Services and Offerings
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HBPS has over 20 years of experience developing monoclonal
antibodies from a wide variety of peptides and proteins. Our
flexible service allows for the development of up to 3, 5 or 10
positive clones. Our scientists will work with you to develop a
procedure that will best meet your needs. In addition, our
production process is broken down into 3 phases which allows you to
pay for only those phases that are completed. Once the desired
clones are developed, we will scale up some material for your use,
either as in vivo or in vitro. Our schedule for
producing up to 3 positive clones is as follows.
STAGE I: Immunization
- Ten virus antibody-free mice will be immunized with the
customer's antigen. The mice will be housed in an isolator during
the immunization period. We immunize 10 mice to increase our chance
of an immunogenic reaction.
- In order to get a better antibody response the antigen is
prepared with an adjuvant such as FCA( Freund's complete adjuvant)
for the primary immunization. This emulsion contains heat-killed
mycobacterium that ensures the antigen is released slowly into the
animal's circulatory system. The bacteria in the adjuvant
stimulates the animals immune system. Further boost's or
immunizations are necessary for high antibody levels. FIA (Freund's
incomplete adjuvant) is used. Common routes for injection are i.p.,
i.m., i.v., s.c.. For hybridoma development, s.q. is typically
used. 5-100 ug of antigen are used for each immunization. We ask
the client for 2-3 mg of antigen.
- At six weeks, following the primary immunization and the first
boost, the mice will be tested for antibody titer. The antibody
will be determined by ELISA assay unless the customer has an
alternative assay. If additional boosts are required, they will be
administered and the mice will be retested for antibody titer.
- Once immunization is complete, the mice will be held for
three-to-four weeks before fusion. Three-to-five days before the
fusion, a mouse will be selected for the pre-fusion boost.
STAGE II
1) Fusion
- The spleen from the mouse will be harvested and all spleen
cells fused with a myeloma cell line. We generally use NS-1 for the
fusion.
- Generally six-to-ten 96-well panels will be plated.
- Cells will be grown in HAT ( Hypoxanthine, aminopterin and
thymidine) selection medium. This selectively kills cells deficient
in the enzyme (HGPRTase), which is essential for the operation of
the salvage pathway for making nucleic acids. The myeloma and
spleen cells will die off.
- A maximum of three separate fusions will be performed.
2) Screen and Select
- All 96 well panels will be screened by ELISA for antibody
positive wells.
- The standard production will be screened with a single ELISA
antigen. Additional ELISA antigens may be added as an option
- About 15 positive wells from the ELISA assay will be selected
to be expanded and frozen for possible cloning.
- Tissue culture supernatant (0.2 ml per clone) can be sent to
the customer for further testing.
STAGE III
1) First Cloning
- A maximum of 6 positive wells will be selected for
cloning.
- They will be cloned by the limiting dilution method. In this
method cells are diluted so that they can be individually pipetted
into separate wells.
- Isolated colonies will be tested by ELISA and positives
selected to be expanded for the next cycle of cloning. Colonies
should be visible after 10-14 days.
- Cells from these positive clones will be frozen as backup.
2) Final Cloning
- Positive clones from the first cloning cycle will be selected
for the second cycle.
- Isolated colonies will be tested by ELISA and positives
selected to expand. Several cycles of cloning may be required in
order to obtain true, stable clones. Cells from these positive
clones will be frozen as backup.
3) Selection For Non-HT
Requirement
- A maximum of three positive clones will be further selected for
clones, which grow in medium lacking HT.
4) Frozen Stock and
Ascites
- The final three clones selected will be expanded and a small
stock of each will be frozen. A small amount of tissue culture
supernatant (2-5 ml) will be prepared for each clone. Each clone
will be isotyped.
- In addition, several mice will be inoculated with each clone
for ascites production. Supernatant production can also be
performed. Ascites fluid, supernatant and/or vials of cells will be
shipped to the customer for evaluation.
Contact Information
- To receive a
Hybridoma Development Information Sheet, pricing, or for more
information about our capabilities, contact our Customer Services
Center at 800-972-4362 or email us at hbps@harlan.com. Our technical
staff is also available to assist you in determining the type of
production system that best meets your needs.