ENZYME LINKED IMMUNOSORBENT

ASSAY(ELISA)

DEFINITION AND TYPES

• Is a quantitative immunological procedure in which the Ag-Ab reaction is monitored by enzyme

measurements.

• It is a plate- based assay technique designed for detecting and quantifying substances such as
peptides,proteins,antibodies,and hormones.

• The term ELISA was first used by Engvall and Perlmann in 1972

ELISA BUFFERS

Standard buffers

• Several different buffers are used during an ELISA:

a. coating
b. blocking
c. washing

Coating buffers

• Process where a suitably diluted Ag/Ab is incubated until adsorbed to the surface of the well
 • Adsorbtion occurs passively as the result of hydrophobic interactions between the aminoacid
side chains on the antibody or antigen used for coating the plastic surface

• It is dependent upon the temperature,ph of the coating buffer,concentration of coating agent

Blocking buffers

Washing buffers

• To remove unbound materials

• Usually,PBS with a small concentration of a non-ionic detergent such as Tween-20 is used

• Washing is typically repeated 3-5times between each step in the ELISA

ELISA Antibodies

• Can be monoclonal,polyclonal or combination of both

• Ag-Ab interactions is described in three ways:

SPECIFICITY: is an indication of whether an antibody binds solely to a unique antigen

AFFINITY:describes the strength of binding of an antibody to a single antigen

AVIDITY:accounts for the total stability of the antibody-antigen interaction

ENZYMES

The most commonly used enzyme labels are:

1. Horse radish peroxidase(HRP)

2. Alkaline phosphatase(AP)
3. β-galactosidase
4. acetylcholinesterase
5. catalase
 Plate characteristics

• It maintains consistency,minimizing edge effects and providing optical conditions for data
collection.

• Low to medium binding type(100-200ng of IgG/sqcm)

• High-binding plates (400-500 ng of IgG/sqcm)

• Antigen or antibody pre-coated plates are also commercially available

The technique is divided into :

I. Direct ELISA

II. Indirect ELISA

III. Sandwich ELISA

IV. competitive ELISA

V. Reverse ELISA

Direct ELISA

• A buffered solution of antigen to be tested for, is added to microtiter plates

• A solution of non reacting protein,such as bovine serum albumin or casein is added to each well

• A primary antibody with an attached (conjugated) enzyme is added which binds specifically to
test antigen

• A substrate for this enzyme is added

 ADVANTAGES:

a) Detection is much faster than other Elisa techniques

b) Less prone to error since fewer reagents

c) Best for analyzing the immune response to an antigen

DISADVANTAGES:

a) Antigen immobilization is not specific

b) Less flexible

c) No signal amplification-reduces assay sensitivity

INDIRECT ELISA

 The protein antigen to be tested for is added to each well of ELISA plate, where it is given time
to adhere to the plastic through charge interactions.

 A solution of non-reacting protein is added to block any plastic surface in the well that remains
uncoated by the protein antigen.

 Then the serum is added, which contains a mixture of the serum antibodies, of unknown
concentration, some of which may bind specifically to the test antigen that is coating the well.

 Afterwards, a secondary antibody is added, which will bind to the antibody bound to the test
antigen in the well.

 This secondary antibody often has an enzyme attached to it

 A substrate for this enzyme is then added.

 Often, this substrate changes colour upon reaction with the enzyme.

 The colour change shows that secondary antibody has bound to primary antibody, which
strongly implies that the donor has had an immune reaction to the test antigen.

 The higher the concentration of the primary antibody that was present in the serum, the
stronger the colour change.

 Often a spectrometer is used to give quantitative values for colour strength

ADVANTAGES:

a) Economical

b) High sensitivity

c) Greater flexibility

d) Best for determining total antibody concentration in samples

DISADVANTAGES:

a) Longer procedure than direct ELISA technique

b) Additional incubation step for secondary antibody is needed

SANDWICH ELISA

• The ELISA plate is coated with Antibody to detect specific antigen

METHOD

 Prepare a surface to which a known quantity of capture antibody is bound.

 Block any non specific binding sites on the surface

 Apply the antigen-containing sample to the plate.

 Wash the plate, so that unbound antigen is removed.

 A specific antibody is added,and binds to antigen(hence the sandwich)

 Enzyme linked secondary antibodies are applied as detection antibodies

ADVANTAGES:

• High sensitivity-2 to 5 times more sensitive than direct or indirect ELISA

• High specificity-two antibodies are involved in capture and detection

• Flexibility-both direct and indirect detection can be used

 • Best for analysis of complex samples,since the antigen does not need to be purified prior to
measurement

DISADVANTAGES:

• Antibody optimization can be difficult-cross reactivity may occur between the capture and
detection antibodies

COMPETITIVE ELISA

• Also called as BLOCKING ELISA

• Unlabelled antibody is incubated in the presence of its antigen

• These bound antibody-antigen complexes are then added to an antigen coated well

• The more antigen in the sample, the more Ag-Ab complexes are formed, so less unbound
antibodies available to bind to the antigen in the well, hence COMPETITION

• The secondary antibody coupled to the enzyme is added

• A substrate is added,remaining enzyme elicits chromogenic or florescent signal.

• Wash the plate, so that the unbound antibody-enzyme conjugates are removed.

• Apply a chemical which is converted by the enzyme into a coloured product.

• Measure the absorbency of the plate wells to determine the presence and quantity of antigen
 ADVANTAGES:

• No sample processing is required and crude or impure samples can be used

• More robust-less sensitive to sample dilution and sample matrix effects than the SANDWICH
ELISA

• More consistent-less variability between duplicate samples and assays

• Maximum flexibility-it can be based on direct,indirect,Sandwich ELISA

• Commonly used when only one antibody is available for the antigen of interest

• Suitable for detecting small antigens that cannot be bound by two different antibodies such as in
the sandwich ELISA technique

REVERSE ELISA

• Doesnot use the traditional wells,this test leaves the antigens suspended in the test fluid

Procedure :

• Unlabelled Ab is incubated in the presence of its Ag

• A sufficient incubation period is allowed

• The sample is then passed through the “scavenger container”(this can be a test tube or a
specifically designed flow through channel)

• Surface of scavenger container has “scavenger antigens” bound to it

• These can be identical or similar to primary Ag.

• Scavenger container must have sufficient surface area and time to allow the scavenger antigens
to bind to all excess antibodies introduced into the sample.

• Now the labelled Ab is added.

• The sample is then passed through the detector-flow cytometer.

Uses:

1. It allows multiple antigens to be tagged and encountered at the same time.

2. Specific strains of bacteria can be identified-by 2 or more different colour tags.

DISADVANTAGES OF ELISA

• Measurement of enzyme activity can be more complex than measurement of activity of some
type of radioisotopes.

• Enzyme activity may affected by plasma constituent.

• Kits are commercially available,but not cheap.

• False positive/negative cases is possible,especially in mutated or altered antigen.

An example of ELISA experiment

• Before starting the work read kit instruction carefully

• The 96 titre plate is labelled carefully and the first wells are used to draw the standard curve

• An example of an ELISA experiment-Cont

• The sample is added to plate in duplicate or triplicate and then the mean result is calculated

• The quality control sample which is provided with the kit is treated as the test samples

Results

• After reading the results the standard curve is drawn were the concentration is blotted on the
X-axis and the absorbance on the Y-axis

• The standards concentrations is specified on the x-axis and the reading of each standard is
specified on the y-axis and the standard curve is drawn
• This standard curve is used to determine the unknown concentration of each sample by finding
the opposite concentration to the absorbance

• The quality control sample concentration is determined from the standard curve and if the
result is in the range given by the kit manufacturer the results could be accepted

Applications of ELISA

• For determining Serum Antibody Concentrations

• Monoclonal antibody screening

• Disease outbreaks- tracking the spread of disease

e.g. HIV, bird flu, commoncolds, cholera, STD etc

• Detections of antigens

e.g. drug allergen, mad cow disease

• Toxins in the food-pesticides,botulism

• Hormone levels-hcG,testosterone,ACTH,pregnancy hormones

• Specific disease factors-clotting factor VII

• Detection of antibodies in blood sample for past exposure to disease e.g. Lyme Disease,
trichinellosis, HIV, bird flu

• Detection of Rota virus,E coli in faeces

IN VETERINARY

• Indirect elisa have been developed for the detection of anti-nipah and anti-Hendra IgG
Immunoglobulin

• Capture elisa for detection of anti-nipah IgM immunoglobulin

• ELISA-using excretory-secretory Ag’s from the infective stage larvae-diagnostic test of choice for
Toxocariasis

• Sensitiviy of serum elisa in paratuberculosis is medium to high

• Gold standard test for detecting influenza viral infection in the respiratory system by using anti-
H3N2 antibody

• Serological diagnosis of canine sarcoptic mange

• Determination of anti-aspergillus IgG in the serodiagnosis of canine aspergillosis

• Evaluation of Leishmania recombinant K39 antigen as a diagnostic marker for canine

leishmaniasis.

- submitted by
P.SUMA,
TVM/2019-50,
Department of veterinary biochemistry,
SVVU,Tirupati.