IMMUNOLOGICAL PURIFICATION

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.

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 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.

Antibody Structure
1.Fab 2.Fc 3.heavy chain 4.light chain 5.antigen binding site 6.hinge regions

 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. Polyclonal antibodies represent the antibodies from multiple clones of B lymphocytes, and therefore bind to a number of different epitopes .

Polyclonal antibodies

Protein Immunize Antibodies

Immune Response
Epitopes
A mixture of antibodies - all bind to epitopes of the original antigen. Some bind with higher affinity than others.

Polyclonal antibodies

Monoclonal antibodies
Monoclonal antibodies are antibodies that are identical because they were produced by one type of immune cell (B cell). Reproducible, Predictable & Potentially inexhaustible supply of Ab with exquisite specificity enable the development 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.

Harvest Ab

Monoclonal antibodies

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).

source

Antibody

Conmtaminats

Immunized animal serum Purified serum Ig G

polyclonal

Other serum proteins,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

 Purification methods range from very crude to highly specific and can be classified as follows: Physicochemical fractionation differential precipitation, size-exclusion or solid-phase binding of immunoglobulins based on size, charge or other shared chemical characteristics of antibodies in typical samples. Class-specific affinity solid-phase binding of particular antibody classes (e.g., IgG) by immobilized biological ligands (proteins, lectins, etc.) that have specific affinity to immunoglobulins. This purifies all antibodies of the target class without regard to antigen specificity.

 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. 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.

 . By contrast, for polyclonal antibodies (serum samples), antigen-specific affinity purification is required to prevent copurification 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 general properties are sufficiently different from most other abundant proteins in serum, such as albumin and transferrin, that the immunoglobulins can be selected and enriched on the basis of these differentiating physicochemical propertiestion

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, and will elute late .

 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

Ammonium Sulfate Precipitation

Ammonium sulfate precipitation is frequently used to enrich and concentrate antibodies from serum, ascites fluid or cell culture supernatant. As the concentration of this lyotropic salt is increased in a sample, proteins and other macromolecules become progressively less soluble until they precipitate; the lyotropic effect is called "salting out

 Antibodies precipitate at lower concentrations of ammonium sulfate than most other proteins and components of serum. At 40 to 50% ammonium sulfate saturation (100% saturation equals 4.32M), immunoglobulins will precipitate . The usual method involves very slowly adding an equal volume of saturated ammonium sulfate solution to a neutralized antibody sample, followed by incubation for several hours at room temperature or 4C.

 After centrifugation and removal of the supernatant, the antibody-pellet is dissolved in buffer, such as phosphate-buffered saline (PBS).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 .

The diagram shows two proteins, with their hydrophilic regions coloured blue.The protein on the left has relatively few hydrophilic regions, and hence will aggregate and precipitate at a relatively low concentration of ammonium sulphate - perhaps around 20 - 30% saturation. By contrast, the protein on the right has considerably more hydrophilic regions, and hence will remain in solution until the concentration of ammonium sulphate is considerably higher - perhaps around 50 - 60% saturation.

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, conditions for IEC can be determined that bind and release the target antibody with a high degree of specificity. Conversely, conditions can be found that bind nearly all other sample components except antibodies. Once so optimized, IEC is a cost-effective, gentle and reliable method for antibody purification.

 Most of the charged impurities are usually anions such as nucleic acids and endotoxins. ]Either cation exchange chromatography is used at a low enough pH that the desired antibody binds to the column while anions flow through, or anion exchange chromatography is used at a high enough pH that the desired antibody flows through the column while anions bind to it. 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, a Quaternary amine; and DEAE resin, DiEthylAminoEthane .

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 adsorption depends upon a high concentration of lyotropic salt (e.g., potassium sulfate as opposed to sodium chloride). With typical antibody samples that have been equilibrated with potassium sulfate, binding is quite specific to antibodies. After non-bound components are washed away, the antibodies are easily recovered with gentle elution conditions (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.

 The adsorbent has a high binding capacity and broad specificity toward imunoglobulins from various animal species.

 Melon Gel is a proprietary resin chemistry (and optimized buffer system) for purifying antibodies by chemical-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 allowing purified IgG to be collected in the flow-through fraction. 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 Chromatography

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

Class-specific Affinity Purification of Antibodies

Their are certain pathogenic bacteria which have evolved proteins having specific antibody-binding functions. proteins have been identified and isolated from certain species of bacteria. These native anti-Ig proteins are useful as affinity ligands for antibody purification. Protein A, Protein G and Protein L are three bacterial proteins whose antibody-binding properties have been well characterized.

 A genetically-engineered recombinant form of Protein A and Protein G, called Protein A/G, is also widely available.
Protein A Protein G Protein L

Species

Staphylococcus aureus
Component of human body flora; cause of "Staph" infections 40 to 60kDa

Streptococcus spp.
(Group C and G) Orig. 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. constant region (Fc) region (Fc) of IgG of IgG

kappa light chains of Igs

Binding sites of antibody-binding proteins

Antibody Purification with Protein A, G and L

Antibody purification with Protein A, Protein G, Protein A/G or Protein L, they are covalently immobilized onto porous resins (such as beaded agarose) or magnetic beads. These proteins contain several antibody-binding domains, nearly every individual immobilized molecule, 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, the immobilized forms of these proteins can be used in purification scheme.

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. The antibody coated beads specific for desired antigen are attached into the column. A complex mixture of antigens is passed through the beads to allow the antigen that is recognized by the antibody to bind. Unbound molecules are washed away and the bound

 Antigen is eluted by changing the ph or by exposure to a chemical that breaks the antigen-antibody bonds. It can similarly used for the purification antibodies by attaching antigen to beads and passing through supernatants.

 Antigen being tested are labelled and antibody is added , which binds only to its specific antigen The complexes are precipitated by addition of coprecipitating agents ,such as anti-immunoglobulin antibodies. The insoluble complexes are spun down and washed to remove any unbound labelled antigens. The precipitate is resolubilized for example in SDS and the components separated on analytical gels.After running ,the fixed gels are autoradiographed to show the position specific antigen.

IMMUNOPRECIPITATION

 IgM Purification Protein A and Protein G bind IgM very poorly or not at all, in part because binding sites on the Fc regions of IgM are sterically hindered by its pentameric structure. . For IgM (class M antibodies) that possess the appropriate type of light chains , Protein L can be used for purification; however, IgGs having the same type of light chains will co-purify. IgM antibodies are usually purified by a combination of techniques, including ammonium sulfate precipitation followed by gel filtration, ion exchange chromatography or zone electrophoresis

 IgA Purification Jacalin is an a-D-galactose binding lectin extracted from jackfruit seeds (Artocarpus integrifolia). The lectin is a glycoprotein of approximately 40kDa composed of four identical subunits. Jacalin immobilized on supports such as agarose has been useful for the purification of human serum or secretory IgA. more yield when combined with ion exchange chromaography

 Ig G is purified by precipitation with sodium sulphate or ammonium sulphate. Precipitation with ammonium sulphate followed by ion exchange chromatography. Chromatography on immobilized protein A or protein G. Precipitation with caprylic acid.

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

 Peptide Antigens and Affinity Ligands Most antibodies are produced using peptide antigens that were synthesized and conjugated to an immunogenic carrier protein. 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.

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 experimental animals and in several human diseases, including systemic lupus erythematous, rheumatoid arthritis, viral hepatitis and acute forms of glomerulonephritis. immune complexes usually contained all three immunoglobulin classes IgG, Ig M and Ig A.

 Material and method: Preparation of single stranded DNA which is labelled by I125. Precipitation of immuno complexes by PEG: serum 0.1 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 of PBS and Ig G, Ig M and Ig A measured by RADIAL IMMUNODIFFUSION. Immunoglobins in unreacted sera is also determined

 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.