Applications of Antibodies in purification

Presented by

Ankur SharmaM.Sc Ist year (ABTC)

INTRODUCTION

Use of highly specific interaction between antigen & antibody to purify the protein present in small quantities as a mixture with several types of molecules is called immunopurification.

Due to customized avidity and specificity, monoclonal antibodies have become indispensable tool for both protein characterization and purification.

IMMUNOAFFINITY CHROMATOGRAPHY

Immunoaffinity chromatography is a process in which affinity of an antigen to specific antibody is utilized as basis of separation.Affinity chromatography enables purification of biomolecules on the basis of their chemical structure or biological function.

The method was invented in 1968 by Pedro Cuatrecasas and Meir Wilchek.

The ligand ( here antibody) is immobilized covalently to chromatographic matrix to yield an active immunosorbent.

The use of antibodies as the immobilized ligand has been exploited in isolation and purification of a range of proteins including membrane proteins of viral origin.

Mechanism of immunoaffintiy chromatography :

The Monoclonal antibodies are fixed to insoluble matrix like dextran or agarose bead by cross linking agents like cyanogen bromide, N-hydroxysuccinamide (NHS),carbonyl di imidazole(CDI)

Another technique uses a solid support coated with an antibody binding protein, such as Protein A or G, which captures and immobilizes the antibodies.

The antibody is then covalent linked to the resin with the aid of a chemical cross linker. 

Procedure:-

The bacterial proteins, protein A and protein G are the most commonly used antibody binding proteins.

Protein A is a 42 kDa protein that is a normal constituent of S. aureus bacteria cell walls.

  Protein G is a 30-35 kDa protein that is

found in the cell wall of β-hemolytic streptococci of the C and G strains.

 

The beads are packed in a column through which the solution containing the protein to be purified is passed.

The interaction of Ag and Ab form Ag-Ab complex which is retained in the column while remaining molecules freely pass through the column.

Suitable washing procedure allow collection of purified antigen held as Ag-Ab complex in column.

The reversible interaction between Ag and Ab can be disrupted to yield a highly purified product in the column eluate.

Elution of the bound protein quite often requires forceful conditions because very tight binding with antibody.

Elution procedure include use of high salt concentrations with or without

detergent and the use of urea or guanidine hydrochloride, both of which cause denaturation of protein.

To avoid denaturation some chaotropic agents such as thiocynate, perchlorate and trifluoroacetate are used.

Another option is lowering the pH near about 3

Immunoaffintiy chromatography

APPLICATIONS Purification of Human Chorionic Gonedotropin (hCG) from pregnancy urine by immunoaffinity chromatography using a monoclonal antibody anti b chain hCG.

Identification of carcino embryonic antigen(CEA) related Ag in mecmium and colorectal carcinoma tissue by western blotting and differential immunoaffinity chromatography.

Immunoaffinity purification of factor IX (Christmas factor) by using conformation-specific antibodies directed against the factorIX-metal complex:-

Factor IX is a vitamin K dependent zymogen that is functionally defective or absent in patients with hemophilia B.

A method of imnmunoaffinity chromatogiaphy has been developed for a one-step high yield purificationof factor IX directly fromplasma.

The technique utilizes conformation-specific antibodies that bind solely to the metal-stabilized factor IX conformer, but not to the conformer of factor IX found in the absence of metal ionsActive eukaryotic RNA polymerase II (RNAP II) is purified by immunoaffinity chromatography, using a monoclonal antibody (mAb) that reacts with the highly conserved hepta peptide repeat of the largest subunit.

This MAb (designated 8WG16) is conjugated to CNBr-activated Sepharose and used to purify RNAPII from wheat germ and calf thymus.

FLOW CYTOMETRYINTRODUCTION

Flow cytometer was designed to automate the analysis and separation of cells stained with fluorescent antibody.Flow cytometer which is also known as Fluorescence activated cell sorter(FACS) is a technique for counting, examining and sorting microscopic objects suspended in a fluid.In 1944 Albert Coons gave the concept of technique immunofluorescence

Principle:-

Flow cytometer is based on immuno fluorescence.According to it, antibodies could be labeled with fluorescent molecules called fluorochrome which absorb light of one wavelength(excitation) and emit light of another wavelength(emission).Most commonly used fluorochromes are:

Fluorescein :- absorbs blue light(490nm) and emits intense yellow-green light(517nm).

Rhodamine :- absorbs yellow-green(515nm) and emits deep red(546nm)

Pycoerythrinh :- it is efficient absorber of light(~30 fold greater than fluorescein) and emits red light.

It may be more convenient to use a second non specific fluorescent anti Ig to bind to the unlabeled specific Ab bound to cell surface Ag.

the antibody conjugate will bind to only those cells that exibit the concerned Ag on their surfaceSuch cells are therefore identified and separated due to their fluorescence.

MECHANISM:-The flow cytometer uses a laser beam and light detector to count single intact cell in suspension.The mixture containing cells to be purified is loaded in the sample chamber of the flow cytometer.The cells are expelled, one at a time, from the vibrating nozzle that generates micro droplets.

As it leaves the nozzle each droplet receive small electric charge and attached computer can detect the exact time at which a particular droplet will pass through the deflection platesBy applying a momentary charge to the deflection plates when a droplet is passing between them it is possible to deflect the path of a particular droplet into one or another collecting vessels.This allows sorting of population of cells into subpopulations having different profile of surface markers.

Flow cytometer

Kuby IMMUNOLOGY SIXTH EDITION

APPLICATIONSDNA Measurement and Cell Cycle Analysis

by Flow Cytometry.

Two of the most popular flow cytometric applications are the measurement of cellular DNA content and the analysis of the cell cycle.The nuclear DNA content of a cell can be quantitatively measured at high speed by flow cytometry . Initially, a fluorescent dye that binds stoichiometrically to the DNA is added to a suspension of permeabilized single cells.

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The principle is that the stained material has incorporated an amount of dye proportional to the amount of DNA.

The stained material is then measured in the flow cytometer and the emitted fluorescent signal yields an electronic pulse with a height (amplitude) proportional to the total fluorescence emission from the cell.

Thereafter , such fluorescence data are considered a measurement of the cellular DNA content.

Flow cytometry assay for intracellular rabies virus detection

A suspensions of BHK-21 and C6 cells were infected with fixed rabies virus strain (Pasteur Virus

strain—PV) or a wild rabies virus strain (WRS) and a time course of virus infection was established.

Infected and control uninfected cells were fixed, permeabilized, and stained with a rabies virus-specific antibody conjugate.

This was achieved byre suspending cells in a solution containing p-formaldehyde in FACS lysis fluid, which allowed the detection of intracellular virus with flourescein-coupled antibodies by flow cytometry

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