Custom antibodies

Polyclonal antibody production - Monoclonal antibody production - Antibody packages - Immunisation schedule - Antibody services

Polyclonal antibody production

Epytop raises polyclonal antibodies using a variety of host animals (rabbit, chicken, rat, guinea-pig and mice) immunised with proteins or immunogenic peptides.

We offer a comprehensive range of services including peptide design, synthesis, immunisation and affinity purification of the antibodies. The services are provided separately or you can order the complete Antipeptide Antibody Package. We offer a flexible service and will happily modify our standard immunization schedule and the number of host animals used.

Epytop has extensive experience in the identification of specific antigenic peptides. Our free peptide
design service includes the analysis of different parameters such as hydrophilicity, antigenicity and
accessibility, identification of peptide signals, transmembrane domains and sequence alignment. Our recommendations are subjected to a BLAST search to assess the specificity of peptides.

Epytop also advises on the best possible coupling strategy with carrier proteins, including KLH, BSA, and OVA, or with MAP resin (Multiple Antigen Peptide).

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Monoclonal antibody production

Epytop provides the following hybridoma development program to produce monoclonal antibodies:

Immunogen design and immunization

• Preparation of protein antigen by customer or design and synthesis of peptide immunogen by Epytop
• Pre-immune bleed of group of 4 mice (6 to 8 weeks)
• 5 to 8 injections for a 8 weeks protocol
• Determination and qualification of the titer of the best responding mouse
(ELISA)
• Shipment of a detailed report including injection schedule and blood sampling

Cell fusion and Screening

• Fusion of murine myeloma Sp2/Oag-14 cell line and splenocyte from the immunized mice
• Expansion of clones
• Screening for IgG producing clones
• Amplification of the positive colonies (three vials of every hybridoma are stored in liquid nitrogen)
• 15 to 20 ml of supernatant of cloned hybridoma are shipped to the customer for the hybridoma(s) to be cloned.

Cloning & isotyping

• Cloning by limiting dilution
• Expansion of cells
• Screening and amplification of positive clone
• Isotyping of clones
• Storage of three vials of selected hybridoma in liquid nitrogen
• Shipment of 2-5 ml of every single hybridoma supernatant and of a detailed report

Storage of selected hybridomas

At the end of the contract, Epytop propose to store the selected hybridomas.
Vials are frozen in liquid nitrogen (-170°C) in order to preserve cells.

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Antibody packages

Standard Service: Peptide Synthesis - 10-20 Amino Acids - 5-10 mg - Peptide purity >70% - HPLC & Mass Spectrometry Analysis - Delivery time 2-3 weeks Conjugation to a carrier protein - BSA, OVA, KLH… of 3mg of peptide Immunization of 2 Rabbits - 81 days immunization protocol - 60-80 ml serum per rabbit		Service Package 1: « Minimum titer guaranteed » Selection of the most antigenic sequences (data bank screening and homology searching)*. Peptide Synthesis - 10-20 Amino Acids - 20-30 mg - Peptide purity >70% - HPLC & Mass Spectrometry Analysis - Delivery time 2-3 weeks Conjugation to a carrier protein - BSA, OVA, KLH… of 3mg of peptide Immunization of 2 Rabbits - 81 days immunization protocol - 60-80 ml serum per rabbit - Titration with PeptiPlak™ of serum antibody titers - Immunopurification of antipeptide antibodies (50µg to 100µg)
Service Package 2: « Minimum titer guaranteed » Selection of the 2 most antigenic peptides (data bank screening and homology searching)*. Peptide Synthesis - 10-20 Amino Acids - 20-30 mg - Peptide purity >70% - HPLC & Mass Spectrometry Analysis - Delivery time 2-3 weeks Conjugation of the 2 peptides to a carrier protein - BSA, OVA, KLH… of 3mg of peptide Immunization of 2 Rabbits - 81 days immunization protocol - 60-80 ml serum per rabbit - Titration with PeptiPlak™ of serum antibody titers - Immunopurification of antipeptide antibodies (50µg to 100µg)		Service Package 3: «guaranteed antipeptides **» Selection of the 3 most antigenic peptides (data bank screening and homology searching)*. Peptide Synthesis - 10-20 Amino Acids - 20-30 mg - Peptide purity >70% - HPLC & Mass Spectrometry Analysis - Delivery time 2-3 weeks Conjugation of the 3 peptides to a carrier protein - BSA, OVA, KLH… of 3mg of peptide Immunization of 2 Rabbits - 81 days immunization protocol - 60-80 ml serum per rabbit - Titration with PeptiPlak™ of serum antibody titers - Immunopurification of antipeptide antibodies (50µg to 100µg)

* Service free of charge except if the project is cancelled. In this case, the cost for this service is 160 €.
**An antipeptide against the native protein in Western Blot

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Immunisation schedule

Standard Schedule-Rabbit in 89 days
Animals: Specific Pathogen Free New Zealand White Rabbits

The final bleed is shipped to our customer on day 89 except if the project continues on a month-to month basis.
*ELISA tests of all sera and affinity purification of 1-2 ml from titre serum for Packages 1, 2 and 3.

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Antibody services

ELISA test on PeptiPlaks™
Epytop uses an original method to covalently coat the peptide on PeptiPlaks™. PeptiPlaks™ immunoassays are performed within 2 hours and preimmune and immune serum are tested at 8 dilutions. A detailed ELISA report is provided with every test regarding antibody titer of each animal.

Affinity Purification
Epytop offers the following antibody purification services:

- Antibody purification on Protein A column

- Antipeptide antibody immunopurification

. Sepharose-peptide
. ELISA testing
. SDS-PAGE analysis

- Supply of the affinity column