Enzyme Linked Immuno-Sorbant Assay

• This test was first described by VanWeeman in 1971 and by

Engval & Perlman in the same year.
• This test is qualitative as well as quantitative.
• This test is used for the detection of antigen or antibodies.

• Principle:
– When enzyme conjugated specific antibodies react with specific antigen,
substrate present in the system will give color to the system due to
availability of enzyme.
– This reaction produces a specific color, which indicates a positive result.
 Components of ELISA
1. Solid Phase

2. Specific Abs

3. Enzymes

4. Substrate

5. Elisa Reader
 Solid Phase
• It consists of nitrocellulose membrane and ELISA plates just like
micro-titration plates, having 12 columns and 8 rows and may be
made up of polystyrene/polyvinyl material.
• These materials have charged surfaces.
• They may be V, U or flat-shape bottom.
 Specific Antibodies
• These antibodies are of 2 types:
– Antibodies against a particular Ag, which are conjugated with enzymes.

– Anti-species Abs are the antibodies which are raised against the
antibodies of a particular species like Rabbit anti-chicken antibodies
means antibodies raised in rabbit against chicken antibodies (Globulins).

• These antibodies are available in commercial forms.

 Method of raising Anti-species Abs
• Days Injections

• 1 0.2 ml
• 3 0.4 ml
• 5 0.6 ml
• 7 0.8 ml
• 9 1.0 ml
 Enzymes
• Enzymes commonly used in ELISA are:
– Horse Reddish PerOxidase (HRPO)
– Alkaline phosphatase
• Other enzymes used in ELISA are:
– β-D-glactosidase
– Urease
– Penicillinase
• Enzyme should have following characteristics:
– High turnover rate
– Readily coupled to proteins
– Cheap
– Resistant to high temperatures
 Substrate
• Enzyme substrate reaction is specific.
Substrates produce specific colors.
– ABTS (Blue)

– DiAminoBenzidine (DAB) (Dark Brown)

– TetraMethyleneBenzedine (TMB) (Blue)

– p-NitroPhenyl Phosphate (pNPP) (Yellow)

 ELISA Reader
• It is an advanced form of Spectro-photometer. The principle of it
is that concentration of solution is directly proportional to the
absorbance.

• Concentration α Absorbance

• Qualitative ELISA
• Quantitative ELISA
 Direct ELISA
S S S
4
S S S

3
3

1 1

2 2
Indirect ELISA (Ag-Detection)
S S S

3
1
2
Indirect ELISA (Ab-Detection)
S S S

3
1

2
 Competitive ELISA (Ab-Detection)
• Competitive ELISA is a technique used for the estimation of antibodies
present in a specimen, such as serum.
• Principle of the test is that two specific antibodies, one conjugated with
enzyme and the other present in test serum are used.
• Competition occurs between the two antibodies for the same antigen.
• Appearance of color indicates a negative test (absence of antibodies)
• Absence of color indicates a positive test (presence of antibodies)
• The central event of competitive ELISA is a competitive binding process
executed by original antigen (sample antigen) and add-in antigen.
• The procedures of competitive ELISA are different in some respects
compared with other forms of ELISA.
Competitive ELISA (Ab-Detection)