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Enzyme Linked Immunosorbent Assay
- 1. ELISA ( ENZYMELINKED IMMUNOSORBENT ASSAY) Minu Treeza M 1st Mpharm
- 2. IMMUNOASSAY • Immunoassays arebioanalytical methods in which the quantification of the unknown antigen/antibody(analyte)based on antigen-antibody reaction. • Quantification is done by using a specific known labelled antigen/antibody. • Label used- Enzymes , radioactive compounds, chemiluminiscent agents, etc. • Widely used – diagnosis of diseases, TDM, clinical pharmacokinetic and bioequivalence studies,etc.
- 3. ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA) Principle : • Based on the formation of Ag- Ab complex ,which is detected by chromogenic detection using enzyme conjugated secondary antibody/antigen. • An enzyme conjugated with an antibody/antigen reacts with a colourless substrate to generate a coloured reaction product . Such a substrate is called a chromogenic substrate. • The amount of compound corresponds to the substrate converted to coloured product by the enzyme linked antibody. • Light absorption of the product formed after substrate addition is measured and converted to numeric values.
- 5. DIRECT ELISA • Usedfor the detection of antigen in the given biological sample. • Microtiter wells are initially coated with Ag to be detected which is followed by an Ab linked to an enzyme conjugate • This follows addition of substrate which produces color detected using ELISA detector.
- 6. Advantages: • Simple andquick to perform due to minimal steps required Disadvantages: • Specificity of the primary antibody may be affected by the enzyme- linking • Linking 1' antibody for each specific ELISA experiment is expensive and time-consuming • Minimal signal amplification
- 7. INDIRECT ELISA Used forthe detection of antibody in the given sample.
- 8. • Microtiter wellsare initially coated with antigen specific for antibody to be detected. • 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. • 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.
- 10. Advantages: • Specificity ofthe 1' antibody is retained • Many enzyme-linked 2' antibodies are commercially available • Better signal amplification since multiple polyclonal 2' antibodies can bind to each 1' antibody Disadvantages: • Potential cross reactivity with 2' antibody leading to non-specific signal
- 11. SANDWICH ELISA Used forthe detection of antigen in the given biological sample.
- 12. • Antibody iscoated on the microtiter well. • A sample containing antigen is added to the well and allowed to react with the antibody attached to the well, forming antigen-antibody complex. • After the well is washed, a second enzyme-linked antibody specific for a different epitope on the antigen is added and allowed to react with the bound antigen. • Then after unbound secondary antibody is removed by washing. Finally substrate is added to the plate which is hydrolyzed by enzyme to form colored products.
- 13. Advantages: • High specificitysince the signal detection requires the binding of two 1' antibodies • Crude sample can be used: target antigen-antibody complex is immobilized to the plate, and everything else can be washed off Disadvantages: • Commercially-prepared kits may not be available
- 14. COMPETITIVE ELISA • Thistest is used to measure the concentration of an antigen in a sample.
- 15. • Antibody isfirst incubated in solution with a sample containing antigen • The antigen-antibody mixture is then added to the microtitre well which is coated with antigen. • The more the antigen present in the sample, the less free antibody will be available to bind to the antigen-coated well. • After the well is washed, enzyme conjugated secondary antibody specific for isotope of the primary antibody is added to determine the amount of primary antibody bound to the well. • The higher the concentration of antigen in the sample, the lower the absorbance.
- 16. Advantages: • High sensitivity •Crude sample can be used • Signal can be quantified by comparing to a serial dilution standard curve Disadvantages: • Requires 1' antibodies with high specificity to the antigen
- 17.
- 19. ENZYMES AND SUBSTRATES •ABTS – 2,2’-azino-(3- ethylbenzothiazoline-6-sulphonic acid) • TMB (3,3',5,5'- Tetramethylbenzidine) TMB is one of the most versatile, most widely used substrates for ELISA assays. It is typically used in conjunction with HRP and results in a blue color reaction. • pNPP (Para- Nitrophenylphosphate) pNPP is used to detect alkaline phosphatase in ELISA kits.
- 20. Data analysis • Afteradding stop solution, keep the microtiter plate in microtiter reader. • Standard curve of the known concentration of the sample is plotted.
- 21. • For quantitativeand semi-quantitative ELISAs, a Standard Curve is generated using a serial dilution of Standard with a known concentration. • The signal reading (optical density, fluorescence) of each concentration is graphed vs log concentration to produce a sigmoidal curve . • The relatively long linear region of the curve is most accurate and reproducible and is used to generate a linear equation. • The intensity of a sample with an unknown analyte concentration can then be applied to this linear equation to determine analyte concentration.
- 23. ELISAs can bedesigned to yields three different types of results, quantitative, qualitative and semi-quantitative. Quantitative The precise amount of analyte in a test sample can be determined by comparing its values to those generated from a serial dilution of a known, purified analyte (a Standard Curve). This type of result is particularly useful when measuring the effect on a system in response to changing experimental conditions. Qualitative Qualitative assessment simply indicates whether an analyte is present in the sample or not, as compared to a negative and positive control run in parallel. This type of result is commonly used for diagnostic tests, such as detecting the presence of immune response to a particular pathogen. Semi-quantitative Semi-quantitative results indicate the relative amount of analyte within a sample because the strength of the signal obtained will vary proportionately with analyte concentration.
- 24. • ADVANTAGES Reagentsare relatively cheap & have long shelf life. Highly specific and sensitive No radiation hazards occuring during labelling or disposal of waste. Easy to perform Equipment can be inexpensive and widely available. • DISADVANTAGES Mesurement of enzyme activity can be more complex than measurement of some types of radioisotopes. Enzyme activity may be affected by plasma constituents.
- 25. APPLICATIONS • Presence ofantigen or the presence of antibody in a sample can be evaluated. • Determination of serum 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.
- 26. REFERENCE • https://microbiologynotes.com/elisa-principle-types-and-applications • https://www.lsbio.com/resources/devkitfaq#competitive-elisa •https://blog.benchsci.com/elisa-principle • Darwish I. A (2006) Immunoassay methods and their applications in pharmaceutical analysis: Basic methodology and recent advances. International Journal of Biomedical Science.