Pocono Rabbit Farm and Laboratory Inc.

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PRF&L Immunochemistry Services

PRF&L offers complete Immunochemistry services, from antigen development thru antibody purification, labeling, and packaging.

ANTIBODY PROGRAM DESIGN

ASSISTANCE IN PEPTIDE SEQUENCE SELECTION
In this FREE service, PRF&L staff uses computer algorithms to predict antigenic regions within an amino acid (or translated DNA) sequence, for customers interested in anti-peptide antibody production. The customer will receive a minimum of three antigenic peptide sequences corresponding to these optimal antigenic domains; the pI, MW, and calculated peptide charge for each peptide. Also included in this service are strategies for the bioconjugation to larger carrier proteins and a suggested antibody host. There is NO CHARGE for this service providing PRF&L produces a peptide and generates antibodies to the protein sequence evaluated, otherwise a $250 consultation fee will apply for each protein evaluated.

ADVANCED POLYPEPTIDE CHARACTERIZATION SERVICE
This "consultation fee" based service is available to provide a more detailed polypeptide evaluation. Peptide domains selected for the project may be "modified" in the cells of the organism. A summary of any specific domains, points of post-translation modifications or protease or cleavage sites, may indicate a necessity to select another peptide sequence. Our goal is to produce anti-peptide antibodies that are functional in the final assay. Additionally with this service, you can request a complex homology sequence analysis between family proteins from different or same species. These analyses are used when the goal of the antibody project is to produce a Unique or a Common domain-specific antibody. Here customers can maximize their antibody usage in evaluating the expression of all or each of the target family proteins. Please call for additional information and pricing on this service.

CUSTOM PEPTIDE SYNTHESIS
PRF&L outsources peptide synthesis to one select supplier, pricing can vary depending upon sequence, purity, and amount needed. We will be glad provide you with a custom quotation after discussing your exact needs and the intended use of the antibodies you modified to mimic most naturally occurring secondary modifications.

CONJUGATION OF PEPTIDES, POLYPEPTIDES, AND SMALL MOLECULES BY SEQUENCE SPECIFIC METHODS
Since peptides are smaller molecular weight polypeptides pieces, when introduced as an antigen on their own, they elicit poor immunological responses. Therefore, bioconjugation of these peptides to larger carrier proteins such as KLH (keyhole limpet hemocyanin), OVA (ovalbumin), BSA (bovine serum albumin) or others is necessary to make them a more functional immunogen. When PRF&L is designing and selecting amino acid peptide sequences the chemistry of this bioconjugation is taken into account. A variety of amino acids can be selected as cross-linking points where the peptide is joined to the pre-activated carrier proteins. In one example, sulfo-SMCC-activated KLH protein is mixed with peptides (containing Cys amino acids) and a cross-link via -SH functional group occurs. Other reactions specific to the sequence are used as well as other modifications can be preformed if necessary.

If you are planning on making your own peptide(s) and having us do the conjugations, please feel free to contact us in advance if you would like assistance in determining what sequence or modifications may be better suited for a successful bioconjugation and development of a functional antibody. PRF&L provides percent incorporation results on all conjugations we perform. This type of validating assures our customers of a well characterized antigen at the start of their antibody program.

ANTIBODY TITRATION/QUANTITATING BY ELISA
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. All results samples 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.

SDS POLYACRYLAMIDE GEL ELECTROPHORESIS
Antigens imbedded in polyacrylamide gel samples can be further purified by electrophoresis in order to provide either a soluble form of the antigen for further use as an immunogen by methods other than subcutaneous injection. Or, couple the antigen to solid phase chromatography for use in affinity purification of your specific antibodies.

ANTIGEN PURIFICATION FROM SDS POLYACRYLAMIDE GEL
Customer supplied PAGE (gel slice) embedded protein samples purified by electrophoresis. Customer supplied mixed protein samples. Protein antigen is sized fractionated on SDS PAGE gels, excised, and purified by electrophoresis.

WESTERN BLOT ASSAY
Though most investigators do their own blots, we are willing to do this validation step for you as well. Please feel free to contact us for more information on this service or to incorporate this into a custom Antibody Validation Program specific to your studies needs.

ANTIBODY PURIFICATION SERVICES
PRF&L can purify your monoclonal and polyclonal antibodies using a variety of purification methods. Depending on the application of the antibody, purity and recovery will likely determine the strategy of purification. Our staff will be happy to discuss your purification options and develop the type of purification that will provide the best balance between yield, purity, and cost.

Antibody purification can be dived into two main groups: precipitation methods and chromatographic methods. The latter is grouped further into non-affinity and affinity chromatography.

The choice of purification methods available include:

• Polyethylene Glycol (PEG) and /or Ammonium Sulphate Precipitation

• Protein A Affinity Chromatography

• Protein G Affinity Chromatography

• Protein A/G Affinity Chromatography

• Ig Affinity

• Antigen Affinity

• Size Exclusion Chromatography or Ion Exchange Chromatography

Polyethylene Glycol and/or Ammonium Sulphate Precipitation

This method is useful for concentration and partial purification of antibodies from most sources and all species including purification of IgY’s from chicken egg yolks. Although, on its own the yields of antibodies are impure, it is the method of choice when combined with ion exchange for large volumes of antisera and or followed by Antigen Affinity purification to enrich for specific antibodies. It is not recommended for purification of tissue culture supernatant.

Protein A

This process binds the Fc region of major subclasses of mouse and rat IgG to varying degrees. The results with Protein A are usually high purity and high yields. It is useful for large-scale purifications of tissue culture supernatants.

Protein G

Protein G can be used for purifying antibody from most sources. Like Protein A, the results are usually of high purity and high yield. It is applicable to a wider range of immunoglobulins than Protein A, but elution procedures may be harsher.

Protein A/G

Protein A/G is a recombinant or genetically engineered protein that combines both Protein A and Protein G binding properties. Protein A/G is designed to contain four Fc binding domains from Protein A and two from Protein G.

Ig Affinity

This method can be used to isolate monoclonal antibodies from supernatants which contain high levels of serum Ig. It is the choice for secondary antibody production.

Antigen Affinity

This is the method of choice when specific antibody is required from polyclonal antisera. For best results this method requires pure antigen for the preparation of the affinity columns.

Size Exclusion

This method is used when IgM antibodies need to be separated from polyclonal IgG’s in antisera. It is not a single stage purification.

Ion Exchange Chromatography

This method is used for all sources of antibodies. It is applicable to all IgG isotypes. It is usually the method of choice when purifying antibodies from ascites, as the monoclonal can be resolved from the host immunoglobulins.

CUSTOM IMMUNOAFFINITY COLUMN PREPARATIONS
Specific affinity purified IgG or IgY antibodies will be covalently linked to affinity matrices. Linkage methods used maximize the functional orientation of the antibodies for optimal antigen capture.

ENZYME & TAG LABELING FITC, DIG, BIOTIN, HRP, ALK PHOS & other tags

Direct labeling of antibodies to FITC, DIG, BIOTIN, HRP, ALK PHOS as well as other tags is available please contact us for a quotation specific to your needs.

TOTAL RNA ISOLATION / ARCHIVING
If you get a great animal that produces a wonderful antibody, why not save the Genetic information that codes for the antibody until the time a recombinant antibody can be generated. Isolation of RNA from both bone marrow and spleen is available at the end of your project

CUSTOM “PHAGE DISPLAY” LIBRARY DEVELOPMENT
Acting as a subcontract extension of your laboratory with you being responsible for all end use licensing agreements, we can provide you with a Custom Library developed from your animals enriched genetic material that was collect after completion of your antibody project. Both very successful antibody production projects as well as ones that showed either poor or no serum antibody presence can be either enhanced or rescued by the use of Phage Display Technology.

Allow us to provide you with as much assistance as you may need in the development of a functional Custom Library and downstream in vitro antibody production.