


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.

Details


Capture Antibody

Adsorption


Sandwich ELISA, III

• After incubation of the capture antibody, the wells are washed to remove any unbound antibody.

Details


3 Washes


Sandwich ELISA, IV

• Next, ‘blocking’ solution is added. This is non- specific protein that binds to the open sites in the well and keeps the non-specific proteins in the sample from binding to the well.

Details


Blocking Protein


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.

Details


Unknown #1 Unknown

#2 Unknown #3

Antigen Binding


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 don’t recognize each other, or the same site on the antigen of interest.

Details


‘Detection’ Antibody

Sandwich


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.

Details


Conjugated Tertiary Antibody


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.

Details


Substrate

Signal

(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 enzyme- linked 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 can’t find what you are looking for! Email: [email protected]


mailto:[email protected]


Competitive Competitive ELISAELISA


Protein


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.

Details


AntibodySample Combined Sample

Complex

Antigen

Antibody


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.

Details


Combined Sample


Competitive ELISA, III

• After sample incubation, the wells are washed to remove any unbound protein or antibody/antigen ‘complex’.

Details


3 Washes


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

COMPETITION

• 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.

Details


Conjugated Secondary Antibody


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.

Details


Substrate

Signal

(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 ProblemsCommon Problems

Common Problems

• The negative controls can give positive results when the blocking solution isn’t 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.

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Indirect ELISA Protocol, II

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



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.



Indirect ELISA Protocol, IV

5. 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.



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.



Indirect ELISA Protocol, VI


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.



Indirect ELISA Protocol, VII13. 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 end- point measurement after 1hr.




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.



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.



Sandwich ELISA Protocol, III

4. Wash three times by filling each well with 300l PBST.



Sandwich ELISA Protocol, IV


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



Sandwich ELISA Protocol, V


8. Add 50l of the antigen solution to each well, in serial dilution, with at least duplicate wells for each dilution.




Sandwich ELISA Protocol, VI10. Wash three times by filling each well with 300l


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!

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Sandwich ELISA Protocol, VI, Continued




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


14. Add 50l of enzyme-conjugated antibody, which will recognize your ‘detection’ antibody, at the manufacturer’s suggested dilution.




Sandwich ELISA Protocol, VIII


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.




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 manufacturer’s 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.



Competitive ELISA Protocol, II

4. Add 50l from each competition vial created, to a well.




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.




Competitive ELISA Protocol, IV





Competitive ELISA Protocol, V


11. Add 50ml of the enzyme-conjugated secondary antibody to each well, at the manufacturer’s suggested dilution.




Competitive ELISA Protocol, VI13. Wash three times by filling each well with 300l


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.




ELISA Guide

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