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PRESENTED BY: HARNEET –KAUR L-2011-V-87-M
ANTIBODY AND ANTIGEN
• Antibodies, or Y-shaped immunoglobulins , are proteins found in the blood that help to fight against foreign substances 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 by B lymphocytes . • Great diversity and specificity: >109 different antibodies.
• 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 immune regulation 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
• Each Y contains four polypeptides---two identical copies of a polypeptide knownas the heavy chain and two identical copies of a polypeptide called the light chain. • The two heavy-chain polypeptides in the Y structure are identical and are about 55kDa. The two light chains are also identical and are about 25 kDa. • The four polypeptide chains are held together by disulfide bridges and noncovalent bonds.
hinge regions .antigen binding site 6.Fab 2.Antibody Structure 1.heavy chain 4.light chain 5.Fc 3.
• Antisera generated by injection of antigen into an animal will contain a mixture of antibodies directed against different antigens determinants on the molecule. • Such antisera which contain mixture of antibodies that was exclusively directed against the immunogen to which they are raised is known as polyclonal antibodies. and therefore bind to a number of different epitopes . • Polyclonal antibodies represent the antibodies from multiple clones of B lymphocytes. Polyclonal antibodies .
Protein Immunize Antibodies Immune Response Epitopes A mixture of antibodies . Some bind with higher affinity than others. Polyclonal antibodies .all bind to epitopes of the original antigen.
. Predictable & Potentially inexhaustible supply of Ab with exquisite specificity enable the development of secure immunoassay systems clones of a single parent cell. • Reproducible.Monoclonal antibodies • Monoclonal antibodies are antibodies that are identical because they were produced by one type of immune cell (B 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.
Harvest Ab Monoclonal antibodies B B B B B B B B .
Introduction: • Antibody purification involves selective enrichment or specific isolation of antibodies from serum (polyclonal antibodies). . ascites fluid or cell culture supernatant of a hybridoma cell line (monoclonal antibodies).
other Ig classes IgG of different specificity polyclonal Affinity purified Ig G polyclonal IgG of different specificity Possibly bovine Ig G Tissue culture supernatant with 10% fetal calf serum Ascitic fluid monoclonal monoclonal Other mouse Ig .source Antibody Conmtaminats Immunized animal serum Purified serum Ig G polyclonal Other serum proteins.
• Purification methods range from very crude to highly specific and can be classified as follows: • Physicochemical fractionation – differential precipitation. . • Class-specific affinity – solid-phase binding of particular antibody classes (e. This purifies all antibodies of the target class without regard to antigen specificity. lectins. size-exclusion or solid-phase binding of immunoglobulins based on size. charge or other shared chemical characteristics of antibodies in typical samples. IgG) by immobilized biological ligands (proteins. etc.g..) that have specific affinity to immunoglobulins.
. • Antibodies that were developed as monoclonal antibody hybridoma cell lines and produced as ascites fluid or cell culture supernatant can be fully purified without using an antigen-specific affinity method (third type) because the target antibody is (for most practical purposes) the only immunoglobulin in the production sample.• Antigen-specific affinity – affinity purification of only those antibodies in a sample that bind to a particular antigen molecule through their specific antigen-binding domains. This purifies all antibodies that bind the antigen without regard to antibody class or isotype.
IgM) share the same general structure. such as albumin and transferrin. that the immunoglobulins can be selected and enriched on the basis of these differentiating physicochemical propertiestion .• . These general properties are sufficiently different from most other abundant proteins in serum.. antigen-specific affinity purification is required to prevent copurification of nonspecific immunoglobulins. By contrast.g. for polyclonal antibodies (serum samples). • Physicochemical Fractionation Antibody Purification • The main classes of serum immunoglobulins (e. including overall amino acid composition and solubility characteristics. IgG.
. and will elute late .Size Exclusion Chromatography (SEC) • high-molecular weight cut-offs (MWCO) can be used to separate immunoglobulins (>140kDa) from small proteins and peptides • Thus. a small molecule that can penetrate every corner of the pore system of the stationary phase "sees" the entire pore volume and the interparticle volume.
• Whereas a very large molecule that cannot penetrate the pore system "sees" only the interparticle volume (~35% of the column volume) and will elute earlier when this volume of mobile phase has passed through the column. • these techniques alone cannot purify antibodies from other proteins and macromolecules that are present in typical antibody samples .
Size-exclusion chromatography .
ascites fluid or cell culture supernatant.Ammonium Sulfate Precipitation • Ammonium sulfate precipitation is frequently used to enrich and concentrate antibodies from serum. proteins and other macromolecules become progressively less soluble until they precipitate. As the concentration of this lyotropic salt is increased in a sample. the lyotropic effect is called "salting out .
• The usual method involves very slowly adding an equal volume of saturated ammonium sulfate solution to a neutralized antibody sample. • At 40 to 50% ammonium sulfate saturation (100% saturation equals 4. immunoglobulins will precipitate . .• Antibodies precipitate at lower concentrations of ammonium sulfate than most other proteins and components of serum. followed by incubation for several hours at room temperature or 4°C.32M).
.• After centrifugation and removal of the supernatant. such as phosphate-buffered saline (PBS). the antibody-pellet is dissolved in buffer.proteins remain in solution . • Ammonium sulfate precipitation provides sufficient purification for some antibody applications. but most often it is performed as a preliminary step before column chromatography or other purification method .
perhaps around 20 .The diagram shows two proteins. with their hydrophilic regions coloured blue. the protein on the right has considerably more hydrophilic regions. .30% saturation. By contrast. and hence will aggregate and precipitate at a relatively low concentration of ammonium sulphate . and hence will remain in solution until the concentration of ammonium sulphate is considerably higher .60% saturation.The protein on the left has relatively few hydrophilic regions.perhaps around 50 .
Ion Exchange Chromatography • Ion exchange chromatography (IEC) uses positively or negatively charged resins to bind proteins based on their net charges in a given buffer system (pH) • Especially in commercial operations involving production of monoclonal antibodies. • Conversely. • Once so optimized. IEC is a cost-effective. gentle and reliable method for antibody purification. . conditions for IEC can be determined that bind and release the target antibody with a high degree of specificity. conditions can be found that bind nearly all other sample components except antibodies.
Various proteins can also be separated out along with the anions based on their isoelectric point(pI) • Commonly used anion exchange resins are Q-resin. and DEAE resin. DiEthylAminoEthane . or anion exchange chromatography is used at a high enough pH that the desired antibody flows through the column while anions bind to it. a Quaternary amine. • ]Either cation exchange chromatography is used at a low enough pH that the desired antibody binds to the column while anions flow through. .• Most of the charged impurities are usually anions such as nucleic acids and endotoxins.
Ion-Exchange chromatography + + If pH mobile phase =7.2 Then charge of the proteins: (-) (-) (+) (+) + + + + + + + - + - + + + + + + + + + - + Anion exchange column = + charged .
. .Immobilized Metal Chelate Chromatography • Immobilized metal chelate chromatography (IMAC) uses chelate-immobilized divalent metal ions (usually nickel. Ni2+) to bind proteins or peptides that contain clusters of three or more consecutive histidine residues. • mammalian IgGs are one of the few abundant proteins in serum (or monoclonal hybridoma cell culture supernatant) that possess histidine clusters capable of being bound by immobilized nickel.
Thiophilic Adsorption • Thiophilic adsorption is a highly selective type of protein-ligand interaction that combines the properties of hydrophobic interaction chromatography (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.
• Thiophilic Adsorbent (also called T-Gel) is 6% beaded agarose modified to contain the sulfone-thioether ligand. 50mM sodium phosphate buffer. the antibodies are easily recovered with gentle elution conditions (e. • With typical antibody samples that have been equilibrated with potassium sulfate. • After non-bound components are washed away. pH 7 to 8).• thiophilic adsorption depends upon a high concentration of lyotropic salt (e..g. .g.. binding is quite specific to antibodies. potassium sulfate as opposed to sodium chloride). ).
.• The adsorbent has a high binding capacity and broad specificity toward imunoglobulins from various animal species.
while allowing purified IgG to be collected in the flow-through fraction. Melon Gel Chromatography . ascites fluid and culture supernatants. • In the specified mild buffer condition. Melon Gel resin binds most non-IgG proteins found in serum. • the Melon Gel system uses negative selection and requires no elution steps. it also provides a convenient and effective method for removing bovine serum albumin (BSA).• Melon Gel is a proprietary resin chemistry (and optimized buffer system) for purifying antibodies by chemical-based fractionation.
convenient and gentle purification of IgG.• The various Melon Gel kits are optimized for rapid. .
.Class-specific Affinity Purification of Antibodies • Their are certain pathogenic bacteria which have evolved proteins having specific antibody-binding functions. Protein G and Protein L are three bacterial proteins whose antibody-binding properties have been well characterized. • These native anti-Ig proteins are useful as affinity ligands for antibody purification. • Protein A. • proteins have been identified and isolated from certain species of bacteria.
called Protein A/G.• A genetically-engineered recombinant form of Protein A and Protein G. isolated from pharyngitis patients (tonsils or blood) 40 to 65kDa Peptostreptococcus magnus Commensal and/or pathogenic anaerobic Grampositive bacteria 76kDa Human Pathology Native Size(s) Ig-binding Target heavy chain heavy chain const. is also widely available. Protein A Protein G Protein L Species Staphylococcus aureus Component of human body flora. (Group C and G) Orig. constant region (Fc) region (Fc) of IgG of IgG kappa light chains of Igs . cause of "Staph" infections 40 to 60kDa Streptococcus spp.
Binding sites of antibody-binding proteins .
. G and L • Antibody purification with Protein A. Protein A/G or Protein L. nearly every individual immobilized molecule.Antibody Purification with Protein A. • These proteins contain several antibody-binding domains. they are covalently immobilized onto porous resins (such as beaded agarose) or magnetic beads. the immobilized forms of these proteins can be used in purification scheme. no matter its orientation maintains at least one functional and unhindered binding domain. • The proteins bind to antibodies at sites other than the antigen-binding domain. Protein G.
• The antibody coated beads specific for desired antigen are attached into the column.AFFINITY CHROMATOGRAPHY • In affinity chromatography antibody is attached to a solid phase particle eg an agarose bead either by direct chemical coupling or by indirect coupling may achieved by means of an anti-antibody. • Unbound molecules are washed away and the bound . • A complex mixture of antigens is passed through the beads to allow the antigen that is recognized by the antibody to bind.
. • It can similarly used for the purification antibodies by attaching antigen to beads and passing through supernatants.• Antigen is eluted by changing the ph or by exposure to a chemical that breaks the antigen-antibody bonds.
the fixed gels are autoradiographed to show the position specific antigen.such as anti-immunoglobulin antibodies.After running . • The insoluble complexes are spun down and washed to remove any unbound labelled antigens. which binds only to its specific antigen • The complexes are precipitated by addition of coprecipitating agents .• Antigen being tested are labelled and antibody is added . IMMUNOPRECIPITATION . • The precipitate is resolubilized for example in SDS and the components separated on analytical gels.
ion exchange chromatography or zone electrophoresis .• IgM Purification • Protein A and Protein G bind IgM very poorly or not at all. including ammonium sulfate precipitation followed by gel filtration. in part because binding sites on the Fc regions of IgM are sterically hindered by its pentameric structure. IgGs having the same type of light chains will co-purify. however. Protein L can be used for purification. • IgM antibodies are usually purified by a combination of techniques. . • For IgM (class M antibodies) that possess the appropriate type of light chains .
• more yield when combined with ion exchange chromaography . The lectin is a glycoprotein of approximately 40kDa composed of four identical subunits.• IgA Purification • Jacalin is an a-D-galactose binding lectin extracted from jackfruit seeds (Artocarpus integrifolia). • Jacalin immobilized on supports such as agarose has been useful for the purification of human serum or secretory IgA.
. • Precipitation with caprylic acid. • Chromatography on immobilized protein A or protein G.• Ig G is purified by precipitation with sodium sulphate or ammonium sulphate. • Precipitation with ammonium sulphate followed by ion exchange chromatography.
Antigen-specific Affinity Purification of Antibodies • This can be accomplished by immobilizing the particular antigen used for immunization so that only those antibodies that bind specifically to the antigen are purified in the procedure. • Antigen Immobilization and Presentation: • affinity purification of antibody depends on effective presentation of the relevant epitopes on the antigen to binding sites of the antibody .
• Such antigens can be customized to contain a unique functional group (handle) for both conjugation and immobilization • Protein Antigens and Affinity Ligands • protein antigens are usually most easily immobilized for affinity purification by targeting primary amines. . which typically occur in several locations at the outer surface of protein structure.• Peptide Antigens and Affinity Ligands • Most antibodies are produced using peptide antigens that were synthesized and conjugated to an immunogenic carrier protein.
• viral hepatitis and acute forms of glomerulonephritis. Ig M and Ig A. .POLYETHYLENE GLYCOL(PEG) • Polyethylene glycol (PEG) was used to isolate immune complexes from sera. • immune complexes usually contained all three immunoglobulin classes IgG. rheumatoid arthritis. including systemic lupus erythematous. • The pathogenic role of circulating immune complexes (IC) is now well established in both experimental animals and in several human diseases.
Ig M and Ig A measured by RADIAL IMMUNODIFFUSION.• Material and method: • Preparation of single stranded DNA which is labelled by I125. • The washed pellets were resuspended in 0.1 ml of 8% PEG or 4% PEG in phosphate buffered saline and then incubated for 1 hrs at 4c .1ml of PBS and Ig G. Immunoglobins in unreacted sera is also determined . • The pellets were then washed with 0.5 % PEG. • Mixture were centrifuged at 1000g for I hr at 4 c. • Precipitation of immuno complexes by PEG: serum 0.1 ml was mixed with o.
• Therefore the percentage of serum immunoglobins and complement components precipitated by 4% PEG in excess 2 standard deviationms from the normal mean was considered to be in immune complexes. .
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