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PRF&L Standard Protocols for Custom Polyclonal Production
in Chickens

STANDARD PROTOCOL for PROTEIN and PEPTIDE ANTIGENS

Printable pdf format PRF&L Chicken Standard Protocol for
Protein and Peptide Antigens

Day 0 Preimmune Egg Collection - We will collect one preimmune egg per chicken unless you request more or none. Preimmune Eggs are cracked and the yolk is separated and stored here at -80 degrees C until we send to you, usually at the time of the first bleed

Preimmune Bleed - about 10ml plasma. Preimmune will be stored here at -80 degrees C until the first immune bleed.

Inject 100-200ug intramuscular using Complete Freund's Adjuvant (CFA). We will inject intramuscular and subcutaneous if the concentration of antigen is less than 0.4mg/ml. Acrylamide gel can only be injected subcutaneous.
Day 14 Inject 50-100ug intramuscular in Incomplete Freund's Adjuvant (IFA)
Day 28 Inject 50-100ug intramuscular with IFA.
Day 42 Bleed - about 10ml plasma - The Tuesday following Day 42 we will ship via Federal Express the preimmune bleed and this first immune bleed on dry ice. It is then up to the investigator to test the serum, determine the quality of antibodies, and let us know if any changes are necessary in the protocol. PRF&L now offers antibody titration by ELISA and can now check titers for you. There is an additional charge for this service.
Day 56 Inject 25-50ug intramuscular or subcutaneous with IFA. We will usually do intramuscular unless you specify subcutaneous.
Day 70 Bleed -We will do bleeds every 28 days unless you specify. The Tuesday following Day 70 we will ship via Federal Express this bleed. It is up to the investigator to test the serum, determine the quality of antibodies, and let us know if any changes are necessary in the protocol. PRF&L now offers antibody titration by ELISA and can now check titers for you. There is an additional charge for this service.

We can begin immune egg collection at anytime. Many of our customers have found it easier to test the bleeds and upon determining that the bleeds are sufficient quality, then request that we begin to collect eggs. Chickens lay about six eggs per week. You will need to tell us for how long or how many eggs to collect. Immune eggs are stored in the shell at 4 degrees C. The day we ship eggs, they are cracked, yolk separated, and packaged in one week pools, by chicken unless you have a specific request. We send them to you via Federal Express on wet ice or ice packs. We now offer egg yolk purification (IgY) purification service.
After Day 70 See Continuing your Protocol below.


STANDARD PROTOCOL for FUSION PROTEIN ANTIGENS

 Printable pdf format PRF&L Chicken Standard Protocol for
Fusion Protein Antigens

Day 0 Preimmune Egg Collection - We will collect one preimmune egg per chicken unless you request more or none. Preimmune Eggs are cracked and the yolk is separated and stored here at -80 degrees C until we send to you, usually at the time of the first bleed

Preimmune Bleed - about 10ml plasma. Preimmune will be stored here at -80 degrees C until the Day 63 bleed.

Inject 100-200ug intramuscular using Complete Freund's Adjuvant (CFA). We will inject intramuscular and subcutaneous if the concentration of antigen is less than 0.4mg/ml. Acrylamide gel can only be injected subcutaneous.
Day 28 Inject 50-100ug intramuscular in Incomplete Freund's Adjuvant (IFA)
Day 42 Inject 50-100ug intramuscular with IFA.
Day 56 Bleed - about 10ml plasma

Inject 25ug intramuscular using IFA

Inject 25ug intravenous (antigen must be soluble in PBS or water) using no adjuvant. If this is not available, we will skip the IV injection.
Day 63 Bleed. The Tuesday following Day 63 we will ship via Federal Express the Preimmune bleed the Day 56 bleed and this bleed. It is then up to the investigator to test the serum, determine the quality of antibodies, and let us know if any changes are necessary in the protocol. PRF&L now offers antibody titration by ELISA and can now check titers for you. There is an additional charge for this service.
Day 70 Inject 25-50ug intramuscular using IFA We will usually do intramuscular unless you specify subcutaneous.
Day 84 Bleed. We will do these bleeds every 28 days unless you specify. It is up to the investigator to test the serum, determine the quality of antibodies, and let us know if any changes are necessary in the protocol. PRF&L now offers antibody titration by ELISA and can now check titers for you. There is an additional charge for this service.

We can begin immune egg collection at anytime. Many of our customers have found it easier to test the bleeds and upon determining that the bleeds are sufficient quality, then request that we begin to collect eggs. Chickens lay about six eggs per week. You will need to tell us for how long or how many eggs to collect. Immune eggs are stored in the shell at 4 degrees C. The day we ship eggs, they are cracked, yolk separated, and packaged in one week pools, by chicken unless you have a specific request. We send them to you via Federal Express on wet ice or ice packs. We now offer egg yolk purification (IgY) service.
After Day 84 See Continuing your Protocol below.

CONTINUING for BOTH PROTOCOLS

We will continue give boosts every four weeks with 25-50ug of antigen intramuscular or subcutaneous with IFA. We will do a bleed two weeks after each boost and send to you the following Tuesday. We will continue to boost and bleed every four weeks until you contact us. Keep in mind that you have several other options depending on your needs and the success of the project.
When the antisera contains sufficient antibody titer, we can start production bleeds. We can do production bleeds every seven days. Production bleeds with chickens provide you with the same volume as any other bleed as we only offer one size bleed with chickens. Please contact us if you want to change the bleeding and/or boosting schedule. You may also request that we not do any boosts after the initial immunization scheme outlined above since some antigens and some animals may respond negatively to boosts during the period of peak antibody activity. Boosts may be required only when titer drops. It is up to the investigator to decide and let us know if we should discontinue boosts as our standard schedule provides for boosts every 28 days.
The volume of plasma collected for production bleeds depends on the weight and the general health of the Chicken. We generally take 20-25ml of blood yielding 8 to 15ml of plasma. We have found that drawing plasma gives a high usable yield for each bleed. However, you may request that we bleed serum.
Our experience shows that the peak antibody titer occurs at an average of 120 days, however we have experienced the onset of peak activity as early as six weeks and as long as a year. If results are not as you expect, please consult with us and we can make recommendations on how to change your protocol.
When you have collected enough antisera, you will need to contact us to terminate the project. You may request that we perform an exsanguination (terminal bleed) or if no additional sera is required, the animal(s) will be humanely euthanized (discard). When you instruct us to terminate your animal(s), you must tell us if you want exsanguination or discard, species, and the animal(s) number. Written confirmation by email or fax may be requested for terminal procedures.

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