immunological purification
TRANSCRIPT
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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 .
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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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