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Run an ELISA:
Indirect, Sandwich, and Competitive!
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.
2007 NOVUS BIOLOGICALS
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.
2007 NOVUS BIOLOGICALS
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.
2007 NOVUS BIOLOGICALS
Indirect ELISA
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2007 NOVUS BIOLOGICALS
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.
Original Sample
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
2007 NOVUS BIOLOGICALS
Details
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
2007 NOVUS BIOLOGICALS
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
2007 NOVUS BIOLOGICALS
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
Secondary Binding
Details
Substrate
(Click: NovaLume)
2007 NOVUS BIOLOGICALS
Sandwich ELISA
Protein
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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.
Original Sample
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
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
2007 NOVUS BIOLOGICALS
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
Details
Substrate
(Click: NovaLume)
2007 NOVUS BIOLOGICALS
Competitive ELISA
Protein
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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
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.
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)
2007 NOVUS BIOLOGICALS
Just as in other ELISAs, known standards can be run to determine concentration, but remember the inverse rule.
2007 NOVUS BIOLOGICALS
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.
2007 NOVUS BIOLOGICALS
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.
2007 NOVUS BIOLOGICALS
13.
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.
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2007 NOVUS BIOLOGICALS
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.
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2007 NOVUS BIOLOGICALS
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.
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2007 NOVUS BIOLOGICALS
5.
7.