PRINCIPLES ANDPRINCIPLES AND
APPLICATION OFAPPLICATION OF
ELISAELISA
Dr. S. Parasuraman
Faculty of Pharmacy, AIMST Univers...
Enzyme-Linked Immunosorbent Assay
(ELISA)
 ELISA
 Principles
 Types
 Applications
96-well polystyrene plate
What is ELISA?
Enzyme-Linked Immunosorbent Assay (ELISA) is
biochemical assay technique used mainly in
immunology.
It is...
Principle of ELISA
• A sensitive immunoassay that uses an enzyme linked to
an antibody or antigen as a marker for the dete...
Principle of ELISA
 The sensitivity of detection depends on amplification of
signal during the analytic reaction. In some...
Types of ELISA
 Qualitative ELISA
 Postive or Negative results
 Quantitative ELISA
 optical density or fluorescent uni...
Methods/ELISA Methods
 Direct ELISA protocol
 Indirect ELISA protocol
 Sandwich ELISA protocol
 Competitive ELISA prot...
Steps in ELISA
 A general ELISA is a five-step procedure
 coat the microtiter plate wells with antigen
 block all unbou...
General ELISA protocol
Plate Preparation Working concentration of antibody/antigen coated
in 96-well microplate & incubate...
General ELISA protocol
Calculation of Results
Calculate the mean absorbance
for each set of STD, control and
sample and s...
Methods/ELISA Methods
 Direct ELISA protocol
 Direct ELISA is suitable for the detection of
proteinaceous antigens and m...
Methods/ELISA Methods
 In-direct ELISA protocol
 If the primary antibody is not conjugated, then indirect
ELISA is requi...
Apply a sample of known antigen
to a surface
Apply a sample of known antigen
to a surface
Direct ELISADirect ELISA
coated ...
Sandwich ELISA protocol
 The sandwich ELISA measures the amount of
antigen between two layers of antibodies (i.e.
capture...
Sandwich
ELISA protocol
Steps in sandwich ELISA
Prepare a surface to which a known quantity of antibody is
bound.
Apply ...
Methods/ELISA Methods
Applications of ELISA
 Serum Antibody Concentrations
 Detecting potential food allergens
 (milk, peanuts, walnuts, almo...
Furtherreading
 http://www.elisa-antibody.com/ELISA-Introduction/ELISA-
Principle
 http://www.sigmaaldrich.com/life-scie...
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Principles and Applications of ELISA

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Principles and Applications of ELISA

  1. 1. PRINCIPLES ANDPRINCIPLES AND APPLICATION OFAPPLICATION OF ELISAELISA Dr. S. Parasuraman Faculty of Pharmacy, AIMST University, Malaysia
  2. 2. Enzyme-Linked Immunosorbent Assay (ELISA)  ELISA  Principles  Types  Applications 96-well polystyrene plate
  3. 3. What is ELISA? Enzyme-Linked Immunosorbent Assay (ELISA) is biochemical assay technique used mainly in immunology. It is a plate-based assays designed for detecting and quantifying substances such as peptides, proteins, antibodies and hormones. First and most basic test to determine if an individual is positive for a selected pathogen, such as HIV. Dimension of ELISA plant:  8 cm x 12 cm plastic plate which contains an 8 x 12 matrix of 96 wells, each of which are about 1 cm high and 0.7 cm in diameter.
  4. 4. Principle of ELISA • A sensitive immunoassay that uses an enzyme linked to an antibody or antigen as a marker for the detection of a specific protein, especially an antigen or antibody. • ELISA involves detection of “analyte” in a liquid sample using liquid reagent (wet lab) or dry strips (dry lab). • In dry analysis, strip can be read in reflectometry. The quantitative reading usually based on detection of intensity of transmitted light by spectrophotometry at specific wavelength.
  5. 5. Principle of ELISA  The sensitivity of detection depends on amplification of signal during the analytic reaction. In some enzymatic reaction, the signal generated by enzyme which are linked to the detection reagents in fixed proportions to allow accurate quantification (enzyme linked).  There are two main variations on ELISA method is  ELISA can be used to detect the presence of antigens that are recognized by an antibody or  it can be used to test for antibodies that recognize an antigen.
  6. 6. Types of ELISA  Qualitative ELISA  Postive or Negative results  Quantitative ELISA  optical density or fluorescent units of the sample is interpolated into a standard curve, which is typically a serial dilution of the target.
  7. 7. Methods/ELISA Methods  Direct ELISA protocol  Indirect ELISA protocol  Sandwich ELISA protocol  Competitive ELISA protocol or ELISPOT protocol
  8. 8. Steps in ELISA  A general ELISA is a five-step procedure  coat the microtiter plate wells with antigen  block all unbound sites to prevent false positive results  add primary antibody (e.g. rabbit monoclonal antibody) to the wells  add secondary antibody conjugated to an enzyme (e.g. anti- mouse IgG)  reaction of a substrate with the enzyme to produce a colored product
  9. 9. General ELISA protocol Plate Preparation Working concentration of antibody/antigen coated in 96-well microplate & incubate overnight at 4℃ (100 L/ well).μ  Aspirate WITH 300 l washμ buffer(3 times) Aspirate with 300 l wash buffer(3 times) –μ Step-2 Blockplates by adding 300 L of blocking bufferμ  incubate foran hour Aspiration/wash as in step 2 Assay Procedure 100 L of sample/standards in dilution bufferμ  incubate for2 hours at roomtemp. Aspiration/wash as in step 2 Add 100 L of the detection antibodyμ  incubate foran hour Repeat wash  Add 200 L ofμ substrate solution (each well) incubate for20 min at roomtemperature  50 L of stop solutionμ  Avoid placing the plate in direct light
  10. 10. General ELISA protocol Calculation of Results Calculate the mean absorbance for each set of STD, control and sample and subtract the mean zero STD from each. Calibration curve plotted using absorbance (y-axis) against the concentration (x-axis). Unknown concentration determined by interpolation method.
  11. 11. Methods/ELISA Methods  Direct ELISA protocol  Direct ELISA is suitable for the detection of proteinaceous antigens and may require pre- purification of sample. Direct ELISA can be performed when desired antibody is available in a pre- conjugated sate (fluorometric, colorimetric, enzymatic)  Advantages:  Fast  Eliminates possible non-specific binding of secondary antibody  Disadvantages  Reactivity of primary antibody may be reduced conjugation.  Little signal amplification.
  12. 12. Methods/ELISA Methods  In-direct ELISA protocol  If the primary antibody is not conjugated, then indirect ELISA is required in which a conjugated secondary antibody is targeted to the isotype (e.g. e.g., mouse IgG1, goat IgM, rabbit IgG1,k, chicken IgY) of the primary antibody.  Advantages:  Wide variety of secondary conjugates are available for detection  Immunoreactivity of the primary antibody is not compromised.  Multiple binding of the secondary affords some signal amplification  Disadvantages  Extra step in the protocol.  Some non-specific binding of the secondary may cause high
  13. 13. Apply a sample of known antigen to a surface Apply a sample of known antigen to a surface Direct ELISADirect ELISA coated with blocking buffercoated with blocking buffer Detecting antibody added & incubated Detecting antibody added & incubated Wash to remove unbound antibody Wash to remove unbound antibody Apply a substrate which is converted by the enzyme to elicit a chromogenic/ fluorescent signal Apply a substrate which is converted by the enzyme to elicit a chromogenic/ fluorescent signal View/quantify the result using a spectrophotometer or other optical device View/quantify the result using a spectrophotometer or other optical device Apply a sample of known antigen to a surface Apply a sample of known antigen to a surface coated with blocking buffercoated with blocking buffer Detecting antibody added & incubated Detecting antibody added & incubated Wash, second antibodies (to form antigen-antibody complexes), wash Wash, second antibodies (to form antigen-antibody complexes), wash Apply a substrate which is converted by the enzyme to elicit a chromogenic/ fluorescent signal Apply a substrate which is converted by the enzyme to elicit a chromogenic/ fluorescent signal View/quantify the result using a spectrophotometer or other optical device View/quantify the result using a spectrophotometer or other optical device In-direct ELISAIn-direct ELISA
  14. 14. Sandwich ELISA protocol  The sandwich ELISA measures the amount of antigen between two layers of antibodies (i.e. capture and detection antibody).  The antigen to be measured must contain at least two antigenic sites capable of binding to antibody, since at least two antibodies act in the sandwich.
  15. 15. Sandwich ELISA protocol Steps in sandwich ELISA Prepare a surface to which a known quantity of antibody is bound. Apply the antigen-containing sample to the plate. Wash the plate, so that unbound antigen is removed. Apply the enzyme-linked antibodies which are also specific to the antigen. Wash the plate, so that unbound enzyme-linked antibodies are removed. Apply a chemical which is converted by the enzyme into a fluorescent signal. View the result: if it fluoresces, then the sample contained antigen.
  16. 16. Methods/ELISA Methods
  17. 17. Applications of ELISA  Serum Antibody Concentrations  Detecting potential food allergens  (milk, peanuts, walnuts, almonds and eggs)  Disease outbreaks- tracking the spread of disease  e.g. HIV, bird flu, common, colds, cholera, STD etc  Detections of antigens  e.g. pregnancy hormones, drug allergen, GMO, mad cow disease  Detection of antibodies in blood sample for past exposure to disease  e.g. Lyme Disease, trichinosis, HIV, bird flu
  18. 18. Furtherreading  http://www.elisa-antibody.com/ELISA-Introduction/ELISA- Principle  http://www.sigmaaldrich.com/life-science/cell- biology/antibodies/antibodies- application/protocols/elisa.html  http://www.nj.gov/drbc/library/documents/PMP_Resources/ ELISA/definition.pdf

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