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 $9,000.00 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.

References from academics who have worked with CMI in producing new hybridomas using the production outline above will be provided if requested.

 

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