Topic to be cover Introduction Antibodies Nomenclature of AntibodiesProduction of Monoclonal Antibodies Application of Monoclonal Antibodies Conclusion

ANTIBODIES Antibodies are a

group of glycoproteins secreted by the plasma cells in the serum and tissue fluids of all the mammals. They may occur free in the serum or else occur on the surface of B cell.

Structure of Immunoglobulin1.Four (4) polypeptide chains: 2 identical LIGHT chains and 2 identical HEAVY chains.2.Both light and heavy chains are held together by COVALENT DISULFIDE BONDS.3.Heavy chains are interconnected by DISULFIDE LINKAGES in the HINGE region.4.Ig has 2 terminal regions:

Carboxyterminal - with constant amino acid sequence (constant region). Aminoterminal - with varying antibody specificity (variable region)

Types of Antibodies

Monoclonal antibodies (mAb)

Polyclonal antibodies

POLYCLONAL. MONOCLONAL.

Derived from different B

Lymphocytes cell linesBatch to Batch

variation affecting Ab reactivity & titre

NOT Powerful tools for clinical diagnostic

tests

Derived from a single B cell clone

mAb offer Reproducible, Predictable &

Potentially inexhaustible supply of Ab with exquisite

specificityEnable the

development of secure immunoassay

systems.

DISCOVERY OF MONOCLONAL ANTIBODIES

Immortal Monoclonal antibodies were found in the patients suffering from a disease called Multiple myeloma(a cancer of

B-lymphocytes) by Georges Kohler and Ceaser Milstein in 1975

HYBRIDOMA TECHNOLOGY

Myeloma cell culture applied to fused cells resulting due to fusion of following two types of cells:

an antibody producing B-lymphocyte cell

a single myeloma cell

HYBRIDOMA CREATES MONOCLONAL ANTIBODIES

• Monoclonal antibodies are typically made by fusing myeloma cells with the B cells from a mouse that has been immunized with the desired antigen

• Recent advances have allowed the use of rabbit B-cells.

1) Immunize animal (mouse or rabbit)

2) Isolate spleen cells (containing antibody-producing B cells)

3) Fuse spleen cells with myeloma cells (e.g. using PEG - polyethylene glycol)

4) Add HAT(hypoxanthine-aminopterin-thymidine) culture to kill unfused myeloma cells

5) Clone remaining cells (place 1 cell per well and allow each cell to grow into a clone of cells)

6) Screen supernatant of each clone for presence of the desired antibody (ELISA)

7) Grow the chosen clone of cells in tissue culture indefinitely.

8) Harvest antibody from the culture supernatant.

PROCEDURESTEP -1 Immunization of a mouse.

STEP -2 Culture separately spleen cells (B lymphocytes) that produce specific antibodies.

STEP -3(A) Preparation of Myeloma Cells

- treated with 8-azaguanine to ensure sensitivity to HAT

STEP -3(B) myeloma cells have two features :

(i) Unable to synthesize antibodies and

(ii) Deficient in hypoxanthine guanine phosphoribosyl transferase enzyme or HGPRT.

STEP -4 Fusion of cells using polyethylene glycol (PEG).

STEP -5 Selection for the hybrid cells:

Selectively grow hypoxanthine aminopterin thymidine (HAT) medium

Aminopterin block nucleotide synthesis pathway

Another pathway needs HGPRT enzyme.

STEP -6 Selection of desired hybridoma cell:

Only one in several hundred cell hybrids will produce antibodies of the desired specificity

Facilitated by preparing single cell colonies

STEP -7 Culture of selected hybridoma

Monoclonal antibodies in large quantity.May be frozen for future use.May also be injected in the body of an animal.

STEP -8 Harvesting:

Can be done later from the body fluid.

PURIFICATION OF MONOCLONAL ANTIBODIES

In-vitro contaminants – growth factors and hormones etc.

In-vivo contaminants – host antibodies, protease, nuclease, nucleic acids etc.

Techniques are:

(i) Centrifugation and filteration – 0.45µm.(cell, cell debris, lipids and clotted material).

(ii) Ion exchange chromatography – charged impurities.Anionic: nucleic acids and endotoxins

(iii) Size exclusion chromatography – Transferrin.

iv)AFFINITY CHROMATOGRAPHY

- Specific binding interaction- Ligand coupled to solid

support- Unbound components

washed away- Bound molecules stripped

by low pH buffer

Problems with using mouse monoclonal antibodies

For ethical reason humans cannot be immunized against Pathogen.

Limited therapeutic use in humans Severe allergic response .The fused human lymphocytes-mouse myeloma cells are very unstable.

There are no suitable myeloma cells in human that can replace mouse myeloma cells.

References:http://www.searchpdfengine.com/HYBRIDOMA-

TECHNOLOGY-FOR-PRODUCTION-OF-MONOCLONAL-ANTIBODIES.html

grants.nih.gov/grants/policy/antibodies.pdf http://www.searchpdfengine.com/Therapeutic-

monoclonal-antibodies.htmlBiotechnology by U Satyanaryana (Pages:214-

219)