ELISA.ppt
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This document summarizes ELISA (enzyme-linked immunosorbent assay), an immunoassay technique used to detect the presence and quantify antigens or antibodies. ELISA uses enzyme-labeled antibodies to detect the presence of an antigen or antibody in a solid-phase. It is a sensitive and quantitative technique that can be used to measure levels of antigens or antibodies in samples. There are different types of ELISA including direct, indirect, sandwich, and competitive ELISA depending on the antigen or antibody being detected.
![Chemiluminescent Immunoassay
• Chemiluminescent immunoassays (CLIAs) use
chemiluminescence generating molecules as
labels, such as luminol derivatives, acridinium
esters, or nitrophenyl oxalate derivatives and
ruthenium tri-bipridyl[Ru(bpy)32+] with
tripropylamine for electrochemiluminescence
• Oxidation-reduction reactions generate
light(OH−/H2O2 as a chemical trigger)](https://image.slidesharecdn.com/elisa-230125094052-d925df47/85/ELISA-ppt-37-320.jpg)
ELISA.ppt
- 1. ELISA
- 2. ELISA ELISA= enzyme linked immunosorbant assay ELISA or EIA is an immunoassay which uses enzyme labeled antibodies for the quantitatve measurement of antigen or antibody mostly in a solid phase
- 3. Immunoassay An immunobiochemical reaction with the help of which we can measure(both qualitatively or quantitaively) the levels of an antigen or an antibody in a test sample.
- 4. Antigen
- 5. Hapten
- 6. Antibody
- 8. Solid phase • Agarose gel • Polyacrylamide • Plastic beads • Polystyrene • Iron oxide coated particles • Streptavidin • NeutrAvidin
- 9. StreptAvidin • Streptavidin is a tetrameric protein, which can bind four biotins per one molecule; each monomer binds one molecule. (Figure 1) • Biotin binds to streptavidin with a very high affinity ( Kaff = 1013 M-1 ) • Streptavidin/biotin complexes are very stable over a wide range of temperature and pH • Streptavidin doesn’t have any electrical charge in neutral or slightly basic; it doesn’t cause for example unspecific electrostatic binding • Because the binding of compounds on streptavidin plates is based on the strong affinity of streptavidin towards biotin, the compounds must be biotinylated prior to use
- 10. NeutrAvidin • Near-neutral isoelectric point (6.3) and no glycosylation, unlike avidin • No RYD recognition sequence like streptavidin • Generally lower nonspecific binding than avidin and streptavidin
- 11. Enzyme labeling Peroxidase Horseradish plant MW= 40,000 Turnover rate = 10,000 Periodate oxidation(Nakane method) TMB =Tetramethylbenzidine ABTS =Azino-bis ethylbenzthiazoline sulfonic acid HPPA=Hydroxyphenylpropionic Acid , Alkaline phosphatase Bovine intestine MW=100,000 Turnover rate=250,000 Glutaraldehyde method pNPP = p-Nitrophenyl phosphate MUP = 4-Methylumbelliferyl phosphate Beta-galactosdase E. Coli MW=530,000 Turnover rate=318,000 Dimaleimide method
- 12. Classification of immunoassays • Precipitation IA • Particle IA • Radioimmunoassay • Enzyme linked IA • Fluorescence IA • Chemiluminescence IA
- 13. Types of ELISA Heterogenous • Direct • Indirect • Sandwich • Competitive • Multiplex • Fluorescent Enzyme Immunoassay • Chemiluminescent Enzyme Immunoassay(CL-EIA) Homogenous • Mention ahead
- 14. Direct ELISA
- 15. Direct ELISA Advantages • Quick • No cross reactivity Disadvantages • Reduced immunoreactivity • Expensive
- 16. Indirect ELISA
- 17. Indirect ELISA Advantages • More sensitive • More imunoreactive primary antibodies • Versatile as labelled secondary ab shoudnt be antigen specific Disadvantages • Less specific due to cross reactivity by sec. ab. • Time consuming bcz of extra incubation step
- 18. Sandwich ELISA
- 19. Sandwich ELISA Advantages 1.Can measure very small amount of an antigen 2.Antigen purification is not required 3.Very specific 4. Signal amplification Disadvantages 1.Multivalent antigens only 2.Match pairing should be done to avoid hindrance of binding sites.
- 21. Competitive ELISA Advantages • Suitable for complex samples, since the antigen does not require purification prior to measurement • Flexibility and sensitivity, since both direct and indirect detection methods can be used • No hook effect • No match pairing is required Disadvantages • Cross reactivity
- 22. Standard curve for noncompetitive ELISA
- 23. Fish Hook effect Tumor markers hCG Ferretin CRP Dilution Competitive Beer’s law
- 25. The Matrix effect • Serum or plasma sample is a complex compound of lipids, proteins, carbohydrates, salt and water. The sum of interferences of all sample components (with exception of analytes), which affect the target analyte to be measured is known as “the matrix effect” • Example–Heparin therapy in patients with acute myocardial infraction (AMI) affects the result of determination of troponin I concentration
- 26. The Edge effect • Temperature differences occur between wells in the middle and at the edge of the microtiter plate • Temperature difference can cause change in reaction kinetics • A difference of 2 degree has been noted • Use incubators
- 27. Homogenous immunoassay COMPETETIVE CONJUGATE Modulation manner EMIT Ag + lysozyme or G6PD Steric hindrance SLFIA Ag + substrate ‘’ ARIS Ag + prosthetic group ‘’ ENZYME CHANNELING Ag + G6PD or hexokinase Enhancement to proxmity BIOTIN ENZYME AVIDIN Ag + avidin Steric hindrance CEDIA Ag + fragments of subunits ‘’ NONCOMPETETIVE HYBRID ANTIBODY Hybrid ab specific to ag or to inhibitor ‘’ PROXIMAL LINKAGE Antibody to G6PD & hexokinase Substrate cascade by proxmity EHIA Ab to amylase Steric hindrance ENZYME ENHANCEMENT Ab to beta galactosidase Charge effect AEST Ab to peroxidase stabilization
- 28. Enzyme Multiplied IT Rubenstin1972 Enzyme act= free haptens G6PD LYSOZYME
- 29. Substrate Labeled Fluoroscent IA Burd 1977 Substrate=B-galactosylumbeliferone Enz=beta galactosidase
- 30. Apoenzyme Reactivation IS MORRIS1981 APOGLUCOSE OXIDASE
- 31. Cloned Enzyme Donor IA Handerson 1986 Beta-galactosidase genetically engineered
- 32. Enzyme Inhibitory Homogenous IA Ashihara1988 HMW antigen=ferretin,AFP Alfa-amylase,Dextranase noncompetitive
- 33. Advantages and Disadvantages of Enzyme Immunoassay A. Advantages 1. Sensitive assays can be developed by the amplification effect of enzymes. 2. Reagents are relatively cheap and can have a long shelf life. 3. Multiple simultaneous assays can be developed. 4. A wide variety of assay configurations can be developed. 5. No radiation hazards occur during labeling or disposal of wastes. B. Disadvantages 1. Measurement of enzyme activity can be more complex than measureme of the activity of some types of radioisotopes. 2. Enzyme activity may be affected by plasma constituents. 3. Homogeneous assays at the present time have the sensitivity of 10−9 M and are not as sensitive as radioimmunoassays.
- 34. ELISPOT
- 37. Chemiluminescent Immunoassay • Chemiluminescent immunoassays (CLIAs) use chemiluminescence generating molecules as labels, such as luminol derivatives, acridinium esters, or nitrophenyl oxalate derivatives and ruthenium tri-bipridyl[Ru(bpy)32+] with tripropylamine for electrochemiluminescence • Oxidation-reduction reactions generate light(OH−/H2O2 as a chemical trigger)
- 41. Preis Biochemische Analytik, Munich, April 1976.