IBA : Custom Services

A fast and flexible service according to your needs

Requirements and procedure

Phase I / Immunization
For immunization we require 1 mg of purified protein* or 3 mg of purified peptide. As a general procedure we immunize three Balb/c mice with 90 to 150 µg each. If required, we can switch to other mouse strains if the provided antigen is critical concerning its immunogenicity (e.g. mouse proteins or peptides).
Mice are immunized over a period of 17 days every second day and one day before fusion. Depending on the antigen we can use different immune stimulating agents like Gerbru or incomplete Freund's Adjuvant combined with CpG oligonucleotides (Krieg, AM 2002: Annu.Rev.Immunol. 20, 709-760).

* if a Western blot is requested(cat. no. 2-2601-004), an additional mg protein is required

Phase II / Fusion
After immunization, cells from knee lymph knots and spleen of all three mice will be prepared and fused with Ag8 myeloma cells in the presence of PEG 1500. As a positive control we also collect immune serum from the immunized mice. At the end of cell fusion the cells are plated on 24-well plates which results in 360 oligo clones each usually consisting of 5 - 10 clones. Cells are cultivated for 10 days in selective and conditioned RPMI medium selecting for fused cells only.

Phase III / Screening and subcloning
Within the next two days, the clones of interest have to be selected. All 360 oligo clone supernatants are screened by ELISA for antibody positive wells.

For the next screening step we offer three options:

A maximum of 72 oligoclone supernatants are tested in Western blots at IBA.
A maximum of 360 oligoclone supernatants are screened by immunofluorescence at IBA.
A maximum of 20 oligoclone supernatants (5-10 ml) are shipped to the customer for screening under his own conditions.

Usually screening is completed within a period of three weeks. The selected oligoclone will be subcloned to monoclonality. Therefore, positive clones are selected for cloning by the limiting dilution method. Isolated colonies will be tested by ELISA, and positives selected to be expanded for the next cycle of cloning. Several cycles of cloning may be required in order to obtain a true, stable clone, which is then used to produce 100 ml supernatant of the monoclonal antibody. Cells from the positive clones are frozen as back up. As part of our service, we are storing the clones until the next production.

Antibody production
Different production scales are available starting at 100 ml antibody including supernatant up to 100 mg purified antibody or more.
We also offer antibody production services from the hybridoma provided by the customer. Please contact us for further information.
Communication with our clients is a priority. Therefore, we take a collaborative approach in establishing effective working relationships with our customers. If you have any comments, questions or suggestions, please get in touch with us at services@iba-go.com.

	- immunization of 3 mice - fusion of B-lymphocytes with myeloma cells, generation of hybridoma cells - ELISA screening of 360 hybridoma supernatants (oligoclones) to select for the presence of antigen specific antibodies
	-max. 20 positive supernatants are shipped to customer for tests under his own conditions or -max. 72 positive supernatants are tested at IBA in a Western blot against antigen provided by the customer or -max. 360 positive supernatants are tested at IBA in an immuno- fluorescence assay (IF) against target cells provided by the customer
	-customer decides which oligoclone should be subcloned -100 ml supernatant of the subcloned hybridoma will be shipped to the customer (purification on request) -large scale antibody production (under GMP) on request

Standard protocol
mAB generation	description	cat. no.
Phase I/Immunization	3 Balb/c mice, 17 days immunization, pre-bleeding, immune serum	2-2601-001
Phase II/Fusion	Preparation of lymphocytes, fusion with myeloma cell line, selection	2-2601-002
Phase III/Screening and subcloning, ELISA screening and production	ELISA screening, subcloning by limiting dilution, second ELISA screening, production of 100 ml supernatant of selected antibody	2-2601-003
Optional Western blot screening	Screening of max. 72 ELISA positive oligoclones in a Western blot against the antigen provided by the customer	2-2601-004
Optional immunofluorescence screening	Screening of max. 360 oligoclones in an immunofluorescence assay against target cells provided by the customer	2-2601-005

Order information
for a quote please contact services@iba-go.com

In order to develop a specific immunization and screening strategy our customer is requested to fill out a questionnaire. Please inquire at services@iba-go.com.

Custom monoclonal antibody service

A fast and flexible service according to your needs

Requirements and procedure