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HYBRIDOMA TECHNOLOGY

Suman Kumar Mekap

CUTM A

HISTORY:

In 1975, these technology developed by

Georges J.F.Kohler and Cesar Milstein.

And in 1984, they shared a Nobel prize forthis discovery.

They make a hybrid cell that will make anumbers of monoclonal antibodies againstantigen .

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PRINCIPLE:

The hybrid cell has the capacity of antibodyproduction derived from B-cells (spleen cell ).

At the same time it can divide continuously bythe quality derived from myeloma cell.

By combining the desired qualities of both thecells, the technology ensures large, antibodyproduction of single specificity.

Specific hybridomas(spleen cell and myelomacell) obtain monoclonal antibodies in artificialmedia, this technology called as HYBRIDOMATECHNOLOGY.

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MONOCLONAL ANTIBODY:

Monoclonal antibodies (mAb) areantibodies that are identical because theyare produced by one type of immune cell, allclones of a single parent cell.

Basically produced by white blood cellwhich is called as plasma cell.

Is used for treatment of cancerous cells andas anti-venom( anti snake venom)

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PROCEDURE:

1. Immunization of specific animal whichgenerate hybridoma cell with spleen cell.

2. Isolation of myeloma cells.

3. Fusion between spleen cell and myelomacell.

4. Selection of HAT medium.

5. Isolation of hybridoma cell.

6. Screening of hybridoma cell.

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1. Immunization of specific animal.

An antigen immunized to an animal (like mice) viaintravenously(directly to blood) by injection.

Where in spleen it activate B-cell which produceplasma cell (spleen cell).

Plasma cell to produce monoclonal antibodies

Isolation of plasma cell from spleen of animal.

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2. Isolation of myeloma cells.

Myeloma cells are cancerous cells which isisolated from bone-marrow.

Myeloma cells are generally immortal innature (that which never dies) and hasmultiplication property.

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3. Fusion of spleen cell and myeloma cell.

It requires PEG (poly ethylene glycone)medium for fusion

It can also done by electro fusion.

Fusion between spleen cell and myelomacell produced five different types of cells.

Fused plasma

Fused myeloma

Hybridoma

Unfused plasma

Unfused myeloma

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4. Selection of HAT medium. ( Hypoxanthine, Aminopterin, Thymidine)

Before multiplication of Anti-body, it has tosynthesize new copy of DNA and for that itrequire synthesis of nucleotide.

For synthesis of nucleotide mainly two pathwaysare there:1. Salvage pathway2. De-novo Synthesis

In 1 , Salvage pathway it requires degraded partof old nucleotide to produce new nucleotide.

In 2, De-novo synthesis it synthesizedcompletely new nucleotide by small molecules(sugar, amino-acid).

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So in HAT medium, Cells not synthesized byDe-novo synthesis due to presence ofAminopterin in HAT medium which blocksDi-hydro follate enzyme which isnecessary for these synthesis.

For synthesis in salvage pathway it mustrequires HGPRT enzyme (HypoxanthineGuanine Phospho-Ribosyl Transferase).

Where hypoxanthine and thymidine areused as precursors.

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5. Isolation of hybridoma cell

Myeloma cell doesn’t have HGPRT enzyme

Spleen cell have HGPRT enzyme

1. Fused plasma present2. Fused myeloma absent3. Hybridoma present4. Unfused plasma present5. Unfused myeloma absent

HGPRT

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Fused myeloma and unfused myelomadidn’t have HGPRT enzyme so, can’t survivein HAT medium.

Fused plasma and unfused plasma haveHGPRT enzyme but didn’t have long-life.

Hybrid cell has HGPRT enzyme from spleencell as well as they have the ability tomultiply repeatedly as myeloma cell.

So, isolation of hybrid cell because is onlycell which survive in HAT medium.

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6. Screening of hybridoma cell.

ELISA screening method which done byincubating hybridoma culture in whichsecondary enzyme gets conjugate andformation of colored product shows positivehybridoma.

Used for multiplying the hybridoma cells

o In-vivo

o In-vitro

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In-vivo procedure involves introduction ofhybridoma cells into the peritoneal cavity ofthe animal , then from ascetic fluidantibodies are isolated.

In-vitro method involves culturing ofhybridoma cells in suitable culture mediaand then antibodies are isolated andpurified.

Once a hybridoma colony is established, itwill continually grow in culture mediumlike RPMI-1640 and produce antibodies.

Storage: liquid nitrogen.

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APPLICATION OF HYBRIDOMA TECHNOLOGY

Serological:

Identification of ABO blood group

Diagnosis:

Detection of pregnancy by assaying of hormones withmonoclonal.

Separation of one substance from a mixture of very similarmolecules.

Immunopurification:

Purification of individual interferon using monoclonal.

Inactivation of T-lymphocytes responsible for rejection of organtransplants.

Therapy:

Removal of tumor cell from bone marrow.

Treatment of acute renal failure.

Treatment malignant leukemic cells, B cell lymphomas, and avariety of allograft rejections after transplantation.

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THANK YOU

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