Ex7_Enzyme- Linked ImmunoSorbent Assay(ELISA)

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Ex7_Enzyme- Linked ImmunoSorbent Assay(ELISA)에 대한 보고서 자료입니다.

본문내용

ill 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 a 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 that contains the standard concentration of analyte will be prepared. Unknowns that generate a signal that is stronger than the known sample are "positive." Those that generate weaker signal are "negative."
③ Can ELISA assay be applied for analyze the specific protein-protein interactions?
A detection enzyme or other tag can be linked directly to the primary antibody or introduced through a secondary antibody that recognizes the primary antibody. It also can be linked to a protein such as streptavidin if the primary antibody is biotin labeled. The most commonly used enzyme labels are horseradish peroxidase (HRP) and alkaline phosphatase (AP). Other enzymes have been used as well, but they have not gained widespread acceptance because of limited substrate options. These include β-galactosidase, acetylcholinesterase and catalase. A large selection of substrates is available for performing the ELISA with an HRP or AP conjugate. The choice of substrate depends upon the required assay sensitivity and the instrumentation available for signal-detection (spectrophotometer, fluorometer or luminometer).
④ Indirect ELISA와 Sandwich ELISA의 장단점
*Indirect ELISA 장점
- A wide variety of labeled secondary antibodies are available commercially.
- Versatile because many primary antibodies can be made in one species and the same labeled secondary antibody can be used for detection.
- Maximum immunoreactivity of the primary antibody is retained because it is not labeled.
- Sensitivity is increased because each primary antibody contains several epitopes that can be bound by the labeled secondary antibody, allowing for signal amplification.
- Different visualization markers can be used with the same primary antibody.
*Indirect ELISA 단점
-Cross-reactivity might occur with the secondary antibody, resulting in nonspecific signal.
-An extra incubation step is required in the procedure.
*Sandwich ELISA 장점
- 분석하기 전에 sample을 purify하지 않아도 된다.
- direct나 indirect방법에 비해서 2-5배 정도 민감하다.
- 시료 내 항원의 양이 적거나 다른 간섭물질의 양이 분석하려는 항원의 양보다 훨씬 많을 때 유용하게 활용된다.
*Sandwich ELISA 단점
- 사용해야 할 antibody pair를 test해 보고 최적화 시켜야 하기 때문에 그 과정이 다른 실험보다 어려울 수 있다. 같이 사용하는 antibody pair는 서로 다른 epitope 를 detection 해야 하고, antibody가 binding 하는데 방해하지 말아야 한다.
Reference
http://en.wikipedia.org/wiki/ELISA
http://www.elisa-antibody.com/index.php?page=applications
http://www.dawinbio.com/nlbc/essential/read.html?board_code=a1135&num=12&page=1&list_num=7

키워드

Enzyme-Linked ImmunoSorbent Assay,   ELISA,   에쎄이,   ELISA