Immunochemical techniques

[Department of Pharmacy (Pharmaceutics)] | Sagar savale

1 December 24, 2015

Immunochemical Techniques Mr. Sagar Kishor Savale

[Department of Pharmacy (Pharmaceutics)]

2015-016

[email protected]

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

mailto:[email protected]


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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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Example - rotavirus in foecus sample (ag) Goat antibody (ab) Enzyme: alkaline phosphatase

Subs: paranitrophenyl phosphate.

http://en.wikipedia.org/wiki/File:ELISA_diagram.png


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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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3. Competitive assay


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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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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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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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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.


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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.



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



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

http://medical-dictionary.thefreedictionary.com/Protein+Electrophoresis

http://medical-dictionary.thefreedictionary.com/Protein+Electrophoresis

http://medical-dictionary.thefreedictionary.com/Multiple+Myeloma





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

http://www.elisa.wiki.org.in/

http://www.immunoassay.wiki.com/

http://www.immnoprecipitationreaction.org.in/

http://www.electrophrosis.com/

http://www.immunochemical/

http://www.sciencediarect.com/

http://www.mediscape.com/

http://www.pubmed.com/

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Immunochemical techniques