The document discusses various immunochemical techniques used for identifying and analyzing proteins, peptides, antigens, and antibodies. Key techniques include ELISA, radioimmunoassay, immunoprecipitation, and immunoelectrophoresis, each important for diagnosing conditions such as cancer and infections. It highlights the principles, mechanisms, and applications of these techniques in clinical and research settings.

1. [Department of Pharmacy (Pharmaceutics)] | Sagar savale
1December 24, 2015
Immunochemical Techniques
Mr. Sagar Kishor Savale
[Department of Pharmacy (Pharmaceutics)]
2015-016
avengersagar16@gmail.com
1. Immunochemical Techniques
The Technique which are used for identification, Characterization, Analysis, Optimization of
Protein, Peptide, Antigen and Antibody Reactions is known as Immunochemical Technique.
It can mainly include,
1. ELISA
2. RADIOIMMUNOASSAY
3. IMMUNOPRECIPITATION
4. IMMUNOELECTROPHOROSIS
1. ELISA [Enzyme Linked Immunosorbent Assay]
It is important type of technique is used for determination of antigen and antibody specificity,
reaction, interaction, studies.
 It is important for antigen and antibody reaction.
 It is important type of technique is based on Immunological assay.
 It is important type of Diagnosis Technique for Cancer and AIDS.
 It is important type of technique for Hepatitis A, B, and C
 It is important type of technique for bacteria, virus, fungus, and Protozoa’s

2. [Department of Pharmacy (Pharmaceutics)] | Sagar savale
2December 24, 2015
Principle – Antigen and Antibody reaction.
Mechanism - mechanism is based on Immunochemical Technique.
Types
1. Indirect assay - example used in detection of Rotavirus antigen in feces.
2. Sandwich assay - example used in detection of HIV antibody in serum
3. Competitive assay - example used in detection of HIV antibody in serum
1. Indirect assay
Result: color change indicate positive result ,
no color change indicate negative result.
finally substrate is added .
Substrate binds to enzyme and forms a color.
Now well should be washed because free ag and ab are removed
Antibody along with enzyme is added.
If there is suitable antigen , antibody binds and form complex along with enzyme.
The well was coated with sample (antigen)

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3December 24, 2015
Example - rotavirus in foecus sample (ag) Goat antibody (ab) Enzyme: alkaline phosphatase
Subs: paranitrophenyl phosphate.

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4December 24, 2015
2. Sandwich assay
Again well should be washed (to remove free ab-enzyme ) finally substrate was added . Color
change indicates positive result.
Now ab along with enzyme is added .
This ab binds to another end of the know ag
Wash the well – free ag / ab are removed
The known ag was added to the well. if there is a suitable ab it binds to ag and form ag-ab complex
The well was coated with unknown antibody (ab)

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5December 24, 2015
3. Competitive assay

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6December 24, 2015
2. RADIOIMMUNOASSAY
The Technique which are used for determination of antigen and antibody reaction.
Principal – it is important for serum plasma insulin level.
Mechanism – Antigen – antibody reaction and separation of protein molecule.

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7December 24, 2015
The technique of radioimmunoassay has revolutionized research and clinical practice in many
areas,
It is also used in,
 blood banking
 diagnosis of allergies
 endocrinology
Radioimmuno assay system

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8December 24, 2015
Assay Procedure
The unlabeled antigen concentration is more so it free the labeled
antigen . This labeled antigen will float in supernatant is measured using
gamma counter
This causes the unlabeled (or "cold") antigen from the serum to compete
with the radiolabeled antigen ("hot") for antibody binding sites
Sample contain unknown antigen is added
then mixed with a known amount of antibody for that antigen, and as a
result, the two chemically bind to one another
a known quantity of an antigen is made radioactive, example: gamma-
radioactive isotopes of iodine attached to tyrosine

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9December 24, 2015
Advantages
 Highly specific: Immune reactions are specific
 High sensitivity : Immune reactions are sensitive
 Possible to detect picograms of Ag
 Sepharose beads used in RIA are reuseable
Application
 Analysis of hormones, vitamins,metabolites, diagnostic markers
 Therapeutic drug monitoring
 Diagnostic procedures for detecting infection
 Narcotic drug detection
 Tracking of leukemia virus
 Diagnosis and treatment of peptic ulcers
 Research with Neurotransmitters
Difference between Radioimmunoassay and ELISA Technique
Parameter RIA EIA
Sensitivity Nanomolar to picomolar Millimolar
Cost More Less
Time duration More Less
Ease of handling Tedious Easy
Radiation hazards More No
Disposal Care has to be taken Easy
Equipment Complex Less
Mechanism Based on the measurement of
radioactivity associated with
immune complexes
Based on measurement of
enzymatic reaction associated with
immune complexes.

10. [Department of Pharmacy (Pharmaceutics)] | Sagar savale
10December 24, 2015
3. IMMUNOPRECIPITATION
 Antibody and soluble antigen interacting in aqueous solution form a lattice that
eventually developed into visible precipitate.
 Antibodies that aggregate soluble antigens are called precipitins.
Procedure
 A quantitative precipitation reaction can be performed by placing a constant amount of
antibody in a series of tubes and adding increasing amount of antigen to the tubes.
 After the precipitate forms each tube is centrifuged to pellate, the precipitate is
measured.
 Plotting the amount of precipitate against increasing antigen concentration yields a
precipitin curve.

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11December 24, 2015
Types of immunoprecipitation techniques
1. Immunodiffusion
2. Immunoelectrophoresis
3. Countercurrent electrophoresis
4. Rocket immunoelectrophoresis
5. Crossed Immunoelectrophoresis
Application
 Determining presence of protein
 Determining the size/molecular weight of a protein
 Monitoring post-translational modification
 Determining protein-protein interaction
 Determining specific enzymatic activity
4. IMMUNOELECTROPHOROSIS
Definition
This technique involves combination of two different methods, electrophoresis of sample
followed by Immunodiffusion to identify precipitin bands.& it is also called as gamma
globulin electrophoresis.
Importance

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12December 24, 2015
The increased resolution is of benefit in the immunological examination of serum proteins.
Immunoelectrophoresis aids in the diagnosis and evaluation of the therapeutic response in
many disease states affecting the immune system. It is usually requested when a different
type of electrophoresis, called a serum protein electrophoresis, has indicated a rise at the
immunoglobulin level. Immunoelectrophoresis is also used frequently to diagnose multiple
myeloma, a disease affecting the bone marrow.
Uses
 To check the purity of Isolated serum components like Ag/Ab.
 Identification of plasma/serum components for presence or absence of certain proteins
 It is qualitative technique &useful In quantitation of Ag levels as low as 0.2µg/ml and
can detect Ab conc. Of 3-20µg/ml
Reference
1. microbiology by michael j.pelczar fifth edition textbook of microbiology and
immunology by c.k.j paniker seventh edition
2. Yalow R, Berson S. Immunoassay of endogenous plasma insulin in man. J. Clin. Invest
1960; 39: 1157-1175.
3. Abraham G. Radioimmunoassay of steroids in biological fluids. J. Steroid
Biochemistry 1975; 6: 261-270.
4. Associate Professor Dr. Özhan Eyigör, Uludag University College of
Medicine,Department of Histology & Embryology Radioimmunoassay

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13December 24, 2015
5. www.ELISA.wiki.org.in
6. www.Immunoassay.wiki.com
7. www.immnoprecipitationreaction.Org.in.
8. www.electrophrosis.com
9. www.immunochemical assay.co.in
10. www.sciencediarect.com
11. www.mediscape.com
12. www.pubmed.com