HYBRIDOMA TECHNOLOGY

1975, Georges Köhler and Cesar Milstein

devised a
method for preparing monoclonal antibody
called hybridoma
 HYBRIDOMA TECHNOLOGY
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)
HGPRT+
4) Allow unfused B cells to die

5) Add HAT culture to kill unfused myeloma cells

6) Clone remaining cells (place 1 cell per well and

allow each cell to grow into a clone of cells)

7) Screen supernatant of each clone for presence

of the desired antibody (ELISA)

8) Grow the chosen clone of cells in tissue culture

indefinitely.

9) Harvest antibody from the culture supernatant.

 Monoclonal antibodies

• MAbs produced from a single clone of B cells

• Monoclonal antibodies all have identical antigen-

binding sites. Thus they all bind to the same epitope
with the same affinity

• Mostly produced by fusing a B cell secreting the

desired antibody with a myeloma cell capable of
growing indefinitely in tissue culture
 Polyclonal antibodies Monoclonal Antibodies

Produced by: Many B cell clones A single B cell clone

Bind to: Multiple epitopes of all A single epitope of a single

antigens used in the antigen
immunization

Antibody class: A mixture of different All of a single Ab class

Ab classes (isotypes)

Ag-binding sites: A mixture of Abs with All Abs have the same antigen
different antigen-binding binding site
sites

Potential for cross-reactivity: High Low

 Production of mAb
• Step 1: Immunization of mice
• Mice are immunized with an antigen (attached to adjuvant). The antigen
can be whole cells, membrane fragment, or complex molecules.
• Mice sera are screened using various techniques such as ELISA.
• When sufficient titer is reached the mice are euthanized and spleen is
removed as a source of cells for cell fusion.

• Step 2: Preparation of Myeloma Cells

• Myeloma cells are immortalized cells that are capable of dividing
indefinitely.
• These cells are treated with 8-azaguanine to ensure sensitivity to HAT
 Production of mAb
Step 3: Fusion of myeloma cells with Spleen cells
• Spleen cells harvested from mice are fused with myeloma
cells.
• polyethylene glycol facilitates fusion
• Cells are plated in selection medium
• hypoxanthine-aminopterin-thymidine (HAT) selection–
inhibitor of aminoterin which blocks nucleotide synthesis
• Only fused cells with grow on HAT
• Cells are distributed on feeder cells (murine bone-marrow)
to promote growth of the hybridoma cells.
 The HAT-trick
In normal cells, nucleotide synthesis occurs from
simple sugars.
In HAT medium, aminopterin blocks DNA de
novo synthesis, but cells can survive by synthesising
DNA using hypoxanthine and thymidine provided in
the HAT medium by alternative pathway (the
"salvage pathway"), provided that they have the
right enzymes, which means having functioning
copies of the genes that encode them.
 Production of mAb
Step 4: Cloning of Hybridoma cells.
• A mouse is inoculated with the cell and thereby becomes a factory for
producing the mAb.
• Ascites are collected from the mouse.

Step 5: Ab are screened and Purified

• Ab are screened using specific Ag binding.

Advantage of in vivo process

• Relatively inexpensive and easy
Disadvantage:
• Ethical concerns with using animals.
 Production of mAb
Step 6: Desired Ab are cloned
• This is done in vitro on culture bottles

Reclone and cultivate positive clones

•Monoclonal antibodies can be
produced in cell culture or in
animals.
•Unethical to inject
hybridoma cells in mice!!!
 Uses
Uses
Measuring protein and drug levels in serum
Typing tissue and blood
Identifying infectious agents
Identifying clusters of differentiation for the
classification and follow-up therapy of leukemias
and lymphomas
Identifying tumor metastasis
Identifying and quantifying hormones
Immunoaffinity Purification
Immunotoxins- delivery of therapeutic molecules to
target tumour cells
Problems with using mouse mAb
• The therapeutic use of rodent monoclonal antibodies in humans is
limited by their immunogenic, short circulating half-life, and
inability to efficiently trigger human effectors mechanisms.

• This is due to genetic differences between the mouse and humans.

• Severe allergic response in human when mouse mAb are

introduced to a patients.

• Constant region of murine mAb are not effective in interacting with

human effector molecules.