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IMMUNOASSAY

Name : marwa kamal

ghanim
 IMMUNE & ASSAY

• Immuno refers to an immune response that causes the body to generate

antibodies.
• Assay refers to a test.
• Thus immunoassay is a test that utilizes immunocomplexing when
antibodies and antigens are brought together.
 INTRODUCTIONI IMMUNOASSAYI
• measures the presence or concentration of a macromolecule or a small
molecule in a solution through the use of an antibody (usually) or
an antigen (sometimes). The molecule detected by the immunoassay is often
referred to as an "analyte" and is in many cases a protein, although it may be
other kinds of molecules, of different sizes and types, as long as the proper
antibodies that have the required properties for the assay are developed.
 PRINCIPLE

• Immunoassays rely on the ability of an antibody to recognize and bind a specific

macromolecule in what might be a complex mixture of macromolecules. In immunology the
particular macromolecule bound by an antibody is referred to as an antigen and the area on
an antigen to which the antibody binds is called an epitope.
• In some cases, an immunoassay may use an antigen to detect for the presence of antibodies,
which recognize that antigen, in a solution. In other words, in some immunoassays, the
analyte may be an antibody rather than an antigen.
 CLASSIFICATIONS AND FORMATS

• Competitive, homogeneous immunoassays

In a competitive, homogeneous immunoassay, unlabelled analyte in a sample competes with
labeled analyte to bind an antibody. The amount of labelled, unbound analyte is then measured.
In theory, the more analyte in the sample, the more labelled analyte gets displaced and then
measured; hence, the amount of labelled, unbound analyte is proportional to the amount of
analyte in the sample
 CLASSIFICATIONS AND FORMATS

• Competitive, heterogeneous immunoassays

• As in a competitive, homogeneous immunoassay, unlabelled analyte in a sample competes
with labelled analyte to bind an antibody. In the heterogeneous assays, the labelled, unbound
analyte is separated or washed away, and the remaining labelled, bound analyte is measured.
• One-site, noncompetitive immunoassays
• The unknown analyte in the sample binds with labelled antibodies. The unbound, labelled
antibodies are washed away, and the bound, labelled antibodies are measured. The intensity
of the signal is directly proportional to the amount of unknown analyte.
 CLASSIFICATIONS AND FORMATS

• Two-site, noncompetitive immunoassays

• The analyte in the unknown sample is bound to the antibody site, then the labelled antibody
is bound to the analyte. The amount of labelled antibody on the site is then measured. It will
be directly proportional to the concentration of the analyte because the labelled antibody will
not bind if the analyte is not present in the unknown sample. This type of immunoassay is
also known as a sandwich assay as the analyte is "sandwiched" between two antibodies
 ANTIBODIES
• Antibody is a protein that is produced by body in response to an invading (foreign)
substance.
• These are the key reagents on which the success of any immunoassay depends .The
antibodies can be either polyclonal or monoclonal.
• For immunoassay development ,monoclonal antibodies are more advantageous than
polyclonal ones because they differ from polyclonal antibodies in that they are
highly specific for a single epitope on a monovalent antigen.
 CONTINUED...

• Polyclonal antibodies- antiserum contains a mixture of antibodies that recognise and bind to
the same antigen , but they may attach to different epitopes. An antigen that has multiple
sites for antibodies to bind is called a multivalent antigen.These types of antibodies, present
as a diverse mixture, are called polyclonal antibodies.
• But many successful immunoassays were developed using polyclonal antibodies because it
was possible to generate the antibodies with high affinity to the analyte.
 CONTINUED..

• Antibodies posses high

1. Specificity
2. Affinity For a specific antigen
• lt is the specific binging of an antibody to an antigen that allows the detection of analytes by
a variety of immunoassay methods.
 ANTIGEN

• Antigen is a substance capable of causing an immune response leading to the production of

antibodies and are also the targets to which antibodies will bind.
• The area on an antigen to which the antibody binds is called an epitope.
• Antibodies are antigen specific and only bind to the antigen that initiated their production.
 GENERAL PROCEDURE FOR IMMUNOASSAY

• When these immunoanalytical reagents are mixed and incubated, the analyte is bound to the
antibody forming an immune complex.
• This complex is separated from the unbound reagent fraction by physical or chemical
separation technique.
• Analysis is achieved by measuring the label activity (e.g. radiation,fluorescence or enzyme)
in either bound or free fraction. II
ADVANTAGES OF IMMUNOASSAYS

• High sensitivity-Low detection limit

• High specificity -Detect specific compound ·
• Safe and simple ·
• Fast Tests (between 5 minutes and 1h،our) ·
• Cost effective ·
• Tests can yield quantitative or qualitative data
 DISADVANTAGE OF IMMUNOASSAYS

• Negative results don't always rule out the presence of a drug

• May not be sensitive to be certain compounds
• Some chemists are reluctant to use immunoassay due to its biological basis and their
unfamiliarity with it
 USES OF IMMUNOASSAY
• These measures the presence or concentration of macromolecule or a small molecule in a
solution through the use of an antibody or an antigen. For ex. in analyte fluids urine and
serum.
• These are used in sports anti-doping laboratories to test athletes, blood samples for
prohibited recombinant human growth hormone.
• These are used in analysis of metabolites or biomarkers which indicate disease diagnosis.
• Used in measurements of very low concentrations of low molecular weight drugs.
• Used in therapeutic drug monitoring.
• In clinical pharmacokinetic.
• Used in bioequivalence studies in drug discovery and pharmaceutical industries.
TECHNIQUES OF IMMUNOASSAY

• ELISA
• RIA
• FIA
MAGLUMI 600 IMMUNOASSAY ANALYZER
BY:LIFE CARE ASSOCIATES, CHENNAI