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.
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.
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.
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.
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.
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.
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.
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.
PRF&L introduced the Mighty Quick Protocol during November 2007. This protocol can make your antibody in as fast as 28 days.
Yes, you can! See the next question below.
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.
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.
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.
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.
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.
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.
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.
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.