Custom Monoclonal Antibody Service - Mouse and Rat Monoclonals

Synaptic Systems offers specialized services providing individual solutions for our customers' specific requirements. A complete service from peptide synthesis or protein expression to antibody production and longterm storage of hybridoma cells is available.

Antibody Development

A standard protocol includes the following steps:

Phase I / Immunization:

For immunization 3 mg of purified peptide or 1 mg of purified protein is required (2 mg protein if a Western blot screening is requested). As a general procedure three Balb/c mice or Lou/c rats are immunized with 90 to 150 µg each. If the immunogenicity of the antigen is critical (e.g. mouse/rat proteins or peptides) special strains can be used.
Animals are immunized over a period of 17 days every second day and one day before fusion.
Depending on the antigen different immune stimulating agents are used like Gerbu or incomplete Freund's Adjuvant combined with CpG oligo-nucleotides (Krieg AM 2002. Annual Review of Immunology 20: 709-60).

Phase II / Fusion:

After immunization cells from knee lymph knots and spleen of all three animals are prepared and fused with Ag8 myeloma cells in the presence of PEG 1500. After fusion cells are plated on 24-well plates resulting in 360 oligo clones each usually consisting of 5 - 10 clones. Cells are cultivated for 10 days in selective and conditioned medium selecting for fused cells only.

Phase III / Screening and subcloning:

Within the next two days, all 360 oligo clone supernatants are screened by ELISA for antibody positive wells.
For the next screening step we offer four options:

1. up to 72 oligo clone supernatants are tested in Western blots at Synaptic Systems.
2. up to 360 oligo clone supernatants are tested by immunofluorescence at Synaptic Systems.
3. up to 360 oligo clone supernatants are screened by a specific sandwich ELISA for antibody microarray at Synaptic Systems.
4. up to 20 oligo clone supernatants (3-5 ml each) are shipped to the customer for screening under his own conditions.

Usually screening is completed within a period of three weeks. After customer's decision which oligo clone should be subclones the selected oligo clone is subcloned to monoclonality by the limiting dilution method. Subclones are screened according to the options (a-d) chosen by the customer, and positives 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 will be frozen as back up and stored for up to 12 months if desired. Longterm storage is possible for a small fee.

It is our goal to provide our customers with monoclonal antibodies that fit to their assays in the best possible way. Hence, close collaboration during the course of the project is our first priority.

For further information please contact antibodyservice@sysy.com.