Editing ELISA (section)

==Types==
There are many ELISA tests for particular molecules that use the matching antibodies. ELISA tests are broken into several types of tests based on how the analytes and antibodies are bonded and used.<ref>{{Cite book|title=ELISA: theory and practice|last=Crowther|first=John R. |chapter=Basic Immunology |series=Methods in Molecular Biology|date=1995|volume=42|pages=1–218|publisher=Humana Press|isbn=978-0896032798|location=Totowa, N.J.|doi=10.1385/0-89603-279-5:1|pmid=7655571|oclc=32130600}}</ref><ref>{{Cite book|title=ELISA|editor-first=Robert|editor-last=Hnasko|date=2016|publisher=Humana|isbn=978-1493953851|edition=1|location=New York, NY|oclc=960834982|doi=10.1007/978-1-4939-2742-5|series=Methods in Molecular Biology |volume=1318 }}</ref> The major types are described here.<ref>{{cite web |title=What is an ELISA? |url=https://www.rndsystems.com/resources/what-is-an-elisa-and-elisa-types |website=R&D Systems |access-date=31 January 2020}}</ref>

=== Direct ===
[[File:ELISA diagram.png|200px|thumb|Direct ELISA diagram]]

The steps of direct ELISA<ref>{{cite book |editor1-last=Berg |editor1-first=Jeremy M. |editor2-last=Tymoczko |editor2-first=John L. |editor3-last=Gatto Jr. |editor3-first=Gregory J. |editor4-last=Stryer |editor4-first=Lubert |title=Biochemistry |date=2015 |publisher=W.H. Freeman/Macmillan |location=New York, NY |isbn=9781464126109 |edition=8 |url=https://archive.org/details/JeremyM.BergJohnL.TymoczkoGregoryJ.GattoJr.LubertStryerBiochemistry_201708}}</ref>{{Page numbers needed|date=April 2025}} follows the mechanism below:
* A buffered solution of the antigen to be tested for is added to each well (usually 96-well plates) of a [[microtiter plate]], where it is given time to adhere to the plastic through charge interactions.
* A solution of non-reacting protein, such as [[bovine serum albumin]] or [[casein]], is added to each well in order to cover any plastic surface in the well which remains uncoated by the antigen.
* The [[primary antibody]] with an attached (conjugated) enzyme is added, which binds specifically to the test antigen coating the well.
* A substrate for this enzyme is then added. Often, this substrate changes color upon reaction with the enzyme. 
* The higher the concentration of the primary antibody present in the serum, the stronger the color change. Often, a spectrometer is used to give quantitative values for color strength.

The enzyme acts as an amplifier; even if only a few enzyme-linked antibodies remain bound, the enzyme molecules will produce many signal molecules. Within common-sense limitations, the enzyme can go on producing color indefinitely, but the more antibody is bound, the faster the color will develop. A major disadvantage of the direct ELISA is that the method of antigen immobilization is not specific; when serum is used as the source of test antigen, all proteins in the sample may stick to the microtiter plate well, so small concentrations of analyte in serum must compete with other serum proteins when binding to the well surface. The sandwich or indirect ELISA provides a solution to this problem by using a "capture" antibody specific for the test antigen to pull it out of the serum's molecular mixture.{{citation needed|date=July 2020}}

ELISA may be run in a qualitative or quantitative format. Qualitative results provide a simple positive or negative result (yes or no) for a sample. The cutoff between positive and negative is determined by the analyst and may be statistical. Two or three times the standard deviation (error inherent in a test) is often used to distinguish positive from negative samples. In quantitative ELISA, the optical density (OD) of the sample is compared to a standard curve, which is typically a serial dilution of a known-concentration solution of the target molecule. For example, if a test sample returns an OD of 1.0, the point on the standard curve that gave OD&nbsp;= 1.0 must be of the same analyte concentration as the sample.{{citation needed|date=July 2020}}

The use and meaning of the names "indirect ELISA" and "direct ELISA" differ in the literature and on websites depending on the context of the experiment. When the presence of an antigen is analyzed, the name "direct ELISA" refers to an ELISA in which only a labeled primary antibody is used, and the term "indirect ELISA" refers to an ELISA in which the antigen is bound by the primary antibody which then is detected by a labeled secondary antibody. In the latter case, a sandwich ELISA is clearly distinct from an indirect ELISA. When the "primary" antibody is of interest, e.g. in the case of immunization analyses, this antibody is directly detected by the secondary antibody and the term "indirect ELISA" applies to a setting with two antibodies.{{citation needed|date=July 2020}}

===Sandwich===
[[Image:ELISA-sandwich.svg|thumb|300px|'''A sandwich ELISA'''. (1) Plate is coated with a capture antibody; (2) sample is added, and any antigen present binds to capture antibody; (3) detecting antibody is added, and binds to antigen; (4) enzyme-linked secondary antibody is added, and binds to detecting antibody; (5) substrate is added, and is converted by enzyme into a detectable form.]]

A "sandwich" ELISA is used to detect sample antigen.<ref name="Schmidt2012">{{Cite book |last1=Schmidt |first1=SD |last2=Mazzella |first2=MJ |last3=Nixon |first3=RA |last4=Mathews |first4=PM |chapter=Aβ Measurement by Enzyme-Linked Immunosorbent Assay |title=Amyloid Proteins |series=Methods in Molecular Biology |volume=849 |pages=507–27 |year=2012 |pmid=22528112 |doi=10.1007/978-1-61779-551-0_34 |isbn=978-1-61779-550-3 }}</ref> The steps are:

# A surface is prepared with a known quantity of capture antibody.
# Any nonspecific binding sites on the surface are blocked.
# The antigen-containing sample is applied to the plate, and captured by antibody.
# The plate is washed to remove unbound antigen.
# A specific antibody is added, and binds to antigen (hence the 'sandwich': the antigen is stuck between two antibodies). This primary antibody could be in the serum of a donor, to be tested for reactivity towards the antigen.
# Enzyme-linked secondary antibodies are applied as detection antibodies, which bind specifically to the antibody's Fc region (nonspecific).
# The plate is washed to remove the unbound antibody-enzyme conjugates.
# A chemical is added to be converted by the enzyme into a color, fluorescent, or electrochemical signal.
# The absorbance, fluorescence, or electrochemical signal (e.g., current) of the plate's wells is measured to determine the presence and quantity of the antigen.

The image to the right includes the use of a secondary antibody conjugated to an enzyme, although, in the technical sense, this is not necessary if the primary antibody is conjugated to an enzyme (which would be direct ELISA). However, the use of a secondary-antibody conjugate avoids the expensive process of creating enzyme-linked antibodies for every antigen one might want to detect. By using an enzyme-linked antibody that binds the Fc region of other antibodies, this same enzyme-linked antibody can be used in a variety of situations. Without the first layer of "capture" antibody, any proteins in the sample (including serum proteins) may competitively adsorb to the plate surface, lowering the quantity of antigen immobilized. Use of the purified specific antibody to attach the antigen to the plastic eliminates a need to purify the antigen from complicated mixtures before the measurement, simplifying the assay, and increasing the specificity and the sensitivity of the assay. Therefore, a sandwich ELISA used for research often needs validation, to reduce the risk of false positive results.<ref>{{cite journal|last1=Kragstrup|first1=Tue W|last2=Vorup-Jensen|first2=Thomas|last3=Deleuran|first3=Bent|last4=Hvid|first4=Malene|title=A simple set of validation steps identifies and removes false results in a sandwich enzyme-linked immunosorbent assay caused by anti-animal IgG antibodies in plasma from arthritis patients|journal=SpringerPlus|date=2013|volume=2|issue=1|pages=263|doi=10.1186/2193-1801-2-263 |pmid=23875127|pmc=3695686 |doi-access=free }}</ref>

===Competitive===
A third use of ELISA is through competitive binding. The steps for this ELISA are somewhat different from the first two examples:

Unlabeled antibody is incubated in the presence of its antigen (sample).
# These bound antibody/antigen complexes are then added to an antigen-coated well.
# The plate is washed, so unbound antibodies are removed. (The more antigen in the sample, the more Ag-Ab complexes are formed and so there are less unbound antibodies available to bind to the antigen in the well, hence "competition".)
# The [[secondary antibody]], specific to the primary antibody, is added. This second antibody is coupled to the enzyme.
# A substrate is added, and remaining enzymes elicit a chromogenic or fluorescent signal.
# The reaction is stopped to prevent eventual saturation of the signal.

Some competitive ELISA kits include enzyme-linked antigen rather than enzyme-linked antibody. The labeled antigen competes for primary antibody binding sites with the sample antigen (unlabeled). The less antigen in the sample, the more labeled antigen is retained in the well and the stronger the signal.

Commonly, the antigen is not first positioned in the well.

For the detection of HIV antibodies, the wells of microtiter plate are coated with the HIV antigen. Two specific antibodies are used, one conjugated with enzyme and the other present in serum (if serum is positive for the antibody). Cumulative competition occurs between the two antibodies for the same antigen, causing a stronger signal to be seen. Sera to be tested are added to these wells and incubated at 37&nbsp;°C, and then washed. If antibodies are present, the antigen-antibody reaction occurs. No antigen is left for the enzyme-labelled specific HIV antibodies. These antibodies remain free upon addition and are washed off during washing. Substrate is added, but there is no enzyme to act on it, so a positive result shows no color change.

===Indirect===
A fourth ELISA test does not use the traditional wells, rather leaves the antigens suspended in the test fluid.<ref>{{cite patent|country=US|number=7767404|title=Apparatus and method for single-step immunosorbent assay for single and multiple analytes|issue-date=August 3, 2010|inventor-last=Charbonnet|inventor-first=Derrick}}</ref><ref>{{cite patent|title=Systems and methods for immunosorbent assays for single and multiple analytes|country=US|number=8735142|issue-date=May 27, 2014|inventor-last=Charbonnet|inventor2-last=Evans|inventor-first=Derrick|inventor2-first=Norman Scott}}</ref>

# Unlabeled antibody is incubated in the presence of its antigen (sample)
# A sufficient incubation period is provided to allow the antibodies to bind to the antigens.
# The sample is then passed through the Scavenger container. This can be a test tube or a specifically designed flow through channel. The surface of the Scavenger container or channel has "Scavenger Antigens" bound to it. These can be identical or sufficiently similar to the primary antigens that the free antibodies will bind.
# The Scavenger container must have sufficient surface area and sufficient time to allow the Scavenger Antigens to bind to all the excess Antibodies introduced into the sample.
# The sample, that now contains the tagged and bound antibodies, is passed through a detector. This device can be a [[Flow cytometry|flow cytometer]] or other device that illuminates the tags and registers the response.<ref>{{cite journal |last1=Mahmoudi Gomari |first1=Mohammad |last2=Saraygord-Afshari |first2=Neda |last3=Farsimadan |first3=Marziye |last4=Rostami |first4=Neda |last5=Aghamiri |first5=Shahin |last6=Farajollahi |first6=Mohammad M. |title=Opportunities and challenges of the tag-assisted protein purification techniques: Applications in the pharmaceutical industry |journal=Biotechnology Advances |date=December 2020 |volume=45 |pages=107653 |doi=10.1016/j.biotechadv.2020.107653 |pmid=33157154 |s2cid=226276355 |url=https://www.sciencedirect.com/science/article/abs/pii/S0734975020301555 |language=en |issn=0734-9750|url-access=subscription }}</ref>

This test allows multiple antigens to be tagged and counted at the same time. This allows specific strains of bacteria to be identified by two (or more) different color tags. If both tags are present on a cell, then the cell is that specific strain. If only one is present, it is not.

This test is done, generally, one test at a time and cannot be done with the [[Microplate|microtiter]] plate. The equipment needed is usually less complicated and can be used in the field.