酶联免疫吸附试验（又称酵素免疫分析法，Enzyme-linked immunoassay，简称ELISA）利用抗原抗体之间专一性键结之特性，对检体进行检测；由于结合于固体承载物(一般为塑胶孔盘)上之抗原或抗体仍可具有免疫活性，因此设计其键结机制后，配合酵素呈色反应，即可显示特定抗原或抗体是否存在，并可利用呈色之深浅进行定量分析。根据待测样品与键结机制的不同，ELISA可设计出各种不同类型的检测方式，主要以双抗体夹心法（sandwich）、间接法（indirect）、以及竞争法（Competitive）三种为主，以下为各种方法之介绍。

Enzyme-linked immunosorbent assay, also called ELISA, enzyme immunoassay or EIA, is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample. The ELISA has been used as a diagnostic tool in medicine and plant pathology, as well as a quality control check in various industries. In simple terms, in ELISA, an unknown amount of antigen is affixed to a surface, and then a specific antibody is washed over the surface so that it can bind to the antigen. This antibody is linked to an enzyme, and in the final step a substance is added that the enzyme can convert to some detectable signal. Thus in the case of fluorescence ELISA, when light of the appropriate wavelength is shone upon the sample, any antigen/antibody complexes will fluoresce so that the amount of antigen in the sample can be inferred through the magnitude of the fluorescence.

Performing an ELISA involves at least one antibody with specificity for a particular antigen. The sample with an unknown amount of antigen is immobilized on a solid support (usually a polystyrene microtiter plate) either non-specifically (via adsorption to the surface) or specifically (via capture by another antibody specific to the same antigen, in a “sandwich” ELISA). After the antigen is immobilized the detection antibody is added, forming a complex with the antigen. The detection antibody can be covalently linked to an enzyme, or can itself be detected by a secondary antibody which is linked to an enzyme through bioconjugation. Between each step the plate is typically washed with a mild detergent solution to remove any proteins or antibodies that are not specifically bound. After the final wash step the plate is developed by adding an enzymatic substrate to produce a visible signal, which indicates the quantity of antigen in the sample.

Traditional ELISA typically involves chromogenic reporters and substrates which produce some kind of observable color change to indicate the presence of antigen or analyte. Newer ELISA-like techniques utilize fluorogenic, electrochemiluminescent, and real-time PCR reporters to create quantifiable signals. These new reporters can have various advantages including higher sensitivities and multiplexing[1][2]. Technically, newer assays of this type are not strictly ELISAs as they are not “enzyme-linked” but are instead linked to some non-enzymatic reporter. However, given that the general principles in these assays are largely similar, they are often grouped in the same category as ELISAs.
Because the ELISA can be performed to evaluate either the presence of antigen or the presence of antibody in a sample, it is a useful tool for determining serum antibody concentrations (such as with the HIV test[3] or West Nile Virus). It has also found applications in the food industry in detecting potential food allergens such as milk, peanuts, walnuts, almonds, and eggs.[4] ELISA can also be used in toxicology as a rapid presumptive screen for certain classes of drugs.

The ELISA, or the enzyme immunoassay (EIA), was the first screening test widely used for HIV because of its high sensitivity. In an ELISA, a person’s serum is diluted 400-fold and applied to a plate to which HIV antigens are attached. If antibodies to HIV are present in the serum, they may bind to these HIV antigens. The plate is then washed to remove all other components of the serum. A specially prepared “secondary antibody” — an antibody that binds to other antibodies — is then applied to the plate, followed by another wash. This secondary antibody is chemically linked in advance to an enzyme. Thus, the plate will contain enzyme in proportion to the amount of secondary antibody bound to the plate. A substrate for the enzyme is applied, and catalysis by the enzyme leads to a change in color or fluorescence. ELISA results are reported as a number; the most controversial aspect of this test is determining the “cut-off” point between a positive and negative result.

A cut-off point may be determined by comparing it with a known standard. If an ELISA test is used for drug screening at workplace, a cut-off concentration, 50 ng/mL, for example, is established, and a sample will be prepared which contains the standard concentration of analyte. Unknowns that generate a signal that is stronger than the known sample are “positive”. Those that generate weaker signal are “negative.”

ELISA can also be used to determine the level of antibodies in faecal content…specifically the direct method
Before the development of the EIA/ELISA, the only option for conducting an immunoassay was radioimmunoassay, a technique using radioactively-labeled antigens or antibodies. In radioimmunoassay, the radioactivity provides the signal which indicates whether a specific antigen or antibody is present in the sample. Radioimmunoassay was first described in a paper by Rosalyn Sussman Yalow and Solomon Berson published in 1960[5].

Because radioactivity poses a potential health threat, a safer alternative was sought. A suitable alternative to radioimmunoassay would substitute a non-radioactive signal in place of the radioactive signal. When enzymes (such as peroxidase) react with appropriate substrates (such as ABTS or 3,3’,5,5’-Tetramethylbenzidine), this causes a change in color, which is used as a signal. However, the signal has to be associated with the presence of antibody or antigen, which is why the enzyme has to be linked to an appropriate antibody. This linking process was independently developed by Stratis Avrameas and G.B. Pierce[6]. Since it is necessary to remove any unbound antibody or antigen by washing, the antibody or antigen has to be fixed to the surface of the container, i.e. the immunosorbent has to be prepared. A technique to accomplish this was published by Wide and Jerker Porath in 1966.[7]

In 1971, Peter Perlmann and Eva Engvall at Stockholm University in Sweden, and Anton Schuurs and Bauke van Weemen in The Netherlands, independently published papers which synthesized this knowledge into methods to perform EIA/ELISA.[8][9]
双抗体夹心法
双抗体夹心法常用于检测大分子抗原，一般之操作步骤为:

1.将具有专一性之抗体固著（coating）于塑胶孔盘上，完成后洗去多余抗体
2.加入待测检体，检体中若含有待测之抗原，则其会与塑胶孔盘上的抗体进行专一性键结
3.洗去多余待测检体，加入另一种对抗原专一之一次抗体，与待测抗原进行键结
4.洗去多余未键结一次抗体，加入带有酵素之二次抗体，与一次抗体键结
5.洗去多余未键结二次抗体，加入酵素受质使酵素呈色，以肉眼或仪器读取呈色结果
双抗体夹心法分别以两种抗体对检体中的抗原进行两次专一性辨认，因此专一性相当高，但此待测抗原必须是多价抗原，如此才可获得两种以上的专一性抗体，以分别进行夹心；而且此法需要足够的表位空间以进行抗原抗体的夹心，所以并不适用于半抗原或小分子抗原等分子量较小之标的。
间接法
间接法常用于检测抗体，一般之操作步骤为：

1.将已知之抗原固著于塑胶孔盘上，完成后洗去多余之抗原
2.加入待测检体，检体中若含有待测之一次抗体，则其会与塑胶孔盘上的抗原进行专一性键结
3.洗去多余待测检体，加入带有酵素之二次抗体，与待测之一次抗体键结
4.洗去多余未键结二次抗体，加入酵素受质使酵素呈色，藉仪器（ELISA reader）测定塑胶盘中的吸光值（OD值），以评估有色终产物的含量即可测量带测抗原的含量。

竞争法
竞争法是一种较少用到的ELISA检测机制，一般用于检测小分子抗原，其操作步骤为：

1.将具有专一性之抗体固著于塑胶孔盘上，完成后洗去多余抗体
2.加入待测检体，使检体中的待测抗原与塑胶孔盘上的抗体进行专一性键结
3.加入带有酵素之抗原，此抗原也可与塑胶孔盘上的抗体进行专一性键结，由于塑胶孔盘上固著的抗体数量有限，因此当检体中抗原的量越多，则带有酵素之抗原可键结的固著抗体就越少，亦即，两种抗原皆竞相与塑胶孔盘上抗体键结，即所谓竞争法之由来。
4.洗去检体与带有酵素之抗原，加入酵素受质使酵素呈色，当检体中抗原量越多，代表塑胶孔盘内留下之带有酵素的抗原越少，显色也就越浅。
当需要侦测无法获得两种以上单一性抗体的抗原，或是不易得到足够的纯化抗体以固著于孔盘上时，一般会考虑使用竞争法ELISA。