ELISA

types of ELISA

1. ELISA
 ELISA is an immunological technique used for the quantitative
determination of the concentration of certain antigens or
antibodies
 First introduced in 1971 by engvall and perlmann
 Is similar in principle to RIA but depends on an enzyme rather
than a radioactive label
 An enzyme conjugated with an antibody reacts with a colorless
substance to generate a colored reaction product such a
substance is called chromogenic substrate
 Alkaline phosphatase, horseradish peroxisase urease and beta
galactosidase are the enzymes used in ELISA
 Used for the qualitative and quantitative measurement of either
antigen or antibody



Indirect ELISA:’
 Used to detect and measurement of antibody
 The antigen of interest is first coated on the well
 Washed to remove excess antigen
 Test antiserum is added
 Specific antibody will bind to the antigen forming complex
 Washed to remove excess antibodies
 Enzyme conjugated antibody(secondard antibody) is added
 Secondary antibody will bind to the primary antibody
 Washed to remove excess secondary antibody
 Substrate is added
 Colour changes due to the hydrolysis of substrate by the enzyme
conjugated to secondary antibody

2. 
 Cross reactivity may occur due to the secondary antibody-main
disadvantage
Direct ELISA or sandwich ELISA:
 Used to detect and measurement of antigen
 Antibody of interest is first coated on the well
 Washed to remove excess antibody
 Test antigen is added
 Specific antigen will bind to the specific antibody forming
complex
 Washed to remove excess antigens
 Enzyme conjugated secondary antibody is added
 Secondary antibody will bind to the specific antigen
 Washed to remove excess antibody
 Substrate is added
 Colour changes as a result of hydrolysis of substrate
 The amount of product is measured by spectrophotometric plate
readers

3.  High specificity, since two antibodies are used the antigen is
specifically captured and detected.

Competitive ELISA:
 used to measure the concentration of an antigen in an sample
 in this test antibody is first incubated in solution with a sample
containing antigen
 the antigen antibody mixture is then added to the well which is
coated with antigen
 more the antigen present in the sample the less free antibody
will be available to bind to the antigen coated well
 then enzyme conjugated secondary antibody specific for primary
antibody is added to determine the amount of primary antibody
bound to the well
 higher the concentration of antigen in the sample the lower the
absorbance


application of ELISA:
 presence of antigen or the presence of antibody in a sample can
be evaluated
 determination of serum antibody concentration in a virus test
 used in food industry when detecting potential food allergens
 tracking the spread of disease e.g, HIV, bird flu, cholera, STD
 detection of antibodies in blood sample for past exposure to
disease