TECHNIQUES:

•FLOWCYTOMETRY

•IMMUNOFLUORESCENCE

•AGGLUTINATION INHIBITION

ASSAY

•RIA(RadioImmunoassay)

•RAST(Radioallergosorbent test)

FLOWCYTOMETRY

Flow cytometry is a laser-based,

biophysical technique employed in cell

counting, cell sorting, biomarker

detection and protein engineering, by

suspending cells in a stream of fluid

and passing them by an electronic

detection apparatus.

The flow cytometer uses a laser beam

and light detector to count single intact

cells in suspension.

A

Applications:

1.Determine the kind and number of white blood

cells in blood samples.

2. How many cells express the target antigen as

an absolute number and also as a percentage of

cells passing the beam.

3. The size of cells. This information is derived

from analysis of the light-scattering properties of

members of the cell population under

examination.

IMMUNOFLUORESCENCEFluorescent molecules absorb light of one

wavelength(excitation) and emit light of another

wavelength (emission).

If antibody molecules are tagged with a fluorescent dye,

or fluorochrome, immune complexes containing these

fluorescently labeled antibodies (FA) can be detected by

colored light emission when excited by light of the

appropriate wavelength.

The emitted light can be viewed with a fluorescence

microscope.

In this technique, fluorescent compounds such as

fluorescein and rhodamine are conjugated to the Fc

region of an antibody molecule without affecting the

specificity of the antibody.

Fluorescent-antibody staining of cell

membrane molecules or tissue sections

can be direct or indirect.

In direct staining, the specific antibody (the

primary antibody) is directly conjugated

with fluorescein.

In indirect staining, the primary antibody is

unlabeled and is detected with an

additional fluorochrome-labeled reagent.

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ADVANTAGES OF INDIRECT

OVER DIRECT:

1. The primary antibody does not need to be

conjugated with a fluorochrome. As the supply of

primary antibody is often a limiting factor, indirect

methods avoid the loss of antibody that usually

occurs during the conjugation reaction.

2. Indirect methods increase the sensitivity of

staining because multiple molecules of the

fluorochrome reagent bind to each primary

antibody molecule.

APPLICATIONS:

1.Identifification a number

of subpopulations of lymphocytes, notably the

CD4

and CD8 T-cell subpopulations.

2. Detecting Ag-Ab complexes in autoimmune

disease.

3. Localization of antigens in tissue sections or

in sub cellular compartments.

AGGLUTINATION INHIBITION

ASSAY

A modification of the agglutination reaction, called

agglutination inhibition, provides a highly sensitive

assay for small quantities of an antigen.

In this assay the presence or absence of an

antigen can be determined based on the formation

of agglutination or not.

For example it was used in early pregnancy kits

and also to find out if a person is on certain kind of

drugs.

APPLICATIONS:

1.Pregnancy test

2.To test presence of certain drugs in

the body

3.Exposure to certain

viruses(hemagglutination test).

RADIOIMMUNOASSAY

The principle of RIA involves competitive binding

of radiolabeled antigen and unlabeled antigen to

a high-affinity antibody.

The labeled antigen is mixed with antibody at a

concentration that saturates the antigen-binding

sites of the antibody.

Then test samples of unlabeled antigen of

unknown concentration are added in

progressively larger amounts.

USES:

RIA is widely used for

measuring hormones, serum

proteins, drugs, and vitamins at

concentrations of 0.001

micrograms per milliliter or less.

RASTIt detects the serum level of IgE specific for a

given allergen.

The allergen is coupled to beads or disks, the

patient’s serum is added, and unbound antibody

is washed away.

The amount of specific IgE bound to the solid-

phase allergen is then measured by adding 125I

labeled rabbit anti-IgE, washing the beads, and

counting the bound radioactivity

RAST can quantify nanogram amounts of

serum IgE specific for a particular allergen.

Contrary to RIST, this method

is used to detect specific IgE

antibodies, to determine the

substance a subject is allergic

to.

In RIST the total serum IgE

levels are determined.