Immunoassays

Immunoassays

Laboratory techniques that makes use of the binding between an antigen and its homologous antibody in order to identify and quantify the specific antigen or antibody in a sample.

ELISA technique Is a biochemical technique used mainly in

immunology to detect the presence of an antibody or an antigen in a sample.

• The technique is divided into

1- Competitive ELISA 2- Sandwich ELISA (also called direct ELISA)3- Indirect ELISA

ELISA

• An ELISA test uses components of the immune system and chemicals to detect Ag-Ab reactions

• The ELISA test involves an enzyme (a protein that catalyzes a biochemical reaction). It also involves an antibody or antigen (immunologic molecules).

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Types of ELISA

• Qualitative ELISA– Positive or Negative results

• Quantitative ELISA– optical density or fluorescent units of the sample is

interpolated into a standard curve, which is typically a serial dilution of the target.

ELISA• Principle:

– Bound antigen is detected by an antibody that is covalently coupled with an enzyme such as peroxidase or alkaline phosphatase

– Quantification of antigen-antibody binding is achieved by measuring the colour intensity of the coloured product generated by the enzyme and the added substrate.

– The intensity of the colour is proportional to the amount of the labelled antibody bound to the antigen.

• Direct detection of an antigen with the labelled antibody is not popular because of low sensitivity.

• In the indirect method, the antibody with specificity for the desired antigen is unlabelled, and a second enzyme labelled antibody with a specificity for the first antibody (an antibody to an antibody) is then added to the mixtur.e

Basic principles of ELISA• the antibodies fixed to a

solid surface, such as the inner surface of a microtitre plate;

• a preparation of the same antibodies coupled to an enzyme. that produces a coloured product after reaction with a colourless substrate.

Direct ELISA

Other types of ELISA’s…

Outline of procedure e.g Sandwish ELISA • Coat an appropriate surface with a known quantity of capture

antibody and allow time for binding• Wash to remove any unbound antibody• Block any non specific binding sites(empty sites ) on the surface with

a an irrelevant protein• Apply the antigen-containing sample(TEST sample) to the plate. • Wash the plate, so that unbound antigen in the test sample is

removed. • Apply enzyme linked primary antibodies as detection antibodies which

also bind specifically to the antigen captured by first antibody. • Wash the plate, so that the unbound antibody-enzyme conjugates are

removed. • Apply a chemical substrate which reacts with enzyme in the conjugate

to form a a coloured product.• Measure the absorbance of the plate wells to determine the presence

and quantity of antigen

Sandwich ELISA• The ELISA plate is coated with Antibody to

detect specific antigen

Results-ELISA• This standard curve is used to determine

the unknown concentration of each sample by finding the opposite concentration to the absorbance

Concentration ng/ml

Absorption nm

Advantages of ELISA• ELISA tests are generally relatively accurate

tests. • They are considered highly sensitive and

specific and compare favorably with other methods used to detect substances in the body, such as radioimmuno assay (RIA) tests.

• They have the added advantages of not needing radioisotopes (radioactive substances) or a costly radiation counter (a radiation-counting apparatus).

APPLICATIONS OF ELISA• Diagnostic Assays

– Serum Antibody Concentrations– Disease outbreaks- tracking the spread of disease HIV,– Detections of antigens in body fluids e.g pregnancy

hormones, drug allergens• Detecting potential food allergens

– (milk, peanuts, walnuts, almonds and eggs)• Research and industrial applications

ELISPOT-

Set similar to ELISA but used to identify antigen secreting cells e.g lymphocytes secreting a particular cytokine using anticytokine labelled monoclonal antibody.

ELISPOT Assays• PBMC are plated on a filter-bottom 96-well plate coated

with anti-cytokine antibody.• The plate is cultured 24-48 hours to allow cytokine

secretion and capture on the plate.• Cells are washed off and detector antibody is added,

followed by enzyme substrate.• Cytokine-secreting cells are identified as spots of

secreted cytokine.

SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis

• SDS-PAGE, is a technique widely used in biochemistry, forensics, genetics and molecular biology:

• Separate proteins according to their electrophoretic mobility (a function of length of polypeptide chain or molecular weight).

• Separate proteins according to their size

SDS

• SDS (sodium dodecyl sulfate) is a detergent (soap) that can dissolve hydrophobic molecules but also has a negative charge (sulfATE) attached to it

• Therefore, if a cell is incubated with SDS, the membranes will be dissolved, all the proteins will be solubIlized by the detergent and all the proteins will be covered with NET negative charges.

PAGE( polyacrylamide gel electrophoresis ).

• If the proteins are denatured and put into an electric field, they will all move towards the positive pole at the same rate, with no separation by size.

• However, if the proteins are put into an environment that will allow different sized proteins to move at different rates e.g polyacrylamide

• Small molecules move through the polyacrylamide faster than big molecules.

• Big molecules are slower and remain close to the starting point

• The entire process is called polyacrylamide gel electrophoresis (PAGE). ddd

 Protein gel (SDS-PAGE) that has been stained with Coomassie

Blue.

Sample of SDS- PAGE(Silver stain)

After electrophoresis?

• 1. Fix the proteins in the gel followed by staining

• 2. Electrophorectic transfer to a membrane and then probe with antibodies- (Western blotting)

..Western blotting

• Western blot analysis can detect specific protein in a mixture of any number of proteins while giving you information about the size of the protein.

• This method is, however, dependent on the use of a high-quality antibody directed against a desired protein.

• This antibody is used as a probe to detect the protein of interest.

Western Blot followed by SDS• Proteins are separated using SDS-polyacrylamide gel

electrophoresis which separates proteins by size. • Separated components transferred from gel to

nitrocellulose paper by “blotting”• Nitrocellulose membrane is placed on the gel. The actual

blotting process may be active (electroblotting) or passive (capillary).

• Electroblotter is used for faster and more efficient transfer of protein from gel to membrane

• Sandwich of filter paper, gel, membrane and more filter paper is prepared in a cassette, which is placed between platinum electrodes.

• An electric current is passed through the gel causing the proteins to electrophorese out of the gel and onto the nitrocellulose membrane.

..Western Blot• The blot is incubated with a generic

protein (such as milk proteins) to bind to any remaining sticky places on the nitrocellulose.

• An antibody is then added to the solution which is able to bind to its specific protein.

• The antibody has an enzyme (e.g. alkaline phosphatase or horseradish peroxidase) or invasible dye attached to it

..Western Blot• The location of the antibody is revealed

by incubating it with a colourless substrate which reacts with the attached enzyme and is concerted to a colored product that can be seen and photographed.

Western blotting

Western Blot

Radioimmunoassay (RIA),-A very sensitive method, but is becoming less attractive,

because it is potentially hazardous and tedious.-The assay was originally developed to detect trace amounts of

peptides and hormones in plasma or biological fluids.-In this assay, antibodies are immobilized and the radiolabeled

(usually 125I) and cold antigen are allowed to compete for binding to the immobilized antibodies

Radioimmunoassay (RIA)Very sensitive test; used for measuring hormones,

serum proteins, drugs, etc. at low [C]’s (≤ 0.001ug/ml)

measures “competitive binding” of radiolabelled Ag + unlabelled (test) Ag to high affinity Ab

Other immunological assays:• Immunoprecipitation.

– Provides a quick and sensitive test for identifying small amounts of antigens (low conc) in a complex mixture. Antigen is isolated from a radiolabelled mixture by specific precipitation with antibody and analysed by polyacrylamide gel electrophoresis followed by auto radiography

• Chemiluminescence– Set up similar to ELISA technique but uses a different detection

system.– The system uses Luminol, hydrogen peroxide and horseradish

peroxidase which react to emit light.• Complement Fixation – Uses the ability of Ab-Ag complexes to fix

complement because an Ag/Ab complex will "consume" complement if it is present whereas free Ag's or Ab's do not. Test only works with complement fixing antibodies.