Custom Services
- Antibody Production
- Polyclonal Antibody Development
- Ascites Production
- Monoclonal Antibody Development
- Peptide Synthesis
- Cloning & Gene Expression
- Assay Development
- Antibody Conjugation Service
Millipore develops custom immunological and molecular tools to expedite research, product development, and drug discovery efforts. The proprietary custom molecular technology and 18 years of antibody manufacturing experience can significantly shorten the path from RESEARCH to RESULTS. Millipore offers expert consulting as well as molecular assay, antibody, and immunological assay development services in a GMP manufacturing facility. Our services are confidential, fast, efficient and well-documented. The contract services extend from immunological to molecular, providing tremendous flexibility in assay design, and customized services to meet your specific needs.
Antibody Production
In polyclonal antibody production, antisera are commonly produced by injection of the immunogen (antigen) of interest into an animal, often in combination with an adjuvant to increase the immune response. The antibody response can be enhanced by subsequent booster injections of the antigen with or without adjuvant. Blood samples are obtained from the animal to assess the level of antibodies produced, and once a sufficiently high titre has been reached, the antiserum is prepared by blood collection followed by serum preparation, with subsequent purification of antibodies from the serum if required.The level and quality of the antibodies produced will vary from animal to animal and from a single animal over time. Careful consideration should be given to the appropriateness of the species and strain chosen. The investigator should consider the following factors: 1) The quantity of antibody or antiserum required (larger animals should be considered when larger quantities of antibody are required); 2) The phylogenetic relationship between the species from which the protein antigen originated and the species used to raise the antibody and 3) The intended application use of the antibodies. The rabbit is the most commonly used animal for the production of polyclonal antibody, as it is easy to handle and to bleed, and for most applications will produce an adequate volume of high-titre, high affinity, antiserum.
Polyclonal Antibody Development
Millipore has one of the largest polyclonal antibody development facilities in the world. We offer complete project design including:immunogen production, testing, purification, and conjugation. Projects are carried out using your protocol or our standard procedures. Our expert researchers, technicians, and project managers, coupled with twenty-three years of antibody development experience, ensure the greatest chance of successful completion of your custom projects.
Millipore's Facilities
- Accreditation Includes: NIH Assurance, USDA Class B, USDA Class R, and ISO®13485
- Capacity: Our facilities can house greater than 10,000 rabbits, 715 Goats, and 500 sheep.
- Projects from one to thousands of antibodies are welcome. IgY antibody production from chickens is also available.
- Peptides
- 1–50 amino acid, phospho, cyclic, MAP and more
- Proteins, cells, cell lysates
- Conjugations: KHL, BSA, OVA
- Polyclonal antibodies are tested by ELISA against antigen.
- Western Blot against antigen, IHC, and FACS are also available.
- Protein A chromatography
- Protein G chromatography
- Ion exchange chromatography
- Antigen Affinity chromatography
- All custom polyclonal antibodies can be conjugated to FITC, DTAF, Biotin, Peroxidase, Alkaline Phosphatase,
- Rhodamine, Cy3, Cy5, Photoproteins, R-PE, R-PE/Cy5, and AlexaFluorâ dyes.
- New Zealand White Rabbits
- White Leghorn Chickens
- Liester Rams crossed with Sheep
- Guinea Pigs
- Nubian or Boer Goats
- Balb/c Mice
Monoclonal Antibody Development
Monoclonal antibodies, following the development of this technology in 1975, have become an extraordinarily important resource for medical research, diagnosis, therapy, and basic science. They are antibodies which have a single, selected specificity and which, usually, are secreted continuously by “immortalized” hybridoma cells. A hybridoma is a biologically constructed hybrid of an antibody- producing lymphoid cell and a malignant (immortal) myeloma cell. The first step in the production of MAbs is to immunize a mouse with an antigen. When the mouse begins to produce antibodies to the antigen, its spleen is removed. Antibody-producing cells from the spleen are then fused with a myeloma cell line, one which is not antibody-producing and has been maintained in culture. The new fused cell line, which does produce antibodies, is grown briefly in culture and then re-injected into another mouse’s peritoneum. Finally, the ascites fluid which contains monoclonal antibodies is harvested from the mouse.Millipore has developed and manufactured thousands of monoclonal antibodies. We provide custom hybridoma development, ascites production, cell culture production, cell cloning, hybridoma screening and clone banking. Our state of the art facilites are ISO13485,cGMP, and TGA certified. We offer small and large scale (multigram) production of monoclonal antibodies.
Custom Hybridoma Development Occurs in Phases as Follows:
Millipore’s custom monoclonal antibody services include: design concept development, antigen conjugation, cloning, screening and clone banking.
Phase I: Immunization
Balb/c mice/rats or other rodent strain, are immunized with antigen provided by the client or developed by Millipore. Between five and ten animals will be immunized to optimize the probability of success.Phase II: Cell Fusion
Cell fusion utilizes the spleenocytes from the hyperimmunized animals and an appropriate myeloma cell line (SP2/O, NS-1, or equivalent). Fusion is performed using PEG, and viable hybridoma colonies are grown out in 96 well plates.Phase III: Screening & Selection of Clones
Cells are screened and selected by ELISA testing and/or Western blot. Up to five clones are selected. If additional clones exist, they will be made available for purchase.Phase IV: Subcloning
Clones that are approved by the client are subcloned. Additional subclones will be made available for purchase.PhaSe V: Colony Storage
Millipore will store the positive colonies that were not subcloned for a period of six months. Millipore can provide the client with additional colonies if desired. The remaining clones will be destroyed within 6 months or stored and banked per the client’s request.Phase VI: Supernatant and Ascites Production
25 mLs of ascites will be provided for each clone. Hybridoma supernatant can be manufactured at an additional cost per clone, 500 mLs of supernatant is the standard volume provided.
Quality Control Measures
The following quality control measures are employed to create the highest quality monclonal antibodies commercially available:Standard Purity Checks Include
- ELISA against the antigen
- SDS/PAGE
- IEF
- HPLC
- Western blot
- Flow cytometry analysis
- Immunohistochemistry
- Immunoprecipitation
Ascites Production and In Vitro Monoclonal Production
Ascites Production- Balb/c,nude, rats and other rodent strains are available upon request
- Initial expansion, animal priming, and subclass typing
- Number of animals is dependent on the required ascites volume:
- 20 mice (60–120 mL)
- 50 mice (150–300 mL)
- 100 mice (300–600 mL)
- 200 mice (600–1200 mL)
To meet the needs of our customers world wide, Millipore has invested in a cell factory facility that is dedicated to in vitro production of monoclonal antibodies. Our facility utilizes hollow fiber systems, CL units, and large profusion systems providing us with the capacity to produce as little as 10 mg to greater than 10 g of monoclonal antibody.
Purification Services
Monoclonal and polyclonal antibodies can be purified using:
- Protein A or G chromatography
- Ion Exchange chromatography
- Immuno-affinity chromatography
- Ammonium sulfate precipitation
- Hydrophobic interaction chromatography (HIC)
- Gel Filtration
- Client dedicated columns
Antibody Fragmentation Services
- Fab and F(ab)’2 preparation and purification (10-50 mg antibody per process)
Millipore has the manufacturing capacity to accommodate your bulk polyclonal and monoclonal antibody requirements. Our production facilities can produce in kilogram quantities,with sufficiently large lot sizes to lessen your manufacturing logistics. All manufacturing is performed according to cGMP guidelines, with corresponding documentation and quality oversights.
Protein Development
Antigen is obtained by synthesis of peptides and conjugation of these peptides to carrier proteins and production of recombinant protein. For synthesis of peptides, analysis of the protein sequence for immunogenic regions of the protein is essential. To generate antibodies to a peptide, the peptide must be covalently conjugated to a carrier protein prior to immunization. Protein expression is a subcomponent of gene expression. The genetic sequence coding for the target protein must be isolated and introduced into a suitable expression vector. The vector is placed in a suitable expression system (bacterial, insect, or mammalian cells, for example). The protein is produced by these cells and purified from the culture media in which the cells are grown.
The final purity depends upon the experiments, >70% pure for non-sensitive screening assays, >90% pure for immunological applications and >95% pure for receptor/ligand studies, bioassay studies or cellular assays.
Peptide Synthesis
Millipore provides custom peptide synthesis services to meet all of your research needs. A number of labels and peptide modifications are available as are unnatural amino acids, which can be incorporated at any position and in any combination. Custom peptides are synthesized by the FMOC solid phase method. All peptides are supplied lyophilized with a complete quality control package including HPLC chromatograms to ensure purity and Mass Spectral analysis to confirm identity.
Peptide Production Scale
- 5mg
- 15mg
- 25mg
- 10mg
- 20mg
- 30+ mg
- Crude
- Immunograde (>70%)
- Pure Grade (>90%)
- Ultra Pure (>95%)
- N-terminal
- Biotin
- Palmitylation
- C-terminal FITC
- N-terminal Acetylation
- C-terminal Amidation
- D-form (or other unnatural)
- amino acids
- Cyclic
- MA
Cloning & Gene Expression
The molecular and cell biology group at Millipore has the experience and know-how to clone, sequence, and express your gene of interest in either prokaryotic or eukaryotic systems. Our patent portfolio includes the well known Glutathione S-Transferase (GST) fusion protein expression/purification system. This technology allows for the rapid and efficient method of expressing and purifying high quality proteins.We offer the following services:
Cloning
- Gene isolation and cloning
- Sequence confirmation
- Construct development
- Expression optimization
- PAGE Analysis
- High purity through affinity, ion exchange, and gel filtration chromatography
- Endotoxin removal
- DNA cloning and preparation
- Sequence confirmation for accuracy and orientation
- Transfection
- Selection and cloning (All cell lines are clonal)
- Analysis of cell lines for protein expression and functionality (low, medium, & high expressing cell lines can be generated)
- Cell line cryopreservation
Assay Development
An antibody is only useful if the assay you are using is appropriate for your antigen. Assay development is often a challenge and time consuming. But reliable, robust assays are key to every phase of pharmaceutical development. Developing immunoassays are usually used for antigen detection, analysis, or quantification. Assay development begins with the design goals which you provide.- Establish assay parameters
- Evaluate preliminary reagent stability
- Determine detection limits
- Data review
- Develop prototype for evaluation
Put Millipore’s extensive experience in assay design, validation, and production to work for you. Our GMP manufacturing environment utilizes accepted design controls, ensuring that your resulting assay will be developed systematicaslly, thoroughly documented and readily transferred to Iargescale screening and lot manufacturing. Our custom assay development team will communicate results in a timely manner, tailoring development to meet your requirements and giving you the freedom to focus on your work. Assays are available with colorimetric, fluorescent, or bioluminescent readouts for improved sensitivity and dynamic range.
Assay development begins with the design goals provided by you – alternatively, design goals can be generated through a joint development program. If all of the components are available, then the assay development proceeds quickly. Alternatively, Millipore can supply reagents,work with outside sources, or develop new antibodies, antigens, or nucleic acids to meet your assay requirement needs. Once the assays are developed and documented, we can provide large scale manufacturing or ensure a complete technology transfer to your facility. Reliable, reproducible assays are the main objective. Assay validation and standardization services are available.
Pilot Studies
- Conjugation with detection molecules
- Determine ability to detect target
- Develop assay format
- Establish assay parameters
- Evaluate preliminary reagent stability
- Determine detection limits
- Data review
- Develop prototype for evaluation
- Determine reagent stability
- Measure inter and intra-assay variability
- Screen with samples
- Write specifications
- Provide complete technical transfer
Bioluminesence Labeling Ultra Sensitive Detection with Aqualite
AquaLite is a sensitive bioluminescent label for universal use in assays and conjugations. AquaLite® is available for custom conjugation to any antibody or fragment, as well as to streptavidin, biotin, or any oligonucleotide. Millipore can also design a custom assay using AquaLite ultra-sensitive detection.
Antibody Conjugation Service
New Antibody Tools for Cell Based AssaysMillopore has partnered with Amersham and Evident Technologies to provide a broad range of conjugated antibody possibilities. CyDye®, EviTag™ Quantum Dot, fluorescein (FITC), rhodamine, or biotin conjugation can accelerate your research, facilitate multiplexed analysis, and help make findings not readily possible using Western blotting.
These conjugates span a variety of applications, colors, and emission ranges. Of particular note are the conjugates provided by our partners, Evident Technologies and GE Healthcare.
The EviTag Quantum Dots from Evident Technologies offer photo-stability and multicolor fluorescence in a variety of wavelengths, with the advantage over organic fluorophores of improved photostability, color multiplexing, and single source excitation. Each Evitag generates a sharp emission wavelength making them ideal for multiplexing in intact cell environments.
The Amersham® brand CyDyes offer superior photostability at a broad range of pH. They have been used in a wide range of applications. Cy®5® is the market leading red dye used today in fluorescent applications. Most antibodies in the Millipore catalog can be conjugated and delivered in 1mg or greater quantities within 2 weeks. Simply select a Millipore antibody and select one of the conjugated antibody labels from below.
Many of these labels can be used with multiple antibodies that do not cross-react to create custom multiplexed assays.
Each of these labels can be conjugated in a 1mg minimum with any Millipore antibody. Millipore can also label any antibody with a fluorescent dye that you supply.
