Custom polyclonal antibody production

Transcription

1 EUROGENTEC 2008 Catalogue > > Custom polyclonal antibody production 231 General overview 232 Selecting a polyclonal programme 233 Peptide antibodies versus protein antibodies 235 Single peptide versus Double X strategy 235 Anti-Protein packages 237 Super Speedy protocol 237 CLASSIC protocol 238 FasT protocols 239 Anti-Peptide packages 240 Super Speedy protocol 240 CLASSIC protocols 241 FasT protocol 242 Double X & XP protocols 243 Post-translational modification specific antibody production 244 Sequence specific detection of PTM sites 244 PTM-specific purification protocol 245 The First Class programme 246 Create your own polyclonal programme 247 Additional services 248 Order forms 249

2 Custom polyclonal antibody production General overview Eurogentec works with a team of skilled veterinarians and technicians to produce your custom antibodies. We offer modern animal facilities that house over 3000 animals at any given time. We propose a number of different species adapted to all your antibody needs and you benefit from our broad experience in immunization techniques. Contact us with your project goals. Animals available on a regular basis: mouse, rat, guinea pig, chicken, rabbit, goat, pig, sheep, horse, cow, llama Low background serum Constant quality (ISO 9001:2000) Sterility guaranteed Specialist project design advice Bulk quantities High quality production under ISO 9001:2000 conditions This guarantees you: Safety is assured prior to blood sampling by anaesthetising the animal and serum is drawn with a sterile, haemolysis-preventing syringe. Our animal facilities comply with the highest quality standards and to the European Community guidelines for animal housing and in vivo experiments. Contact us for details. We offer a complete range of antibody services Anti-peptide antibodies (incl. peptide synthesis) Anti-protein antibodies DNA immunization antibodies Monoclonal antibodies In vitro antibody production Antibody labelling (enzymes, biotin, fluorescence probes, etc.) Affinity purification ELISA testing Assay development services Flexibility is our service Constant, high quality animals for immunization Standard procedures for immunization and blood sampling Excellent animal maintenance record Traceability Starting from your project goals we can provide you with a support team for all your production and post-production related antibody projects. Send your contact details to info@eurogentec.com and a specialist will be pleased to discuss your project goals with you. Full confidentiality guaranteed Online services Antibody production can be done under the terms of a secrecy agreement if requested. Eurogentec guarantees full confidentiality regarding antigen identity and project results. Specific Pathogen Free (SPF) quality SPF animals are tested for exposure to various viruses, bacteria, parasites etc as recommended by FELASA. SPF animals offer reduced background signals and higher success rates in antibody production. Eurogentec offers SPF rabbits for your custom antibody production. You may request the following services free of charge and with no obligation via Peptide design Anti-protein polyclonal quotation Anti-peptide polyclonal quotation Promoting the ethical treatment of animals Animals are maintained in an environment designed to minimize their stress and exposure to danger. Animals are provided water and feed according to their desire, are housed in groups to promote animal interaction, and are maintained in a climate-controlled environment. EUROGENTEC 2008 Catalogue > > order@eurogentec.com

3 233 Selecting a polyclonal programme The generation of a polyclonal antibody is dependent on two main factors, after which biology takes over. 1. The first parameter involves foreignness a sequence alignment between your antigen s sequence and the host animal is highly recommended before starting an immunization programme. 2. The second parameter is availability of pure antigen in sufficient quantity to elicit an immune response in the host. Please use the following flowchart to select the best polyclonal strategy. What s a standard antibody programme? Unfortunately antibody suppliers all have their own standard programmes. Our standard programme exceeds those of other suppliers. Our rabbit polyclonal standard programme: 87-day protocol 4 immunizations 4 bleeds shipping included As antibody production is a natural phenomenon that follows a welldescribed time course, there is a direct dependence on antibody titre and programme duration. In general, the longer the programme, the better the antibody titre and antibody affinity. The following flowchart should help you select a polyclonal protocol Antigen sequence NO Homology with isoforms? YES Have antigen? MW > 10 kda, purified to > 80 %, 1 mg (rabbit Ab) NO Homology undesired? YES NO YES Send sequences to peptide.design@eurogentec.com for best peptide selection help Select best host based on - homology to antigen - amount of Ab required Select best host based on - homology to peptide seq - amount of Ab required Complete anti-protein antibody order form Complete anti-peptide antibody order form EUROGENTEC 2008 Catalogue > > order@eurogentec.com

4 Custom polyclonal antibody production Animal host characteristics Host Min. antigen quantity / injection Bleed volumes Comment Antigen MW < 18 kda Antigen MW > 18 kda Mouse 40 µg 15 µg 30 µl in 270 µl of PBS Pre-immune Small Bleed Large bleed Final bleed - - ± 300 µl Good to test antigenicity Guinea pig 50 µg 30 µg 1 ml 1 ml 2-3 ml 5-7 ml For small serum volumes Rat 50 µg 30 µg 1 ml 1 ml 2-3 ml 5-7 ml For small serum volumes Chicken 200 µg 100 µg 1 egg 1 egg ± 10 eggs ± eggs For mammalian antigens and large quantities of Abs, 1 egg = 50 ml rabbit serum Rabbit 200 µg 100 µg 2 ml 2 ml 20 ml + 2 ml ml + 2 ml For most applications Goat 400 µg 200 µg 2 ml 2 ml 250 ml + 2 ml 1000 ml + 60 ml For large batch volumes pig 400 µg 200 µg 2 ml 2 ml 250 ml + 2 ml 1000 ml + 60 ml For large batch volumes Sheep 400 µg 200 µg 2 ml 2 ml 250 ml + 2 ml 1000 ml + 60 ml For large batch volumes Llama 400 µg 200 µg 2 ml 2 ml 250 ml + 2 ml 1000 ml + 60 ml Single chain antibodies and for large batch volumes Horse 400 µg 200 µg 2 ml 2 ml 250 ml + 2 ml 1000 ml + 60 ml For large batch volumes Other Contact us for recommendations Standard package overview Type Host Peptide Days Inj Bleed ELISA Purif Ship Reference Anti-Prot Guinea pig (2) incl. AS-PNOR-2MOGPG Anti-Prot Rabbit (2) incl. AS-PNOR-2MORAB Anti-Prot Rabbit (2) incl. AS-PNOR-3MORAB Anti-Prot Rabbit (2) incl. AS-SUPR-ANTIGEN Anti-Prot Your host Your protocol incl. Contact info@eurogentec.com Anti-Pep Mouse (3) 1 x 25 mg incl. AS-PCAP-MOUSE Anti-Pep Guinea pig (2) 1 x 25 mg incl. AS-PCAP-GUIPG Anti-Pep Hen (2) 1 x 25 mg incl. AS-PCAP-HEN Anti-Pep Hen (2) 1 x 25 mg Yes Yes incl. AS-PAFF-HEN Anti-Pep Hen (2) 2 x 25 mg Yes incl. AS-DOUB-LXHEN Anti-Pep Rabbit (2) 1 x 25 mg incl. AS-PCAP-RABBIT Anti-Pep Rabbit (2) 1 x 25 mg Yes incl. AS-PELI-RABBIT Anti-Pep Rabbit (2) 1 x 25 mg Yes Yes incl. AS-PAFF-RABBIT Anti-Pep Rabbit (2) 2 x 25 mg incl. AS-DOUB-LX Anti-Pep Rabbit (2) 2 x 25 mg x 5 ml incl. AS-DOUB-LXP Anti-Pep Rabbit (2) 1 x 25 mg incl. AS-SUPR-SINGLE Anti-Pep Rabbit (2) 2 x 25 mg incl. AS-SUPR-DX Anti-Pep Rabbit (2) 2 x 25 mg x 5 ml incl. AS-SUPR-DXP Anti-Pep Rabbit (2) 2 x 25 mg Yes 2 x 50 ml incl. AS-PSPE-CIFIC Anti-Pep Rabbit (2) 2 x 25 mg Yes 2 x 50 ml incl. AS-PTMA-LYSME1 Anti-Pep Rabbit (2) 2 x 25 mg Yes 2 x 50 ml incl. AS-PTMA-LYSME2 Anti-Pep Rabbit (2) 2 x 25 mg Yes 2 x 50 ml incl. AS-PTMA-LYSME3 Anti-Pep Rabbit (2) 2 x 25 mg Yes 2 x 50 ml incl. AS-PTMA-ARGME1 Anti-Pep Rabbit (2) 2 x 25 mg Yes 2 x 50 ml incl. AS-PTMA-ARGME2SYM Anti-Pep Rabbit (2) 2 x 25 mg Yes 2 x 50 ml incl. AS-PTMA-ARGME2ASY Anti-Pep Rabbit (2) 2 x 25 mg Yes 2 x 5 ml incl. AS-FIRS-TCLASS Anti-Pep Your host Your protocol incl. Contact info@eurogentec.com

5 235 Additional services overview Optional Services Reference Pre-immune sera from 5, 10, or 20 hosts to reserve your host Pre-immune serum AS-PREI-05 AS-PREI-10 AS-PREI-20 ELISA 1 antigen, 1 host, 3 bleeds AS-ELIS-01 1 antigen, 1 host, 2 bleeds AS-ELIS-LL 2 antigens, 2 hosts, 2 bleeds AS-ELIS-LLDX Affinity purification 5 ml AS-PURI-SM 20 ml AS-PURI-MED Second 20 ml AS-PURI-MED02 50 ml AS-PURI-01 Second 50 ml AS-PURI-02 Total IgG / IgY purification IgG: 50 ml serum AS-PURI-PCIGG050 IgY: 2 egg yolks AS-PURI-IGY20 IgY: additional yolk AS-PURI-IGYADD Peptide antibodies versus protein antibodies The best antibody results are obtained from antigens that present a large number of epitopes. This can mean a purified protein if one is not limited by homology with protein isoforms. Protein antigens offer many epitopes, providing and increased chance of generating antibodies that are useful in a wide range of applications. The prerequisites are that the antigen be purified to > 80 % purity and that sufficient material be provided for immunization. For our standard 87-day rabbit programme, we recommend at least 100 µg of purified protein per injection per rabbit, which translates into 800 µg of purified antigen. Peptide antibody programmes are particularly useful for the selective targeting of an antigen isoform, by selecting a peptide region that is unique to the desired isoform, or for antibody programmes that are designed to detect post-translational modifications. Peptide antibodies are today a very viable alternative to anti-protein programmes. A peptide antibody programme offers the advantages of being able to immunize with highly purified material and to affinity purify the antibodies against the peptide. Single peptide versus Double X strategy The single peptide strategy is best suited for reproducing the previously documented success of a polyclonal production with a known peptide sequence. If you have already successfully generated antibodies using a particular peptide or if the scientific literature suggests that a particular peptide is useful for generating specific antibodies, Eurogentec recommends the single peptide approach. For the first-time generation of antibodies against a particular antigen, we recommend the Double X strategy. The Double X strategy involves the design of two peptides from the same antigen and immunizing the host animals with both peptides at the same time. This co-immunization more closely mimics the protein than a single peptide strategy and we have success rates in the 90 % in Western Blots using this technique.

6 Custom polyclonal antibody production Antibody titre evolution Antibody affinity evolution Exposure to a specific antigen will initiate the immune response and in the first phase will result in the production of IgM class antibodies. IgM antibodies are responsible for the first attempt at clearance by the host animal. Initially the serum antibody titre is composed mainly of low affinity and low titre of predominantly IgM type antibodies. A second exposure will trigger a faster response, on the order of hours to days, and will generate longer lasting IgG type antibodies. IgG antibodies are of higher titre and affinity than IgM antibodies. With the generation of IgG antibodies is the generation of memory cells to quickly inactivate further exposure to the same antigen. Each immune response is different however a general schematic of IgM and IgG antibody titre as a function of time is represented below. The second immune response is not only characterized by a faster immune reaction but also by antibodies with up to a 10,000 fold increased affinity for the antigen. This increase is due to random somatic hypermutation in the variable region of the light chain and the heavy chain during expansion of memory cells. The diversity of the specificity will increase amongst a single clone. A second mechanism occurs following the antigen binding: isotype class switching. It refers to a rearrangement of the genes coding for the heavy chains in memory cells. The loss of coding region causes a class switch from IgM to IgG, IgA and IgE. A combination of these two mechanisms, somatic mutation and clonal selection leads to affinity evolution. Affinity evolution can be monitored by a number of methods, below are the results from serum drawn at different time points during a 3-month polyclonal rabbit programme. The flatter the curves, the tighter the binding affinities. Evolution of IgM and IgG titres as a function of immunization and time Evolution of affinity during an 87-day anti-peptide polyclonal programme

7 237 Anti-Protein packages Anti-Protein programmes Super Speedy protocol Rabbit - 28 days - your antigen Immunization schedule Please note Our antibody production service is designed to minimize all problems. We work with a team of skilled veterinarians and technicians. We offer no guarantees concerning the utility of antibodies in your application. These immunization protocols are sold for experimental use only. Contact us for diagnostic and therapeutic applications. We now offer a 28-day rabbit polyclonal antibody programme that provides similar titres and antibody affinities to an 87-day protocol. You send us Antigen Your antigen lyophilized or in solution, Nitro-cellulose blots and polyacrylamide gel slice will not be accepted for 28-day protocols. Antigen purity must be > 80 % to ensure a quality antibody production. Antigen quantities should exceed 800 µg of protein. The Super Speedy protocol (28-days) Type Host Reference Antigen Rabbits (2) AS-SUPR-ANTIGEN ELISA 1 peptide, 2 hosts AS-ELIS-LL Pre-immune 5 test sera AS-PREI-05 Pre-immune 10 test sera AS-PREI-10 Pre-immune 20 test sera AS-PREI-20 You receive Immunization protocol: 2 New Zealand White Rabbits (SPF) as described above. Pre-immune serum (2 ml) and first bleed (2 ml) for testing Medium bleed ( ml per rabbit) for testing Final bleed (50-70 ml per rabbit) for your application All shipping costs on dry ice included.

8 Custom polyclonal antibody production Anti-Protein programmes Classic protocol rabbit - 87 days your antigen Immunization schedule You send us Antigen Your antigen lyophilized, in solution or blotted on nitro-cellulose or in polyacrylamide gel slices. Antigen should be at least 80 % pure Antigen quantities should exceed 800 µg of protein (4 injections in 2 rabbits, 100 µg / inj / rabbit) You receive Immunization protocol: 2 New Zealand White Rabbits (SPF) as shown in the above figure. If the antibody titre is insufficient for your application, it is possible to continue boosting with an additional injection every 28 days and one bleed per month 10 days after the injection. Pre-immune serum (2 ml) and Small test bleed (2 ml) day 38, per host Large test bleed (20 ml + 2 ml aliquot) day 66, per host Final bleed (60-80 ml + 2 ml aliquot) day 87, per host All shipping costs on dry ice are included in this package. Please note Our antibody production service is designed to minimize all problems. We work with a team of skilled veterinarians and technicians. We offer no guarantee concerning the utility of antibodies in your application. These immunization protocols are sold for experimental use only. Contact us for diagnostic and therapeutic applications. Small Serum Volumes SPF Rabbits, 87-day protocol, your antigen, shipping included Type Host Reference Antigen Rabbits (2) AS-PNOR-3MORAB ELISA 1 antigen, 2 hosts AS-ELIS-LL Pre-immune 5 test sera AS-PREI-05 Pre-immune 10 test sera AS-PREI-10 Pre-immune 20 test sera AS-PREI-20

9 239 Anti-Protein programmes FAST protocol rabbit - 65 days your antigen Immunization schedule Anti-Protein programmes FAST protocol guinea pig - 65 days your antigen Immunization schedule You send us Antigen Your antigen lyophilized, in solution blotted on nitro-cellulose or in polyacrylamide gel slices. Antigen should be at least 80 % pure Antigen quantities should exceed 800 µg of protein (4 injections in 2 rabbits, 100 µg / inj / rabbit) You send us Antigen Your antigen lyophilized, in solution, blotted on nitro-cellulose or in polyacrylamide gel slices. Antigen should be at least 80 % pure Antigen quantities should exceed 400 µg of protein (4 injections in 2 guinea pigs, 50 µg / inj / guinea pig) You receive Immunization protocol: 2 New Zealand White Rabbits (SPF) as shown in the above figure. If the antibody titre is insufficient for your application, it is possible to continue boosting with an additional injection every 28 days and one bleed per month 10 days after the injection. You receive Immunization protocol: 2 guinea pigs (SPF) immunized as shown in the above figure. If the antibody titre is insufficient for your application, it is possible to continue boosting with an additional injection every 28 days and one bleed per month 10 days after the last injection. Pre-immune serum (2 ml) and Small test bleed (2 ml) day 38, per host Final bleed (60-80 ml + 2 ml aliquot) day 65, per host All shipping costs on dry ice are included in this package. Pre-immune serum (1 ml) and Small test bleed (1 ml) from day 38, per host Final bleed (7-10 ml) from day 65, per host All shipping costs on dry ice are included in this package. Please note Our antibody production service is designed to minimize all problems. We work with a team of skilled veterinarians and technicians. We offer no guarantee concerning the utility of antibodies in your application. These immunization protocols are sold for experimental use only. Contact us for diagnostic and therapeutic applications. Please note Our antibody production service is designed to minimize all problems. We work with a team of skilled veterinarians and technicians. We offer no guarantee concerning the utility of antibodies in your application. These immunization protocols are sold for experimental use only. Contact us for diagnostic and therapeutic applications. Standard serum volumes SPF rabbits, 65-day protocol, your antigen, shipping included Type Host Reference Antigen Rabbits (2) AS-PNOR-2MORAB ELISA 1 antigen, 2 hosts AS-ELIS-LL Pre-immune 5 test sera AS-PREI-05 Pre-immune 10 test sera AS-PREI-10 Pre-immune 20 test sera AS-PREI-20 Small serum volumes SPF guinea pigs, 65-day protocol, your antigen, shipping included Type Host Reference Antigen Guinea pig (2) AS-PNOR-2MOGPG ELISA 1 antigen, 2 hosts AS-ELIS-LL Pre-immune 5 test sera AS-PREI-05 Pre-immune 10 test sera AS-PREI-10 Pre-immune 20 test sera AS-PREI-20

10 Custom polyclonal antibody production Anti-Peptide packages Anti-Peptide programmes Super Speedy protocol rabbit/guinea pig 28 days one peptide Immunization schedule You receive Peptide, mg, uncoupled together with QC data for positive control, purification, ELISA etc. One peptide for single programmes, two peptides for Double X/XP programmes. Pre-immune serum (2 ml) and first bleed (2 ml) for testing Medium bleed ( ml per rabbit) for testing Final bleed (50-70 ml per rabbit) for your application Double XP: Affinity purified serum We now offer a 28-day rabbit polyclonal antibody programme that provides similar titres and antibody affinities as an 87-day protocol. We provide A selection of potential antigenic sequences, a determination of the antigenicity and hydrophilicity profile of your peptide. Peptide synthesis: mg, > 70 % purity, amino acids (2-3 weeks). One peptide for single programmes, two peptides for Double X/XP programmes. Carrier protein conjugation: coupling of 5 mg of peptide to either KLH, OVA, BSA, THY. Other carriers available upon request (1 week) Immunization protocol: 2 New Zealand White Rabbits (SPF) as described above. Double XP: Affinity purification of 5 ml of serum from each rabbit against each peptide. Please note Our antibody production service is designed to minimize all problems. We work with a team of skilled veterinarians and technicians. Nevertheless, some animals could fail to respond to certain peptides. For this reason, we offer no guarantees concerning the utility of antibodies in your application. These immunization protocols are sold for experimental use only. Contact us for diagnostic and therapeutic applications. Standard package Type Host Reference Speedy - antigen Rabbit (2) AS-SUPR-ANTIGEN Speedy - 1 peptide Rabbit (2) AS-SUPR-SINGLE Speedy - 2 peptides Rabbit (2) AS-SUPR-DX Speedy - 2 peptides + Affinity purification Rabbit (2) AS-SUPR-DXP Speedy - antigen Guinea pig (2) AS-SUPR-GPANTIGEN Speedy - 1 peptide Guinea pig (2) AS-SUPR-GPSINGLE Speedy - 2 peptides Guinea pig (2) AS-SUPR-GPDX Speedy - 2 peptides + Affinity purification Guinea pig (2) AS-SUPR-GPDXP ELISA 1 peptide AS-ELIS-LL ELISA 2 peptides AS-ELIS-LLDX Affinity purification 5 ml AS-PURI-SM Affinity purification 20 ml AS-PURI-MED All shipping costs on dry ice included. Affinity purification 50 ml AS-PURI-01 Additional amino acid beyond 16 aa, per aa AS-ADDA-A

11 241 Anti-Peptide programmes Classic protocol rabbit - 87 days one peptide Immunization schedule Anti-Peptide programmes Classic protocol guinea pig - 87 days one peptide Immunization schedule We provide A selection of potential antigenic sequences, a determination of the antigenicity and hydrophilicity profile of your peptide. We provide A selection of potential antigenic sequences, a determination of the antigenicity and hydrophilicity profile of your peptide. Peptide synthesis: mg, > 70 % purity, amino acids (2-3 weeks) Peptide synthesis: mg, > 70 % purity, amino acids (2-3 weeks) Carrier protein conjugation: coupling of 5 mg of peptide to either KLH, OVA, BSA, THY. Other carriers available upon request (1 week) Immunization protocol: 2 New Zealand White Rabbits (SPF) as described above. If the antibody titre is insufficient, it is possible to continue boosting with one injection every 28 days and a bleed 10 days later. All shipping costs on dry ice included. You receive Peptide, mg, uncoupled together with QC data for positive control, purification, ELISA etc Pre-immune serum (2 ml) and first bleed (2 ml) for testing Large bleed ( ml per rabbit) for testing Final bleed (50-70 ml per rabbit) for your application Please note Our antibody production service is designed to minimize all problems. We work with a team of skilled veterinarians and technicians. Nevertheless, some animals could fail to respond to certain peptides. For this reason, we offer no guarantees concerning the utility of antibodies in your application. These immunization protocols are sold for experimental use only. Contact us for diagnostic and therapeutic applications. Carrier protein conjugation: coupling of 5 mg of peptide to either KLH, OVA, BSA, THY. Other carriers available upon request (1 week) Immunization protocol: 2 Guinea pigs as described above. If the antibody titre is insufficient, it is possible to continue boosting with one injection every 28 days and a bleed 10 days later. All shipping costs on dry ice included. You receive Peptide, mg, uncoupled together with QC data for positive control, purification, ELISA etc Pre-immune serum (1 ml) and first bleed (1 ml) for testing Large bleed (2-3 ml per guinea pig) for testing Final bleed (7-10 ml per guinea pig) for your application Please note Our antibody production service is designed to minimize all problems. We work with a team of skilled veterinarians and technicians. Nevertheless, some animals could fail to respond to certain peptides. For this reason, we offer no guarantees concerning the utility of antibodies in your application. These immunization protocols are sold for experimental use only. Contact us for diagnostic and therapeutic applications. Standard package Type Host Reference Anti-Pep Rabbit (2) AS-PCAP-RABBIT Anti-Pep + ELISA Rabbit (2) AS-PELI-RABBIT Anti-Pep + ELISA + Affinity purification Additional amino acid beyond 16 aa, per aa Rabbit (2) AS-PAFF-RABBIT AS-ADDA-A For medium serum volumes Type Host Reference Anti-Pep Guinea pig (2) AS-PCAP-GUIPG Additional amino acid beyond 16 aa, per aa AS-ADDA-A

12 Custom polyclonal antibody production Anti-Peptide programmes Classic protocol hen - 87 days one peptide Immunization schedule Anti-Peptide programmes Fast protocol mice - 38 days one peptide Immunization schedule We provide A selection of potential antigenic sequences, a determination of the antigenicity and hydrophilicity profile of your peptide. Peptide synthesis: mg, > 70 % purity, amino acids (2-3 weeks) Carrier protein conjugation: coupling of 5 mg of peptide to either KLH, OVA, BSA, THY. Other carriers available upon request (1 week) Immunization protocol: 2 Chickens as described above. If the antibody titre is insufficient, it is possible to continue boosting with one injection every 28 days and an egg yolk collection 10 days later. All shipping costs on wet ice included. You receive Peptide, mg, uncoupled together with QC data for positive control, purification, ELISA etc Pre-immune egg (1 yolk) and first egg yolk collection for testing (days 14-38) Second egg yolk collection for testing (days 39-52) Final egg yolk collection (day 53-87) for your application Please note Our antibody production service is designed to minimize all problems. We work with a team of skilled veterinarians and technicians. Nevertheless, some animals could fail to respond to certain peptides. For this reason, we offer no guarantees concerning the utility of antibodies in your application. These immunization protocols are sold for experimental use only. Contact us for diagnostic and therapeutic applications. We provide A selection of potential antigenic sequences, a determination of the antigenicity and hydrophilicity profile of your peptide. Peptide synthesis: mg, > 70 % purity, amino acids (2-3 weeks) Carrier protein conjugation: coupling of 5 mg of peptide to either KLH, OVA, BSA, THY. Other carriers available upon request (1 week) Immunization protocol: 3 mice as described above. If the antibody titre is insufficient, it is possible to continue boosting with one injection every 28 days and a bleed 10 days later. All shipping costs on dry ice included. You receive Peptide, mg, uncoupled together with QC data for positive control, purification, ELISA etc Pre-immune serum 30 µl in 270 µl of PBS for testing Final bleed 300 µl of serum for your application Please note Our antibody production service is designed to minimize all problems. We work with a team of skilled veterinarians and technicians. Nevertheless, some animals could fail to respond to certain peptides. For this reason, we offer no guarantees concerning the utility of antibodies in your application. These immunization protocols are sold for experimental use only. Contact us for diagnostic and therapeutic applications. For conserved mammalian antigens Type Host Reference Anti-Pep Hen (2) AS-PCAP-HEN Anti-Pep + Affinity purification Additional amino acid beyond 16 aa, per aa Hen (2) AS-PAFF-HEN AS-ADDA-A Test your epitope Type Host Reference Anti-Pep Mouse (3) AS-PCAP-MOUSE Additional amino acid beyond 16 aa, per aa AS-ADDA-A

13 243 Anti-Peptide programmes Double X & XP protocols (the high success formula) Please note Our antibody production service is designed to minimize all problems. We work with a team of skilled veterinarians and technicians. Nevertheless, some animals could fail to respond to certain peptides. For this reason, we offer no guarantees concerning the utility of antibodies in your application. These immunization protocols are sold for experimental use only. Contact us for diagnostic and therapeutic applications. Immunization schedule Standard package Type Host Reference Anti-Pep Rabbit (2) AS-DOUB-LX Anti-Pep + 2 x Affinity purification Rabbit (2) AS-DOUB-LXP ELISA 2 peptides, 2 rabbits AS-ELIS-LLDX Statistically, our single peptide immunization protocols have a success rate of about 75 % in WB. The Double X protocol increases this success rate. By designing two peptides from your antigen sequence and immunising two rabbits with a mixture of these peptides, the success rate increases to ( ) = %. This programme is ideal for the first time generation of an antibody. Additional amino acid beyond 16 aa, per aa AS-ADDA-A We provide A selection of potential antigenic sequences, a determination of the antigenicity and hydrophilicity profile of your peptide. Peptide synthesis: mg, > 70 % purity, amino acids (2-3 weeks) Carrier protein conjugation: coupling of 5 mg of peptide to either KLH, OVA, BSA, THY. Other carriers available upon request (1 week) Immunization protocol: 2 New Zealand White Rabbits (SPF) as described above. If the antibody titre is insufficient, it is possible to continue boosting with one injection every 28 days and a bleed 10 days later. Double XP: Affinity purification of 5 ml of serum from each rabbit against each peptide. All shipping costs on dry ice included. You receive Peptide, mg, uncoupled together with QC data for positive control, purification, ELISA etc Pre-immune serum (2 ml) and first bleed (2 ml) for testing Large bleed ( ml per rabbit) for testing Final bleed (50-70 ml per rabbit) for your application Double XP: Affinity purified serum

14 Custom polyclonal antibody production Post-translational modification specific antibody production Phospho-specific PTM antibody production Type Reference AS-PSPE-CIFIC Sequence specific detection of ptm sites Immunization protocol Phospho-specific Super Speedy 28-day protocol Methyl lysine (mono) specific Methyl lysine (-di) specific Methyl lysine (-tri) specific Methyl arginine specific Methyl arginine (-di, sym) specific Methyl arginine (-di, asy) specific AS-SUPR-PSPEC AS-PTMA-LYSME1 AS-PTMA-LYSME2 AS-PTMA-LYSME3 AS-PTMA-ARGME1 AS-PTMA-ARGME2SYM AS-PTMA-ARGME2ASY Eurogentec is a GMP and ISO 9001 certified provider of research tools and services in the fields of genomics, proteomics and biologicals. We have been producing antibodies for academic, biotech and top pharmaceutical companies since Our PTM antibody programs are designed to detect the presence of modified amino acids in a sequence specific context. These antibodies are double purified against the sequence and the PTM modified sequence to ensure specificity. Includes Peptide selection expertise mer Synthesis of mg of modified peptide Synthesis of mg of unmodified peptide Conjugation of the modified peptide to your carrier of choice 3-month programme in SPF rabbits Vacutainer bleeds Pre-immune, small, large and terminal bleeds ELISA testing of pre-immune and large bleed sera to determine best responding animal Purification of 50 ml of serum from the best responding animal against the modified peptide Re-purification of above serum against unmodified peptide ELISA test of purification Remaining crude sera (about 100 ml) Remaining free peptide All shipping on dry ice

15 245 PTM-specific purification protocol Column 1 Column 2 Crude serum, 50 ml A + B Column with modified peptide Second column is coupled with non-modified peptide sequence. Sequence specific (A) and modification sequence specific (B) antibody are loaded onto 2 nd column. Non-modification, sequence specific antibody is captured. Sequence specific (A) and modification sequence specific (B) antibodies are captured. Unrelated antibodies are washed off and the sequence specific antibodies are recovered (A + B). Desired modification-specific antibody is recovered and shipped.

16 Custom polyclonal antibody production The First Class programme Antibodies that work in your application, guaranteed Custom peptide antibodies adapted to your application B-cell epitopes are selected based on the antigen s predicted 3D structural features in your application conditions The right antibody for your immunoassay or your money back We have improved antigenic sequence prediction algorithms. The antigenic surface of a protein is a 3-D patchwork of small linear antigenic sequences. Individually, each sequence is antigenic but, depending on the number of accessible residues in the interaction, the affinity will be either acceptable or too low. Currently, antibody affinities of 10-8 to 10-9 M can be expected for linear epitope, by calculating and analyzing the structure of each potential epitope. We will propose the most appropriate antigenic fragments that should exhibit such affinities. An epitope for each application As the structure of your protein may vary with each assay, an assay antigen specific analysis must be made to determine structural and interaction properties. Major factors leading to changes in the epitope structure are (1) denaturation by detergent and (2) chemical modification of the amino acids of the epitope, for example with fixatives or other chemicals. For your immuno-assay technique the most exposed epitope may not be your best choice. Depending on whether the antibody will be used on a native, a detergent-denatured or a fixative-modified structure of your protein, we shall select either a flexible or a more rigid region of the protein such as a turn or a helix. Increased flexibility is reported to lead to improved affinity, however, rigid structures can provide good epitopes more resistant to detergent denaturation. Additionally, sequences containing aromatic residues rather than highly polar amino acids may be selected if application conditions will involve the use of chemicals that react with polar moieties. Indeed, as long as they do not impair the fragment accessibility, hydrophobic residues could improve the affinity. The prediction of the best epitope is closely linked to the application for which the antibody will be used. The First Class programme what do we do? Using a consensus of epitope prediction procedures we characterize all potential linear epitopes. Then, in a second step, we calculate their structures and keep those whose characteristics are adequate to raise high affinity antibodies. In the third step, we select the fragment(s) that are the most likely to be useful in your immunoassay. What do we need? The accession number of the protein of interest and a complete description of the application in which you will use the antibody. Please complete the questionnaire on the corresponding order form. We include : Application specific peptide design 5 pre-immune rabbit sera for animal selection Synthesis of 2 peptides, mg each Coupling of each peptide to a carrier protein Double X protocol in a 4-month program ELISA testing to monitor titer evolution Affinity purification of 5 ml of serum against each peptide Delivery of mg of free peptide Remaining antisera All shipping costs Please note 100 µl of the different bleeds and 100 µg of the purified antibody will be shipped for testing prior delivery of the total volumes. The First Class programme Type Reference First Class programme AS-FIRS-TCLASS

17 247 Create your own polyclonal programme You may of course select the number and type of hosts for your own custom polyclonal programme, as well as the immunization and bleed schedule for each animal. The creation of a custom protocol is recommended for particular antigens that are refractory to the standard 87-day protocol or for bulk serum protocols. The following table will help to identify the host that best corresponds to the amount of antigen available and the amount of serum required. Anti-protein programme Host Min. antigen quantity / injection Bleed volumes Comment Antigen MW < 18 kda Antigen MW > 18 kda Mouse 40 µg 15 µg 30 µl in 270 µl of PBS Pre-immune Small Bleed Large bleed Final bleed - - ± 300 µl Good to test antigenicity Guinea pig 50 µg 30 µg 1 ml 1 ml 2-3 ml 7-10 ml For small serum volumes Rat 50 µg 30 µg 1 ml 1 ml 2-3 ml 7-10 ml For small serum volumes Chicken 200 µg 100 µg 1 egg 1 egg ± 10 eggs ± eggs For mammalian antigens and large quantities of Abs, 1 egg = 50 ml rabbit serum Rabbit 200 µg 100 µg 2 ml 2 ml 20 ml ml For most applications Goat 400 µg 200 µg 2 ml 2 ml 250 ml 1000 ml For large batch volumes Pig 400 µg 200 µg 2 ml 2 ml 250 ml 1000 ml For large batch volumes Sheep 400 µg 200 µg 2 ml 2 ml 250 ml 1000 ml For large batch volumes Llama 400 µg 200 µg 2 ml 2 ml 250 ml 1000 ml Single chain antibodies and for large batch volumes Horse 400 µg 200 µg 2 ml 2 ml 250 ml 1000 ml For large batch volumes Other Contact us for recommendations You send us Antigen Your antigen lyophilized, in solution, Nitro-cellulose blots or polyacrylamide gel slice. Antigen purity must be > 80 % to ensure a quality antibody production Antigen quantities should exceed the minimum quantities described above. Or your antigen sequence for peptide design and synthesis We provide Host Your desired host Protocol Your desired immunization protocol Our FAST (65-day) protocol can be applied to any host Our CLASSIC (87-day) protocol can be applied to any host All programmes can be prolonged with an additional immunization every 28 days, and an additional bleed 10 days after the immunization. Programme prolongations will be invoiced an additional animal maintenance fee. Purchase of animal (quarantine included) Available animals Reference Mouse (min 3) AS-MOUS-E Guinea pig AS-GUIN-EAPIG Rat AS-RAT Chicken AS-CHIC-KEN Rabbit AS-RABB-IT Goat AS-GOAT Pig AS-PIG Sheep AS-SHEE-P Llama AS-LLAM-A Horse AS-HORS-E Dry ice shipment Mouse (min 3) Guinea pig Rat Chicken Rabbit Goat Pig Sheep Llama Horse Monthly maintenance of animal Injection and bleeds included Available animals Reference AS-MOUS-E-/T AS-GUIN-EAPIG/T AS-RAT/T AS-CHIC-KEN/T AS-RABB-IT/T AS-GOAT/T AS-PIG/T AS-SHEE-P/T AS-LLAM-A/T AS-HORS-E/T

18 Custom polyclonal antibody production Additional services ELISA, affinity purification, pre-immune serum test, labelling To complement your polyclonal antibody production we offer a number additional services. Pre-immune serum test Select the best host We offer you the ability to select the host that provides the lowest background signal. Just tell how many sera you would like to test and based on your results we will reserve those hosts for your immunization programme. Pre-immune test serum Number Reference 5 sera AS-PREI sera AS-PREI sera AS-PREI-20 ELISA tests - Determine the progress of your programme We offer 3 ELISA formats : 1. Testing of pre-immune, small bleed and large bleed, one antigen, one host on 1 ELISA plate 2. Testing of pre-immune and large bleed, one antigen, two hosts on 1 ELISA plate 3. Testing of pre-immune and large bleed, Double X programmes, two hosts, two antigens, on two ELISA plates Affinity purification of serum against purified peptide Our peptide packages include sufficient additional peptide for affinity purification of your serum. You can opt to purify your own serum or if you prefer, you can have it purified by us. Affinity purification are offered in three sizes : 1. Small purification 5 ml of serum 2. Medium purification 20 ml of serum 3. Large purification 50 ml of serum Each purification includes the coupling of the peptide to the matrix material (however does not include peptide synthesis). Should you wish to have a second serum purified against the same peptide as the first serum, a reduced cost reference (AS-PURI-xx02) has been created for this purpose. Affinity purification of serum Size Reference 5 ml AS-PURI-SM 20 ml AS-PURI-MED 2 nd 20 ml AS-PURI-MEDO2 50 ml AS-PURI-01 2 nd 50 ml AS-PURI-02 Affinity purification of egg yolk against purified peptide Our peptide packages include sufficient additional peptide for affinity purification of your egg yolks. You can opt to purify your own egg yolk or if you prefer, you can have it purified by us. ELISA tests Format Reference 1 antigen, 1 host, 3 bleeds AS-ELIS-01 1 antigen, 2 hosts, 2 bleeds AS-ELIS-LL 2 antigens, 2 hosts, 2 bleeds AS-ELIS-LLDX Total IgG purification against protein A/G We also offer an antibody concentration service based on the affinity of protein A and protein G for the constant region of antibodies. This method of purification removes non-igg type molecules however does not provide only antigen specific antibodies. IgY purifications involve a two-step process, firstly the total IgY purification followed by affinity purification against the peptide. Each egg yolk corresponds to approximately 20 ml of rabbit serum in terms of antibody content. Affinity purification includes the coupling of the peptide to the matrix material (however does not include peptide synthesis). Should you wish to have a second serum purified against the same peptide as the first serum, a reduced cost reference (AS-PURI-xx02) has been created for this purpose. Affinity purification of egg yolk Description Reference AS-PURI-PCIGG050 Total IgG purification Reference Total IgY 2 eggs AS-PURI-IGY20 Additional egg AS-PURI-IGYADD Affinity purification AS-PURI-01 2 nd affinity purify AS-PURI-02

19 249 Custom anti-protein polyclonal antibody order form Your purchase order reference**: VAT Nr. : EGT quotation number: If VAT exempt please include a copy of certificate** INVOICE ADDRESS Institute/Company: Department/Lab.: DELIVERY ADDRESS (Please fill in delivery information if different from invoicing information) Institute/Company: Department/Lab.: Contact Person: Address: Postal Code: Country: Phone: Fax: City: Contact Person: Address: Postal Code: Country: Phone: Fax: City: Antigen name: ( 1 order form per antigen please) Number of hosts to immunize: (Number in parentheses is the recommended amount of antigen per injection) q Rabbit (100 µg) q Mouse (15 µg) q Guinea pig (30 µg) q Rat (30 µg) q Hen (100 µg) q Goat (200 µg) q Sheep (200 µg) q Horse (200 µg) q Llama (200 µg) q Other Antigen is sent in the following format (please aliquot the samples on a per injection basis): q In solution, each tube contains mg/ml of antigen in ml of buffer. Number of tubes: q Lyophilized, each tube contains mg of antigen to be resuspended in ml of buffer. Number of tubes: q PAGE slice*, each band contains µg of antigen for mg of gel piece. Number of tubes: Total amount of antigen shipped: µg. Required storage temperature of antigen: Protocol to use: q Eurogentec s standard protocol q Use all the available antigen q Use µg / injection q Do final bleed as scheduled (animals will be automatically bled 3 weeks after the last test bleed) q Wait for my written confirmation before doing final bleed (additional fees for animal maintenance will be charged) q My custom protocol Adjuvant: Number of injections per host: Quantity of antigen per injection: Injection schedule: Days Injection method: Volume of injections (including adjuvant max 1ml): Number of bleeds: Bleed schedule: Days Additional services: q Screen pre-immune serum: q 5 animals (AS-PREI-05) q 10 animals (AS-PREI-10) q 20 animals (AS-PREI-20) q ELISA testing against your antigen: q AS-ELIS-LL (1 antigen / plate, testing pre-immune and 2nd bleed from 2 animals + carrier response) q IgG purification (AS-PROG-01) q Affinity purification (amount of antigen required): q 5 ml (5 mg) AS-PURI-SM q 20 ml (8 mg) AS-PURI-MED q 50 ml (10 mg) AS-PURI-01 Animal Welfare Statement: I hereby certify that the antigen is not dangerous for people or animals. The sample does not contain HIV or Hepatitis virus or purified protein from such viruses. Recombinant proteins from human viruses will be accepted only if the production conditions are explained. I certify that the antigen does not contain sodium azide. Signature: Fax to * not suitable for Super Speedy 28-day protocols. ** note: please provide us with these information (if applicable) as soon as possible in order to avoid any delay

20 Custom polyclonal antibody production Custom anti-peptide-antibody package order form Your purchase order reference*: VAT Nr. : EGT quotation number: If VAT exempt please include a copy of certificate* INVOICE ADDRESS Institute/Company: Department/Lab.: DELIVERY ADDRESS (Please fill in delivery information if different from invoicing information) Institute/Company: Department/Lab.: Contact Person: Contact Person: Address: Address: Postal Code: City: Postal Code: City: Country: Country: Phone: Phone: Fax: Fax: Standard peptide immunization packages: q AS-PCAP-RABBIT Protocol I 1 peptide, 2 rabbits, 3-month programme q AS-PCAP-HEN Protocol II 1 peptide, 2 hens, 3-month programme q AS-PCAP-MOUSE Protocol III 1 peptide, 3 mice, 1-month programme q AS-PELI-RABBIT 1 peptide, 2 rabbits, 3-month programme, ELISA q AS-PAFF-RABBIT 1 peptide, 2 rabbits, 3-month programme, ELISA, affinity purified, SDS-PAGE q AS-PSPE-CIFIC Anti-phosphopeptide Ab 2 peptides, 2 rabbits, 3-month programme, double affinity purified q AS-DOUB-LX Double X 2 peptides, 2 rabbits, 3-month programme q AS-DOUB-LXP Double XP 2 peptides, 2 rabbits, 3-month programme, double affinity purified q Other Peptide selection (Double X/XP & Phosphospecific, please provide 2 sequences, indicate phosphorylation with [P] before AA) q Peptide sequence #1 (10 16 mer): q Peptide is the protein s real q N-terminus q C-terminus q Neither q Peptide sequence #2 (10 16 mer): q Peptide is the protein s real q N-terminus q C-terminus q Neither Conjugate to the following carrier: q KLH q OVA q BSA q THY q Other: Additional services q AS-ADDA-A Peptides longer than 16 amino acids will be invoiced an additional charge per AA over 16 AA q Screen pre-serum prior to initializing immunization programme q 5 animals (AS-PREI-05) q 10 animals (AS-PREI-10) q 20 animals (AS-PREI-20) q ELISA test titre determination q AS-ELIS-LL 1 plate, 1 peptide per plate, testing of pre-immune, 2 nd bleed from 2 animals q AS-ELIS-LLDX 2 plates, 1 peptide per plate testing of pre-immune,2 nd bleed from 2 animals q IgG purification (AS-PROG-01) q Affinity Purification against the peptide q 5 ml (AS-PURI-SM) q 20 ml (AS-PURI-MED) q 50 ml (AS-PURI-01) * note: please provide us with these information (if applicable) as soon as possible in order to avoid any delay Fax the completed order form to

21 251 Custom modification-specific antibody order form Your purchase order reference*: VAT Nr. : EGT quotation number: If VAT exempt please include a copy of certificate* INVOICE ADDRESS Institute/Company: Department/Lab.: DELIVERY ADDRESS (Please fill in delivery information if different from invoicing information) Institute/Company: Department/Lab.: Contact Person: Contact Person: Address: Address: Postal Code: City: Postal Code: City: Country: Country: Phone: Phone: Fax: Fax: Peptide selection Programme name: (1 per order form please) Protein Accession #: Species: Peptide selection (Please indicate site of phosphorylation with [P] before the amino acid) q Peptide sequence (10 mer): Peptide is the protein s real q N-terminus q C-terminus q Neither Conjµgate to the following carrier: q KLH q OVA q BSA q THY q MAP-8 q Other: Additional services q AS-ADDA-A Peptides longer than amino acids will be invoiced an additional charge per AA over AA q Screen pre-serum prior to initializing immunization programme q 5 animals (AS-PREI-05) q 10 animals (AS-PREI-10) q 20 animals (AS-PREI-20) Comments / Remarks: How to order 1. your antigen sequence to peptide.design@eurogentec.com, you shall receive your peptide selections in 2-3 days. 2. Fax the completed order form with the peptide selection to For questions please contact antibodies@eurogentec.com * note: please provide us with these information (if applicable) as soon as possible in order to avoid any delay Fax the completed order form to

22 Custom polyclonal antibody production First Class programme evaluation form Your purchase order reference*: VAT Nr. : EGT quotation number: If VAT exempt please include a copy of certificate* INVOICE ADDRESS Institute/Company: Department/Lab.: DELIVERY ADDRESS (Please fill in delivery information if different from invoicing information) Institute/Company: Department/Lab.: Contact Person: Contact Person: Address: Address: Postal Code: City: Postal Code: City: Country: Country: Phone: Phone: Fax: Fax: Protein accession #: 1. For which experimental method do you want a guarantee? (only one technique please) q ELISA in the absence of detergent or fixative q Western Blot q Immunoprecipitation with no detergent q Immunoprecipitation in the presence of detergent Specify the concentration and the name of the detergent q Staining of chemically fixed tissues Specify the fixative q Staining of frozen-fixed tissues q Other technique, please specify 2. Are you working with: q Isolated cells q Subcellular fractions q Other 3. Is your protein post-translationally modified? q Yes q No q Not known If yes, please indicate the amino acid number and the corresponding type of modification: 4. Are you working under denaturing conditions? q Yes q No 5. Is your protein a membrane protein? q Yes q No q Not known 6. Have you already tried an immunization experiment for this protein? q Yes q No If yes, which peptides/protein sequences were used? In which type of experiments was the antibody used? What were the results? 7. Remarks: * note: please provide us with these information (if applicable) as soon as possible in order to avoid any delay Fax the completed order form to