Editing ELISA (section)

=== 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}}