PRF&L offers complete Immunochemistry services, from antigen development thru antibody purification, labeling, and packaging.
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. Also included in this service are strategies for the bioconjugation to larger carrier proteins and a suggested antibody host. This service is offered in conjunction with our peptide synthesis service.
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.
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.
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.
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 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 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.
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.
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.
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.
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.
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.
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 PEPTIDE SELECTION
PRF&L staff is able to predict antigenic regions within an amino acid (or translated DNA) sequence, for customers interested in anti-peptide antibody production. Also included in this service are strategies for the bioconjugation to carrier proteins and a suggested antibody host. A report of our findings is produced, many times offering several different peptides. This service is offered in conjunction with our peptide synthesis service.
Many investigators find this services to be more comprehensive than other free peptide selections services offered by our competitors.
CUSTOM PEPTIDE SYNTHESIS & CONJUGATION
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.
PURIFICATION OF SERA
PRF&L can purify your sera 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.
Generally, our recommendation is to try the raw sera in your application before requesting purification. In our experience, raw sera can work very well, saving you the time and expense of purification.
You can choose between affinity purification and protein A / protein G purification.
We can affinity purify against either peptide or protein. For peptide antibodies, free peptide (not conjugated) must be used. For protein antibodies, pure protein should be used. For example, if we use a GST protein for immunizations, cleaved protein should be used for affinity column. Usually 2 to 5mg of peptide or protein should be reserved for the affinity column.
Protein A or Protein G purification can be utilized if pure antigen is not available.
As a bonus, the affinity columns we use will be good for several rounds of purification. When we are finished with a purification project, we supply you with the affinity column we used so you can continue to purify more antibody on your own!
PURIFICATION OF EGG YOLKS
IgY can be purified from egg yolks. We use a polyethylene glycol precipitation method. Egg yolks do not store well, therefore, for long term storage, we suggest that egg yolks be at least IgY purified. The IgY can be then affinity purified using our usual affinity purification protocol.
IMMUNOCHEMISTRY SERVICES INCLUDE
- RNA Isolation from Spleen and Bone marrow
- Tissue Harvesting
- Sterile Whole Blood Collection
- Blood collection in anticoagulant such as alsevers, sodium citrate, and heparin
- Defibrinated Blood Preparation
- Reticulocyte Lysate Preparation
- Hybridoma Developement