CUSTOM ANTIBODIES. Fully customised services: rat and murine monoclonals, rat and rabbit polyclonals, antibody characterisation, antigen preparation

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1 CUSTOM ANTIBODIES Highly competitive pricing without compromising quality. Rat monoclonal antibodies for the study of gene expression and proteomics in mice and in mouse models of human diseases available. Fully customised services: rat and murine monoclonals, rat and rabbit polyclonals, antibody characterisation, antigen preparation APPLICATIONS Antibodies are powerful tools used extensively in research, as diagnostics, and increasingly as therapeutics. Monoclonal antibody can be generated from phage libraries, but those with the highest affinities are produced in animals, predominantly mice, but also rats. The completion of the human genome project and parallel projects on mice, rats and a variety of plants and micro-organisms provides information on gene organisation and the primary structure of encoded proteins, but scant information on individual gene function or the more complex biological information on protein-protein interactions and modification in specialised cells and tissues. The vast majority of marketed antibodies are of mouse origin. However mice in particular transgenic mice carrying mutated or abnormally expressed genes provide unique insight into the location, function and interaction of proteins in vivo. Since murine antibodies cannot be raised against most mouse proteins, they remain ineffective tools to locate target murine proteins in mouse tissues and cells in culture. Rat monoclonal antibodies as the alternative source is therefore required. High affinity rat monoclonal antibodies can be produced using robust technology, at low cost, and in a similar timeframe to their murine counterparts. Rat monoclonal antibodies can be used to study complex protein-protein interactions in mice and in particular mouse models of human diseases.

2 Rat monoclonal antibodies can also be used in combination with murine antibodies and different secondary antibody combinations for multiple analyses in single solutions using for example FRET and similar technologies. Rat monoclonal antibodies can be used as research or diagnostic tools in an identical manner to any murine antibody. CUSTOM ANTIBODIES Rat monoclonal antibodies We make monoclonal antibodies to protein antigens or fusion proteins. For customers who do not have ready to use antigens, we provide antigen preparation services that include cdna expression, peptide design, synthesis and cross-linking peptides onto carrier proteins. Producing monoclonal antibodies normally takes weeks, which can be divided into three stages: immunisation and serum tests, fusion and ELISA screening, subcloning and isotyping. We shall report our progresses to customers at each stage so that customers will know exactly what is going on just like they do in their own labs. At the screening stage, many positive clones may be found. Customers will decide how many cell lines they want to have further subcloned. At the end of each project, we shall provide customers stable subcloned cell lines. On requests by customers, we shall do mycoplasma tests, antibody further purification and characterisation. We charge 5700 euros as dissected below for the first cell line and 75 euros for each of the additional cell lines. Customers are asked to pay 50% upfront initial charge. Mycoplasma tests cost 45 euros for each cell line. Immunisation and serum tests 1950 euros Fusion and ELISA screening 1950 euros Subcloning and antibody isotyping 1800 euos Total 5700 euros Murine monoclonal antibodies We make monoclonal antibodies to protein antigens or fusion proteins. For customers who do not have ready to use antigens, we provide antigen preparation services that include cdna expression, peptide design, synthesis and cross-linking peptides onto carrier proteins. Producing monoclonal antibodies normally takes weeks, which can be divided into three stages: immunisation and serum tests, fusion and ELISA screening, subcloning and isotyping. We shall report our progresses to customers at each stage so that customers will know exactly what is going on just like they do in their own labs. At the screening stage, many positive clones may be found. Customers will decide how many cell lines they want to have further subcloned. At the end of each project, we shall provide customers stable subcloned cell lines. On requests by customers, we shall do mycoplasma tests, antibody further purification and characterisation. We charge 4800 euros as dissected below for the first cell line and 75 euros for each of the additional cell lines. Customers are asked to pay 50% initial charge. Mycoplasma tests cost 45 euros for each cell line.

3 Immunisation and serum test s 1500 euros Fusion and ELISA screening 1800 euros Subcloning and antibody isotyping 1500 euros Total 4800 euros Polyclonal antibodies We make rat or rabbit polyclonal antibodies to protein antigens or fusion proteins. Customers who require goat or sheep antibodies please contact us. For customers who do not have ready to use antigens, we provide antigen preparation services that include peptide design and synthesis, cdna expression and cross-linking peptides onto carrier proteins. Producing polyclonal antibodies normally takes 12 weeks. Antibody titre in serum is checked by double immunodiffusion or by ELISA. On requests by customers, we shall further affinity purify antibodies. We charge 135 euros for ml rat anti-serum and 375 euros for 35 ml rabbit anti-serum. Antigen preparation Antigens provided by customers Antigens provided by customers should be soluble, not toxic, not infectious and at least 80% pure judged by SDS- PAGE. Molecular mass of antigens should preferably be over 20 kda. Customers may provide us with fusion proteins. Small peptides or organic molecules need to be cross -linked onto carrier proteins first. The amount of antigens required is listed below. Rat monoclonals Murine monoclonals Rat polyclonals Rabbit polyclonals Amount required 1.5 mg 1 mg 0.3 mg 2 mg cdna expression in E. coli We offer antigen expression services for customers who provide us with cdna inserted expression vector for E. coli. We charge 1050 euros for each antigen expressed. The price includes transformation of the construct in E. coli, expression and purification of the antigen. Peptide synthesis GeneCust synthetize peptide for your project. Peptides are characterised by HPLC and mass spectroscopy. GeneCust provides antigenic peptides prediction from existing protein sequences free of charge. However, we do not guarantee that the peptide predicted will be immunogenic and antibodies raised against a peptide will recognise the native predicted protein. The costs for peptide synthesis must be settled before antibody generation can be initiated.

4 Coupling peptides onto carrier proteins We normally use bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH) as carrier proteins. Peptides should contain cysteine residues. We charge 150 euros for coupling 2-5 mg peptide onto BSA or KLH. Antibody production and purification Ascites production We make ascites from murine hybridomas. Customers should order at least 30 ml of ascites fluid. The whole procedure takes 4 weeks to complete. We charge 22,5 euros for each millilitre ascites fluid delivered. Antibody purification Murine and rat monoclonal antibodies from tissue culture supernatant or ascites are purified using protein A or protein G columns. Polyclonal antibodies are affinity purified using an antigen coupled Sepharose beads column. Growing cells or making ascites, then purify 5 mg antibody using protein G or protein A columns, running SDS-PAGE for the purified antibody. Coupling less than 5 mg antigen onto Sepharose beads,then purifying 10 mg antibody by the affinity column, running SDS-PAGE for the purified antibody. 300 euros 450 euros Antibody modification and characterisation Antibody digestion to Fab or F(ab') 2 fragments We use pepsin for F(ab')2 fragment production and papain for Fab fragment production. Before digesting large amount of antibodies, we perform an analytical digestion to optimise conditions. Undigested antibodies will be removed by protein A columns. Customers should provide minimum of 10 mg antibodies. Normally 3 mg of Fab or F(ab') 2 fragments can be obtained from 10 mg antibodies after absorption with Protein A. The resulted fragments should remain their binding abilities to antigens and show one band (about 100 kda for F(ab') 2 fragments and 50 kda for Fab fragments) under non-reducing conditions and two bands (both are around 25 kda) on SDS-PAGE under reducing conditions. We charge 75 euros for analytical digestion by pepsin and 150 euros for analytical digestion by papain. We charge 270 euros for digestion of 10 mg antibody by pepsin and 375 euros for digestion of 10 mg antibody by papain. Antibody labelling GeneCust provides peroxidase and FITC conjugates of antibody. We charge 195 euros for labelling peroxidase or FITC onto 5 mg of antibodies. Selection of antibodies for Western blotting and flow cytometry In monoclonal antibody production, positive cells are normally selected by ELISA. If customers require, we can go further to check antibody reactivates in Western blotting and flow cytometry. We make 150 euros basic charge for screening up to 20 antibodies for Western blotting. For each positive antibody found, we charge additional 75 euros.

5 Providing cells are available, we make 225 euros basic charge for screening up to 20 antibodies for flow cytometry. For each positive antibody found, we charge additional 75 euros. Selection of capture and detection antibody pairs for sandwich ELISA Usually several monoclonal antibodies with different specificities can be generated against a single antigen. Some of them have epitopes far apart so that they will not compete each other on binding of the antige n. Such pairs can be used for the quantative detection of antigens in solution by sandwich ELISA, one acts as capture antibody and the other labelled with peroxidase acts as detection antibody. Customers who require this service please contact us. PRICING Custom antibody services Note: All the prices exclude charges on package and postage and import taxes, which will be paid by customers. Custom monoclonal antibody from rats 5700 euros for the first cell line and 75 euros for each additional cell line. Custom monoclonal antibody from mice 4800 euros for the first cell line and 75 euros for each additional cell line. Custom polyclonal antibody 375 euros for 35 ml rabbit antiserum and 135 euros for rat antiserum. Affinity purification of 5 mg antibodies from culture 300 euros supernatant or ascites by protein G or protein A columns. Affinity purification of 10 mg antibodies using antigen 450 euros coupled Sepharose beads. Coupling peptides onto carrier proteins 150 euros for coupling 2-5 mg peptide onto BSA or KLH. Antibody digestion to Fab fragments by papain 150 euros for analytical digestion, 425 euros for digestion of 10 mg antibody Antibody digestion to F(ab')2 fragments by pepsin 75 euros for analytical digestion, 350 euros for digestion of 10 mg antibody FITC labelling 195 euros for 5 mg antibody Peroxidase labelling 195 euros for 5 mg antibody Peptide synthesis Enquire Selecting antibody pairs Enquire CONTACT Headquarter : GENECUST EUROPE Laboratoire de Biotechnologie du Luxembourg S.A. 30b rue Dominique Lang L-3505 Dudelange Luxembourg Tva Intra-communautaire : LU Laboratories and offices : GeneCust Europe 8 route de Burange L-3429 Dudelange Luxembourg TEL: FAX: info@genecust.com Website :