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ELISA
- 1. ELISA- Principle, Types and Applications ELISA- 牀牆牆牀橿牀牆牆牀橿, 牀朽牆牆牀橿 牀牀園牀園牀牆 牀牀牀牆牀牆牀牆牆牀橿
- 2. ELISA stands for Enzyme Linked Immunosorbent Assay (牀 牆牀む-牀牀牆牀牆牀牀牆牀牀牆牀 牀 牆牀牆 牀む牆牀牆牀牆牀園牀園迦 牀牀牆牀朽)
- 3. Immunology - 牀む牆牀牆牀牆牀園牀園迦逗牀迦; 牀む牆牀牆牀牆牀園牀園逗牀迦; 牀む牆牀牆牀牆牀む牀む逗園牀逗牀迦 Immune system - 牀 牆牀牆 牀牀む逗萎牀牆牀牆 牀牀牆牀牀迦牆;牀 牆牀牆 牀牀む逗萎牀牆牀む 牀牀む牀牆牀む Antigen - 牀牀牀園牀牆牀牆牀牆 牀牀牆牀牀; 牀牀む逗萎牀牆牀牆牀牀; 牀牀逗園牀牆牀萎牀牆牀む逗萎逗牆牀牆牀牀; 牀牀む逗萎逗牆牀牆牀牀 Antibody - 牀牀牀園牀牆牀牆牀牆 牀牆牀迦牆; 牀牀む逗萎牀牆牀牀牆牀萎牀橿; 牀牀逗園牀牆牀萎牀牆牀む逗萎 Immunity - 牀 牆牀牆 牀牀む逗萎牀牆牀牆牀む 牀む逗園牆; 牀 牆牀牆牀む 牀む牆牀む逗園牆; 牀 牆牀牆牀む 牀む牀牀牆 牀牆牀牆牀牆
- 6. 牀牀朽牀 牆牀萎 牀 牆牀む逗牀逗迦牀牆 牀牀逗 牀牆牀園逗牆牀牀逗牆牀 牀牀牀逗 牆 牀牀牀逗迦む 牀牀む牀牆牀鉦牀牀橿 牀牆牆牀鉦牀牆牀む 牀朽逗 牀牀牆牀牀牆牀萎牀橿 (牀牆牀巌牀牆牀牀牀) 牀牆牀む 牀牀逗牆 牀む園牀牆 牀蹍園牀園牀園牀牀牆牀迦 牀牀萎 牀牆牀牆牀牀萎逗牆牆 牀牀牀牀牆牀牀 牀牀萎 牆牀牆牀牆牀牀逗牆牀 . 牀牀朽牀朽逗牀牀 牀牀牀牆牀牀逗牀牆 牀牀迦牀迦む 牀牀牀逗萎牀牆牀牆 牀牀逗牀迦牀牆 (Active Site) 牀 牀牆牀牀牆牀牆. 牀牀む 牀む牀牆 牀牀萎 牀 牆牀む逗牀逗牆 牀む牀逗む牀む牆牀牀牀牀 (specificity) /牀む牀迦牀迦逗牀む牀む牆牀牀牀牀 牀牆牀牀逗朽 牀牆牀牆牀牀逗牆牀 .
- 7. ELISA is an antigen antibody reaction It is a common laboratory technique which is usually used to measure the concentration of antibodies or antigens in blood.
- 8. ELISA 牀牀牆牀牀む 牀牀逗園牀牆牀萎牀牆牀む逗萎逗牆牀牆牀牀(Antigen) 牀牀逗園牀牆牀萎牀牆牀む逗萎(Antibody) 牀牀む逗萎牀朽逗 牀牀牆牀牆. 牀牀む 牀巌牆牀牀牆牀 牀牀萎む牀むむ牀む逗迦 牀牀逗園牀牆牀萎牀牆牀む逗萎逗牀橿 牀牀迦牀迦む 牀牀逗園牀牆牀萎牀牆牀む逗萎逗牆牀牆牀牀逗牀橿逗牆 牀牆牀園逗 牀牆牀朽逗牆 牀む園牀牆 牀牀牀牆牀牀牆牀む牀む牆牀牀牆牀牆 牀牀萎 牀牀牆牀む 牆 牀牀牆 牀 牀牆牀牆牀牀牆牀牆牀牆.
- 9. ELISA is a plate based assay technique which is used for detecting and quantifying substances such as peptides, proteins, antibodies and hormones. ELISA 牀牀牆牀牀む 牀牀萎 牀む牆牀牆 牀牀牀逗牆牀牀牀牀牀逗迦 牀牀む逗牆牀牆牀牆牀牆 牀牆牀牆牀牀牆牀牆牀牆, 牀牀む 牀牀牀牆牀牀牀牆牀伍, 牀牆牀萎む牆牀牀橿, 牀牀逗園牀牆牀萎牀牆牀む逗萎逗牀橿 牀牀園牀園牀牆 牀項牀萎牀牀牆牀牆牀牀橿 牀牀牆牀牆牀 牀牀牆牀萎牀牆牀牀牆 牀牀牆牀牀園逗 牀牀園牀園牀牆 牀牆牀朽逗牆 牀む園牀牆牀牆 牀牀牀牆牀牀牆牀む牀む牆牀牀牆牀牀逗園む.
- 10. An enzyme conjugated with an antibody reacts with colorless substrate to generate a colored product. Such substrate is called chromogenic substrate. 牀牀萎 牀牀逗園牀牆牀萎牀牆牀む逗萎逗牆牀牀牆 牀牀牆牀牆牀 牀牀萎 牀 牆牀む,牀牀逗園牆牀牀牆牀萎牀橿 牀牀牆牀園逗 牀牀萎 牆牀牆牀 牀牀逗園牀園牀 牀牆牀巌牀牆牀牀牀牀牆牀牀牆 牀牆牀牀迦牀牀牆牀牀逗園む. 牀牀む牀む牀牀 牀牆牀巌牀牆牀牀牀, 牀牆牀牀萎牀牀牆牀牀逗牆 牀牆牀巌牀牆牀牀牀 牀 牀牀牀巌牆牀牀牆牀牀牆牀牀逗園む.
- 11. A number of enzymes have been used for ELISA such as alkaline phosphatase, horse radish peroxidase and beta galactosidase. Specific substrates such as ortho-phenyldiamine dihydrochloride (for peroxidase), paranitrophenyl phosphate (for alkaline phosphatase) are used which are hydrolysed by above enzymes to give colored end product.
- 12. Principle ELISAs are typically performed in 96-well polystyrene plates. ELISA牀牀橿 牀牀牆牀む 牆牀 96 牀む牀牆牀牀橿 牀牀牆牀牆牀 牀牆牀迦牀牆牀牀逗萎逗牀萎逗牆 牀む牀牆牀牀橿逗迦 牀牀逗牀巌牀む牀む牆牀牀牆牀牀逗牆牀 . The serum is incubated in a well, and each well contains a different serum.
- 13. A positive control serum and a negative control serum would be included among the 96 samples being tested. 牀牀萎 牀 牀萎牀牀牀園牆 牀牀牆牀牆牀牆牀牆牀牆牀牆 牀牆牀萎牆 牀牀園牀園牀牆 牀牀萎 牀牀む逗萎牀牀牀 牀牀牆牀牆牀牆牀牆牀牆牀牆 牀牆牀萎牆 牀牀牀逗牀 96 牀牆牀む逗萎逗牀橿逗迦 牀牀橿牆牀牀牆牀牆牀牆. Antibodies or antigens present in serum are captured by corresponding antigen or antibody coated on to the solid surface.
- 14. After some time, the plate is washed to remove serum and unbound antibodies or antigens. To detect the bound antibodies or antigens, a secondary antibodies that are attached to an enzyme are added to each well. 牀牀逗牆牀牆牀牀牆牀牀牆牀 牀牀逗園牀牆牀萎牀牆牀む逗萎逗牀橿 牀牀迦牀迦む 牀牀逗園牀牆牀萎牀牆牀む逗萎逗牆牀牆牀牀逗牀牆 牀牀牆牀牀園逗, 牀牀萎 牀 牆牀む逗牆牀牆 牀牀牆牀牆牀牀牆牀牀牆牀牆牀橿牆 牀牀萎牆牀牆牀牆 牀牀逗牀 牀牀逗園牀牆牀萎牀牆牀む逗萎逗牀橿 牀牀朽牀 牆牀牆牀園逗迦牀牆 牀牆牀萎牀牆牀牀牆牀牀牆牀牀逗牆牀
- 15. After an incubation period (牀牀牀牀牆牀牆牀牆牀牆 牀牆牀迦牆), the unbound secondary antibodies are washed off. When a suitable substrate (牀牆牀巌牀牆牀牀牀) is added, the enzyme (牀 牆牀む逗牀牆) reacts with it to produce a color. This color produced is measurable as a function or quantity of antigens or antibodies present in the given sample.
- 16. Types of ELISA Frequently there are 3 types of ELISA on the basis of binding structure between the Antibody and Antigen. Indirect ELISA (牀牀園牀牆牀 ELISA) Sandwich ELISA (牀牀鉦牆牀牆牀朽逗牆 ELISA) Competitive ELISA (牀 牀鉦牆牀牀 ELISA)
- 17. Indirect ELISA (牀牀牀園牆牀 ELISA)
- 18. In this technique, antigen is coated on the microtiter well. Serum or some other sample containing primary antibody is added to the microtiter well and allowed to react with the coated antigen. Any free primary antibody is washed away and the bound antibody to the antigen is detected by adding an enzyme conjugated secondary antibody that binds to the primary antibody.
- 19. Unbound secondary antibody is then washed away and a specific substrate for the enzyme is added. Enzyme hydrolyzes the substrate to form colored products. The amount of colored end product is measured by spectrophotometric plate readers that can measure the absorbance of all the wells of 96-well plate.
- 20. Sandwich ELISA (牆牆牀牆牀牆牀朽逗牆 ELISA)
- 21. In this technique, antibody is coated on the microtiter well. A sample containing antigen is added to the well and allowed to react with the antibody attached to the well. After the well is washed, a second enzyme- linked antibody is allowed to react with the bound antigen.
- 22. Then after unbound secondary antibody (牀牀萎牆牀牆牀牆牀牀逗牀 牀牀逗園牀牆牀萎牀牆牀む逗萎) is removed by washing. Finally substrate (牀牆牀巌牀牆牀牀牀) is added to the plate which is hydrolyzed(牀牆牀萎牀牆牀牀牆牀牆牀牆) by enzyme to form colored products.
- 23. Competitive ELISA (牀牀牆牀牆牀牀 ELISA) Antibody (牀牀逗園牀牆牀萎牀牆牀む逗萎) is first incubated in solution with a sample containing antigen (牀牀逗園牀牆牀萎牀牆牀む逗萎逗牆牀牆牀牀) The antigen-antibody (牀牀逗園牀牆牀萎牀牆牀む逗萎逗牆牀牆牀牀 - 牀牀逗園牀牆牀萎牀牆牀む逗萎 )mixture is then added to the microtitre we which is coated with antigen.
- 24. The more the antigen present in the sample, the less free antibody will be available to bind to the antigen- coated well. 牀牀逗園牀牆牀萎牀牆牀む逗萎逗牆牀牆牀牀逗牀橿 牀牆牀朽 牀牀む逗牀牆牀 牀牆牆牀牆牀牀牆牀牆 牀牀牆牀む ,牀 牀園牀園牀牀牆 牀牀牆牀牆牀 牀牀逗 牀牀牆牀萎牀橿 牀牀む逗萎逗牀橿逗牆 牀牆牀朽 牀牆牀牀 牆牀 牀牆牆牀牆牀牀牆牀牆 After the well is washed, enzyme conjugated secondary antibody specific the primary antibody is added to determine the amount of primary antibody bound to the well. For competitive ELISA, the higher the sample antigen concentration, the weaker the eventual signal.
- 26. Application Presence of antigen or the presence of antibody in a sample can be evaluated. Determination of serum (牀牆牀萎牀む逗牆牀萎牀牆牀牆牀牀牆) antibody concentrations in a virus test. Used in food industry when detecting potential food allergens . Applied in disease outbreaks- tracking the spread of disease e.g. HIV, bird flu, common, colds, cholera, STD etc.