Hybridoma TechniqueHybridoma TechniqueHybridoma TechniqueHybridoma Technique

In 1975, Kohler and Milstein first fused lymphocytes to produce a cell line which was both immortal and a producer of specific antibodies. The two scientists were awarded the Nobel Prize for Medicine in 1984 for the development of this "hybridoma." The value of hybridomas to the field was not truly appreciated until about 1987, when MAbs were regularly produced in rodents for diagnostics.

In a specific immune response, only those T and B cells that can bind to the antigensof the pathogen are selected to participate in the response.

Clonal selection of lymphocytes during the specific immune response

An antigen with 2 epitopes - red epitope, blue epitope

Mixture of T and B cellswith different antigenspecificities

Proliferation of cells with receptors capableof binding epitopes ofthe antigen

Polyclonal antibodies:

If an animal is immunized with a protein, a wide array of B cells will be stimulated to produce anti-protein antibodies.

Antibodies may be made to a number of different epitopes of the protein.

Even antibodies that bind to the same epitope may have different antigen-binding sites and bind the epitope with different affinity.

The mixture of antibodies produced in response to an antigen are referred to as polyclonal antibodies (they are produced by many different clones of B cells).

Epitopes

Immune ResponseAntibodies

A mixture of antibodies - all bind to epitopes of the original antigen. Some bind with higher affinity than others.

Polyclonal antibodies

Protein

Immunize

B

B

B

B

Polyclonal antibodies:

Polyclonal antibodies are a mixture of antibodies with different antigen binding sites that may bind to different epitopes or antigens of the immunizing agent with varying affinities. They may be of different antibody classes.

The serum obtained from an immunized animal is referred to as a polyclonal antiserum.

A polyclonal antiserum contains antibody to different epitopes and different antigens that were present in the immunizing inoculum.

From http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/A/AffinityChrom.html

Affinity chromatography - antibody purification.

Antigen can be bound to the support matrix in order to purify antigen-specific antibody from a polyclonal antiserum.

B

B

B

B

Polyclonal antibodies (Polyclonal antiserum)

B

B B B B B B B B

Harvest AbMonoclonal antibodies

- B lymphocytes can mutate into tumor cells that result in a type of cancer termed myeloma.

- Myeloma cells become “immortal” and will grow indefinitely in culture.

- Fusion of a single activated B cell and a myeloma cell will create a hybridoma that can grow indefinitely in culture.

Hybridomas Technique

B

Harvest AbMonoclonal antibodies

M

M

M

M

M

M

M

M

MMyeloma cells

Grow indefinitely in cell culture but don't secrete the desired antibody

FUSE Hybridoma cells

Secrete antibody but don't grow in tissue culture

Grow indefinitely in cell culture AND secrete antibody

Monoclonal antibodies:

Antibodies produced from a single clone of B cells.

Produced by fusing a B cell secreting the desired antibody with a myeloma cell capable of growing indefinitely in tissue culture.

Monoclonal antibodies all have identical antigen-binding sites. Thus they all bind to the same epitope with the same affinity. They are all of the same antibody class (isotype).

Inverted microscope

Myeloma cells have been genetically engineered such that they can not use hypoxanthine, aminopterin, and thymidine (HAT medium) as a source for nucleic acid biosynthesis and will die in culture.

Only B cells that have fused with the engineered myeloma cells will survive in culture when grown in HAT medium.

Hybridoma SelectionThe “HAT Trick”

Practical steps in monoclonal antibody production:

1) Immunize animal

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

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

4) Allow unfused B cells to die

5) Add aminopterin to culture to kill unfused myeloma cells

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

7) Screen supernatant of each clone for presence of the desired antibody.

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

9) Harvest antibody from the culture supernatant.

10) (If you’re a biotech company) charge about $1,000-$2,000 per mg.

Kuby Figure 4-22

Rasing ascitic fluid

In vitro cultivation

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 singleantigens used in the antigenimmunization

Antibody class: A mixture of different All of a single Ab classAb classes (isotypes)

Ag-binding sites: A mixture of Abs with All Abs have the same antigendifferent antigen-binding binding sitesites

Potential for cross-reactivity: High Low

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 hormonesImmunoaffinity Purification

Uses Uses

99mTc-mAb

in vitro in vivo

Tumor diagnosis

Figure 6.24

Affinity chromatography:

1. Bind antibody to a support matrix (e.g. sepharose gel)

2. Add protein mixture - antigen binds to antibody on support

3. Wash to remove unbound material

4. Lower pH - antibody releases the antigen - which is now free of contaminants

From http://ntri.tamuk.edu/fplc/pursammat.html

Affintity chromatography - protein elution profile

Potential Future Uses:

The remarkable specificity of antibodies makes them promising agents for human therapy.

Imagine, for example, beingable to make an antibody that will bind only to the cancer cells in a patient coupling a cytotoxic agent (e.g. a strong radioactive isotope) to that antibody, and then giving the complex to the patient so it can seek out and destroy the cancer cells (and no normal cells).

Mouse monoclonal antibodies have been genetically engineered to replace all of the antibody molecule with human counterparts except the hypervariable regions directly involved with antigen binding.

Humanized monoclonal antibodies are currently be tested in human clinical trials.

Humanized Monoclonal Antibodies

Figure 6.21

Genetic engineering methods can be used to modify monoclonal antibodies

e.g. creation of “humanized” monoclonal antibodies.

Figure 6.22

Antibodies of single specificity (monoclonal) can be obtained by cloning the appropriate heavy and light chain genes.

Figure 6.23d

Engineering to create “immunotoxins”