RADIOIMMUNO ASSAY AND ENZYME LINKED IMMUNOSORBANT
ASSAY
SUBMITTED BY:
PANDEY AJAY SANJAY
M.PHARMA 1st Year
PHARMACEUTICS
SUBMITTED TO:
Dr. KAISAR RAZA
ASSISTANT PROFESSOR
DEPARTMENT OF PHARMACY
CENTRAL UNIVERSITY OF RAJASTHAN

• Introduction
• RadioImmunological Assay
• Advantages and Disadvantages Of RIA
• Enzyme linked immunosorbant Assay
• Types Of ELISA
• Advantages and Disadvantages of ELISA
• Detection Of Results
• Aplication of ELISA and RIA
• Referenes

• IMMUNOLOGICAL ASSAY:
• It is a Highly selective Bio-analytical method that measures the Presence or
Concentration of analyte ranging from small molecule to macromolecule in a
solution through an Antigen or Antibody as biorecognition agenet.
• Play a critical role in Clinical Diagnostic, Bio-Pharmaceutical techniques, Food
Testing etc
• The investigation on the antigenicity of small molecules by Landsteiner in 1917
has provided the framework for the development of immunological assays for
detection of low molecular weight compounds.
• In typical immunoassay the antibody act as a reagent to detect analyte(antigen)
of intrest.

• RIA (1959) by Solomon,Belson,and yalow for estimation
of Insulin in Human Serum.
• This technique is used for the biological fluids that are
found in very low concentration(nanogram or picogram).
• PRINCIPLE:
It involves a combination of three principles.
• An immune reaction i.e. antigen, antibody binding.
• A competitive binding or competitive displacement
reaction. (It gives specificity)
• Measurement of radio emission.

Advantages of RIA (Radioimmunoassay)
• It is an extremely sensitive assay as it can measure antigen up to picogram
quantities.
• It is a highly specific test as the antibody-antigen reaction is highly specific.
• A large number can be processed.
• It is an indirect method of analysis.
Disadvantages of RIA (Radioimmunoassay)
• Radiation hazardous .
• Require special arrangements for storage of radioactive material.
• The high cost of waste disposal.
• Lengthy counting time
• There are some difficulties in the automation of this assay.
• The reaction time is long due to the use of highly diluted reagent.

• ELISA works on the principle that specific antibodies bind the target
antigen and detect the presence and quantity of antigens binding.
• The enzyme-linked immunosorbent assay (ELISA) is an immunological
assay commonly used to measure antibodies, antigens, proteins and
glycoproteins in biological samples.
• Some examples include: diagnosis of HIV infection, pregnancy tests,
and measurement of cytokines in cell supernatant or serum.
• ELISA assays are generally carried out in 96 well plates, allowing
multiple samples to be measured in a single experiment.
• These plates need to be special absorbant plates (e.g. NUNC Immuno
plates) to ensure the antibody or antigen sticks to the surface.

TYPES OF ELISA
DIRECT ELISA INDIRECT ELISA SANDWITCH ELISA COMPETITIVE ELISA
(antigen-coated
plate; screening
antibody)
(antigen-coated
plate; screening
antigen/antibody)
(antibody-coated plate;
screening antigen)
(screening antibody
andAntigens)
TYPES OF ELISA

DETECTION:
• Horseradish peroxidase (HRP)-
Blue color
• Alkaline phosphatase (ALP)-
Yellow color
FOR WASHING:
Phosphate-buffered saline (PBS)
FOR SAMPLING
• Polystyrene plates, typically in
96-well plates.

Blood sample collected( SERUM In PHOSPHATE
BUFFER SOLUTION)
Added to Microtiter plates(Antigens adhered to
plates)
BLOCKING is done by adding BOVINE SERUM
ALBUMIN
ENZYME linked PRIMARY ANTIBODIES
added(binds to antigens if present)
SUBSTRATE is added which binds to ENZYME to
give product as generation of COLOUR
ANTIGENS adhered plate is collected
BLOCKING is done by adding BOVINE SERUM ALBUMIN
Blood sample collected ( SERUM is placed In PHOSPHATE
BUFFER SOLUTION)
( PRIMARY ANTIBODY adhered to ANTIGENS ALREADY
PRESENT IN WELL)
ENZYME linked antibodies added against Primary
Antibodies
(Primary Antibodies acts as a Antigen for Enzyme lined
Ab)
SUBSTRATE is added which binds to ENZYME to give
product as generation of COLOUR

CAPTURED ANTIBODY(specific to HIV) is added to
Microtitere plate
BLOCKING is done by adding BOVINE SERUM ALBUMIN
Blood sample collected ( SERUM is placed In PHOSPHATE
BUFFER SOLUTION)
Added to wells( Antigens specific to HIV Antibodies binds
with them
Different detector protiens are present on antigens
surface(EPITOPES)
ENZYME linked secondary antibodies(DETECTION
ANTIBODIES) added
Binds to other epitope present on antigens surface
SUBSTRATE is added which binds to ENZYME to give
product as generation of COLOUR
DONE WHEN IT IS CONFIRMED THAT PATIENT HAS SPECIFIC
ANTIGENS(CONCENTRATION DETERMINATION)
CAPTURED ANTIBODY(specific to HIV) is added to
Microtitere plate
BLOCKING is done by adding BOVINE SERUM ALBUMIN
Blood sample Colleted ( SERUM is placed In PHOSPHATE
BUFFER SOLUTION)
ENZYME linked antigens specific to antibodies added
Both antigens gets adhered to antibodies depending on
their concentration
SUBSTRATE is added which binds to ENZYME to give
product as generation of COLOUR
Depending on the colour produced, concentration of
antigens were determined.

• Data gathered from ELISA tests can be quantitative, qualitative, or
semiquantitative.
• The quantitative concentration results are plotted and compared to a standard
curve.
• The qualitative results confirm or deny the presence of a particular
antigen/antibody in a sample.
• The semiquantitative results compare the intensity of the signals, which can
compare relative antigen levels in a sample.
• Once color changes are measured from the assay, the results are graphed either
on paper or software.
• Typically, the graph compares optical density to log concentration, which gives a
sigmoidal curve.

• It used to check the plasma level of
hormones.
• Also used in the analysis of vitamins
• Also used in the analysis of anti-
DNA antibody in systemic lupus
erythematosus.
• It is also used in the early detection
of cancer.
• Also used in the research of brain
chemicals called neurotransmitter.
• The presence of antibodies and
antigens in a sample can be
determined.
• It is used in the food industry to detect
any food allergens present.
• To determine the concentration of
serum antibody in a virus test.
• During a disease outbreak, to evaluate
the spread of the disease, e.g. during
recent COVID-19 outbreak, rapid testing
kits are being used to determine
presence of antibodies in the blood

• Aydin S. A short history, principles, and types of ELISA, and our
laboratory experience with peptide/protein analyses using ELISA.
Peptides. 2015 Oct;72:4-15. [PubMed]
• Weng X, Gaur G, Neethirajan S. Rapid Detection of Food Allergens by
Microfluidics ELISA-Based Optical Sensor. Biosensors (Basel). 2016 Jun
07;6(2):24. [PMC free article]
• Journals of Immunoassaand Immunochemistry 25(3) 241-258
• www.google.comimages
• www.slideshare.Net

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