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The answers provided in the Frequently Asked Questions (FAQ) are provided to the research community as a service of Pocono Rabbit Farm & Laboratory, Inc.(PRF&L) The questions are typical of information requests we receive. The answers are based on the 55 years of experience PRF&L has in the antibody production industry. The answers provided are true for most but not all projects. The variability of antigens and immune systems in animals precludes guaranteed results.

Who uses your services?

Investigators from universities, biotechnology firms, and pharmaceuticals worldwide have made antibodies with us, including many Nobel Prize winners. We take care of all the work and worries of caring, injecting, and bleeding the animals. Your valuable time is spent on research.

How many animals should I use?

We recommend that at least two, preferably three animals of each species for antibody production. We have found variability of the antigenic response in different individuals. Using more than one animal allows a more diverse response in terms of quantity of antibodies, specificity, and affinity. However, one animal (maybe) is appropriate if the (same immunogen was used previously in multiple animals which all produced a similar response) antigen has been used in the past successfully to raise antibodies.

How much antigen do you need?

Our package antibody programs need 500ug per rabbit, chicken, sheep, and goat to get through the initial 28 / 70 / 91 day period.  If you go for the project extension, we need another50ug per 28 day cycle per animal.  If you send us one milligram per animal at the start of the protocol, we will have plenty of antigen for one year of antibody production in rabbits, chickens, sheep, and goats. One-half milligram is recommended for guinea pigs and rodents. You cannot make that much? We have alternative protocols available when the total amount of antigen available is limited.  Also, you can send us antigen additional antigen if you do not send enough antigen at the start of the protocol.

What antigen concentration is required?

We recommend that antigen be supplied at 1mg per ml or higher for the first three injections. Other boosts can be 0.25mg per ml. If the concentration is too dilute, we may need to use alternative protocols.

The lowest recommended concentration for the initial injection is 0.8mg/ml. If the concentration is between 0.2mg/ml and 0.7mg/ml, we will inject the recommended amount of antigen using two different routes. This usually does not present a problem. However, if the concentration is less than 0.2mg/ml, we will inject less than the recommended amount of antigen and may need to alter the protocol. Please ask us for recommendations if you are unable to concentrate the antigen preparation to more than 0.2mg/ml.

What is the best way to send my antigen?

The best form is soluble or slightly insoluble. Please try to aliquot the antigen in eppendorf tubes or other suitable containers. By doing so, this helps eliminates potential Freeze / Thaw damage and contamination risks when all antigens are stored in one container. We recommend that 100ug of antigen be place in each tube along with 100ul of PBS or other suitable buffer. Send as many 100ug aliquots as you want. It is not necessary to send all the antigen at the start of the project; more can be sent later. Package the aliquots and send them to us using a reliable overnight carrier such as Federal Express or UPS. We will store the aliquots either in our -80 degree Celsius freezer or in our refrigerator @ 4 degrees Celsius, as directed by the investigator.

Other forms are acceptable. Please inquire.

Be sure to read our Suggestions for Shipping Perishable Antigens - from your lab to ours page.

My antigen is not soluble. What should I do?

Usually we can inject an insoluble antigen. If the particles can fit through an eighteen-gauge needle, it will work. We recommend not using detergents such as SDS and Urea to make the antigen soluble. We will vortex the antigen / buffer mixture and make an emulsion with the adjuvant to ensure a homogeneous preparation.

What injection and bleeding protocols do you follow?

Our protocols are on this website. Exceptions to our protocols should be submitted in writing and are subject to review by the technical staff and our IACUC.

What about the Mighty Quick Protocol?

PRF&L introduced the Mighty Quick Protocol during November 2007.    This protocol can make your antibody in as fast as 28 days. 

Can I make exception to your protocols?

Yes, you can! See the next question below.

I have raised antibodies successfully to this antigen in the past with another company or myself. My supply of antibody is depleted and now I need more. Can you duplicate the procedures?

Yes, we can! If you tell us the protocol used, we will follow your protocol. You will need to submit the protocol to us for our review and comments. The field of custom immunology is constantly evolving and we are eager to try new protocols. Contact us for any protocols that you would like us to try that don't fit into our standardized protocols.

How much do you charge?

Please see our prices under the pricing menu.  We have fixed prices for our package protocols.  If you decide for an open protocol / with project extension after the initial 28 / 70 / 84 / 91 day period, we use an a-la-carte pricing scheme. All of these prices are listed on our Current Price List.   Price estimates/quotations are available upon request. 

Do I need to order animals for my project in advance?

No. We always have animals available and conditioned in our facility and ready to start. If you have a larger group of animals, over ten, to start at one time, please contact us about one week in advance just to make sure of availability.

What happens if my antigen preparation is not pure?

The animal can potentially make antibodies to whatever is injected. If antibodies which may be produced to contaminating proteins or other components could cause a problem, consider an alternative method or additional purification steps. If you have multiple preparations containing different degrees of purity, we recommend that the purest material be injected first. Also consider if affinity purification is required later on, the more pure the antigen that is coupled to your solid phase column more specific antibodies will be in the end.

Can you inject a polyacrylamide gel? Nitrocellulose?

Yes, we can inject a gel slice. Run the antigen on the gel, stain with Coomassie Blue, cut out the band of interest and place in a tube with just enough distilled water or PBS to keep the gel slice moist. Coomassie Blue is not toxic to the animals. Try not to use a protocol that uses much ascetic acid. Please do not freeze the gel prep. Antigens in gel are stable at 4 degrees Celsius. You may send the gel at room temperature or with an ice pack. Estimate how much you are sending. You do not need to aliquot the gel as we emulsify the entire batch and assign a concentration. We only inject gel slices subcutaneously. PRF&L suggests not relying exclusively on injecting gels as the percentage of successful projects is less than injecting a soluble preparation. Contact us for further information.

Antigen on nitrocellulose paper is very stable. It can be sent to us in an envelope! You may send us the paper and we can dissolve here, but please test a small piece at your lab for solubility in DMSO. We can also inject small amounts of nitrocellulose paper (less than 1cm square) directly. This is a good method when only small quantities of pure antigen are available.

I want to prescreen a group of animals to check for background. Is that possible and what is the procedure?

Yes, you can prescreen a group of our stock animals to make sure they will be suitable for your project. You may find that some of our animals may already have generated closely related antibodies to something in their environment or their feed which may be similar to your antigen. This is especially important for yeast proteins in rabbits. Submit your request for this service by submitting a Client Information Form and email us the species and number of animals you wish to screen.

We test bleed a group of our animals, send you serum, and you tell us which animals to use. You only pay the setup fees for the animals that you use for antibody production.

Please have your screening methodologies ready prior to requesting the prescreening bleeds to avoid animal maintenance fees. We reserve the right to charge maintenance fees for animals not chosen within 7 days of receipt of serum.

We can do the ELISA testing for prescreening if you are short on time. See our Immunochemistry Services Page and Price List

I have heard about generating antibodies from Egg Yolks? Please tell me more.

IgY antibodies are available in chicken egg yolks. The availability of antibody in chicken egg yolks can be much greater that serum, up to eight times more than the same antibody in rabbit serum. We can even do the purification of the IgY from egg yolks.

I think I need to conjugate my peptide. What should I use?

We recommend that you conjugate your antigen if it is a non-antigenic hapten or of low molecular weigh, under 30kDa. We found that there is usually no harm for conjugating larger peptides or proteins.

There are many conjugates. The most popular seems to be keyhole limpet hemocyanin (KLH). Others are bovine serum albumin (BSA), thyroglobin, and ovalbumin. There is also a Multiple Antigenic Peptide (MAP peptide) method that works well. We recommend that peptides, small proteins, and nonprotein antigens need to be conjugated to a large immunogenic carrier protein.
PRF&L can do the conjugation procedure for you. See our Immunochemistry Services Page and Price List

Conjugation is not a substitute for using an adjuvant. The adjuvant helps concentrate and deliver the antigen to the immune system. Conjugation increases immunogenic properties of the antigen by increasing the size of the molecules.

For greatest specificity, we suggest that you use KLH for the first three injections and then use another carrier such as BSA, thyroglobin, or ovalbumin for further boosts. Our lab highly recommends that when doing ELISAs, that the plates be coated with antigen that is conjugated to a carrier that has not been used for immunizations.

I have heard and read negative comments about Freund's Adjuvant. Please comment. Are other adjuvants available?

The literature has both positive and negative comments about Freund's Complete Adjuvant. Freund's Complete Adjuvant (FCA) was developed way back in the 1930s. It contains killed mycobacteria tuberculosis, paraffin oil, and mannide monoosleate. The mycobacteria elicits a delayed hypersensitivity reaction. The water in oil emulsion using FCA is stable and therefore favorable when injecting novel antigens. The emulsion provides a slow release of antigen and protects the antigen from degradation. The reported trouble with FCA is it can cause granulomas and inflammation at the injection site with an intradermal injection. Through experience and practiced technique, Pocono Rabbit Farm & Laboratory has only a few negative incidents using FCA. FCA is only used once in an animal's lifetime. Titermax is generally kept in stock as an alternative adjuvant and available. DMSO is sometimes used as an adjuvant along with polyacrylamide found in gel preparations.

Pocono Rabbit Farm & Laboratory, Inc. and its IACUC continue to endorse the use of FCA for antibody production. Results of side-by-side studies using novel proteins with FCA and alternative adjuvants have concluded that in most cases, the response with FCA is superior than the alternative adjuvant.

Note that for studies of mycobacterium, using FCA may not be appropriate, use of another adjuvant should be considered.

My institution requires contract labs involving animals to have AAALAC accreditation. Are you fully accredited by AAALAC? What does that mean?

AAALAC stands for the Association for the Assessment and Accreditation of Laboratory Animal Care International. Yes, we are fully accredited (Unit Number 000926). Click here to view our most recent Accreditation letter from AAALAC. This is a voluntary accreditation program that demonstrates that highest standard of excellence in the field of laboratory animal science, using the guidelines set forth in the The Guide for the Use and Care of Laboratory Animals . The standards required by AAALAC are more rigorous than the requirements of USDA's Animal Welfare Act (PRF&L is a registered Reaseach Facility 23-R-0141) and has a NIH OLAW's Animal Welfare Assurance (PRF &L's Animal Welfare Assurance # A3886-01).

 

What is AALAS?

AALAS stands for American Association of Laboratory Animal Science. AALAS is a professional, nonprofit, association concerned with the production, care, and study of laboratory animals. They are the medium for exchange of information through various educational activities and a certification program. Most of the technicians and technical support staff at Pocono Rabbit Farm & Laboratory, Inc. are certified by AALAS and are continually striving to advance their certification.

Do you require any IACUC forms? What is an IACUC anyway?

We have a Polyclonal Antisera Request Form that needs to be completed for each new antibody project. Also, each Principal Investigator needs to have a current Client Information Form on file with us. If any Immunochemistry Services are desired you will need to submit our Immunochemistry Services Request Form. You will find our forms informative and easy to complete because it is geared for antibody production and may even be filled out online and submitted to us via email or simply filled out and printed and submitted with your antigen. The approval process is easy since our standard protocols are already approved by our IACUC. (IACUC stands for Institutional Animal Care and Use Committee. ) The PRF&L IACUC reviews and approves all proposals for research involving animals.

I have an IACUC where I work. Do I need approval from them?

It depends! We recommend that you check with your IACUC, purchasing department, and/or outside funding agency. All projects are reviewed by the Pocono Rabbit Farm & Laboratory IACUC. If you require approval letters, our IACUC can forward them to your institution or funding agency upon request.


Feel free to contact us with any additional questions.