immunological assay

Presented by-

Asha M. Jagtap.

Rajarambapu college of

pharmacy , kasegaon .

M.Pharmacy

(pharmaceutics)

An immunoassay is a biochemical test that

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.

IMMUNOLOGICAL ASSAY.

RIA

ELISA

1.RIA (RADIO IMMUNO ASSAY)

It was developed by “Rosalyan Yalow and S.

A. Berson in the 1950s”.

Development of the ‘RIA for insulin;

DEFINATION –

It is a scientific method used to

test antigens without the need to use a

bioassay.

PRINCIPLE

It involve competition between

labelled and unlabelled antigen for binding with

specific antibodies so antigen-antibody

complex is formed .& radio activity is measured.

INSRUMENTATION

It is an alternative technique employed in

diagnosis of several antigen like hepatitis.

It is competitive binding assay in which serum

sample is combined with radioactively labelled

antigen and antibodies to the antigen .

The radioactive antigen and antigen specimen

will compete for antibody .the antigen-antibody

complex thus formed is isolated and analyzed for its

radioactivity. The concentrations ‘standard dose

response curves;

DIAGRAMMATIC REPRESENTATION OF RIA.

APPLICATION

1. Detection of insulin.

2. Detection of narcotic drug .(morphine and

heroin )

3. Detection of digoxin .

4. Determination hydromorphon and

hydrocondron in human serum .

5. Measurement of ferritin.

6. Thyroid testing .

2.ELISA (ENZYME LINKED IMMUNOSORBENT ASSAYS)

The ELISA is a fundamental tool of clinical

immunology and is used as an initial screen for

HIV detection.

Definition –

Enzyme-linked ImmunoSorbent Assay ,also

called ELISA ,enzyme Immuno Assay or ELA is

a biochemical technique used mainly in

immunology to detect the presence of an

antibody or an antigen in a sample.

PRINCIPLE

Antigen –antibody interaction this test

allows for easy visualization of results and can

be completed without the additional concern of

radioactive material used.

INSTRUMENTATION

In which enzyme like alkaline phosphate

is employed to visualize antigen-antibody

reaction.

TWO TYPE –

1. Direct ELISA ( double antibody sandwich)

2. Indirect ELISA ( direct antigen coating )

DIAGRAMMATIC REPRESENTATION OF ELISA.

1. DIRECT ELISA

It is performed in microtitre

well, in which antibody to specific antigen is

adsorbed to well, on which specimen

containing antigen to be demonstrated is

added. Unbound antigen are washed off and

enzyme –linked antibody specific for test

antigen is added which binds to complexed

antigen forming sandwich.

2. INDIRECT ELISA -

It is also performed in microtitre well, in

which antigen to the antibodies demonstrated is

adsorbed to well ,on to which patient antiserum

specimen is added If serum contains specific

antibody ,it will bind to the adsorbed antigen

.un-reacted serum is washed off and enzyme –

linked anti-HSIG is added, which will bind to

antibodies in the initially formed antigen

antibody complex.

APPLICATION

1. It is a useful tool for determination serum

antibody concentrations.( such as with the HIV

TEST)

2. For detecting the presence of antigen .it has

also found applications in the food industry in

detecting potential food allergens such as milk

,almond, eggs

APPLICATION

3. ELISA can also be used in toxicology as a rapid

presumptive screen for certain classes of drug .

4. Diagnosis of infection .