A. Polyclonal Development

1. Total service include

1. Analysis of protein sequence antigenicity
2. Synthesis of peptide
3. Purification of peptides by HPLC
4. Purity analysis by MALDI
5. Conjugation of the peptide to carrier
6. Delivery the unconjugated peptide
7. Purification
8. Technical assitance for antibody production

This is a complete service for the production of polyclonal antibodies (PcAb) in rabbits.
More information

The service includes the following protocols:

• I. Analysis of the peptide for the selection of regions showing the strongest antigenicity.

Immunostep offers its customers a free protein analysis service in which we include selection of the most antigenic peptide sequence; study of homogeneity with respect to the protein in other living beings, and possible identification of the signal peptide, of transmembrane domains and other structural motifs…

Our company offers a free, non-obligatory, consulting service on behalf of all its scientists for the search for the most antigenic sequences of customers proteins. Our service is directed towards researchers who lack the necessary tools to carry out these analyses, who are inexperienced in the field, or who are simply looking for help and a way round investing too much time in analysing the protein sequence in question.

• Identification of specific antigenic peptides.
• Search for homology with other species
• How to request a sequence analysis
• Confidentiality

Identification of specific antigenic peptides.
We can analyse protein sequences partially or wholly. The parameters analysed by us are: antigenicity (link to image), hydrophobicity (link to image), and accessibility (link to image),. Our programs locate the regions with the greatest antigenicity, hydrophobicity and accessibility (between 10 and 20 amino acids). This information is sent via e- mail or FAX in graphic form for clear understanding. The programs used cannot always ensure success, but they do allow us to have an approximate idea of what is happening, orienting us towards the regions with the best likelihood of success.

Search for homology with other species
The sequences chosen are transferred to databases to determine possible homologies with proteins from other species and thus confirm the specificity of the peptides. With this process, we search for the sequence -or part of it- that has the least similarity with the rest of homologous sequences from other species in order to avoid undesirable cross-reactivity. In the long run it is the researcher who will decide whether the homology shown by the protein might be a problem or not. This information is then sent by e-mail of FAX. The information is reviewed carefully to avoid unwanted recognition of the antibody.

How to request a sequence analysis
Immunostep can analyse the possible secondary structure of the three or four sequences proposed, which together with the above information helps us to discern which peptide sequence we will keep.

Confidentiality

Immunstep maintains all information under the strictest confidentiality. All information and/or materials supplied by the client will be used as directed. Upon completion of the project, all materials will be either returned or disposed of as directed by the client. IPA is willing to execute confidentiality agreements with its clients.

• II. Synthesis of peptide.

This service includes a variety of specialized and modified amino acids as well as an array of different coupling and branching options in a range of different scales and purities. Immunostep will also work with you and assist in the design of your immunogen.

Peptide Pricing

Contact us to other options.

• III. Purification of peptides by HPLC.

High-performance liquid chromatography (HPLC) has proved extremely versatile over the past 25 yr for the isolation and purification of peptides varying widely in their sources, quantity and complexity. After synthesis, the peptide is cleaved from the resin, yielding the crude form of the peptide. Generally, this type of synthesis yields crude peptides with 85% purity, but HPLC purification can be done if higher purity is desired. Average peptide lengths are 10-20 amino acids.
If high purity peptide is required, we offer HPLC purification of peptides. Our HPLC purification consists of taking approximately 100 mg of crude peptide (the amount depends on the complexity of the analytical HPLC trace of the crude product) and injecting that material onto a preparative scale reverse phase column. We collect all column fractions and pool those containing the ideal product. The amount of pure material recovered depends on the sequence but usually is better than 50% of that loaded. The pure peptide is provided along with any crude peptide remaining from the purification process

• IV. Purity analysis by MALDI.

The Facility operates a Micromass TofSpec MALDI/TOF mass spectrometer for the routine molecular weight analysis of biomolecules. This system is particularly suitable for the purity assessment of synthetic peptides and oligonucleotides, the analysis of recombinant proteins, peptide fingerprinting, assessment of peptide labelling reactions (e.g. fluorescent tags), etc.
The tolerance of this technique to contaminating substances renders it an extremely valuable tool in biomolecule characterisation. Low concentrations of general biological buffers, salt solutions, detergents, etc. may be tolerated, although they do make analysis more difficult. Often, if sufficiently concentrated, samples can be diluted to minimise interference from contaminating substances.

• V. Conjugation of the peptide to the KLH carrier, BSA or the Blue carrier through a N- and C-terminal residue of the peptide.

Keyhole Limpet Hemocyanin (KLH) or Blue Carrie and BSA activated with SMCC crosslinker for immunogen preparation with cysteine peptides and other sulfhydryl-containing haptens and EDC crosslinker for preparing immunogens from carboxyl-containing peptide antigens and other haptens.

• VI. Delivery of 5 mg of the unconjugated peptide, except if purification by means of an affinity.

We conjugated the necessary peptide for immunization, the rest will be sent to the client.

• VII. Purification.

Immunostep offers its customers a purification service for monoclonal antibodies and polyclonal sera. This includes different steps, depending on the needs of the customer and the origin of the sample.

Antibody purification
	The usual purification steps are: 1. Preparation, extraction and clarification. With this step, we are able to remove 50-60% of pollutants. It is used when samples are very large or when there are high contents in fats, albumin or other proteins. 2. Capture. Antibody capture allows us to isolate, concentrate and stabilise the samples. The degrees of purity achieved are about 95-98%, depending on the sample in question. The product is concentrated and transferred to an environment that will preserve the activity of the antibody. 3. Polishing. Most impurities such as nucleic acids, endotoxins, viruses …, are removed in this last step. Also, with this process it is possible to remove antibody aggregates or dimers, achieving purities of 99%. 4. Affinity purification. With this technique, we remove all the antibodies unable to recognise our protein or peptide. With this service, Immunostep offers its customers the possibility of improving the recognition of their antibodies. To accomplish this, we first coat the column with the protein or peptide selected and then purify the antibody.

• VIII. Technical assitance for antibody production.

Immunostep has a technical assistance unit to solve any problems or doubts about any of the services we offer.

Our company also has a group of experts who work as external scientific assessors and will also be able to offer advice about any issue that might need clarifying.

2. Inmunization Protocol.

Antigen requirements

• Peptides
• 5 mg of KLH conjugated peptide for immunization
• 2 mg of BSA coupled peptide for screening
• Recombinant or Purified Molecules
• 3mg of Antigen
• Soluble Antibens

Antigens in soltion must be prepare in a PBS buffered solution, pH 7.4 at a concentration of 0.5 – 2 mg/mL

The protocol to be followed can be modified if the customer sees fit:
Protocol: (añadir el protocolo de inmunización)

3. The customer will receive

• 5 mg of purified peptide (only if service include Synthesis of peptide)
•  Between 50 and 100 mL of serum from each rabbit.
• 2 ml of serum from each one of the three extractions performed per rabbit during the immunisation routine
• 2 mg of antibody purified from the final extraction per rabbit.

4. Guarantee

The Service for the Production of Polyclonal Antibodies guarantees the production
of polyclonal antibodies able to recognise a given sequence, or part of it.
All work is completely confidential and the results obtained become the property of the
customer.
Our Production Service has been designed to minimise possible problems. We work with a team of veterinary scientists and technicians, but some animals may fail to respond when challenged with certain peptides. Accordingly, we can offer no guarantees concerning successful antibody production.
All our products are manufactured for experimental use; they are neither for use in
humans nor for diagnostic assays.