HYBRIDOMA TECHNOLOGY

• Hybridoma cells are formed via fusion between a short-lived antibody-producing B-cell and an
immortal myeloma cell.
• Hybridoma technology is a method for producing large number of identical antibodies (also called
monoclonal antibodies), specific to antigens of interest.
• Each hybridoma constitutively expresses a large amount of one specific monoclonal antibodies
(mAb).

A. MONOCLONAL ANTIBODIES

• Each antigen has specific antigen determinants (epitopes) located on it.

• The conventional or polyclonal antibodies are isolated from serum; represent a collection of
antibodies from different B cells that recognize multiple epitopes on the same antigen.

• These antibodies have higher potential of cross reactivity due to recognizing multiple epitopes.

• To overcome the limitations of polyclonal antibodies, a new methodology was invented to produce
monoclonal antibodies.

• Monoclonal antibodies are produced by Hybridoma technique, developed by G Kohler and C Milstein
(1975), for which they were awarded Nobel Prize in 1984.

Table 1: Characteristics of polyclonal and monoclonal antibodies

Monoclonal Antibodies Polyclonal Antibodies

Expensive production Inexpensive production

Long production time Rapid production

Large quantities of specific antibodies Large quantities of non specific antibodies

Recognize a single epitope on an antigen Recognize multiple epitopes on an antigen

Production is continuous and uniform Different batches vary in composition

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Advantages of monoclonal antibodies
• mAbs have very specific targets
• Don’t have intercellular activity-as a result, there are few anticipated side effects and reactions.
• Catabolised within the cells resulting in amino acids, which are recycled within the body.

B. HYBRIDOMA TECHNOLOGY TO PRODUCE MONOCLONAL ANTIBODIES

Principle
A clone of B cell stimulated against a single epitope of antigen (i.e. antibody-producing plasma B-cell)
is fused with malignant antibody-producing myeloma cell to produce a hybridoma cell. This hybridoma
cell has two unique properties:
1. Produces monoclonal antibody of same antigen specificity (due to B-cell component).
2. Multiplies indefinitely producing a clone of identical cells (due to immortal myeloma cell
component).

Steps involved in monoclonal antibodies production

1. Immunization
• Mice are immunized with an antigen that is prepared for injection with adjuvant (intradermally or
subcutaneously).
• Adjuvant is non antigenic in nature, however stimulate the immune response.
• In general, mice are immunized every 2-3 weeks to stimulate immune response.
• When a sufficient antibody titer is reached in serum, immunized mice are euthanized and the spleen
removed to use as a source of cells for fusion with myeloma cells.

2. Screening of mice for antibody production

• After several weeks of immunization, blood samples are obtained from mice for measurement of
serum antibodies.
• Serum antibody titer is determined with various techniques, such as ELISA and flow cytometry.
• If the antibody titer is high, cell fusion can be performed. If the titer is too low, mice can be boosted
until an adequate response is achieved, as determined by repeated blood sampling.
• When the antibody titer is high enough, mice are commonly boosted by injecting antigen without
adjuvant intraperitoneally or intravenously (via the tail veins) 3 days before fusion but 2 weeks after
the previous immunization.

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• Then the mice are euthanized and their spleens which contain antibody-producing plasma cells
removed for in-vitro hybridoma cell production.

3. Preparation of myeloma cells

• As antibody-producing spleen cells cannot remain viable in the cell culture for a long time, so they
must be fused with cells (myeloma cells) that are capable of surviving and multiplying in tissue
culture.
• Myeloma cells are immortalized cells that are cultured with 8-azaguanine to ensure their sensitivity
to the hypoxanthine-aminopterin-thymidine (HAT) selection medium used after cell fusion.

• The HAT medium allows only the fused cells to survive in culture.

4. Fusion of myeloma cells with immune spleen cells

• Fusion is accomplished by co-centrifuging freshly harvested spleen cells and myeloma cells in
polyethylene glycol, a substance that causes cell membranes to fuse.
• The exposure of PEG is only for a short period, as it is toxic.
• PEG is then removed by washing and the cells are kept in a fresh medium.

5. Selection of hybridomas
• Hybridoma selection after fusion of myelomas and spleen cells is a critical step in monoclonal
antibody production. HAT (hypoxanthine-aminopterin-thymidine) method i s used to accomplish
this task.
• During the fusion process, three types of cells are present:
1) Unfused myeloma cells that are deficient in an enzyme called HGPRT,
2) Unfused spleen cells, and
3) Fused hybridoma cells.
• Unfused spleen cells are easily selected against since they do not replicate in culture.
• Unfused myelomas can be selected against using media containing HAT.

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* The aminopterin found in the medium blocks the de novo DNA nucleotide synthesis pathway. Typically
when the de novo pathway is blocked, cells will then utilize the salvage pathway as an alternative means
to replicate (only if hypoxanthine and thymidine are present). However, these myelomas are unable to
do so since they are deficient in an enzyme called HGPRT, which is required for the salvage pathway.
Hence, myelomas are unable to replicate in culture.

Figure 1: De novo and Salvage pathway of DNA production

TK-Thymidine Kinase,
HGPRT- Hypoxanthine-guanine phosphoribosyl transferase

• Only hybridomas survive. Hybridomas inherit a functioning HGPRT enzyme from the spleen cells,
so even though the de novo pathway is blocked, they can still use the salvage pathway to replicate.

6. Screening of products
• The hybridoma must be screened for the antibody of desired specificity.
• The culture medium from each hybridoma culture is periodically tested for the desired antibody
specificity.
• The two techniques ELISA and RIA are commonly used for this purpose.
• The antibody secreted by the hybrid cells is referred as monoclonal antibody.

7. Cloning and propagation

• The single hybrid cells producing the desired antibody are isolated and cloned.
• Two techniques are used for cloning hybrid cells: Limiting dilution and soft agar method.

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1) Limiting dilution method:

✓ Suspension of hybridoma cells is serially diluted and the aliquots of each dilution are put into
micro culture wells.
✓ The dilutions are so made that each aliquot in a well contains only a single hybrid cell. This
ensures that the antibody produced is monoclonal.

2) Soft agar method:

✓ The hybridoma cells are cultured in soft agar.

✓ It is possible to simultaneously grow many cells in semisolid medium to form colonies.

These colonies will be monoclonal in nature.

• In actual practice, both the above techniques are combined and used for maximal production
of mAbs.

8. Production of monoclonal antibodies

1) In Vitro tissue culture technique:
✓ Hybridoma is grown in batches in the culture medium and mAbs are purified from it.
✓ Fetal bovine serum is used mostly as culture media.

2) In Vivo tissue culture technique:

✓ The production of mAbs in the culture bottles is rather low.

✓ The yield can be increased by growing the hybrid cells as ascites in the peritoneal cavity of
mice.

9. Purification of monoclonal antibodies

• During purification process impurities such as host cell protein, endogenous viruses, endotoxins,
aggregates and other species must be removed while an acceptable yield is maintained.
• The various steps involved in purification process are described in figure 2.

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10. Characterization and storage
• The monoclonal antibody has to be subjected to biochemical and biophysical characterization for the
desired specificity.

• The mAb is elucidated for the immunoglobulin class or sub-class, the epitope for which it is
specific and the number of binding sites it possesses.
• The stability of the cell lines and the mAbs are important. The cells (and mAbs) must be
characterized for their ability to withstand freezing, and thawing.
• The desired cell lines are frozen in liquid nitrogen (-196ºC) at several stages of cloning and culture.

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C. TYPES OF MONOCLONAL ANTIBODIES
1. Mouse mАb (-omab): It contains 100% mouse-derived proteins. They can lead to an allergic
reaction in humans.
2. Chimeric mAb (-ximab): It is prepared by recombination of mouse proteins (variable region) and
human proteins (constant region). These can cause an allergy.
3. Humanized mAb(-zumab): Here, only the antigen-binding site (i.e. CDR-complementarity
determining region) is mouse derived (10%) and the remaining part of mAb is human-derived.
4. Human mAb (-umab): Contains 100% human-derived amino acids. It is the best-accepted mAb in
humans.

Figure 3: Different classes of monoclonal antibodies

D. APPLICATIONS OF MONOCLONAL ANTIBODIES

Monoclonal Antibodies

Diagnostic Applications Therapeutic Applications Protein Purification Miscellaneous

Applications
In Biochemical Analysis As Therapeutic Agents
• Pregnancy • In destroying disease causing organisms Catalytic mAbs
• Cancers • In the treatment of cancer
Autoantibody
• Hormonal Disorders • In immunosuppression of organ
• Infectious diseases
Fingerprinting
transplantation
• In treatment of autoimmune diseases
In Diagnostic Imaging
• Cardiovascular Diseases As Targeting Agents in Therapy
✓ Myocardial Infraction • As Immunotoxins
✓ Deep Vein Thrombosis • In Drug Delivery
✓ Atherosclerosis • In Disease Treatment
• Cancers • In the Dissolution of Blood Clots
• In Hematopoietic Malignancies
• Bacterial Infections

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1. Diagnostic Testing
Once mAbs are produced for a specific substance, they can be then used to test for the presence of
that substance in a vessel. This can include toxins, drugs or hormones.

2. Pregnancy Testing
mAbs that have been developed to detect human chorionic gonadotropin (HCG) are now present in
pregnancy test kits.

3. Radioimmunodetection (RID) of Cancer

An imaging technique used to detect the presence of cancerous or cancer-specific cells has been
developed deploying radio-labelled antibodies, which can be produced as mAbs.

4. Radioimmunotheraphy (RIT) of Cancer

RIT uses mAbs to specifically target antigen cells that are associated with tumours, and then blast
these with a lethal dose of radiation, while minimising the level of radiation absorbed by normal cells.

5. Treatment of Cancer through Drugs

Many different drugs are being developed in clinical trials with the ultimate hope of being able to
treat various strains of cancer.
In 1997, a drug named Ritoxin was approved by the FDA for commercial use which is based on mAb
technology.

6. Viral Disease Treatment

Doctors hope that with further research into mAbs and an increased knowledge of their properties,
treatments will become available for diseases previously thought to be incurable, such as AIDS.

7. Identifying Pathogens
mAbs can now be used to identify strains of a single pathogen, for example Neisseria gonorrhoeae.

8. Tracing Specific Cells and their Functions

Scientists can use mAbs to first identify and then track certain cells or molecules in a living thing,
and determine its function.

9. Organ Rejection
A certain mAb named OKT3 (developed as an antibody to the T3 antigen) is able to be used to
alleviate the effects and likelihood of organ rejection when transplanting new organs into a subject.

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10. Rhesus disease Immunisation
Anti-rhesus antiserum is becoming increasingly hard to find, and the UK Blood Products laboratory
has been researching the possibility of substituting mAb rhesus immunisation, with a view to
ultimately replacing the serum.

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