Principles, Practical Aspects and Applications of Primary Ag-Ab Interaction- Equilibrium Dialysis RIA ELISA Immunofluorescence Biotin-Avidin Ab Technique Western Blotting Flow Cytometry

Presented by Jyoti Devendra Adala

The combined strength of the non covalent interaction between a single Ag binding site on the Ab is the affinity of the Ab for that epitope.

where k1 is the forward (association) rate constant and k-1 is the reverse (dissociation) rate constant.

The ratio k1/k-1 is the association constant Ka (i.e., k1/k-1 = Ka), a measure of affinity and can be determined by equilibrium dialysis.

EQUILIBRIUM DIALYSIS

 

Ka=  r/{(n-r)(c)}           r = the ratio of the concentration of bound ligand to the concentration of antibody molecules placed in the system,n = the number of ligand binding sites on the antibody molecule (i.e., its valence), andc = the concentration of unbound ("free") ligand.

RADIO IMMUNO ASSAY (RIA)Principle: Involves competitive binding of radiolabeled Ag and unlabeled Ag to a high affinity Ab.

1) Labeled Ag mixed with Ab.

2) Test sample of unlabeled Ag of unknown concentration are added in progressively large amounts.

DETECTION OF HEPATITIS B VIRUS

ENZYME LINKED IMMUNO SORBENT ASSAY (ELISA)

An enzyme conjugated with an Ab reacts with a colorless substrate to generate a colored reaction product.3 types:a) INDIRECT ELISAb) SANDWICH ELISAc) COMPETITIVE ELISA

IMMUNOFLUORESCENCEAlbert Coons (1941) developed the immunofluorescence techniques for the first time.

Immunofluorescence is an antigen-antibody reaction where the antibodies are tagged (labeled) with a fluorescent dye and the antigen-antibody complex is visualized using microscope.

2 types:Direct immunofluorescenceIndirect immunofluorescence

WESTERN BLOTTINGUsed to detect specific proteins in the given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native proteins and then these proteins are then transferred to a membrane where they are stained with Ab specific to the target protein. The steps are:

1. Tissue preparation

2. Gel electrophoresis

3. Transfer

4. Blocking

5. Analysis

FLOW CYTOMETRYFlow cytometer was designed to automate the analysis and suspension of cells stained with fluorescent Ab.

The flow cytometer uses a laser beam and light detector to count single intact cells in suspension.

As cells passes light is interrupted and signal is recorded.

Fluorescently tagged Ab bound to their cell surface Ag are excited by laser and emitted light is recorded by detector system located at a right angle to the laser beam which is attached to a computer which generates plots.

APPLICATIONS

REFERENCES

IMMUNOLOGY BY KUBY 4TH EDITION BIOPHYSICAL CHEMISTRY BY UPATHYAY