ELISA

          ELISA is a useful and powerful method in estimating ng/mL to pg/mL concentrations of materials in the specimen.Qualitative or quantitative immunological procedures in which the Ag-Ab reaction is monitored by enzyme measurements. Term ELISA was first used by Engvall and Perlma in 1971. The ELISA or EIA was the first screening test commonly employed for HIV

ELISA Qualitative / Quantitative 

• Qualitative - Determines if antigen or antibody is present or absent
• Quantitative -Determines the quantity of antigen and titre of antibody.

Antibody (Ab)

• Protein produced by the immune system which help defend against antigens
• Symbol for an antibody

Antigen (Ag)

• Any molecule that induces production of antibodies when introduced in the body of an animal is called an antigen. 
• Symbol for an antigen 
  
                                                                                                                                

Material needed

• Testing sample
• Antibody/Antigen
• Polystyrene microtitre plate
• Washing buffer
• enzyme,Substrate
• Marker pen
• Tissue paper
• Blotting paper
• Micropipettes
• Micro tips
• Discard bin
• Gloves
• Face mask
• Vials for reconstituted solutions
• Samples and controls grid
• ELISA reader
• Automatic washer
• Incubator

Specemens for ELISA

• Serum
• Plasma
• CSF
• Sputum
• Urine
• Semen
• Stool
• Supernatant of culture

Principle of ELISA reader

Types of ELISA

• Indirect
• Sandwich
• Competitive

Indirect ELISA

• Partially purified, inactivated antigens pre-coated onto an ELISA plate.
• Patient serum which contains antibodies binds to the antigens on the plate.
• Anti-human immunoglobulin coupled to an enzyme (second antibody),binds to  human antibodies.
• Chromogen or substrate changes colour when cleaved by the enzyme attached to the second antibody.
• In indirect ELISA colour change is directly proportional to the concentration of specific antibodies in the specimen. 

Sandwich ELISA 

• Antibodies are coated to microwells
• Antigen in the sample binds to the antibody
• The antibody-enzyme conjugate binds to the antigen to form the double antibody sandwich
• Wells are washed to remove excess conjugate
• Substrate is added and colour development is observed.
• Intense colour indicates that the sample is 'Reactive'
• In sandwich ELISA colour change is directly proportional to the concentration of the antigen in the specimen.

Competitive ELISA

• Used to determine small molecule antigens (T3,T4,Progesterone)
• Antibody coated to microwell
• Native antigen & labelled antigen added
• Bound enzyme conjugate reacts with chromogenic substrate.
• Increased serum antigen results in reduced antigen enzyme conjugate to antibody binding producing less enzyme activity (colour)

Importance of steps

• During the test performance, the incubation time and temperature is crucial for proper binding between antigen & antibody and also for the binding of the conjugate and colour development when reacting with the substrate.
• Proper washing is extremely important for removal of any unbound antigen/antibody
• Incubation and washing determine the quality of ELISA results.

Enzymes used in ELISA

• Horse radish peroxidase (most commonly used)
• Alkaline phosphatase
• β-galactosidase
• Lactoperoxidase
• In case of peroxidase, the substrate hydrogen peroxide is converted into water and O2 in the presence of electron donors like dimethylbenzidine which are themselves oxidized in the reaction.

Enzyme substrate

• Initially the substrate should be colourless 
• After degradation by the enzyme it should be strongly coloured

What is cut-off value ?

• Cut-off value is provided in the kits by the manufacture 

• Nothing but a trade-off between sensitivity and specificity - ROC curves find a cut-off that in some way will minimize the false positive and false negative

• Screening tests may require  maximizing of sensitivity 

• PSA tests- may require maximizing of specificity

• 'Grey zone' cut-off which will identify samples that give a response above background but below the cut-off . Such samples are to be re-tested as if they were reactive.

Advantages of ELISA

• Reagents are economical and have a long shelf life
• Highly specific and sensitive
• No radiation hazards occur during labelling or disposal of waste
• Easy to perform,quick procedures
• Equipment inexpensive and widely available
• can be used to a variety of infections.

Disadvantages of ELISA

• Measurement of enzyme activity can be complex
• Enzyme activity may be effected by plasma components
• Very specific to a particular antigen, won't recognize any other antigen
• False positive/negatives possible,especially with mutated or altered antigen.