enzyme linked immunosorbent assay (elisa)

ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA)

PREPARED BY NAHIMA ANHUM

MSC MICROBIOLOGY 2ND SEMESTERSUBMITTED TO

DR.ALIUNIVERSITY OF HARIPUR

INTRODUCTIONELISA is a widely used method for measuring

the concentration of particular molecule such as antibody or antigen in a fluid,e.g serum or urine.

IMMUNO-refers to an immune response that causes the body to generate antibodies.

ASSAY-refers to test or check amount/purity of a substance in a given mixture.

First used by Engvall and Perlma in 1971.

ANTIGEN (Ag)Any molecule that induces the production of antibodies when introduced in the body of an animal is called antigen. SYMBOL FOR

ANTIGEN

ANTIBODY Proteins produced by the immune system which helps to defend

against antigens.

SYMBOL FOR ANTIBODY

DEFINATIONA ELISA Enzyme linked immunoabsorbent

assay is a specific type of biochemical test that measures the presence or concentration of a antigen or antibody present in a substance.

Enzyme label In ELISA it is necessary to use any marker to

know the antigen-antibody binding.For such purpose we label either antigen or

antibody with some materials that donot interfere with the binding.

Continued….These labels used to change in color,emission of light

or other signal that shows antigen-antibody binding.

The chosen enzyme should not be normally present in the patient samples.

The enzyme converts a colorless substrate to a colored product indicating the presence of Ag-Ab binding.

TYPES OF ELISADIRECT ELISA

SANDWICH ELISA

INDIRECT ELISA

DIRECT ELISAA solution of the antigen to be tested for is

added to each well of microtiter plate, where it is given to adhere to plastic through charge interactions.

A solution of non reacting protein such as casein is added to well in order to cover any plastic surface in the well which remains uncoated by the antigen.

The antibody with an attached enzyme is added which binds specifically to test antigen coating the well.

Continued…A substrate for this enzyme is then added,this

substrate changes the color upon reaction with enzyme.

Higher the concentration of antibody,stronger the color changed.

Often spectrometer is used to give the quantitative value for color change.

INDIRECT ELISAAntigen is absorbed to microtiter plate.

Add serum to test for antibody.

Add secondary antibody.

Primary antibody binds to secondary antibody.

Adds substrate,which reacts with enzyme and change color to show antigen strength.

SANDWICH ELISAIn this procedure plate is coated with known

quantity of antibody.

The primary antigen containing sample is added to plate and captured by antibody.

Plate is washed and removed unbound antigen.

Continued…A specific antibody is added and binds to

antigen.Now antigen is stuck between two antibodies.

Enzyme linked secondary antibodies are applied to detect antibodies.

Substrate is added the enzyme reacts with substrate that changes color.

APPLICATIONS OF ELISA1- Hormones 2- Proteins 3- Infectious Agent ( Viral, Bacterial, 4- For Rapid TestParasitic, Fungal )5- Drug Tests 6- IgG, IgM, IgA7- Tumor Tests 8- Serum Proteins 9- In Clinical Research10-HIV ,Hepatitus B & C test