ELISA

-Dr. Mohammad Aslam Hossain

Enzyme-linked immunosorbent assay, also called ELISA, is a biochemical technique used
mainly in immunology to detect the presence of an antibody or an antigen in a sample. It is also
known as enzyme immunoassay or EIA.
ELISA techniques usually uses an enzyme as a label to determine presence of target protein. The
enzyme linkage or labeling allows to detect the target protein and if present (qualify) and at what
amounts (quantify).
In an ELISA, an antigen must be immobilized to a solid surface. The antigen is then complexes
with an antibody that is linked to an enzyme. Detection is accomplished by incubating this enzyme-
complex with a substrate that produces a detectable product.
Components
Antigen: Any substance that stimulates an immune response. The antigen is the target protein
which comes from the sample extract. The antigen binds to the antibody.
Antibody: An antibody is a protein made in response to an antigen. Each antibody binds only to its
antigen.

Animals are injected with Serum is drawn and antibodies

purified antigen are separated and purified
Enzyme Conjugate
An enzyme conjugate (EC) is an antibody joined with an enzyme. An enzyme conjugate bound or
joined with an antibody which binds with the target protein. This joining of the enzyme to antibody
is often called conjugation. This enzyme labeling is a safe and effective way to track your antibody.
A detection enzyme may be linked directly to the primary antibody or introduced through a
secondary antibody that recognizes the primary antibody.
Enzymes that are used in ELISA
 Horseradish peroxidase
 Calf intestine alkaline phosphatase
 E.coli D -galactosidase
Substrate
A substrate is a compound or substance that undergoes change. A substrate is added after the
antigen antibody reaction. Substrates bind to active sites on the surface of enzymes attached to the
antigen antibody complexes and are converted or changed to color. In ELISA, the specific substrate
are used to change color.
O-phenylenediamine dihydrichloride
P-nitrophenyl phosphate
ELISA Types
• Noncompetitive binding assay or Sandwich methods
• Competitive binding assay

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Noncompetitive binding assay or Sandwich methods: The most commonly used ELISA assay
format is the sandwich assay. This type of assay is called a “sandwich ” assay because the analyte to
be measured is bound between two antibodies– the capture antibody and the detection antibody.
The sandwich format is used because it is sensitive.
Antigen measuring system [Titre wells coated with antibodies; Enzyme labelled antibodies]
Antibody measuring system [Titre wells coated with antigens; Enzyme labelled antiantibodies]
Competitive binding assay: In this method, the antigen is labeled instead of the antibody.
Unlabeled antigen and the labeled antigen compete for binding to the capture antibody, hence
"competition”. The more antigen in the sample, the less antibody will be able to bind to the antigen
in the well and a decrease in signal indicates the presence of the antigen in the sample. Competitive
assays are often used when the antigen is small and has only one epitope, or antibody binding site.
[Titre wells coated with antibodies; Enzyme labelled antigens]
ELISA Methodology
ELISA methods can be designated according to the way in which analytes are captured and
detected.
Capture
Antigen capture ELISA – analyte is captured by the solid phase or microplate
Antibody capture ELISA – an analyte is captured by an antibody attached to the solid phase or
microplate
Detection
Direct ELISA (enzyme conjugate binds directly to the antigen)
Indirect ELISA (enzyme conjugate binds indirectly to the antigen)

Principle of ELISA
To detect antigen, purified antibody specific for that antigen is linked chemically to an enzyme.
The samples to be tested are coated onto the surface of plastic wells to which they bind
nonspecifically; residual sticky sites on the plastic are blocked by adding irrelevant proteins.
The labeled antibody is then added to the wells under conditions where nonspecific binding is
prevented, so that only binding to that antigen causes the labeled antibody to be retained on the
surface.
Unbound labeled antibody is removed from all wells by washing, and the substrate solution is
added. Bound antibody is detected by an enzyme-dependent color-change reaction.

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After a set interval, the reaction is stopped and the concentration of colored product formed is
measured in a spectrophotometer. The intensity of color is proportional to the concentration of
bound antigen.

Principle of Indirect ELISA / sandwich ELISA

• The molecule is detected by antibodies that have been made against it; that is, for which it is
the antigen.
• Antibodies fixed to a solid surface, such as the inner surface of a test tube/micro plate wells
• The tubes/wells are filled with the antigen solution to be assayed. Any antigen molecules
present bind to the immobilized antibody molecules.
• The antibody-enzyme conjugate (a preparation of the same antibodies coupled to an
enzyme) is added to the reaction mixture. The antibody part of the conjugate binds to any

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 antigen molecules that were bound previously, creating an antibody-antigen-antibody
"sandwich".
• After washing away any unbound conjugate, the substrate solution is added.
• After a set interval, the reaction is stopped and the concentration of colored product formed
is measured in a spectrophotometer. The intensity of color is proportional to the
concentration of bound antigen.

PLATE READER
• Machine which measures color density in plate well.
• A specific wave-length of light is chosen (depending upon the color you expect to capture).
• Light is cast from the bottom of the well through the sample.
• No color change wells absorb very little light; wells with color change absorb more light; this is
the optical density or OD.

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Antigen measuring system
Direct Double Antibody Sandwich (DAS)
• Titre wells coated with suitable antibody
• Add sample containing antigen
• Incubate till antigen antibody reaction is complete
• Wash to remove unbound antigen
• Add antibody labeled with Enzyme and incubate till antigen binds labeled antibody
• Wash to remove unbound labeled antibody
• Add substrate & incubate to produce color after enzyme and substrate interaction
• Measure color and color is proportional to antigen in patient sample

Indirect Triple Antibody Sandwich (TAS)

• 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.
• Apply primary antibodies that bind specifically to the antigen.
• Apply enzyme-linked secondary antibodies which are specific to the primary antibodies.
• Wash the plate, so that the unbound antibody-enzyme conjugates are removed.
• Apply substrate which is converted by the enzyme into a color
• Measure the absorbance of the plate wells to determine the presence and quantity of
antigen.

Secondary antibody

Primary antibody

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Antibody measuring system
• Titre wells coated with suitable antigen
• Add sample containing antibody and incubate till antigen antibody reaction is complete
• Wash to remove unbound antibody
• Add antiantibody labelled with Enzyme and incubate till labelled antiantibodies binds
antigen-antibody complex
• Wash to remove unbound labelled antiantibody
• Add substrate & incubate to produce color after enzyme and substrate interaction
• Measure color and color is proportional to antigen in patient sample

Direct Antigen Capture

Indirect Antigen Capture

Competitive binding assay

• Titrewells coated with antibodies
• Add sample containing antigen and antigen labelled with enzyme
• Incubate till antigen antibody reaction is complete
• Wash remove unbound antigens
• Add substrate & incubate to produce color after enzyme and substrate interaction
• Measure color and color inversely related to antigen in sample

Antibody Antigen + Enzyme, Antigen

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Applications of ELISA
• Analysis of hormones, vitamins, metabolites, diagnostic markers – eg. ACTH, FSH, T3, T4,
Glucagon, Insulin, Testosterone, vitamin B12, prostaglandins, glucocorticoids
• Therapeutic drug monitoring – Barbiturates, morphine, digoxin,
• Diagnostic procedures for detecting infection– HIV, Hepatitis A, B etc
• Detection of peptides and other molecules in various diseases
Endocrine diseases: hyperinsulinemia, diabetes, hyperparatyroidism
Tumor antigens (p53 tumor suppressor, PSA, a-foetoprotein)
Antibodies against viral proteins (AIDS, hepatitis)
• Therapeutic:
Neutralizing antibodies: Anti-ErbB2 for breast & ovarian cancer, Anti-CD20 for B-cell non-
Hodgkin's lymphoma
Antisera & antidotes (viruses & venoms)
• Drug discovery: – Identification of therapeutic targets (phage display)
• Detecting allergens in food and house dust
• Measuring toxins in contaminated food

Advantages of ELISA
• Sensitive: nanogram levels or lower
• Reproducible
• Minimal reagents
• Qualitative & Quantitative
– Qualitative eg. HIV testing
– Quantitative assays Eg Ther. Drug Monitoring
• Greater scope : Wells can be coated with Antigens or Antibodies
• Suitable for automation high speed
• No radiation hazards