B.Sc. III: Semester-VI: Microbiology-P-I - PowerPoint Presentation

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B.Sc. III: Semester-VI: Microbiology-P-I

UNIT-

IV

ANTIGEN-ANTIBODY

REACTION

II

(Agglutination Reaction )

Prof.

D.A.Chouhan

M.Sc., NET JRF (CSIR), NET JRF ( UGC), GATE, DMLT

Tagged antibody test:

In previous ppts we discuss about untagged serological reactions. These reactions has following imitations.

Less sensitivityHigh degree of cross reactivityLess stabilityTo overcome from these limitations tagged antibody bases test were discovered.

In these techniques, antigen-antibody complexes are identified by using labels like enzyme, radioisotopes, fluorescent substances etc. 

Enzyme Linked

Immunosorbent

Assay (ELISA):In Enzyme-linked immunosorbent assay, commonly known as ELISA, An

enzyme conjugated with an antibody reacts with a colorless substrate to generate a colored reaction product. Such

a substrate is called a chromogenic substrate

.

A

number of enzymes have been employed for ELISA, including alkaline phosphatase, horseradish peroxidase, and β-

galactosidase

.

These

assays

are being

safer and less costly.

There

are three types of ELISA, namely Indirect,

Sandwitch

and Competitive ELISA.

Direct ELISA:

This is the simplest form of

ELISA. Here an antigen is attached passively to a plastic solid phase by a period of incubation. After a simple washing step, antigen is detected by the addition of an antibody that is linked covalently to an enzyme. After

incubation and washing, the test is developed by the addition of a chromogen/substrate whereby enzyme activity produces a color change.

Color

development is read after a defined time or after enzyme activity is stopped by chemical means at a defined time.

Color is read in a spectrophotometer.

B.

INDIRECT

ELISA: Antibody can be detected or quantitatively determined with an indirect ELISA. Serum or some other sample containing primary antibody (Ab1) is added to an antigen-coated

microtiter well and allowed to react with the antigen attached to the well.

After

any free Ab1 is washed away, the presence of antibody bound to the antigen is detected by adding an enzyme-conjugated secondary anti-

isotype antibody (Ab2), which binds to the primary antibody.  Any free Ab2 then is washed away, and a substrate for the enzyme is added. The amount of colored reaction product that forms is measured by specialized spectrophotometric plate readers, which can measure the absorbance of all of the wells of a 96-well plate in seconds.  

Indirect

ELISA is the method of choice to detect the presence of serum antibodies against human immunodeficiency virus (HIV), the causative agent of AIDS.

C. SANDWICH

ELISA

Antigen can be detected or measured by a sandwich ELISA. In this technique, the antibody (rather than the antigen) is immobilized on a microtiter well.

A sample containing antigen is added and allowed to react with the immobilized antibody.

After

the well is washed; a second enzyme-linked antibody specific for a different epitope on the antigen is added and allowed to react with the bound antigen. 

After any free second antibody is removed by washing, substrate is added, and the colored reaction product is measured.

D. COMPETITIVE

ELISA

Another variation for measuring amounts of antigen is competitive ELISA. In this technique, antibody is first incubated in solution with a sample containing antigen.  The

antigen-antibody mixture is then added to an antigen coated microtiter well.

The

more antigens present in the sample, the less free antibody will be available to bind to the antigen-coated well.

Addition of an enzyme-conjugated secondary antibody (Ab2) specific for the isotype of the primary antibody can be used to determine the amount of primary antibody bound to the well as in an indirect ELISA.

In

the competitive assay, however, the higher the concentration of antigen in the original sample, the lower the absorbance.

Immunofluorescence

Fluorescence

is the phenomenon in which certain substances such as fluorescent dyes having capability to absorb UV light and reemit visible light.Fluorescent dyes show up brightly under ultraviolet light as they convert ultraviolet into visible light. 

Coons and his colleagues (1942) showed that fluorescent dyes can be conjugated to antibodies and that such labeled antibodies can be used to locate and identify antigens in tissue. 

Because

of this reason, immunofluorescence technique is also a type of immunohistochemistry

assay.The most commonly used fluorescent dyes are fluorescein and rhodamine but other highly fluorescent substances such as

phycoerythrin

and phycobiliproteins have also been used. 

Immunofluorescence

Dyes

can be conjugated to the Fc region of an antibody molecule without affecting the specificity of antibody.   Fluorescein, an organic dye that is the most widely used label for immunofluorescence procedures absorb blue light (490nm) and emits an intense yellow green fluorescence (517nm). 

Rhodamine, another organic dye absorb in the yellow green range (515nm) and emits a deep red fluorescence (546nm). 

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.

A

number of reagents have been developed for indirect staining.  The most common is a

fluorochrome

-Labeled secondary antibody raised in one species against antibodies of another species

,

such as fluorescein-labeled goat anti-mouse immunoglobulin

. 

Difference between direct and indirect Immunofluorescence

Questions