Epitomics FAQ and References

General Technology
Custom Antibody Services
Immunogen Design
Immunization
References

General Technology

Q: What is a RabMab ?
A: RabMab stands for Rabbit Monoclonal antibody.

Q: What is the process for making a Rabbit monoclonal antibody ­ how is it different than making a mouse monoclonal ?
A: The general procedure for making a rabbit monoclonal is very similar to the conventional mouse monoclonal. For a general overview of the procedure please see the technology page of our website. Making rabbit monclonals has proven much more difficult than mouse monoclonals which have been around since the late 1970's (1). In fact no one had successfully made a rabbit monoclonal antibody until the work by Dr. Katherine Knight in 1995 (2). Since the work in 1995 and subsequent work by others, Epitomics obtained the exclusive rights to the technology enabling the creation of rabbit hybridomas.

Q: Why make rabbit monoclonals ?
A: Rabbit antibodies are generally high affinity and are an ideal species to choose for making antibodies against mouse and rat proteins as many antibodies made in mice or rats will not react against mouse or rat proteins (2). From our experiences the rabbit immune system appears to be capable of generating affinities against small epitopes which are not recognized by the mouse.

Rabbit polyclonal antibodies have successfully been used as research and even diagnostic applications for many years. However the inherent benefits of monoclonal antibody production quality and reliability have not been available for rabbit antibodies until now. Epitomics RabMab technology thus expands the antibody repertoire available for potential research, diagnostic and therapeutic applications.

Q: Can RabMabs be used in the same applications as other antibodies ?
A: Yes, RabMabs can be used in the same applications as any antibody . RabMabs have been used successfully in western analysis, immunocytochemistry, imunohistochemistry, immunoprecipitation and flow cytometry. A c-kit rabbit monoclonal antibody was also recently approved for use in a diagnostic application. As with any antibody the use of a specific antibody should be validated and a givenantibody may not be appropriate for use in every application. The list of recommended applications for each of our RabMab's can be found with the product specific information.

Q: What kind of affinities can one obtain with Rabbit Monoclonals ?
A: RabMab affinities in the picomolar range, 10^-12 Kd (M) are possible. Typical affinities are in the 10^-9 ­ 10^-10 Kd (M) range.

Q: Do you have any references for rabbit monoclonal antibodies ?
A: Several papers and presentations have been made regarding rabbit moncolonals, please see the list below under references.

Custom Antibody Services

Q: Does Epitomics offer custom rabbit monoclonal antibody services ?
A: Yes. Epitomics offers custom service opportunities that allow customers to create a rabbit monoclonal hybridoma from a specific antigen or peptide of interest.

Q: What is the overall timeline of a typical project?
A: A standard immunization takes 2-3 months, depending on how strong the immune response is. Analysis of the antiserum may take 1-2 weeks. From the day of the fusion, it takes 4-6 weeks to grow up the hybridoma colonies, perform the primary screen, and expand the positives in 24-well plates. After the desired clones have been selected, subcloning and confirming screens take 3-4 weeks. Expansion of subclones and small-scale IgG production (5-20 mg) takes 6-8 weeks. Thus, the overall timeline is 5 to 8 months.

Immunogen Design

Q: Can Epitomics provide help with immunogen design?
A: We can provide assistance with devising the strategy for immunization (synthetic peptide/recombinant protein/cDNA), as well as with peptide design, synthesis, and conjugation.

Q: How does Epitomics select peptides for immunization?
A: For most proteins, the C-terminal peptide is the first choice, followed by the N-terminal peptide. For internal peptides, 3D structure prediction may be utilized in some cases, in order to identify sequences likely to correspond to surface loops or extended flexible regions. Sequences that are known to be phosphorylated or cleaved by proteases may also provide good immunogens, as could state-specific methylated or acetylated proteins.

Q: What types of immunogens can be used?
A: Immunogens can be proteins, peptides coupled to KLH, small molecules coupled to carriers, and cDNA clones expressed in a rabbit cell line.

Q: What about DNA immunization?
A: Epitomics is exploring DNA immunization. So far, superior results have been obtained by transiently expressing the cDNA in a rabbit cell line, and injecting the transfected cells subcutaneously or intraperitoneally.

Q: Which vectors does Epitomics prefer for cDNA immunization (GeneMab)?
A: We have used several CMV-based vectors, such as pcDNA3.1. Vectors without selection markers may be preferable, but the neo selection cassette does not seem to pose a significant problem.

Q: Does Epitomics provide recombinant protein expression service for immunization?
A: At this time, we do not offer protein expression services. However, in the near future we are planning to have in-house capacity for expression and purification of recombinant proteins in different expression systems (bacteria and mammalian).

Immunization

Q: What immunization schedule works best for rabbit monoclonals?
A: Epitomics uses a standard immunization schedule, similar to the schedules commonly used for producing polyclonal antibodies (4 subcutaneous immunizations in 3-week intervals). 3-5 days before splenectomy, a final intravenous booster immunization is administered. In certain cases (insoluble immunogen, immunization with cells), this final immunization can be omitted or replaced by an intraperitoneal immunization.

Q: What about alternative or accelerated immunization schedules?
A: Epitomics has limited experience with accelerated immunization. It is possible to perform fusions with cells obtained from popliteal or inguinal lymph nodes. In this case, the immunogen is injected subcutaneously in the lower leg, and a more rapid immunization schedule (e.g., one-week intervals) can be used.

Q: How much protein is needed for immunization and screening?
A: Epitomics prefers to use a relatively large amount (300 micrograms per rabbit for initial immunization, 100 ­ 200 micrograms for booster immunizations). Altogether, we prefer to have at least 3 mg of protein for immunizing 2 rabbits and performing the ELISA screening. However, in some cases it is difficult to obtain this amount. Depending on the immunogenicity of the protein, good immune responses can be induced with much smaller amounts of protein (in some cases, as little as 10 micrograms per immunization). Injecting into the lower leg and collection of cells from the popliteal lymph node provides an alternative approach that often requires much less immunogen than the standard immunization route.

Q: How much peptide is needed for immunization?
A: Typically, Epitomics synthesizes at least 10 mg of peptide. A portion of this (2-3 mg) is coupled to KLH for immunization; another portion (1 mg) is coupled to BSA for screening. If the antiserum needs to be affinity-purified, another portion of the peptide (2-3 mg) is coupled to Sepharose.

Q: How much antiserum can Epitomics provide?
A: For test bleeds, we typically provide 2-5 ml of serum. Larger amounts (50-100 ml) of antiserum can be collected, if requested. We can also produce affinity-purified polyclonal antibodies for testing in special assays (e.g., IHC, functional assays).

References

1. Kohler G, Milstein C. (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature; 256 (5517): 495-7. *Note ­ mouse monoclonal reference.

2. H Spieker-Polet, P Sethupathi, P Yam, and KL Knight. (1995) Rabbit Monoclonal Antibodies: Generating a Fusion Partner to Produce Rabbit-Rabbit Hybridomas. PNAS USA 92: 9348 - 9352.

3. Cano G, Milanezi F, Leitao D, Ricardo S, Brito MJ, Schmitt FC. (2003) Estimation of Hormone Receptor Status in Fine-Needle Aspirates and Paraffin-embedded Sections from Breast Cancer Using the Novel Rabbit Monoclonal antibodies SP1 and SP2. Diagn Cytopathol, 29(4): 207 -11.

4. S. Rossi, E. Orvieto, S.Chinellato, A. Furlanetto, L.Laurino, F. Facchetti. (2004) Immunohistochemical Analysis of routine paraffin embedded tissue using Rabbit monoclonal Antibodies. Abstract presented at the USCAP, Vancouver 2004. Modern Pathology, 17 (supp. 1): 361A.

5. Wah Cheuk, Kathy O.Y. Wong, Cesar S.C. Wong and John K.C. Chan. (2004) Consistent Immunostaining for Cyclin D1 Can Be Achieved on a Routine Basis using a Newly Available Rabbit Monoclonal Antibody. Am J Surg Path 28(6): 801-807.