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Regulatory issues
Host species available
Submitting an antigen
Amount of antigen required and concentration
Acceptable forms of the antigen
Cocalico Biologicals, Inc. standard protocol
Average cost of our standard protocol
Prescreening of host species
Appropriate number of hosts
Acceptable buffers and concentration for antigen
Peptide conjugation
Initiating an antisera project
Testing of the antisera
Continuation of an antibody project
Shipping and storage of the antisera
Preservation of the antisera
Amount of antisera provided
Antibodies off the shelf
Guarantee
Confidentiality

Regulatory issues

Federal regulations require all organizations producing antibodies to maintain a current USDA research license and a current Animal Welfare Assurance with NIH's Office of Laboratory Animal Welfare. Cocalico Biologicals, Inc. holds the following:

Host species available

Cocalico Biologicals, Inc. offers antibody production in mice, rats, guinea pigs, rabbits, chickens, goats, sheep, donkeys, alpacas and horses.

Submitting an antigen

Please send your antigen to us via any of the common couriers (UPS, FedEx, etc.). We have no specific requirements as to how the materials are to be shipped. We would suggest shipping at a temperature equivalent to the one used to store the antigen (i.e. if frozen ship on dry ice, if refrigerated ship with cold packs). Please include a set of completed Project Forms and the Non-Duplication Statement along with your antigen. You may also complete the Project Forms and submit them online.

Amount of antigen required and concentration

The following are the minimum estimated quantities of antigen need for a standard schedule:

• Mice — Protein antigen = 100ug; Peptide antigen = 400ug
• Rats — Protein antigen = 175ug; Peptide antigen = 700ug
• Guinea pigs — Protein antigen = 175ug; Peptide antigen = 700ug
• Rabbits — Protein antigen = 500ug; Peptide antigen = 2mg
• Chickens — Protein antigen = 500ug; Peptide antigen = 2mg
• Goats — Protein antigen = 2mg; Peptide antigen = 4mg
• Sheep — Protein antigen = 2mg; Peptide antigen = 4mg
• Horses — Protein antigen = 5mg; Peptide antigen = 10mg

Acceptable forms of the antigen

We will accept antigens in a variety of different forms, including a soluble liquid lyophilized, an insoluble precipitate, as a gel slice or on a nitrocellulose membrane.

Cocalico Biologicals, Inc. standard protocol

We will be happy to follow your customized protocol. If one is not provided, we will use the standard protocol.

• Day 0 = Prebleed/Initial Inoculation
• Day 14 = Boost
• Day 21 = Boost
• Day 35 = Test Bleed
• Day 49 = Boost
• Day 56 = Test Bleed

The following procedures are included in our standard protocol. We will not actually schedule them on the listed days unless we are given instructions that your assays of the day 56 test bleed indicate it is appropriate.

• Day 70 = Production bleed
• Day 77 = Boost
• Day 84 = Production bleed
• Day 91 = Exsanguination

Aprocedures are based upon your assay results. Options include monthly boost and test bleed until appropriate response obtained; monthly boost and production bleeds to stockpile sera; final boost and bleed. Our ability to collect large quantities of sera is dependent on the type of host species used.

We will not schedule any additional procedures after the day 56 test bleed until we hear back from you on how the host is responding. We want to truly customize the procedures to meet your specific needs.

You do not have to use our standard protocol. You may submit your own protocol for us to follow. We would have to go through the normal process of IACUC approval of the protocol prior to the start of your project.

Average Cost of our standard protocol

The following are the estimated costs for our standard 91 day protocol which includes 1 host species, 91 days of maintenance, 1 prebleed, 2 test bleeds, 5 injections, 100ml of antisera (prepared as 2 production bleeds and the exsanguinations; 100ml is from a single rabbit, other host species volumes will vary) and the disposal charge:

• Mouse $201.95 /each (includes ~0.5 ml sera)
• Rat $236.40 /each (includes ~6 ml sera)
• Guinea Pig $236.00 /each (includes ~15 ml sera)
• Chicken $308.00 /each (includes ~50 ml sera; additional cost for egg yolks and IgY purification)
• Rabbit $415.00 /each (includes ~100 ml sera)
• Goat $1,379.00 /each (includes ~900 ml sera)

We will be happy to provide a quote for your specific protocol. Click here for quote!

Prescreening of host species

We do offer the prescreening of host species to select the one to immunize with the lowest background. There are no charges associated with this service as long as the results of your assays are reported to us within two weeks of the receipt of the prebleed sera. We will charge daily maintenance for the period of time the animals are maintained after the two weeks without an update.

Appropriate number of hosts

There can be variability between animals in their responses even with the same antigen and following the same immunization schedule. We therefore recommend using a minimum of two (2) animals for each antigen. The higher costs associated with goats, sheep and horse may limit the number of these hosts to just one.

Acceptable buffers and concentration for antigen

Antigens may be submitted in a variety of buffers. The best buffer for the animals is PBS or saline however, we have successfully used such buffers as TRIS, 6-8M Urea, distilled water, HEPES and SDS. It is best to try to keep the concentration of the buffers as low as possible. An ideal concentration of the antigen is 1mg/ml. The most dilute the antigen can be is 100ug/ml.

Gel slices may be stained (usually Coumassie Blue) and destained prior to submission as an antigen. Please rinse the gel well with distilled water to remove any residual acid or alcohol. You need to estimate the amount of protein in the gel slice. Finally, place the gel slice in a tube (no buffer added; do not lyophilize or homogenize) and ship on cold packs by an overnight courier. The use of gel slices is not appropriate in smaller species such as rodents.

Peptide conjugation

We suggest the conjugation of peptides that are less than 20 amino acids in length. We can conjugate the peptide to KLH, BSA, ovalbumin or thyroglobulin. You may wish to add a Cysteine for conjugation if there is no other Cysteine in the native sequence. It is preferable to conjugate to a single point.

Initiating an antisera project

An antibody project can be initiated simply by sending us your antigen along with the completed Project Forms and Non-Duplication Statement. Once we receive your materials we will review the project and secure IACUC approval. We typically can start a project within 48 hours of the receipt of your antigen. Once the project has started, you will receive in the mail a letter from us outlining the project details through the day 56 test bleed. We will not schedule additional procedure after the day 56 test bleed until we hear from you regarding your assay results. The appropriate course of action after day 56 is dependent on how the animals are responding.

Need a project form?

Testing of the antisera

We send the sera from the prebleeds and test bleeds to you for assaying. We do offer an ELISA, at an extra charge. We would need a minimum of 500ug of soluble antigen for the ELISA. IF you wish to use your antibodies in westerns, immunoprecipitation or immunohistochemistry, it is imperative that you screen the sera in those assays even if you ask us to do an ELISA. The ELISA is a good assay to determine if the animals are producing antibodies however it will not predict how those antibodies will work in the other assays.

Continuation of an antibody project

We would be happy to continue your project past the standard protocol. Simply tell us your needs and we will help design a schedule that is appropriate.

Shipping and storage of the antisera

Unless otherwise instructed, we will ship the antisera frozen on dry ice via priority overnight delivery. The antisera should be stored at below -10C. It is important to avoid multiple freeze thaw cycles which will tend to reduce the activity of the antibody. It is best to thaw the antisera once, aliquot it and then store it long term at below -10C. You may store the antisera at refrigerated temperature however it is raw sera and should be filtered or have a preservative added to avoid contamination.

Preservation of the antisera

We do not add any preservative to the antisera prior to its being shipped, unless otherwise instructed.

Amount of antisera provided

The total amount of antisera you can obtain in a project is dependent on the host species and the number of bleeds scheduled. Typically we can collect the following volumes of sera in a standard 91 day protocol if the animals respond well by the second test bleed (some antigens will take longer and therefore the amounts we can obtain will be over a longer period of time):

We can provide even larger quantities of antisera from rabbits, chickens (usually as IgY from egg yolk) and goats when they are assigned to a regular production bleed schedule.

Antibodies off the shelf

All the antibodies we produce are done on a custom basis therefore we do not have any antibodies for sale off the shelf.

Guarantee

We cannot guarantee the performance of any biological product we produce. There are too many variables involved in the production of antibodies to be able to control the outcome of a project. We will guarantee you our best effort in the production process.

Confidentiality

We do maintain confidential the names of our clients and the details of their projects. We would be happy to formalize an agreement when requested. We can provide a standard confidentiality agreement or we will review one submitted by the client.