immunological purification

7/31/2019 Immunological Purification

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IMMUNOLOGICAL

PURIFICATIONPRESENTED BY:

HARNEET KAURL-2011-V-87-M


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ANTIBODY AND ANTIGEN

Antibodies, or Y-shaped immunoglobulins , are proteins

found in the blood that help to fight against foreignsubstances called antigens.

Antigens, which are usually proteins or polysaccharides,

stimulate the immune system to produce antibodies. The antibodies inactivate the antigen and help to remove it

from the body

Antibodies are gamma globulins produced byB

lymphocytes .

Great diversity and specificity: >109 different

antibodies.


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Antibody structure

It has Y shape structure. The two arm domains that carry the antigen

binding sites are known as Fab fragment and the

protein domain that is involved in immuneregulation is termed the Fc fragment.

The region between the Fab and Fc fragments is

called the hinge. The hinge segment allows lateral and rotational

movement of the two antigen binding domains


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Each Y contains four polypeptides---two identicalcopies of a polypeptide knownas the heavy chain andtwo identical copies of a polypeptide called the light

chain.

The two heavy-chain polypeptides in the Y structureare identical and are about 55kDa. The two light

chains are also identical and are about 25 kDa.

The four polypeptide chains are held together bydisulfide bridges and noncovalent bonds.


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Antibody Structure

1.Fab2.Fc3.heavy chain

4.light chain5.antigen binding site6.hinge regions


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Polyclonal antibodies

Antisera generated by injection of antigen into ananimal will contain a mixture of antibodies directedagainst different antigens determinants on themolecule.

Such antisera which contain mixture of antibodies thatwas exclusively directed against the immunogen towhich they are raised is known as polyclonal

antibodies. Polyclonal antibodies represent the antibodies from

multiple clones of B lymphocytes, and therefore bind

to a number of different epitopes .


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Epitopes

Immune Response

Antibodies

A mixture of antibodies - all bind to epitopes ofthe original antigen. Some bind with higher

affinity than others.

Polyclonal antibodies

Protein

Immunize


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Monoclonal antibodies

Monoclonal antibodies are antibodies that are identicalbecause they were produced by one type of immunecell (B cell).

Reproducible, Predictable & Potentially inexhaustiblesupply of Ab with exquisite specificity enable thedevelopment of secure immunoassay systems clones

of a single parent cell. monoclonal antibodies are typically made by fusing

myeloma cells with the spleen cells from a mouse that

has been immunized with the desired antigen.


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B B B B B B B B

Harvest Ab

Monoclonal antibodies


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Introduction:

Antibody purification involves selectiveenrichment or specific isolation of antibodiesfrom serum (polyclonal antibodies), ascites fluid

or cell culture supernatant of a hybridoma cellline (monoclonal antibodies).


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source Antibody Conmtaminats

Immunized animal

serum

polyclonal Other serum

proteins,other Ig classes

Purified serum Ig G polyclonal IgG of differentspecificity

Affinity purified Ig G polyclonal IgG of differentspecificity

Tissue culturesupernatant with 10%fetal calf serum

monoclonal Possibly bovine Ig G

Ascitic fluid monoclonal Other mouse Ig


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Purification methods range from very crude to highlyspecific and can be classified as follows:

Physicochemical fractionation differentialprecipitation, size-exclusion or solid-phase binding ofimmunoglobulins based on size, charge or othershared chemical characteristics of antibodies in typical

samples.

Class-specific affinity solid-phase binding ofparticular antibody classes (e.g., IgG) by immobilized

biological ligands (proteins, lectins, etc.) that havespecific affinity to immunoglobulins. This purifies allantibodies of the target class without regard to antigen

specificity.


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Antigen-specific affinity affinity purification ofonly those antibodies in a sample that bind to aparticular antigen molecule through their specific

antigen-binding domains. This purifies all antibodiesthat bind the antigen without regard to antibody classor isotype.

Antibodies that were developed as monoclonalantibody hybridoma cell lines and produced as ascitesfluid or cell culture supernatant can be fully purified

without using an antigen-specific affinity method(third type) because the target antibody is (for mostpractical purposes) the only immunoglobulin in the

production sample.


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. By contrast, for polyclonal antibodies (serum samples),antigen-specific affinity purification is required to prevent co-purification of nonspecific immunoglobulins.

Physicochemical Fractionation Antibody Purification

The main classes of serum immunoglobulins (e.g., IgG, IgM)share the same general structure, including overall amino acid

composition and solubility characteristics. These generalproperties are sufficiently different from most other abundantproteins in serum, such as albumin and transferrin, that theimmunoglobulins can be selected and enriched on the basis of

these differentiating physicochemical propertiestion


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Size Exclusion Chromatography

(SEC) high-molecular weight cut-offs (MWCO) can be used

to separate immunoglobulins (>140kDa) from smallproteins and peptides

Thus, a small molecule that can penetrate every cornerof the pore system of the stationary phase "sees" theentire pore volume and the interparticle volume, and

will elute late .


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Whereas a very large molecule that cannot penetratethe pore system "sees" only the interparticle volume(~35% of the column volume) and will elute earlier

when this volume of mobile phase has passed throughthe column.

these techniques alone cannot purify antibodies from

other proteins and macromolecules that are present intypical antibody samples


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Size-exclusion chromatography


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Ammonium Sulfate Precipitation

Ammonium sulfate precipitation is frequentlyused to enrich and concentrate antibodies fromserum, ascites fluid or cell culture supernatant.

As the concentration of this lyotropic salt isincreased in a sample, proteins and othermacromolecules become progressively less

soluble until they precipitate; the lyotropic effectis called "salting out


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Antibodies precipitate at lower concentrations ofammonium sulfate than most other proteins andcomponents of serum.

At 40 to 50% ammonium sulfate saturation (100%saturation equals 4.32M), immunoglobulins willprecipitate .

The usual method involves very slowly adding anequal volume of saturated ammonium sulfate solutionto a neutralized antibody sample, followed by

incubation for several hours at room temperature or4C.


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After centrifugation and removal of the supernatant,the antibody-pellet is dissolved in buffer, such as

phosphate-buffered saline (PBS).proteins remain insolution .

Ammonium sulfate precipitation provides sufficient

purification for some antibody applications, butmost often it is performed as a preliminary stepbefore column chromatography or otherpurification method .


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The diagram shows two proteins, with their hydrophilic regions colouredblue.The protein on the left has relatively few hydrophilic regions, andhence will aggregate and precipitate at a relatively low concentration ofammonium sulphate - perhaps around 20 - 30% saturation. By contrast,

the protein on the right has considerably more hydrophilic regions, andhence will remain in solution until the concentration of ammoniumsulphate is considerably higher - perhaps around 50 - 60% saturation.


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Ion Exchange Chromatography Ion exchange chromatography (IEC) uses positively

or negatively charged resins to bind proteins based ontheir net charges in a given buffer system (pH)

Especially in commercial operations involving

production of monoclonal antibodies, conditions forIEC can be determined that bind and release thetarget antibody with a high degree of specificity.

Conversely, conditions can be found that bind nearlyall other sample components except antibodies.

Once so optimized, IEC is a cost-effective, gentle and

reliable method for antibody purification.

M f h h d i i i ll i h


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Most of the charged impurities are usually anions such asnucleic acids and endotoxins.

]Either cation exchange chromatography is used at a low

enough pH that the desired antibody binds to the columnwhile anions flow through, or anion exchangechromatography is used at a high enough pH that the desiredantibody flows through the column while anions bind to it.

Various proteins can also be separated out along with theanions based on their isoelectric point(pI)

Commonly used anion exchange resins are Q-resin, a

Quaternary amine; and DEAE resin, DiEthylAminoEthane .
http://en.wikipedia.org/wiki/Monoclonal_antibodieshttp://en.wikipedia.org/wiki/Monoclonal_antibodies


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Ion-Exchange chromatography

If pH mobile phase =7.2

Then charge of the proteins: (-) (-) (+) (+)

-- + +

+

+

+

+

+

+-

-

---

-+

+

+

+

+

+

+

+

+

+

+

+

Anion exchange column = + charged


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Immobilized Metal Chelate

Chromatography Immobilized metal chelate chromatography (IMAC)

uses chelate-immobilized divalent metal ions (usuallynickel, Ni2+) to bind proteins or peptides that contain

clusters of three or more consecutive histidineresidues.

mammalian IgGs are one of the few abundant

proteins in serum (or monoclonal hybridoma cellculture supernatant) that possess histidine clusterscapable of being bound by immobilized nickel. .

Thi hili Ad i


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Thiophilic Adsorption

Thiophilic adsorption is a highly selective type of

protein-ligand interaction that combines theproperties of hydrophobic interactionchromatography (HIC) and ammonium sulfate

precipitation (the lyotropic effect). The interaction is termed thiophilic because it

involves the binding of proteins to a sulfone group

in close proximity to a thioether.

hi hili d i d d hi h


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thiophilic adsorption depends upon a highconcentration of lyotropic salt (e.g., potassium sulfateas opposed to sodium chloride).

With typical antibody samples that have beenequilibrated with potassium sulfate, binding is quitespecific to antibodies.

After non-bound components are washed away, theantibodies are easily recovered with gentle elutionconditions (e.g., 50mM sodium phosphate buffer, pH

7 to 8). ). Thiophilic Adsorbent (also called T-Gel) is 6% beaded

agarose modified to contain the sulfone-thioether

ligand.

Th d b t h hi h bi di it d b d


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The adsorbent has a high binding capacity and broadspecificity toward imunoglobulins from various animalspecies.


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Melon Gel Chromatography Melon Gel is a proprietary resin chemistry (and

optimized buffer system) for purifying antibodies bychemical-based fractionation.

In the specified mild buffer condition, Melon Gel

resin binds most non-IgG proteins found in serum,ascites fluid and culture supernatants, while allowingpurified IgG to be collected in the flow-throughfraction.

the Melon Gel system uses negative selection andrequires no elution steps, it also provides a convenientand effective method for removing bovine serum

albumin (BSA).

Th i M l G l kit pti i d f pid


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The various Melon Gel kits are optimized for rapid,convenient and gentle purification of IgG.


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A genetically engineered recombinant form of Protein


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A genetically-engineered recombinant form of ProteinA and Protein G, called Protein A/G, is also widelyavailable.

Protein A Protein G Protein L

Species Staphylococcusaureus

Streptococcusspp.

(Group C and G)

Peptostreptococcus

magnus

Human

PathologyComponent of

human body flora;

cause of "Staph"

infections

Orig. isolated from

pharyngitis patients

(tonsils or blood)

Commensal and/or

pathogenic

anaerobic Gram-

positive bacteria

Native

Size(s)40 to 60kDa 40 to 65kDa 76kDa

Ig-binding Target heavy chainconstant region (Fc)

of IgG

heavy chain const.

region (Fc) of IgG

kappa light chains

of Igs


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Binding sites of antibody-binding proteins

Antibody Purification with


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Antibody Purification with

Protein A, G and L

Antibody purification with Protein A, Protein G,Protein A/G or Protein L, they are covalentlyimmobilized onto porous resins (such as beadedagarose) or magnetic beads.

These proteins contain several antibody-bindingdomains, nearly every individual immobilizedmolecule, no matter its orientation maintains at least

one functional and unhindered binding domain.

The proteins bind to antibodies at sites other than theantigen-binding domain, the immobilized forms of

these proteins can be used in purification scheme.


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AFFINITY

CHROMATOGRAPHY In affinity chromatography antibody is attached to a

solid phase particle eg an agarose bead either by directchemical coupling or by indirect coupling may

achieved by means of an anti-antibody. The antibody coated beads specific for desired

antigen are attached into the column.

A complex mixture of antigens is passed through thebeads to allow the antigen that is recognized by theantibody to bind.

Unbound molecules are washed away and the bound

Antigen is eluted by changing the ph or by exposure


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Antigen is eluted by changing the ph or by exposureto a chemical that breaks the antigen-antibody bonds.

It can similarly used for the purification antibodies byattaching antigen to beads and passing throughsupernatants.


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IMMUNOPRECIPITATION Antigen being tested are labelled and antibody is

added , which binds only to its specific antigen

The complexes are precipitated by addition of co-precipitating agents ,such as anti-immunoglobulin

antibodies.

The insoluble complexes are spun down and washedto remove any unbound labelled antigens.

The precipitate is resolubilized for example in SDSand the components separated on analytical gels.Afterrunning ,the fixed gels are autoradiographed to show

the position specific antigen.

IgM Purification


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IgM Purification

Protein A and Protein G bind IgM very poorly or notat all, in part because binding sites on the Fc regionsof IgM are sterically hindered by its pentamericstructure. .

For IgM (class M antibodies) that possess the

appropriate type of light chains , Protein L can beused for purification; however, IgGs having the sametype of light chains will co-purify.

IgM antibodies are usually purified by a combinationof techniques, including ammonium sulfateprecipitation followed by gel filtration, ion exchange

chromatography or zone electrophoresis

IgA Purification


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IgA Purification

Jacalin is an a-D-galactose binding lectin extractedfrom jackfruit seeds (Artocarpus integrifolia). Thelectin is a glycoprotein of approximately 40kDacomposed of four identical subunits.

Jacalin immobilized on supports such as agarose has

been useful for the purification of human serum orsecretory IgA.

more yield when combined with ion exchange

chromaography

Ig G is purified by precipitation with sodium sulphate


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Ig G is purified by precipitation with sodium sulphateor ammonium sulphate.

Precipitation with ammonium sulphate followed byion exchange chromatography.

Chromatography on immobilized protein A or proteinG.

Precipitation with caprylic acid.

A i ifi Affi i


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Antigen-specific Affinity

Purification of Antibodies

This can be accomplished by immobilizing theparticular antigen used for immunization so thatonly those antibodies that bind specifically tothe antigen are purified in the procedure.

Antigen Immobilization and Presentation:

affinity purification of antibody depends oneffective presentation of the relevant epitopeson the antigen to binding sites of the antibody

Peptide Antigens and Affinity Ligands


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Peptide Antigens and Affinity Ligands

Most antibodies are produced using peptide antigensthat were synthesized and conjugated to animmunogenic carrier protein.

Such antigens can be customized to contain a uniquefunctional group (handle) for both conjugation and

immobilization

Protein Antigens and Affinity Ligands

protein antigens are usually most easily immobilizedfor affinity purification by targeting primary amines,which typically occur in several locations at the outersurface of protein structure.

POLYETHYLENE


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POLYETHYLENE

GLYCOL(PEG) Polyethylene glycol (PEG) was used to isolate immune

complexes from sera.

The pathogenic role of circulating immune complexes(IC) is now well established in both experimentalanimals and in several human diseases, includingsystemic lupus erythematous, rheumatoid arthritis,

viral hepatitis and acute forms of glomerulonephritis.

immune complexes usually contained all threeimmunoglobulin classes IgG, Ig M and Ig A.

Material and method:


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Material and method:

Preparation of single stranded DNA which islabelled by I125.

Precipitation of immuno complexes by PEG: serum0.1 ml was mixed with o.1 ml of 8% PEG or 4%PEG in phosphate buffered saline and then

incubated for 1 hrs at 4c .

Mixture were centrifuged at 1000g for I hr at 4 c.

The pellets were then washed with 0.5 % PEG.

The washed pellets were resuspended in 0.1ml ofPBS and Ig G, Ig M and Ig A measured by RADIALIMMUNODIFFUSION. Immunoglobins in

unreacted sera is also determined

Therefore the percentage of serum immunoglobins


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Therefore the percentage of serum immunoglobinsand complement components precipitated by 4%PEG in excess 2 standard deviationms from the

normal mean was considered to be in immunecomplexes.


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immunological purification

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