The use of synthetic peptides as immunogens is generally applied where either the complete protein is not available in sufficient quantities to carry out an adequate immunization protocol, or to obtain antibodies able to recognize only specific regions of a polypeptide chain. The selection of the amino acid sequence of the peptide to be used as an immunogen is a crucial step toward the success of such a strategy. In fact, one can not predict, a priori, that antibodies raised against specific peptide sequences are then able to efficiently recognize the intact protein from which the peptide sequence was originally selected. Nevertheless, it is possible to exploit certain parameters based on hydrophilicity, accessibility, and flexibility criteria, together with considerations of T-cell epitopes to judge the possible immunogenic power of a given peptide, and thus to increase the possibility that anti-peptide antibodies do recognize the complete and often native conformation of the protein chain. Our technical staff will assist our customers with the selection of the best candidate peptide sequences to be used as immunogens. Specific sequences will be proposed to our customers and submitted for their approval prior to the start of the chemical synthesis. In addition, in some cases the use of more than one peptide from a given protein chain is recommended. After synthesis the peptides are combined and used as a single antigen toward the preparation of antibodies able to recognize all the peptides. Through such an approach, the resulting antibodies have a higher capability to recognize the intact and native protein chain. Generally, synthetic peptides used as immunogens are synthesized on a small scale (5 - 10 mg) and are made of approximately 15 amino acid residues.
Small molecules, such as peptides, have low immunogenic potential. To increase their immunogenicity, it is necessary to link, by chemical conjugation, the peptide sequence to a larger protein molecule, which serves as a carrier. Several carrier proteins can be selected to prepare the desired peptide conjugate and, in general, the few required features of the carrier are its molecular weight, which should be higher than 20,000 daltons, and its immunogenic potential.
Most of the immunopeptides produced by Primm are conjugated with carriers such as OVALBUMIN or KLH. Normally we conjugate about 3/5 mg of peptide.
Screening and quality control
Titer and quality of the polyclonal antibodies are verified by ELISA using the free peptide. Results of the test are supplied with the antibodies. Normally, dilutions of the serum, ranging from 1:1,000 to 1:10,000, can be used in most immunological applications.
Primm offers a very competitive service based on the production of recombinant proteins following gene cloning or synthesis. Starting from the nucleotide gene sequence or from a gene ID number/name related to public genomic databases, Primm will proceed to the isolation of the cDNA clone. In case of human gene, Primm has an almost complete and constantly updated gene library from which the desired gene target can be isolated. When 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. Depending on the protein size and features, Primm team will attempt to clone and express the full length gene. Alternatively, a selected domain of 100-300 amino acids will be chosen for expression. For instance, in case of very hydorophobic or transmembrane proteins, the expression and purification of full-length versions is not the preferred choice. In such cases, Primm scientists will select domains of 100-300 aminoacids which in the native protein may be accessible for antibody recognition.
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.
At the end of the immunization protocol, you will also receive an amount of recombinant protein (maximum 500 μg) and a small amount of DNA plasmidic (about 5 μg).
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.
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. This type of purification is useful for relatively non sensitive systems like Western Blotting.
- 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, then the specific antibodies within the pool bind themselves to the antigen-sepharose, while the unbound antibodies are removed by washing. Finally the specific antibodies are eluted from the column and collected in TRIS buffer; their positivity and titer are reconfirmed by ELISA testing. We normally purify half of the total serum and the purified antibodies are then shipped in solution.
Purified antibodies can be labeled by Primm with fluorochromes, biotin, or with enzymes, such as horseradish peroxidase or alkaline phosphatase: please choose the best label for your technique. There are different ways of labeling antibodies. The choice is determined by the technique you are going to use in your experiments. Fluorescent labels are essential for some techniques, such as immunocytochemistry or cell sorting, whereas immunohistology is more successful with enzyme-labeled antibodies. Enzyme-labeled antibodies can also be used in electron microscopic localization, immunoblot, or in immunoassays. Enzyme labels offer the advantage of an instant visual result and high sensitivity but are more difficult to use in a quantitative assay. The biotinylation of antibodies is a very simple and useful technique, and rarely inactivates the antibody. 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.
This service includes the development of specific polyclonal and monoclonal antibodies, the preparation of antigen standards (extractive or recombinant proteins), and the establishment of immunometric assays (i.e. ELISA), with specificity, accuracy and reproducibility.
