Construction of New Hybridomas:
Construction of new hybridomas against native or recombinant antigen (Ag) provided by
the customer, including partial characterization of resulting MAbs, and provision of live,
cloned, hybridomas.
For maximum success this service requires significant direct collaboration with the
research scientist most directly involved with the project.
Details of service:
1. Hybridomas will be constructed from spleens and mesenteric lymph nodes of Balb/c
mice immunized i.p. with 25-100 ug (increasing doses injected at 14 –21 day intervals) of
customer provided Ag. CMI will employ RIBI MPL and TDM emulsion adjuvant.
(Alternative adjuvants will be employed upon customer request and further discussion).
2. Two fusions will be performed 3 days after the third and fourth mouse i.p.
immunizations.
3. The Balb/c myeloma fusion partner will be P-X-63 scII, which is a P8.653 precursor
possessing proven high fusion potential, subsequent hybridoma stability, and therefore
maximum cloning potential.
4. Fusions will be screened by ELISA against the customer provided specific antigen, and
24 – 96 putative positive wells will be selected and expanded from the original 96 well
plates to 1.5 ml cultures in 24 well plates.
5. Expanded hybridoma fusion products will be re-screened by ELISA using the specific
antigen and also a variety of non-specific antigens. A critical step for success is to
screen against the specific Ag required, but also against as many closely related –
but non-specific – Ags as the customer is able to provide (this is a major scientific
focus point).
6. Hybridomas (maximum 15 per fusion) judged to be secreting specific MAbs will be
cloned, and the pre-clone supernatants then provided to the customer for their evaluation.
During the next 7-10 days required for the clones to emerge the customer will have
the chance to evaluate the hybridoma products by their method(s) of choice (e.g.
Western blot; IFA, etc) and to report back on most favorable clones.
7. Surviving cloned hybridomas will be assayed by ELISA on customer antigen for
specific MAb secretion. The best secretor clone from each individual hybridoma will be
selected and expanded to facilitate freezing of 4 vials hybridoma cells into liquid
nitrogen.
8. The customer will then be provided one T-75 flask of viable cells per cloned
hybridoma.
9. The Ig isotype or IgG subclass (plus light chain) will be determined for each selected
hybridoma, and that information also provided to the investigator.
Pricing: Minimum charge of $6,000.00 for services described above, and no guarantee
of success is offered. One non-refundable payment of $2,500.00 is required to initiate this
service, but should no specific hybridomas result from either of the two fusions involved,
then no additional charges to the customer will be incurred.
References from academics who have worked with CMI in producing new hybridomas
using the production outline above will be provided if requested.
Purification of Monoclonal Antibodies from Pre-existing Hybridomas
owned by Customers:
CMI will provide an outline for two different services, the first being the cheaper, and the
second being a more careful approach to maintaining hybridoma integrity. It should be
stressed that the price difference does not influence the quality of the purified MAbs
CMI will provide.
CMI expects customers to provide problem hybridomas, and CMI is proud of a
track record whereby this company has salvaged four individual hybridomas that
were otherwise lost. CMI offers no guarantees for this service, however.
Before commencing either service CMI, will need to know from the customer:
a) The parent mouse myeloma from which the hybridoma was derived, and also the Ig
isotype/subclass and light chain specificity of each specific MAb.
b) A brief description of the culture medium in which the hybridoma is grown – and
particularly if the hybridomas require culture in HAT or if any other special additives are
used (e.g. interleukins, origen etc.).
CMI will expect to sign a customer provided, legally binding document, covering both
confidentiality and ownership of the hybridoma cell-line and secreted products. If the
customer also wishes to supply the hybridoma(s) under simple code – so much the better.
CMI will accept hybridoma cells either frozen on dry-ice, or already growing in T-25 or
T-80 cm2 flasks (the latter method is preferred).
Service 1:
This is a minimum service to provide quality MAb at the lowest price. The minimum
order for this service is 10.0 mg purified MAb from each hybridoma. CMI will:
1. Thaw if necessary, then expand hybridoma cells into larger volume of culture medium.
2. Titrate IgG production from each hybridoma, and further expand the culture to a
volume that will provide the required MAb yield. At harvest of MAb, all viable
hybridoma cells will be destroyed.
3. Isolate the MAb from culture supernatant using:
a) ammonium sulphate precipitation,
b) isolation over an SpA column using a proprietary buffer system which stabilizes the
MAb, and also ensures recovery of all mouse IgG isotypes and IgG subclasses.
4. Concentrate the SpA eluted MAb to >1.0 mg/ml in PBS pH 7.2 with 0.05% sodium
azide. The final MAb concentration will be determined at OD280 by applying an
extinction coefficient of 1.4.
5. Ship purified MAb at ambient temperature to customer address by FedEx.
6. Invoice at $60.00 per mg for 10.0 to 50.0 mg SpA purified MAb per hybridoma.
7. The time required to perform the service is 3-6 weeks.
Service 2:
This is a more detailed service recommended for customers who are likely to repeat their
order in the future, or who have hybridomas that will benefit from the additional cloning
step included. The minimum order for this service is 25.0 mg purified MAb from each
hybridoma. With this service the end-user has input and can request modifications or
variations in the service.
For service 2 the customer is encouraged to supply each hybridoma under simple code,
and the customer must be prepared to supply (also under code) 50-200 ug of specific
antigen relevant for each hybridoma (also under code). CMI will:
1. Thaw if necessary, then expand hybridoma cells into larger volume of culture medium.
2a. Immediately clone each hybridoma by limiting dilution, and when clones have
emerged (10-14 days later) check each clone for activity against specific antigen (by
ELISA).
2b. Briefly report on the cloning efficiency, and on the percentage of specific antibody
secreting cell clones obtained.
2c. Confirm Ig isotype, subclass, and light chain production, of selected, re-cloned,
hybridomas.
2d. Return to the customer 1 X T-80 cm2 flask containing viable, freshly cloned,
hybridoma cells specific for the customer provided Ag.
2e. At the customer’s option, CMI will freeze 4 vials of cells from each cloned
hybridoma and store these in liquid nitrogen under customer code for a period of 3 years
– longer if customer requests. (There is no additional charge for this service).
3. In parallel with hybridoma cloning, CMI will titrate IgG production of each pre-cloned
hybridoma, and then expand each culture to a volume that will provide the required MAb
yield. At MAb harvest all viable pre-cloned hybridoma cells will be destroyed.
4. CMI will then isolate each MAb from culture supernatant by:
a) ammonium sulphate precipitation,
b) isolation over SpA using a proprietary buffer system which stabilizes the MAbs and
ensures recovery of all mouse IgG isotypes and IgG subclasses.
5. Concentrate the SpA eluted MAb to > 1.0 mg/ml (or to a specific concentration as
specified by customer) in PBS pH 7.2 with 0.05% sodium azide. The final MAb
concentration will be determined at OD280 by applying an extinction coefficient of 1.4.
6. Purified MAbs will be shipped at ambient temperature to address provided by FedEx.
7. Invoice at $105.00 per mg for the first 25.0mg purified MAb per hybridoma. If the
order exceeds 25.0 mg then the cost for additional MAb is $60.00 per mg.
The time required to perform the service is 4-7 weeks. The advantages for this service
are:
i. The customer will receive back each hybridoma cell line after re-cloning. This step will
help to ensure the long-term survival and specific MAb secretion of the hybridomas.
ii. The customer has the option to insure hybridoma survival by having the hybridomas
stored at an independent facility (CMI) under the customer code. If the customer then
wishes to repeat the order, initiation requires only an authorizing phone call.
iii. The price for repeat orders is $60.00 per mg, because Service 1 is now all that is
required (i.e. the hybridoma does not require re-cloning).
Discounts for bulk MAb production:
Should a customer require purified MAb quantities significantly in excess of the
minimum orders specified, CMI will be prepared to offer a further discount depending
upon the quantity required.
CMI requires a contract contingency for both Service 1 and Service 2 described above as
follows:
Services as offered and described above as “Purification of Monoclonal Antibodies from
Pre-existing Hybridomas owned by Customers” are contingent upon the customer
provided hybridoma being of good viability and having the potential to secrete a normal
quantity of MAb. Should a hybridoma prove refractory to secreting MAb within the
normal concentration range; should CMI be unable to rectify the problem; should the
customer be unable to provide reagents or information to correct the problem, then CMI
retains the right to terminate the contract at no charge to the customer.