Hybridoma technology mAb production

.and ..

Prsented by .. Abhilasha tiwari Ankita Dwivedi Arijit Biswas Jasmine Kaur

HYBRIDOMA TECHNOLOGY
y 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). y 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. y Therefore ,specific hybridomas are either cultured in vitro or passed through mouse peritoneal cavity to obtain monoclonal antibodies,this is called as hybridoma technology.

PRINCIPLE
y 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 clonal selection .

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. y 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.

y 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. y 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.

Improvements in hybridoma technology

y A continuous cell line was used as a fusion partner for the antibody producing B cell y Feeder layers consisting of extra cells to feed newly formed hybridomas were used for optimal growth and hybridoma production. y The most common feeder layers consisted of the following:
y Murine peritoneal cells y Macrophages derived from mouse,rat,or guinea pigs .. y Human fibroblast peripheral blood monocytes or thymus cells

Immunisation

Mice are immunised with Ag

Spleen cell,lymphocytes suspension is obtained .

Fusion

Lymphocytes are fused with myeloma cells

Myeloma cells are malignant lymphocytes . Cells are fused using PEG(polyethyleneglycol) to promote membrane fusion ..

Culture in HAT
T e e i i ic t e c lt re is

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ec tai s . yp xa t i e,a i pteri ,t y i i e .

Myel

a cells are e gi eere t be eficie t i a e zy e yp xa t i e g a i e p tra sferase(HGPRT) ..

sp

rib syl

He ce t e

yel

a cells

t s rvive i c lt re

less t is e zy e is a

e t t e

e ia

O ly ybri cells s rvive

Splee are s

rt live i.e, t eir life spa is 7-10 ays,s t ey ie gra

HAT kills

yel

a cells ..

ally ..

..

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

The powerful toxins of the media block the metabolic pathway Denovo pathway..essential for purine metabolismin DNA synthesis

Fused cells survive

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

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

Screening
y ELISA y RIA y WESTERN BLOT