.The Nobel Prize In Physiology Or Medicine 1984 Was Awarded Jointly To Niels Jerne , Georges Kohler ,Cesar Milstein “For Theories Concerning The Specificity In Development And Control Of The Immune System And The Discovery Of The Principal For Production Of Monoclonal Antibodies” In 1975 .

A Hybridoma Is A Hybrid Cell Obtained By Fusion Of

B Lymphocyte With Usually A Tumor Cell Of

Antibody Forming System Or B Lymphocyte,(these

Are Called Myelomas).

The Hybrid Cell Thus Formed Posses Ability To

Produce Antibodies Due To B Lymphocyte Genome

And The Capability For The Indefinte Growth In

Vitro Due To Tumor(myeloma)cell Involved In The

Fusion.

Therefore ,Specific Hybridomas Are Either

Cultured In Vitro Or Passed Through Mouse

Peritoneal Cavity To Obtain Monoclonal

Antibodies, This Is Called As Hybridoma Technology.

Antibodies Are Produced By B Lymphocyte , Each B Lymphocyte Being Pre Programmed To Respond To A Single Antigenic Determinant.

Antigenic Determinant Denotes The Region Of Antigen Molecule Which Reacts With An Antibody That Is Specific To It.

When An Antigen Reacts To The Cell Surface Reactor Of B Lymphocyte , It Proliferates Rapidly To Yield A Population Of B Cells ,All Of Which Produces Antibodies Of Same Specificity , This Is Called As “ ClonalSelection”.

Thus A B Lymphocyte Produces Antibodies Of Only One Specificity. In Addition , An Antibody Producing B Lymphocyte Cells Called Plasma Cell Is Fully Differentiated And Does Not Divide When Cultured In Vitro , These Features Are Critical To Hybridoma Technology.

B Lymphocyte Are Isolated From Spleen Of An Animal Eg Mouse Wich Had Been Immunized With Antigen Against Which Monoclonal Antibodies Are To Be Raised.

Immunization Is Achived By Injecting The Antigen Along With The Suitable Adjuvant Either Subcutaneously Or In Peritoneal Cavity Followed By The Booster Doses Of Antigen.

Immunization enhances the population of B lymphocytes producing antibodies specific to antigen used(clonal selection),which greatly increases the chance of obtaining desired hybridoma clone.

A large no. of these b lymphocytes are mixed with cells of selected myeloma and induce to fuse to form hybrid cell.

Myeloma cells are selected for mainly 2 features:

1:These cells must not produce antibodies themselves.

2:They must contain a genetic marker egHGPRT-trait,which permits an easy selection of resulting hybrid cells.

A continuous cell line was used as a fusion

partner for the antibody producing B cell

Feeder layers consisting of extra cells to feed

newly formed hybridomas were used for optimal

growth and hybridoma production.

The most common feeder layers consisted of the

following:

Murine peritoneal cells

Macrophages derived from mouse,rat,or guinea pigs ..

Human fibroblast peripheral blood monocytes or thymus

cells

Spleen cell,lymphocytessuspension is obtained….

Mice are immunised with Ag …

Lymphocytes are fused with myeloma cells

Myeloma cells are malignant lymphocytes…….

Cells are fused using PEG(polyethyleneglycol) to promote membrane fusion…………..

Hence the myeloma cells would not survive in culture…unless this enzyme is added to the media………..

Myeloma cells are engineered to be deficient in an enzyme…hypoxanthine guanine phosphoribosyltransferase(HGPRT)…..

Only hybrid cells survive…….

Spleen are short lived i.e, their life span is 7-10 days,so they die gradually…..

HAT kills myeloma cells…..

The medium in which the culture is done contains….hypoxanthine,aminopterin,thymidine….

Non fused, - HGPRT plasma cells and all non fused B cells die soon…….

Hybridoma cells consisting of immortalized plasma cells fused with B cells will survive in the absence of supplemented HGPRT in culture

medium……

Fused cells survive ………

The powerful toxins of the media block the metabolic pathway “Denovo pathway”..essential for purine metabolism…in DNA

synthesis……

The cells are cultured in HAT medium which is lacking the enzyme HGPRT…

ELISA

RIA

WESTERN BLOT