Antibody Preparation and Related Services

At CBI, we offer comprehensive immunochemistry services. CBI has the ability to synthesize or purify your immunogen, prepare polyclonal or monoclonal antibodies, purify your antibodies from cell culture, ascites fluids, or from whole serum, prepare monospecific polyclonal antibodies by ligand affinity chromatography, or we can help develop novel routes of immunoassay.

Monoclonal antibody production

Our monoclonal antibody contracts are divided into four phases:

Phase I. Immunization of mice and screening of serum (about 7-10 weeks)

The sera from 5 mice are tested by ELISA assay for antibody titer. If the immune response is acceptable, the best mice candidates are boosted again and fusion is performed in four days (Phase II). If no immune response is detected, mice will be boosted at week 9, test bled, and reassayed. If after this last test, none of the mice have produced an acceptable antibody titer, YOU CAN CANCEL THE CONTRACT.

Phase II. Fusion and screening of hybridoma supernatants (about 2-6 weeks)

A fusion procedure is done in the presence of polyethylene glycol between splenocytes from the immunized mouse which shows the highest titer and a murine myeloma cell line. Fused cells are cultured in five 96-well titer plates, and parental hybridoma cultures are selected by growth in HAT medium. A maximum of 10 positive parental cultures will be expanded for further evaluation and will be cryopreserved. In the event that NO positive cultures are produced, one additional fusion will be done with splenocytes from a second mouse, provided the immunized mice have an acceptable ELISA titer. If NO positive cultures are produced in the second fusion, then YOU CAN CANCEL THE CONTRACT.

Phase III. Cloning and ascites production (about 6-8 weeks)

A maximum of 5 parental hybridomas will be cloned by the limiting dilution method. The clones will be examined microscopically for monoclonality. The resulting clones will be analyzed by ELISA and two clones of each parental hybridoma will be expanded, tested by ELISA, and cryopreserved. The highest titer clone from each parental hybridoma will be used for ascites production in three mice per clone. All ascites produced (usually about 5-10 mg antibody per ml) and all cyropreserved cells will be provided to you.

Phase IV. Additional ascites production

Preparation of polyclonal antisera

Synthesis of immunogenic peptides

At CBI, we routinely prepare immunogenic peptides based on predictive analysis of the primary amino acid sequence of your target protein. Please see the description of our Peptide Synthesis services.

Conjugation of the immunogens to carrier proteins

Each conjugation is usually done with 5-10 mgs of peptide and a suitable carrier protein.

Preparation of polyclonal antisera in rabbits

Our standard immunization protocol for production of polyclonal antisera in 2 rabbits is 70 days which includes a pre-immune bleed from each rabbit, 4 immunizations per rabbit, 3 production bleeds from each rabbit, and return of about 100 ml of ELISA titered serum per rabbit.

Purification of the resulting antibodies

We routinely purify antibodies by one of three methods: large scale protein A chromatography, differential precipitation and DEAE chromatography, or gel filtration chromatography.

Preparation of monospecific polyclonal antisera

At CBI, we can immobilize your antigen to a suitable column matrix for preparation of monospecific polyclonal antisera. Typically, peptide is immobilized at a concentration of about 1mM, and then the IgG fraction obtained from 100ml immune serum is purified by peptide affinity ligand chromatography. The titer of the resulting preparation is then determined by ELISA.

Production of polyclonal Ab in chickens

At CBI, we also offer preparation of antibodies in chickens. Here, the immunogen is used to immunize and boost hens which produce IgY, a monomomeric class of antibody comparable to IgG, that is transferred from the hen to her egg. The anitbodies are purified from the egg yolk and are obtained in > 85% purity. If a hen responds to the antigen, it is not unusual to obtain between 1.5 - 2.5 grams of IgY per month. Using chickens to produce antibodies to conserved mammalian proteins is particulary useful because the evolutionary distance between chickens and mammals (as compared to between mammals and rodents) often results in increased immunogenicity. Conjugated and unconjugated anit-chicken secondary anitbodies are commercially available making IgY suitable for use in Western blots, ELISA's, and other applications.

We offer both bulk quantity and research quantity protocols. In the research quantity protocol ($1000 per immunized hen), we usually obtain 0.5 grams antibody within 60 days. In the bulk quantity protocol ($2000 per immunized hen), we usually obtain between 1.5 - 3 grams purified antibody within 90 days. This protocol can also be extended beyond 90 days with monthly antibody production ($750).

Order Form
Price List [pdf]

Please call 800-735-9224 or e-mail for additional information.