Chapter 3.1» Custom Polyclonal Antibodies

180 3 3.1 Custom Polyclonal Chapter 3.1» Custom Polyclonal Antibodies Antibodies 181 General overview 182 Choosing your parameters 184 Selecting your animal host 185 Selecting your immunisation protocol 188 Ready to start an immunisation programme? 189 Post Translational Modifications or Epigenomic Events detection 193 DNA immunisation 195 More for Your Polyclonal Programmes 196 Magnetic beads coupling services

181 General overview For more than 10 years, Eurogentec has acquired a strong experience in the production of custom polyclonal antibodies, proposing quality and exclusive services all over the world. Launching an immunisation programme is an important experimental step that needs care. With Eurogentec, you will always benefit from: k Real support and advice from a dedicated team all along your project k Skilled veterinarians and technicians k Large and modern animal facility k Full range of animal hosts, exclusive immunisation protocols and additional services k Low background sera k Ethical treatment of animals Get ready to start now The evaluation of your project is free-of-charge and with no obligation: k For a general discussion about your project or a tailor-made quotation, please contact your local sales representative or send your request to info@eurogentec.com k do you need free advice to select peptides out of your protein of interest? Send your protein sequence or accession number to peptide.design@eurogentec.com Constant quality you can rely on The whole polyclonal antibody production is based on: k High animal consistency standards leading to quality immunisation k Validated procedures for boosts and blood collections k Excellent record of animal maintenance k Full traceability Guaranteed confidentiality Antibody production can be done under the terms of a Non Disclosure Agreement. Eurogentec guarantees full confidentiality regarding antigen identity and project results. Proprietary policy The polyclonal antibodies we produce for you will always remain your property. Eurogentec will never claim any right on it nor keeping any aliquot for its own use. Nevertheless, if you think that your antibody is of commercial interest, we will be pleased to consider the opportunity to collaborate in its commercialization. Flexibility is our service You do not find what you are looking for in this catalogue? Contact us at info@eurogentec.com. We will be pleased to find the solution that fits your needs. Starting from your project goals, we can provide you with a support team that will guide you through the whole process, taking into account your specific requirements. Proteomic solutions» Custom Polyclonal Antibodies Request your free technical guide and become an expert in immunisation

182 Choosing your parameters Selecting your antigen Antibody titer evolution Antibody affinity evolution Chapter 3.1» Custom Polyclonal Antibodies The generation of a polyclonal antibody depends on two main factors that you should first determine, after which biology takes over: 1. Foreignness. A sequence alignment between your antigen sequence and the animal host proteome is highly recommended before starting any immunisation programme. 2. Pure antigen availability. This is a requirement to elicit an immune response at correct level in the host. In order to select the right protocol, it is also important to clearly understand what is behind two important concepts: antibody titer and affinity evolution. Exposure to a specific antigen initiates the immune response with a first phase resulting 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 titer is composed of predominantly low affinity and low titer 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 titer and affinity than IgM antibodies. Memory cells are also generated to quickly inactivate further exposure to the same antigen. IgM and IgG antibody titers as a function of time may be represented in a general scheme below. Antibody titer IgM IgG 1 st boost s 2 nd boost Time Figure 1: Evolution of IgM and IgG titers as a function of time and immunisation s 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. It can be monitored by a number of methods, below are the results from serum drawn at different time points during a 87-days polyclonal rabbit programme. The flatter the plateau of the curve, the tighter the binding affinities. RU 140 R.I. 120 100 80 60 40 20 0-20 Final bleed Large bleed Small bleed Pre-immune Serum -100 0 100 200 300 400 500 600 Time (s) Times (s) Figure 2: Affinity evolution of an 87-day anti-peptide polyclonal programme (Eurogentec s 3-months standard programme).

183 Protein, Peptide or Genetic Immunisation The best polyclonal antibody results are obtained from antigens that present the most number of desirable epitopes. Protein antigens Protein antigens offer numerous epitopes providing an increased chance of generating antibodies that are useful in a wide range of applications. However uses of antibodies from immunisation with protein antigens can be limited by the homology between protein isoforms. The prerequisites for an immunisation are that the protein be purified to > 80 % purity and that sufficient material be provided. Please check the minimal protein quantity required for all available hosts in the Host characteristics table. Peptide antigens Protein antigens are today a very reliable alternative to anti-protein immunisations. A peptide antibody programme offers the following advantages: k Selection of a peptide region that is unique to a specific protein isoform. k Immunisation with easily synthesized, highly purified material. k Affinity purification of the antibodies against the peptide. k Peptide antigens can be designed to raise antibodies against post-translational modifications (PTM). The single peptide strategy is best suited for reproducing the previously documented success of a polyclonal production with a known peptide sequence. We recommend the Double-X strategy for the first-time generation of antibodies against a particular antigen. The Double-X strategy involves the design of two peptides from the same antigen and immunizing the animal hosts with both peptides at the same time. This co-immunisation more closely mimics the protein than a single peptide strategy. The success rates of this strategy in Western Blots applications reach 90 %. Genetic immunisation Immunisation using the coding sequence of your protein of interest can be done, especially for k Proteins difficult to purify k Proteins with poor antigenicity See page 193 for more details Speedy 28-day polyclonal The new reference in immunisation Titer guaranteed Proteomic solutions» Custom Polyclonal Antibodies See also Eurogentec custom peptides synthesis service, chapter 3.6

184 Selecting your animal host Eurogentec offers a large and modern animal facility that houses over 3000 animals at any given time. Please check the Host characteristics table to properly choose the right animal for your application, with regards to bleed volumes and application of the antibody. k Rabbit should be your first choice for most applications, as it gathers different advantages: easiness of maintainance, efficient immune system and bleed volumes adapted to most needs. Host characteristics Host Min. antigen quantity per injection and per animal Antigen MW < 18 kda Antigen MW > 18 kda SPF Mouse 40 µg 15 µg Preimmune 30 µl in 270 µl of PBS Bleed volumes Small bleed Large bleed Final bleed Comment - - ± 300 µl Good to test antigenicity SPF Guinea pig 50 µg 30 µg 1 ml 1 ml 2-3 ml 5-7 ml For small serum volumes Chapter 3.1» Custom Polyclonal Antibodies k If your application requires only weak quantities of antibody, you may prefer to raise your polyclonals in mouse, rat or guinea pig. In that way you can benefit from lower prices without compromising antiserum quality. k On the other hand, Eurogentec can also raise your antibodies in big animals, allowing you to get higher bleed volumes when required by your application. Please make your choice in the table above with regards to specific bleed volumes. k When considering the animal host, please always check for protein homology. Blast your sequence to learn if your selected host is compatible with efficient immune response. In the case that you work with a protein very conserved into mammals, you should try switching to a non-mammalian system. k Chicken should be your preferred choice in that context. Another big advantage of raising antibodies in chicken is that we collect eggs instead of blood. Egg yolks contain very high titres of the so-called IgY, which are comparable to the mammals IgG, making Chicken another host of choice for application requiring large quantities of polyclonals. SPF 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 ± 10-20 eggs For mammalian antigens and large quantities of Abs, 4 eggs = 50 ml rabbit serum SPF Rabbit 200 µg 100 µg 2 ml 2 ml 2 + 20 ml 2 + 50-70 ml For most applications Goat 400 µg 200 µg 2 ml 2 ml 2 + 250 ml 2 + 1000 ml For large batch volumes Sheep 400 µg 200 µg 2 ml 2 ml 2 + 250 ml 2 + 1000 ml For large batch volumes Llama 400 µg 200 µg 2 ml 2 ml 2 + 250 ml 2 + 1000 ml Single chain antibodies and for large batch volumes Horse 400 µg 200 µg 2 ml 2 ml 2 + 250 ml 2 + 1000 ml For large batch volumes Other Contact us for recommendations Note: For a standard rabbit immunisation programme, at least 100 μg of purified protein per injection per rabbit, translates into 800 μg of purified antigen.

14 12 28-Day Protocol Final Bleed 185 Response Units (RU) 10 87-Day Protocol Final Bleed 8 6 4 2 Negative Control Selecting your immunisation protocol 0 Time (s) 0 10 20 30 40 50 60 Speedy 28-day polyclonal 4,00 3,50 SPF animals Your quality polyclonals raised in only 28 days* Thanks to the strong experience and hard work of our dedicated development team, Eurogentec is proud to provide its customer with the fastest immunisation protocol available on the market. This success is based on the use of a proprietary non-freund s adjuvant: k Helping to quickly elicit a very strong immune response k Accelerating the maturation process that usually takes several weeks k Also able to quickly raise specific antibodies against Post Translational Modifications Comparable titer and affinity as for the classical 87-day approach Response Units (RU) OD (492 nm) 14 12 10 8 6 4 2 0 4,00 3,50 3,00 2,50 2,00 1,50 0 28-Day Protocol Final Bleed 87-Day Protocol Final Bleed Negative Control Time (s) 10 20 30 40 50 60 Figure 3: Antibody affinity - Biacore Affinity Test** OD (492 nm) 3,00 2,50 2,00 1,50 1,00 0.50 0.00 100 300 900 2700 8100 24300 72900 218700 Dilution Figure 4: Antibody titer - ELISA results of the final bleed serum against a good responding peptide** Save two months (4 injections, 3 bleeds) Day Day Speedy 28-day 0 7 10 18 21 28 Classical 87-day TITER GUARANTEE or your money back Time saved 0 14 28 38 56 66 87 A free ELISA at day 21 for each anti-peptide programme*** should give a significant response (2 x the background level) at 1/20 000 dilution for at least one animal. k The guarantee is only valid for peptides approved by Eurogentec. k For Double X programmes, we guarantee the response for at least one of the peptides. k We cannot guarantee the functionality of your antibody in your application. * Does not include the potential peptide production phase ** the same KLH coupled peptide was used to immunise rabbits according to our 87-day and 28-day protocols. *** Anti PTM programme excluded SPF (Specific Pathogen Free) animals are tested for exposure to various viruses, bacteria and parasites that are commonly found in the corresponding species. SPF animals offer reduced background signals and higher success rates in antibody production. Promoting the ethical animal treatment Animals are maintained in an environment designed to minimize their stress. Animals are provided water and are fed according to their desire, are housed in groups to promote animal interaction, and are maintained in a climate-controlled environment. Safety is assured prior to blood sampling by anaesthetizing the animal and serum is drawn with a sterile, haemolysispreventing syringe. Our animal facilities comply with the highest quality standards and to the EU guidelines (FESALA) for animal housing and in vivo experiments. Contact us for details: info@eurogentec.com Proteomic solutions» Custom Polyclonal Antibodies

186 Chapter 3.1» Custom Polyclonal Antibodies All-inclusive packages 1. Peptide design from your antigen sequence 2. Peptide synthesis (> 70 %, 15-25 mg, 16 aa) 3. Peptide coupling (KLH, BSA, OVA) 4. Either 2 SPF rabbits, guinea pigs, rats or one non-spf goat 5. 28-day immunisation protocol (peptide synthesis time not included) 6. All immunisations, pre-immune, large and final bleeds collections and animal care 7. The ELISA guarantee test at day 21 8. Delivery of the free remaining peptide 9. A single shipping on dry ice (in Europe) Note: The points 1, 2, 3, 7 and 8 are peptide programme specific. Point 7 is not included in anti PTM immunisation programmes. For more details on Post Translational Modification specific programmes (PTM), see page 189. Speedy 28-day packages Antigen Synthesized peptide Host Reference Your protein - Rabbit AS-SUPR-ANTIGEN Guinea pig Rat Goat AS-SUPR-GPANTIGEN AS-SUPR-RATANTIGEN AS-SUPR-GOATAG 1 peptide 20-30 mg Rabbit AS-SUPR-SINGLE Guinea pig AS-SUPR-GPSINGLE 2 peptides (Double-X) 2 peptides + 2 affinity purifications (5 ml) 2 peptides / PTM* Rat Goat AS-SUPR-RATSINGLE AS-SUPR-GOATSING 15-25 mg each Rabbit AS-SUPR-DX Guinea pig Rat Goat AS-SUPR-GPDX AS-SUPR-RATDX AS-SUPR-GOATDX 15-25 mg each Rabbit AS-SUPR-DXP 10-20 mg each Guinea pig Rat Rabbit * 2 peptides,1 modified and 1 unmodified, specific for: Phosphorylation (-PSPEC) Acetyl Lysine (-EACK) Mono-, di-, tri-methyl Lysine (-LYSME1 / 2 / 3) Mono-methyl Arginine(-ARGME1) di-methyl Arginine (sym), di-methyl Arginine (asym) Other modifications are available on request. AS-SUPR-GPDXP AS-SUPR-RATDXP AS-SUPR-XXXX

187 The classical 87-day protocol Our traditional immunisation schedules, available for all hosts, are the best choices when: k Your project doesn t require to get your antibody as soon as possible k You need immunisation in big animals (pig, sheep, horse, llama, ) Day 0 14 28 38 56 66 87 For your specific needs, Eurogentec offers the production Day 0 7 10 18 21 28 Time saved of antibodies in chicken. Using a non-mammalian host for immunisation allows you to consider even highly conserved mammalian proteins as targets. Do you know that only 4 eggs contain as much antibody as 50 ml of rabbit serum? Injections Day Collection of eggs Your protein of interest or peptide(s) designed by Eurogentec 0 14 28 38 52 56 66 94 All-inclusive packages 1. Peptide design from your antigen sequence 2. Peptide synthesis (> 70 %, 15-25 mg, 16 aa) 3. Peptide coupling (KLH, BSA, OVA) 4. Either 3 mice* or 2 rabbits, guinea pigs, rats, chickens or 1 goat 5. 87-day immunisation protocol (peptide synthesis time not included) 6. All 4 immunisations, pre-immune, small, large and final bleeds collections and animal care 7. Delivery of the free remaining peptide(s) 8. Three shipping on dry ice (in Europe) Options k ELISA testing k IgG2/ IgY isolation k Affinity purification k Labelling and conjugation For more details, see page 195. Note: The point 1, 2, 3 and 7 are peptide programme-specific 87-day packages Antigen Quantity Host Reference Your protein - Rabbit AS-PNOR-3MORAB Guinea pig AS-PNOR-3MOGPG Rat Chicken AS-PNOR-3MORAT AS-PNOR-3MOHEN 1 peptide 20-30 mg Mouse* AS-PCAP-MOUSE Rabbit AS-PCAP-RABBIT 1 peptide + affinity purification (50 ml/ 4 eggs) 2 peptides (DoubleX) 2 peptides + 2 affinity purifications (5 ml/8 eggs) 2 peptides / PTM, Phosphospecific** Guinea pig Rat Chicken AS-PCAP-GUIPG AS-PCAP-RAT AS-PCAP-HEN 20-30 mg Rabbit AS-PAFF-RABBIT 15-25 mg each 15-25 mg each 10-20 mg each Chicken Rabbit Guinea pig Rat Chicken Rabbit Guinea pig Rat Chicken Rabbit Guinea pig Please contact us for programmes with other animals. Rat * 35 days programme ** 2 peptides, 1 modified and 1 unmodified. Other modifications, available in rabbit only: Acetyl Lysine Mono-, di-, tri-methyl Lysine AS-PAFF-HEN AS-DOUB-LX AS-DOUB-LXGUI AS-DOUB-LXRAT AS-DOUB-LXHEN AS-DOUB-LXP AS-DOUB-LXPGUI AS-DOUB-LXPRAT AS-DOUB-LXHENPL AS-PSPE-CIFIC AS-PSPE-CIFICGUI Proteomic solutions» Custom Polyclonal Antibodies AS-PSPE-CIFICRAT

188 Ready to start an immunisation programme? Anti-peptide antibodies Anti-protein antibodies Construct your own programme Chapter 3.1» Custom Polyclonal Antibodies k Send us your antigen sequence in single amino acid format at peptide.design@eurogentec.com k Please specify your preferred approach: single peptide or Double-X k We will design your peptide(s) for free k Within 3 business days, you will receive our recommended sequence(s) k Send us your complete anti-peptide order form* k You will receive an immunisation schedule depending on the choosen method k We will start the immunisation Host Speedy 87-day PTM (anti P ) Mouse Rat DNA Immunisation (42 days) (42 days) Application Why do customers order these? Monoclonal: Validation, ELISA + LUMINEX, IHC + Duolink, WB; Polyclonal: everything, but low volume just test Validation, ELISA + LUMINEX, IHC + Duolink,WB; IP best after biotinylation Monoclonals Good alternative for rabbit Guinea pig WB, IP, IHC + Duolink, ELISA + LUMINEX Good alternative for rabbit Rabbit (77 days) Goat Sheep, Pig, Horse WB, IP, ELISA (detection) + LUMINEX, IHC + Duolink Validation, WB, ELISA + LUMINEX, IHC + Duolink, IP best after biotinylation Validation, WB, ELISA + LUMINEX, IHC + Duolink, IP best after biotinylation Llama Validation, IP IgG w/o LC Chicken (IgY) k Send us your complete anti-protein order form* k Send us your Protein lyophilized or in solution** k You will receive an immunisation schedule depending on the choosen method k We will start the immunisation Detection of evolutionary conserved proteins, any protocol IP best after biotinylation The most frequently used host Goats are frequently used by industrial customer for high volumes Sheeps are frequently used by industrial customer for high volumes Production of single chain IgG and neutrophiles For plant and bacterial antigens, or projects that failed in other hosts 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. k Select the number and type of hosts k Select the immunisation and bleed schedule for each animal k Send us your complete order form* mentioning either: that you will ship your antigen lyophilized or in solution** your antigen sequence for peptide design and synthesis k You will receive an immunisation schedule depending on the choosen method k You will also receive a quotation taking into account: Animal purchase Animal maintenance, per month (immunisation and bleeds included) ELISA and affinity purifications fees if requested Shipping fees k We will start the immunisation * Order form can be downloaded on www.eurogentec.com/antibody-order-form.html ** Antigen purity must be > 80 % to ensure a quality antibody production. Antigen quantities should exceed the minimum quantities described above.

189 Post Translational Modifications or Epigenomic Events detection Post- and co-translational protein modification and editing events ( PTM in brief) dictate a wide range of protein characteristics or cellular functions. Amino acid modifications to be considered for modification specific immunisations Modification Type Biological Significance Catalogue Antibodies For the understanding of the particular role of any given modification of cellular macromolecules under a certain condition it is crucial to specifically detect modified proteins from a pool. Eurogentec propose you two different options: Acetylation Formylation Histones, N-terminal: 80 % of all proteins N-terminus of bacterial proteins (N-formylmethionine) and related eukaryotic organelles 1. Peptide immunisation protocols Use a custom made approach for the specific detection of the edited amino acids in a PTM protein. 2. Catalogue antibodies See also our catalogue antibodies that focus on the detection of known PTM proteins and modified DNA in various cellular pathways. Biotinylation Myristoylation Palmitoylation Steroylation Hydroxyproline Oxidised cysteine Oxidised methionine Ubiquitination / SUMOylation/ Neddynation (branched peptides) Lysine residues in N-terminal and C-terminal regions of human histone H2A Protein myristoylation is critical in many pathways; e.g. in signal transduction, apoptosis, or alternative extracellular protein export, gene regulation after viral infection (host defence). Protein targeting (NOS2 ER to Golgi transport), lung surfactant composition No concrete hint for terminus, viral proteins Collagen Oxidative stress Oxidative stress Different pathways, histones, protein activation, protein degradation Proteolytic processing N-terminal neo-epitope Protein maturation and degradation C-terminal neo-epitope Protein maturation and degradation Please contact us at info@eurogentec.com if you are interested in getting a quotation for immunisations against the above-mentioned modifications, or if you are looking at a completely different modification. Proteomic solutions» Custom Polyclonal Antibodies Discover the list on www.eurogentec.com or request your brochure at info@eurogentec.com

190 Peptide immunisation protocols for the PTM detection The production of custom-made, PTM specific antibodies is based on either Speedy 28-day or 87-day immunisation protocol. The host is immunized by a modified peptide mimicking the PTM protein. All-inclusive packages Protocol Specificities Column with modified peptide S Column with non modified peptide B A Chapter 3.1» Custom Polyclonal Antibodies k Peptide design from your antigen sequence. k Unmodified and modified peptide synthesis (> 70 %, 10-20 mg, 13 aa) k Peptide coupling (KLH, BSA, OVA) k Either 2 SPF rabbits, guinea pigs or rats k Speedy 28-day or 87-day immunisation protocol (peptide synthesis time not included) k All 4 immunisations, bleeds collections and animal care k ELISA test of sera to determine the best responding animal k Double purification of 50 ml serum from the best responding animal* k Indirect ELISA test against the modified and nonmodified peptides k 1 or 3 shipping(s) on dry ice (in Europe) Isolation of all antibodies specific to the peptide carrying the modified amino acid k After identification of the most promising rabbit during immunisation, Day Delivery 0 of 21 the free 31 remaining 49 peptides 59 and 77 the 5087 ml of serum (S) are applied to an affinity column 1 with the immobilised purifications flow through. modified peptide. During this step, antibodies which are retained are: * 50 ml pooled from both animals available on request Day Day Speedy 28-day 0 7 10 18 21 28 Classical 87-day Time saved 0 21 31 49 59 77 87 FT1 B A) Sequence specific for the peptide B) Specific for the modification in the peptide A Unrelated antibodies will be separated from the desired antibodies by flow through (FT1), and the washing steps after binding. After elution of the mixture of peptide sequence specific antibodies (A) and modification specific antibodies (B) from column 1 (Peak 1), the antibody mix will be characterised by ELISA, and applied to a second affinity column carrying the immobilised non-modified peptide. Peak 1 FT2 B Peak 2 A B A Isolation of modification specific antibodies Column 2 will retain the antibodies, which are specific to the nonmodified peptide (Peak 2). The antibodies of type B, which are specific to the desired modification, will be harvested from the flow through, and characterised by ELISA (Flow through 2, FT2). The antibodies of type A, which are non-specific to the desired modification are eluted (Peak2) Peptide Modification IgG against modified peptide A IgG against nonmodified peptide Day 0 7 10 18 21 28 Time saved

191 Characterisation of Modification specific Antibodies and Sera by Indirect ELISA Test after affinity purification 1 Test after affinity purification 2 The first affinity purification will be carried out against the modified peptide. By this step the serum will be depleted from antibodies that are specific to the modification, and the peptide sequence in general. The serum (S), the flow through 1 (FT1), and the purified antibodies (Peak 1) are tested after this purification by indirect ELISA against the modified peptide and the carrier protein. In general the following results can be expected (Figure 5). OD (492 nm) 4 3.5 3 2.5 2 1.5 1 0.5 Results generally obtained by indirect ELISA against the modified peptide and against carrier after affinity purification against the modified peptide Fraction Cross-affinity purification for modification specific antibodies Purification 1 against the modified peptide and the carrier 0 10 100 1 000 10 000 100 000 1 000 000 Antibody- and serum dilution (x-fold) Purified antibody - (Peak 1) Reactivity against mod. peptide Yes, stronger than S, and much stronger than FT1 Serum (S) Flow through 1 (FT1) Purified antibody (Peak1) Serum (S) Flow through 1 (FT1) Purified antibody (Peak1) Figure 5: Purification 1 against the modified peptide leads to the separation and enrichment of antibodies specific to the whole modified peptide sequence from the serum, flow through 1 (FT1) reflects the serum depleted from antibodies to the modified peptide, and shows as expected low reactivity against the modified peptide, background level at 1:1000, purple line. against modified peptide Reactivity against carrier No, if Yes then removal by dilution quickly possible Non bound antibodies - No, if Yes then much weaker Yes Flow through 1 (FT1) than S and Peak 1 Serum - (S) Yes, weaker than PA Yes against carrier After purification 1 the antibodies against the modified peptide and the peptide sequence itself have to be separated in order to provide you just modification specific antibodies. Eurogentec performs this step by crosspurifying the antibody (Peak 1) from purification 1 against the non-modified peptide. Antibodies just specific to the peptide will be retained by the column (Peak 2) during this step, and antibodies specific to the modification will be in flow through 2 (FT2). Peak 2 and FT2 will be characterised by indirect ELISA against the modified peptide (Figure 6), and against the non-modified peptide (Figure 7). In general the following results can be expected. Please note that antibodies from Peak 2 cannot be considered as antibodies serving in a negative control for the lack of the modification in your protein target. A sample result is reflected in Figure 6 and Figure 7. Please be advised that the population of antibodies against the modification in the deciding fraction FT2 can be very low, due to narrowing the variety of specific molecules towards a single feature of a small epitope. But nonetheless, the antibodies can be used in the most common application types diluted in the same way like other polyclonal antibodies. Results generally obtained by indirect ELISA against the modified peptide and non-modified peptide after crossaffinity purification Fraction Purified peptide specific antibody - (Peak 2) Purified modification specific antibody - Flow through 2 (FT2) Reactivity against mod. peptide Yes, the modified peptide contains a lot of antigens Yes Reactivity against non mod. peptide Yes No, if any it can easily be diluted away OD (492 nm) Figure 6: Antibodies purified by affinity purification 2 (column with 3.5 Purification 2: Indirect ELISA testing against non modified peptide) the non-modified react also peptide with the modified peptide these 3 antibodies 4 cannot be considered as a control for the non-modified 2.5 3.5 Flow through 2 (FT 2) protein target in your experimentation. The flow Ab specific to through the modification 2 (FT2) 2 3 antibodies show a weak reactivity against the Flow modified through 2 (FT 2) Peak peptide Ab specific 2 (P2) 1.5 to the modification 2.5 Ab non-specific to the modification please consider that the modification in the peptide might directly 1 2 Peak 2 (P2) interact with the ELISA plate surface, and therefore Ab non-specific to the modification 0.5 a restricted 1.5 access 0 1 of antibodies to the modified epitope might result, causing a hyperbolical 10 curve 100 shape 1 000 instead 10 000 of 100 sigmoidal 000 1 000 000 kinetics. OD (492 nm) OD (492 nm) Purification 2: Indirect ELISA testing against the modified peptide 4 3.5 3 2.5 Flow through 2 (FT 2) Ab specific to the modification 2 Peak 2 (P2) 1.5 Ab non-specific to the modification 1 0.5 0 10 100 1 000 10 000 100 000 1 000 000 OD (492 nm) 4 Purified antibody dilution (x-fold) Purification 2: Indirect ELISA testing against the modified peptide 0.5 0 Purified antibody dilution (x-fold) 10 100 1 000 10 000 100 000 1 000 000 Purified antibody dilution (x-fold) Purification 2: Indirect ELISA testing against the non-modified peptide 4 3.5 3 Flow through 2 (FT 2) 2.5 Ab specific to the modification 2 Peak 2 (P2) Ab non-specific to the modification 1.5 1 0.5 0 10 100 1 000 10 000 100 000 1 000 000 Purified antibody dilution (x-fold) Proteomic solutions» Custom Polyclonal Antibodies Figure 7: Peak contains the majority of antibodies to the non-modified peptide, which is reflected by the strong reactivity of the antibodies to the target. FT2 might contain a small antibody population also specific for the non-modified peptide. These antibodies can be diluted out or washed off in down stream applications, the soon drop of the yellow curve from FT2 indicates that these antibodies are no good binders to the non-modified peptide.

192 Specific Detection of histone dimethylation A success story in the complex context of histone modifications using the Speedy protocol Chapter 3.1» Custom Polyclonal Antibodies To understand the impact of Histone H3 lysine 9 (H3K9) di and tri-methylation on cellular processes, Eurogentec together with Dr. Christian Seiser (Medical University of Vienna) used the Speedy protocol to develop a modification specific antibody. As shown in the dot blot assay our antibody specifically recognises di-methylated histone H3 peptides in the presence of the neighbouring S10ph and K14ac marks. Histone H3 lysine 9 (H3K9) di and tri-methylation correlate with gene silencing such as HDAC1. While Anisomycin/ TSA cells stimulation induces acetylation of K14 and phosphorylation of S10 that can overcome this effect and allow binding of the transcription promoting histone H3 phosphorylation detector 14-3-3 (Winter et al., The EMBO Journal (2008) 27,88-99). Dot blot 1.0 µg of antigen Peptide modifications (histone H3 peptides, a.a. 1-20 as antigen) K9ac 1 1 K9me2 1 1 1 K9me3 1 K14ac 1 1 1 1 S10ph 1 1 1 1 1 1 A B C D E F G H I J The Speedy polyclonal antibody recognizes Histone H3 K9me2 (E), independently of neighboring modifications (H,I). The antibody specificity has been determined with different peptides derived from histone H3 (amino acids 1-20). Immunisation programmes for PTM in rabbits standard packages Modification Type Classical 87-day Speedy 28-day* Biological Significance Epsilon-acetyl lysine AS-PTMA-EACK AS-SUPR- EACK Cell signalling / histones Phospho-specific (serine, threonine, tyrosine) AS-PSPE-CIFIC AS-SUPR-PSPEC Cell signalling / histones Mono-methyl-lysine specific AS-PTMA-LYSME1 AS-SUPR- LYSME1 Histones Di-methyl-lysine specific AS-PTMA-LYSME2 AS-SUPR-LYSME2 Histones Tri-methyl-lysine specific AS-PTMA-LYSME3 AS-SUPR-LYSME3 Histones Mono-methyl-arginine specific AS-PTMA-ARGME1 AS-SUPR-ARGME1 Histones Di-methyl-arginine (sym) specific AS-PTMA-ARGME2SYM AS-SUPR-ARGME2SYM Histones Di-methyl-arginine (asym) specific AS-PTMA-ARGME2ASY AS-SUPR-ARGME2ASY Histones Nitro-tyrosine AS-PTMA-NITY AS-SUPR-NITY Oxidative stress Protein reference (double X peptide immunisation together with a modification specific programme from above) AS-DOUB-LX AS-SUPR-DX Please contact us at info@eurogentec.com if you wish a quotation for the above-mentioned programmes. *The 28-day Speedy protocol is recommended for modification specific antibody production. Your protein of interest. Please contact peptide.design@eurogentec.com to get sequences suggested that are different from the part where your modification is localised.

193 DNA immunisation Stop scratching your head about how to get your antigen Simply give us its accession number! Use of gene immunisation k Proteins difficult to purify or with structures destroyed by purification. Membrane receptors or channels Post-translational-modified proteins Secreted or intracellular proteins k Proteins of poor antigenicity can be made more immunogenic by fusing their gene to that of a highly immunogenic protein. How does it work? Plasmids harboring genes to be expressed are coated on ICANtibodies nanospheres and injected directly to the host tissues. The ICANtibodies nanosphere delivery system (proprietary of In Cell Art), by enhancing the immunogenicity of plasmid DNA encoding antigen, permits to reduce the needed dose of plasmid for immunisation and displays an excellent safety profile. Immunisation procedure 1 st Step: Building the immunisation synthetic nanocarrier Nucleic acid sequence Design of the oligonucleotides, codon optimisation and gene synthesis (up to 5 kb) atg tcc tag cca tag aca acc gat cca Quality Control 3-4 weeks Cloning Recombinant plasmid construction and cloning HindIII (929) EcoRI (978) HindIII (1139) EcoRI (1390) DNA Restriction map and sequencing data of the recombinant plasmid ICANtibodies formulation To prepare genetic immunisation Proteomic solutions» Custom Polyclonal Antibodies Cell transfection (HeLa cells using ICAFectin 441) to evaluate protein expression and toxicity.

194 2 nd Step: immunisation programme 6-11 weeks Rabbit Mechanism The ICANtibodies nanosphere delivery system (proprietary of In Cell Art), by enhancing the immunogenicity of plasmid DNA encoding antigen, permits to reduce the needed dose of plasmid for immunisation and displays an excellent safety profile. ß - Gal A GFP C Day 0 21 42 56 63 77 91 Chapter 3.1» Custom Polyclonal Antibodies Controls ELISA, WB Delivrables Rat / Mouse Day Preimmune sera 2.5 ml of Antisera (if requested) 2.5 ml of Antisera (if requested) Controls ELISA, WB Delivrables Preimmune sera 0 21 35 42 56 63 Test bleed (if requested) Final bleed Recombinant Plasmid ± 100 ml of Antisera or final bleeds; recombinant plasmid Sera tests k intracellular antigen => ELISA or WB on lysate of transfected cells with ICAFectin 441 and immunisation plasmid k Secreted membrane antigen => one additional recombinant plasmid construction containing an His-cMyc Tag for ELISA or WB on lysate of transfected cells with ICAFectin 441 and His-cMyc Tag plasmid. Naked recombinant expression plasmid ß - Gal B GFP Nanosphere formulated recombinant plasmid In vivo expression of the ß-galactosidase (ß-Gal) (A-B) or Green Fluorescent Protein (GFP) (C-D) gene carried either on nanosphere formulated (B-D) or on unformulated plasmid (A-C) Ready to start? To start discussing your project, please contact your local representative or send us an e-mail at proteomics.services@eurogentec.com D

195 More for Your Polyclonal Programmes 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 immunisation programme, during a maximum of 3 week. Pre-immune test serum Number Reference 5 sera AS-PREI-05 10 sera AS-PREI-10 20 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 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 but however does not provide only antigenspecific antibodies. Total IgG purification Reference AS-PURI-PCIGG050 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 putrification 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 (but 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-SMALL 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. IgY purifications involve a two-step process, firstly the total IgY purification followed by affinity purification against the peptide. 4 egg yolk corresponds to approximately 50 ml of rabbit serum in terms of antibody content. Affinity purification of egg yolk Proteomic solutions» Custom Polyclonal Antibodies Description Reference Total IgY 4 eggs AS-PURI-IGY20 Additional egg AS-PURI-IGYADD Affinity purification AS-PURI-01 2 nd affinity purify AS-PURI-02

196 Magnetic beads coupling services Chapter 3.1» Custom Polyclonal Antibodies Eurogentec has joined forces with a state-of-the-art bead manufacturer, to offer you antibody-magnetic bead complexes, ready-to-go for your application. k Save time and money Don t lose your precious time setting up coupling protocols. Eurogentec does it for you, at an affordable price. k Get the best results Eurogentec has conducted many investigations to choose the best beads available, and select the most efficient coupling protocol. k Focus on your target Don t let pure technical aspects slow you down. Eurogentec has optimised everything to let you focus on your research goals and reach them faster. The coupling principle involves the biotinylation of your antibody followed by linking it to a streptavidin-coated bead. Example of the basic downstream process is described below. Custom antibody made by Eurogentec Your antibody Coupling to biotin Coupling Biotinylated Ab to streptavidine beads Add beads to matrix Technical features k We use 1 µm beads, with the most narrow size distribution k High and constant magnetic content k Perfect compromise between size, magnetic content and sedimentation property k Optimized binding surface k Allows easy and gentle handling of proteins The coupling service is available for any polyclonal or monoclonal custom antibody made by Eurogentec. To learn more about custom antibody production at Eurogentec, and especially our unique 28-day Speedy immunisation, please visit our website www.eurogentec.com We also offer the service for any antibody you provide us. Please contact us to learn about conditions applying to sample format and shipment. Resuspend in liquid Collect beads Remove supernatant Wash SDS PAGE analysis Preperative isolation Bead ELISA Immunoprecipitation Protein Pull-down Magnetic beads Description Coupling of your antibody to magnetic bead includes: Sample preparation Antibody biotinylation Coupling of 60 µg of biotinylated Ab to 3-8.10 8 1 µm magnetic beads QC and report Reference AS-MMCO-0160

197 Proteomic solutions» Custom Polyclonal Antibodies