Establishment of hybridomas

Diatec Monoclonals has many years of experience in establishing of hybridomas from customer delivered antigen. Our hybridoma production project are tailored for each customer: mouse strain, number of immunisations etc. Our programs are modular so the customer can choose how large or small each phase of the program should be. Please contact us, and we will give you an offer based on your special requirements.

Timeline:                 4-6 months

Description of standard project:
One standard hybridoma production project is initiated by immunisation of four BALB/c-mice with immunogen (antigen) supplied by the customer. Next, spleen cells are fused with OURI mouse myeloma cells and successful fusion cells are propagated in selective medium. Fusion wells are screened for reactivity towards the antigen; positive wells are recloned twice. If required, the project may be concluded by establishment of cellbank, small-scale in vitro production and QC according to agreement. Antigen and screening protocols will be developed in close collaboration with the customer.

Serum: For cell culture work, only foetal bovine serum of FDA approved origin is used.

Buffer: Purified antibodies are supplied in phosphate buffered saline containing 0.09% sodium azide.

Standard hybridoma project - general procedure
Time (Week)	Action	Comment
Initial	Arrival of antigen, order of mice	1 weeks acclimatisation of mice before immunisation
0	1st immunisation	4 BALB/c-mice I.P.
2	2nd immunisation	I.P.
3	Test bleed of mice	Investigate reactivity towards antigen by ELISA
+1 week	Opt. extra immunisation	If reactivity towards antigen is poor
4-7	Final boost (immunisation) of mice	I.P. in PBS
5-8	Spleen cells from the mice are fused with mouse myeloma cell line	OURI ( X63-Ag8.653, Ouabain resistant)
7-10	Screening of resulting fusion hybridomas for reactivity towards antigen by ELISA	Screening instructions supplied by customer
8-11	First recloning	Max. 10 good clones are recloned after screening of fusion
10-13	Screening of first recloning for antigen reactivity	Screening instructions supplied by customer
11-14	Second recloning, expansion of selected clones	Max. 6 good clones are recloned after screening of first recloning.
11-12	Expansion of selected clones after screening of first recloning	Cells are expanded, customer is supplied with ca. 10-15 mL supernatant from selected clones
13-16	Screening of second recloning to select good producers	Screening instructions supplied by customer
15-18	Expansion of up to 3 clones selected after 2nd recloning analysis. Making cell banks of 6 ampoules.	Customer supplied with supernatant from cell bank. Subclass analysis of cell bank supernatant.
After customer's analysis	Production of antibody from selected new hybridoma in CL1000	Supply customer with harvests from production. Usually 50-100 mg, dependent on production.
After fulfilled project, customer is supplied with clones, produced supernatant and full documentation report. Total project time: 4-5 months plus one month production

If you have any questions, or are interested in a hybridoma establishment project, please contact us at diatec@diatec.com.