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IMMUNOBLOTTING TECHNIQUE.pptx

This document provides an overview of immunoblotting techniques including Western blotting, Southern blotting, and Northern blotting. It defines immunoblotting as a technique for analyzing proteins, DNA, or RNA in a mixture using electrophoresis and antibodies. It describes the basic process of blotting as transferring separated biomolecules from a gel onto a membrane for detection. It then provides details on the specific procedures and applications of Western, Southern, and Northern blotting. It also discusses ELISA techniques for detecting antigens and antibodies.

Health & Medicine◦
IMMUNOBLOTTING TECHNIQUES BY ASAWE TEJAWINI L. ASSISTANT PROFESSOR SIDHHIS INSTITUTE OF PHARMACY THANE.
DEFINATION  A technique for analyzing or identifying proteins, DNA, RNA in a mixture , involving separation by electrophoresis followed by staining with antibodies.
WHAT IS BLOTTING?  Blotting techniques are applied in the isolation and quantification of specific nucleic acid sequence And in the study of organization gene expression and regulation .  Blots are techniques for transferring DNA , RNA and proteins onto a carrier so they can be separated, and often follows the use of a gel electrophoresis.  The Southern blot is used for transferring DNA, the Northern blot for RNA and the western blot for PROTEIN.
TYPES OF BLOTTING TECHNIQUES Blotting technique Southern Blot It is used to detect DNA. Northern Blot It is used to detect RNA. Western blot It is used to detect protein.
SOUTHERN BLOTTING  The technique was developed by E.M. Southern in 1975.  The Southern blot is used to detect the presence of a particular piece of DNA in a sample.  The DNA detected can be a single gene, or it can be part of a larger piece of DNA such as a viral genome.  This technique is used to identify location of genes and the other sequences on restriction fragment separated by gel electrophoresis.  Restriction fragments are DNA fragment from cutting DNA by restriction enzyme.
GEL ELECTROPHORESIS  GEL electrophoresis is laboratory method used to separate mixture of DNA, RNA or proteins according to molecular size.  Electric current is applied across the gel , one end of gel has positive charge and other end has negative charge.  Movement of charge molecule is called migration and molecules migrates toward positive charge .
 In this technique sample of DNA contains fragments of different sizes are subjected to electrophoresis using either polyacrylamide or agarose gel .  DNA molecule is cut into small fragments by restriction enzyme and pass through the agarose gel electrophoresis method .  DNA molecule separation is based on size.  DNA is denaturated into single stand by adding the gel to alkaline solution  Gel is added on the top of the buffer saturated filter paper and covered with the nitrocellulose filter placed with the dry filter paper.
 Buffer moves from bottom of filter paper by capillary action and DNA trap in Nitrocellulose membrane the process is known as plotting .  Capillary action occurs due to interaction between cohesion (liquid – liquid attraction ) and adhesion (liquid – surface attraction ) between liquids.  Ex. If you deep paper towel in water it magically climbed up and ignore the gravity .  Nitrocellulose membrane is removed from blotting surface and DNA is permanently immobilized on the membrane by baking at 80 °C.  Single standard DNA has high attraction for nitrocellular filter paper.  Membrane is Treated with solution containing 0.5 % each polyvinyl pyrolidone and bovin serum albumin.
 The membrane is placed on the solution of labeled RNA single standard DNA or oligodeoxribonucleotide(probe )  This labeled nucleic acid used to detect and locate the complementary sequence is called probe .  The probe contains sequence is hybridized or join with immobilized DNA on the membrane after hybridization membrane is washed  Washed membrane is kept to X ray film that detects the radioactivity in bound probe  It is used in the DNA fingerprinting & identification of transfer gene etc
NORTHERN BLOTTING TECHNIQUE  Alwin in 1979 discovered a technique in which RNA bands are plot transfer from the gel onto chemically reactive paper  and amino-benzyl-oxymethyl -cellulose paper prepared from whatman paper 540  The hybridization done with redio labeled DNA probes  Hybridized bands are found out by auto radiography.  Thomas in 1980 found that m RNA bands can also be plotted directly on Nitrocellulose paper .  Hybrids are created with S-1 nucleus with RNAasec which digest the single standard DNA or RNA probe  In this technique preparation of reactive paper is not required  Northern Blotting are useful in studies of gene expression.
WESTERN BLOTTING  Tawbin in 1979 developed the western blotting or protein blotting or electro blotting technique to find out proteins by Transformed cell.  Polyacrylamide gel electrophoresis is used for separation and characterization of proteins.  proteins are extracted from transformed cell and separated by using sodium dodecyl sulphate polyacrylamide gel electrophoresis  Sodium dodecyl sulphate act as a denaturant for protein( treat proteins to alter the original state) during electrophoresis  After electrophoresis individual proteins are detected by using specific antibodies and polypeptides are transferred to the nitrocellular membrane  Transfer of protein from gel to Nitrocellulose membrane is called western plotting technique  It is performed by using electric current hence called electrobotting technique
ELECTROBLOTTING OR WESTERN BLOTTING
ELISA Enzyme-Linked Immunosorbent Assay • Enzyme link immunosorbant assay is an important immunological method for detecting and measuring in antigens and antibodies • In enzyme immunosorbant assay antigen or antibody is conjugated or coupled to enzyme. • The enzyme is convert a colorless substance to colored substance in the immune essay is highly sensitive, safe , cost effective and use of simple instrument reaction product. • Hence ELISA is also referred to as a qualitative or quantitative assay for antibodies. • There are two types of ELISA on the basis of binding structure between the antibody and antigen : • 1. Indirect ELISA 2. Sandwich ELISA
Principle - The ELISA is performed in 96-well polystyrene plates. - The serum is incubated in the wells, each containing a different serum. - Positive and negative control serums are included in the 96 samples being tested. - Antibodies or antigens in serum are captured by the corresponding anti gen or antibody coated on the solid surface. - The serum and unbound antibodies or antigens are removed from the plate by rinsing it with wash buffer. - Secondary antibodies attached to peroxidase or alkaline phosphatase enzyme, are added to each well for detecting bound antibodies or antigens. - The unbound secondary antibodies are rinsed off after incubating the wells for a defined period. - On adding an appropriate substrate, it reacts with the enzyme to develop a colour which is measured as a function or amount of antigens or antibody in the sample. - Measurement of the intensity of colour or optical density is done at 450nm, and the colour intensity indicates the number of antigen or antibody.
INDIRECT ELISA - This method is useful in identification and quantitative determination of antibody. - Indirect ELISA is preferred for detecting the presence of serum antibodies against HIV. - In this assay, the recombinant envelope and core proteins of HIV are adsorbed on to micro titre wells as solid –phase antigens. - The HIV infected individuals will produce serum antibodies to epitopes (part of antigens ) on these viral proteins which can be detected within 6 weeks of infection by indirect ELISA.
Procedure of Indirect ELISA: i. The micro titre plate wells are coated with antigen. ii. All unbound sites are blocked to prevent false positive results. iii. Sample containing antibody (e.g. rabbit monoclonal antibody) is added to the wells and incubated at 37°C. iv. Plate is washed so that unbound antibody is removed. v. Secondary antibody conjugated to an enzyme (e.g. anti-mouse IgG) is added. vi. The plate is washed so that unbound enzyme-linked antibodies are removed. vii. Substrate is added, which is converted by the enzyme to produce a coloured product. viii. Coloured product is produced between the reaction of a substrate with the enzyme.
SANDWICH ELISA - This method is useful in detection and measurement of antigen. - In this technique, the antibody is immobilized on a microtitre well. - microtitre is a titer determined by microanalytical titration - Antigen -containing sample is added and allowed to react with the immobilized antibody. - After washing the well, a second enzyme - linked antibody having specificity for a different epitope on the antigen is added and allowed to react with the bound anti gen. - After washing away any free secondary antibody, the substrate is added and the intensity of colour produced is measured.
Procedure of Sandwich ELISA: i. A surface is prepared to which a known quantity of antibody is added and allowed to bind. ii. The antigen-containing sample is added to the plate and incubated at 37°C. iii. The plate is washed, so that unbound antigen is removed. iv. Enzyme-linked antibodies are added which are also specific to the antigen and the incubated at 37°C. v. The plate is washed, so that unbound enzyme-linked antibodies are removed. vi. Substrate is added, which is converted by the enzyme to produce a coloured product. vii. OPD (o- phenylenediamine dihydrochloride is used as a substrate and give blue colour .
APPLICATIONS OF ELISA - Presence of antigen or the presence of antibody in a sample can be evaluated. - Determination of serum antibody concentrations in a virus test. - Used in food industry when detecting potential food allergens. - Applied in disease outbreaks- tracking the spread of disease e.g. HIV, bird flu, common colds, cholera, STD etc. - ELISA test used to detect various kinds of diseases, such as dengue, malaria, and others. - ELISA tests also are used as in in vitro diagnostics in medical laboratories. - The other uses of ELISA include: detection of Mycobacterium antibodies in tuberculosis, rotavirus in feces, hepatitis B markers in serum, hepatitis C markers in serum.
REFERENCES 1. A Textbook of Pharmaceutical Biotechnology by Dr. S. Jayaraman, Dr. Richa Ohri, Dr. Pankaj Verma. Thakur Publication, Page no: 144-148. 2. A Textbook of Pharmaceutical Biotechnology by PeeVee Publication, Page no: 313-318
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