Custom Polyclonal Anitbodies

Custom Polyclonal Antibody Services

Primm provides an efficient service for the production of polyclonal antibodies using rabbits, mice and rats. The service is offered in three different options based on the antigen used for immunization:

Antibody Purification

Once an adequate polyclonal response has been reached, the crude serum can be purified by two distinct methods:

• Protein A (or G) affinity chromatography is used for purifying all the IgGs present in the sample. For better results and to enrich for specific IgG antisera, we recommend the elimination of antibodies with low affinity and specificity but higher binding capacity, such as IgM by purification of the antiserum.
• Immunoaffinity purification is recommended for purifying antigen specific antibodies from a pool of polyclonal antibodies, principally when they are to be used in immunocytochemistry or in immunofluorescence, where a high background would interfere with analysis. In this procedure, the pure antigen (free peptide) is covalently bound to a solid support: CNBr-Sepharose.The specific antibodies within the pool bind the antigen-sepharose, while the aspecific antibodies flow through the column. Finally the specific antibodies are eluted from the column and collected in TRIS buffer; their positivity and titer are reconfirmed by ELISA testing.

Antibody Labeling

Purified antibodies can be labeled by Primm with fluorochromes or biotin. There are different ways of labeling antibodies. Fluorescent labels are essential for some techniques, such as immunocytochemistry or cell sorting. Biotinylated antibodies can be detected using labeled streptavidin or avidin, which have a high affinity for biotin and show low levels of background binding.

At Primm we label antibodies produced by our staff, as well as antibodies supplied by the customer.

Custom polyclonal antibodies against custom delivered proteins 

If you have a purified soluble protein, a partially purified soluble protein, inclusion bodies with the insoluble protein, a protein in a polyacrylamide gel slice, or any other type of antigen, you can send your sample to Primm. For polyclonals development, the total quantity of antigen required for immunization of two rabbits is 1-1.5 mg.
The antigen can be supplied either lyophilized or in solution at a concentration not less than 0.4 mg/ml. In this case, we test the serum in ELISA after the third boosting and we can guarantee a final serum titer of 1:1,000-1:10,000 against the antigen you have supplied.
For an extra charge, there is the possibility for the customer to test the serum simultaneously.

Custom polyclonal antibodies against recombinant proteins

Primm offers a very competitive service based on an optimized fast and efficient technology to produce recombinant protein for immunization. Starting from the nucleotide gene sequence or from a gene ID number/name related to public genomic databases (human, mouse, rat origin), Primm will proceed to the isolation of the cDNA clone. If the gene target is from unusual organism or species and a reliable RNA source is not available, the customer may be requested to supply the gene.

The complete service includes:

• Gene cloning
• Complete DNA sequencing of the clone
• Expression in E.coli as His-tag fusion protein
• Purification of the recombinant protein
• Polyclonal Antibodies generation

Depending on the protein size and features, Primm team will attempt to clone and express the full length gene. Alternatively, a selected domain of 50-300 amino acids will be chosen for expression and immunization, we will then proceed with the form that is best produced.
To select the best domains Primm is open to take into considerations specific needs and preferences of the customer.

Recombinant proteins or protein domains will be produced as His-Tag fusions from E. coli cells and purified in quantities sufficient for the immunization protocol and for ELISA or Western Blot testing; the proteins are produced as “standard grade” in denaturing buffer, which is the typical quality, good to be used as antigens.

At the end of the immunization protocol, you will also receive an amount of recombinant protein (max 300 ug) and a small amount of plasmid DNA (about 3 ug).

Screening and quality control
We guarantee that the polyclonal antibodies are able to specifically recognize the recombinant protein at a dilution not lower than 1:10,000 in Elisa. Results of the test are supplied with the antibodies

Custom Polyclonal Antibodes Against Synthetic Peptides

• For antibodies against specific regions of a protein
• To discriminate between two or more isoforms of a protein
• For antibodies for Western Blot experiments (denatured condition)
• For antibodies for ELISA experiments (indirect, sandwich and competition test)

Custom polyclonal antibodies against synthetic phospho-peptide  

Phosphorylation is the addition of a phosphate (PO4) group to a protein molecule or a small molecule. Its prominent role in biochemistry is the subject of a very large body of research. Phospho specific antibodies can be used as powerful tools to detect whether a protein is phosphorylated at a particular site, binding to and detecting phosphorylation-induced conformational changes in the protein.

Phosphorylation usually occurs on serine, threonine, and tyrosine residues in eukaryotic proteins.
Phospho-specific antibodies are becoming critical reagents both for basic research and for clinical diagnosis. They are generated using, as antigen, a peptide containing a phosphorylated aminoacid residue.

Primm helps you to select the best peptide sequence and suggests to use

• short peptide to increase the relevance of phosphorylated aminoacid
• phosphorylated aminoacid residue either at N- or C-terminus
• Cys residue added for selected carrier conjugation

Primm’s production includes:

• two rabbits immunization with a phospho-synthetic peptide conjugated either to OVA or KLH,
• synthesis of the corresponding non phospho peptide for preparation of depletion column and for differential ELISA test,
• standard immunization schedule (5 injections – about 1.5/2 months), final bleed (40-50 ml/rabbit)

Phospho specific antibodies positivity is guaranteed at a dilution of the purified polyclonals from 1:1,000 to 1:10,000 as compared to the reactivity against the non phospho peptide in differential ELISA test.

Dual species custom polyclonal antibodies 

Primm introduced the Dual Species Custom Polyclonal Antibody Service.
Thanks to this new addition to our list of high performance polyclonal services, you can immunize two species (2 rabbits and 4 mice) obtaining two independent polyclonal preps.
Double species means

• Dual species benefits
• Higher Chances of generating antibody preparations for multiple applications
• Mouse serum is generally very clean, so mice polyclonal antibodies are an excellent choice for immuno histochemistry studies
• Rabbit serum is often very complex, for certain applications rabbit polyclonals need to be affinity-purified. Mouse polyclonals do not need (and cannot) to be purified

Polyclonals from 2 species can be considered as two separate antibody preps, thus double reagents for the customer, double chance to get good and useful antibodies.
Finally this service give to the customer the possibility to go from mouse polyclonals to monoclonal antibodies generation.

Primm specializes in production of the ideal antigen from peptides to recombinant proteins and also accepts custom antigens.
Primm provides two different procedures:

1. PAbs against synthetic peptide 

• peptide design & synthesis
• carrier conjugation (KLH or Ovalbumin)
• deliver of free remaining peptide

2. PAbs against recombinant protein

• choice of different cloning plans
• gene cloning, protein expression and purification
• deliver of free remaining recombinant protein and plasmidic DNA.

For both protocols 2 rabbits & 4 mice immunization (about 45-50 days protocol), ELISA test and minimum titer is guaranteed.

For more information or a custom service quote, please fill out this short form