Pocono Rabbit Farm and Laboratory

28 DAY MIGHTY QUICK PROTOCOL PACKAGE

All 28 Day Mighty Quick Protocols include setup, prebleed, three immunizations (including immune stimulators), one large immune bleed, final bleed, and maintenance. This protocol can be extended if additional sera is required.

49 DAY MIGHTY QUICK PROTOCOL PACKAGE

All 49 Day Mighty Quick Protocols include setup, prebleed, four immunizations (including immune stimulators), four large immune bleeds, final bleed, and maintenance. This protocol can be extended if additional sera is required.

70 DAY POLYCLONAL ANTIBODY PRODUCTION

70 Day Antibody Production Package includes setup, prebleed, four immunizations, one small immune bleed, one large immune bleed, and maintenance. This protocol can be extended if additional sera is required. This protocol is based on the PRF&L Traditional 70 Protocol offered in the past.

84 DAY FUSION PROTEIN ANTIGEN POLYCLONAL ANTIBODY PRODUCTION

84 Day Fusion Protein Protocol, a favorite for many years at Pocono Rabbit Farm & Laboratory. This protocol uses an IV injection at day 56 as a way to improve the antibody. This package includes setup, prebleed, six immunizations, two large immune bleeds, and maintenance. This protocol can be extended if additional sera is required.

91 DAY POLYCLONAL ANTIBODY PRODUCTION

91 Day Antibody Production Package includes setup, prebleed, five immunizations, one small immune bleed, four large immune bleeds, and maintenance. All immunizations are subcutaneous for this package. This protocol can be extended if additional sera is required.

Our services includes cell line preparation (when provided with Mycoplasma free live cell line) for immunization into 5 Balb/c Mice, all necessary procedures and husbandry for a typical production run which is based on removing ascites fluid a maximum of 3 times per mouse followed by euthanasia and disposal. 3 to 10 ml per mouse can be anticipated.

Below are some considerations for using the ascites method for producing monoclonal antibodies.

• Some hybridoma cell lines do not adapt well to in-vitro conditions; the yields are negative or too low.
• Monoclonal antibodies from mouse ascites fluids are essential for experiments when they are injected into mice to determine which components of the immune system are responding to the antigens, e.g., for studies on allograft rejections, autoimmune diseases, histocompatibility complexes, and monocytic antigens.
• Hybridoma cells can be propagated in culture but do not always replicate the appropriate antibody activity; e.g., the monoclonal antibodies do not have high affinity for or binding capacity for the epitopes of some protein antigens.
• The elution of some monoclonal antibodies from purification columns is especially sensitive to denaturation or proteolysis, resulting in the loss of or significant decreases in antigen-binding activity.
• This loss of native conformation can also elicit unwanted immune responses when the monoclonal antibodies are administered therapeutically to humans in-vivo.
• Monoclonal antibodies specific to both viral proteins and for cells of the immune system are critical to understanding the ways the body fights retroviral infections. Moreover, such studies require extremely high concentrations of monoclonal antibodies that cannot be subjected to treatments that may alter their native structure. Purification by salt precipitation or affinity chromatography is often not suitable at high densities since such conditions can be harsh and result in denaturation of a fraction of the antibodies, loss of reactivity with the antigen, and enhanced immunogenicity of the antibodies leading inevitably to a shortening of antibody retention time after in-vivo administration.
• Serum-free, cell culture methods as yet can not produce sufficient amounts of monoclonal antibodies anti-human isotypes crucial for the evaluation of new vaccines against serious bacterial infections, e.g., H. influenza, type b, causing meningitis in infants and children.
• Several different Monoclonal Antibodies (IgM) used to remove tumor cells from autologous bone marrow are needed in the treatment of neuroblastoma in children. The hybridoma lines cannot be adapted to growth in serum-free medium; therefore, the monoclonal antibodies must be harvested from mouse ascitic fluids in order to assess new approaches to myeloablative therapy of the tumors.
• Culture methods sometimes yield populations of monoclonal antibodies that are glycosolated at positions different from those harvested from mouse ascitic fluids, thereby influencing the antigen binding capacity and the outcome of certain biologically important processes, e.g., phagocytosis (Such differences in reactivity are important to investigators who intend to use the monoclonal antibodies in-vivo in human or animal experiments.).
• When myeloma cells producing monoclonal antibodies are contaminated, they often must be passaged through the mouse.
• The hybridoma cell line will not adapt well to in vitro conditions.
• In applications where several different mouse MAb at high concentrations are required for injection into mice, the in vitro method can be inefficient
• MAb from mouse ascitic fluids might be essential for experiments in which MAb are used in vivo in mice
• Rat hybridoma cell lines do not generate ascites efficiently in rats, and usually adapt poorly to in vitro conditions, but usually generate ascites in immunocompromised mice.
• Downstream purification can lead to protein denaturation and decreased antibody activity.
• Serum free or low serum conditions cannot provide sufficient amounts of MAb for some purposes, such as the evaluation of new vaccines against infectious organisms.
• Culture methods sometimes yield populations of IgG MAb that are glycosylated at positions different from those harvested from mouse ascites fluid, thereby influencing antigen
• Binding capacity and important biologic functions.
• When hybridoma cells producing MAb are contaminated with infectious agents, such as yeasts or fungi, the cells often must be passed through mice.
• Some cell lines that do adapt to tissue culture conditions become unable to maintain adequate production of MAb

CUSTOM HYBRIDOMA DEVELOPMENT AND MONOCLONAL ANTIBODY PRODUCTION

PHASE A. Immunization of BALB/c Mice and Serum Screening (approximately 8 weeks)

Eight mice are immunized with antigen(s) provided by customer. Antigen conjugation and purification services are available for an additional charge. At day 35, and later if necessary, mice are test bled and screened for titer by ELISA. If no acceptable response is found during this phase, $1500 will be retained for services up to this point and the remaining balance of deposit will be refunded.

PHASE B. Fusion and Screening of Hybridoma Supernatant (approximately an additional 8-10 weeks)

Upon successful completion of Phase A, selected mice will be euthanized and the spleens harvested. A fusion procedure will be performed and the cells will be distributed into six or eight multiwell plates (96 well) per spleen. Screening will be performed by ELISA using previously supplied antigen. Hybridoma lines from up to ten wells with the highest absorbency by ELISA will be amplified and the supernatants supplied to the investigator. If after the first fusion attempt no acceptable development is observed, a second fusion attempt will be made utilizing previously immunized mice remaining from Phase A. At this point if there is still no response, you the PI or PRF&L has the option to terminate at this point, the charge for completion of Phases A and B will be a total of $5000.

PHASE C. Cloning (approximately an additional 6-8 weeks totaling about 22-26 weeks)

One to five previously developed parental hybridomas will be selected, cloned and then screened by ELISA. Clones with desired titer and specificity will be expanded and cryopreserved. At least five clones will be provided and delivered to the customer depending on the stability of chosen hybridomas.

Total completion cost for phases A, B, & C is $7500 per antigen.

PHASE D. Custom Monoclonal Antibody Production by in vitro methods
Yield of approximately 50 to 100mg per batch
The customer's cell line is amplified using three liter tissue culture polyethylene bags. Monoclonal antibodies are isolated using a protein G column. Available only for IgG producing lines. - $1,450
Other protocols are available and the success with a certain protocol is dependent on the cell line.

No need to order your peptide from another vendor - PRF&L is able to meet your peptide synthesis needs.

Our basic package for antibody production is 10 to 15mg of peptide at 80 percent purity. Some investigators require higher purity or additional peptide, both of which are available.

For routine antibody production, 80 percent purity is sufficient. 85 percent, 90 percent, and 95 percent purity is available.

We are able to produce most common peptide modifications such as phosphorylation and acetylation.

Most peptides for antibody production ranges from 10 to 25 amino acids. If your peptide is over 25 amino acids, please contact us first for pricing as longer peptides can be more difficult to produce.

HPLC and Mass Spec data are available for peptides at no additional charge.

All peptides need to be conjugated to carrier protein. For most successful conjugation, the peptide should have a terminal cystine amino acid.

We are also able to conjugate peptides synthesized by other manufactures. Please make sure you tell us the peptide sequence.

We most commonly recommend conjugation to KLH, keyhole limpet hemocyanin. Other common conjugates BSA (bovine serum albumin) and OVA (ovalbumin).

Our standard conjugation package is to conjugate 4mg. 4mg of conjugated peptide is plenty to immunize several animals.

Unlike most of our competitors, PRF&L provides percent incorporation results on all conjugations we perform.

Free up your time and resources and allow PRF&L to verify specific antibody presence in your projects.

Antigen specific antibodies can be quantitated by Enzyme-Linked Immunosorbant Assay (ELISA). We use a 96 well format that gives an actual titer not just a + or - response and is preformed in duplicate.

Results are normalized back to the preimmune sera or previous bleed. Our staff can typically have the results back to you in electronic format before you would normally receive sera samples if we were to ship them for you to evaluate. This can also save you additional ship costs or simply act as an initial indicator of success prior to further more sensitive applications you may need to perform.

Protein Quantification Services

Protein Purification and Quantification Services

Antibody Services

Protein Services

Biochemistry & Molecular Biology Services

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