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Green Mountain Antibodies

2 Orders Completed
Burlington, Vermont, US

About Green Mountain Antibodies

Green Mountain Antibodies is a recognized leader in the production of rat and mouse monoclonal antibodies. Located in Burlington, Vermont, our purified and highly characterized antibodies are used world-wide in the research, pharmaceutical, and biotech industries.

Our Services (19)


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Custom Monoclonal Antibody (mAb) Production

Price on request

Mouse Monoclonal Antibodies

As an experienced leader in the production of mouse monoclonal antibodies, we can help you make the antibodies you want, at a price you can afford.

Advantages

  • Experience – With over 2 decades of experience in antibody production, we have developed optimized and efficient protocols to... Show more »

Mouse Monoclonal Antibodies

As an experienced leader in the production of mouse monoclonal antibodies, we can help you make the antibodies you want, at a price you can afford.

Advantages

  • Experience – With over 2 decades of experience in antibody production, we have developed optimized and efficient protocols to produce highly specific, high affinity antibodies with a high success rate.
  • Superior Service – A single project manager will coordinate all aspects of your experience and help you to refine the scope and design of your project to achieve your goals.
  • Flexibility – Collaborative data review at each phase allows you to customize your screening process and make decisions on fusion, subcloning, and expansion. Our flexibility allows us to meet the specific design and scheduling needs of both academic and industry scientists.
  • Competitive Pricing – Our highly efficient protocols allow us to be cost-effective in our production of high quality antibodies, which is reflected back to you in pricing.

Schedule for a typical mouse hybridoma project:

Phase I: Immunization (4-6 weeks)

2 mg of antigen is required for standard immunizations (4 injections of 3 mice). Antisera is collected after the 4th immunization and screened by solid-phase ELISA. Data will be provided to you and serum samples can be shipped upon request. The serum titer results will determine when we move to fusion in Phase II.

Phase II: Fusion (2 weeks from fusion to screening)

At this stage, 1 or 2 mice can be selected for fusion. Splenocytes are plated into either 16 (1 mouse) or 32 (2 mice) x 96-well plates, and screened by solid-phase ELISA for antigen specific clones. Fusion positives are expanded into 24-well plates and retested by ELISA against additional counter-screening antigens or with an assay format specific to your end use. Supernatant can be provided to you upon request.

Phase III: Subcloning (2 weeks)

We review all results with you and help you to select which clones to move forward. Fusion positive clones are subcloned by limiting dilution and screened for antigen specific clones by solid-phase ELISA. Top subclone positives are expanded into a 24-well plate and retested by ELISA against additional counter-screening antigens. Supernatant can be provided to you upon request.

Phase IV: Characterization and purification

We offer a wide variety of services including antibody production and purification, antibody characterization, and cell line archival.

Rat Monoclonal Antibodies

Rat monoclonal antibodies offer several advantages over murine antibodies. We use the same murine myeloma partner as our murine hybridoma process so the resulting hybridomas are chimeras. Not only does the rat host allow the development of monoclonal antibodies to murine proteins but rat antibodies pair well with murine monoclonals in sandwich assays. With two different species of antibodies, there is no cross reaction of available anti-mouse secondary antibody binding to the capture antibody.

Advantages

  • Used for murine antigens.
  • Reduced antigen requirement. Single rat immunized versus multiple mice.
  • Host variation offers different antibody responses.
  • Larger spleens yield twice the number of hybridomas per fusion.
  • No special growth media required.
  • Purification methods similar for murine antibodies
  • Pair with existing mouse antibodies in sandwich assays

Schedule for a typical rat hybridoma project:

Phase I: Immunization (4-6 weeks)

Approximately 0.5-1.5 mg of protein is required for standard immunizations. A serum titer will determine when we schedule a fusion.

Phase II: Fusion (2 weeks from fusion to screening)

At this stage, splenocytes are plated into either 32 or 64 x 96 well plates, and screened by solid-phase ELISA for antigen specific clones. Fusion positives are expanded into 24-well plates and can be rescreened using a number of different assays that will help you identify the most desirable clones for your needs.

Phase III: Subcloning (2 weeks)

Fusion positive clones are subcloned by limiting dilution and screened for antigen specific positive clones by solid-phase ELISA.

Phase IV: Characterization and purification

We offer a wide variety of services including antibody production and purification, antibody characterization, and cell line archival.

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Mouse Rat

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Large Scale Antibody Production from Hybridomas

Price on request

One significant advantage of monoclonal antibodies over serum polyclonal antibodies is the ability to produce essentially unlimited amounts of purified antibody with a single specificity.We have designed our standard purification procedure to attain higher antibody yield:

  • We determine production rate before scale-up. This is... Show more »

One significant advantage of monoclonal antibodies over serum polyclonal antibodies is the ability to produce essentially unlimited amounts of purified antibody with a single specificity.We have designed our standard purification procedure to attain higher antibody yield:

  • We determine production rate before scale-up. This is done by a pilot purification of a smaller volume of antibody-containing spent media, or by using a quantitative immunoassay for the antibody during initial culture stages.
  • By knowing a more exact production rates (mg antibody/liter/day) you can more accurately predict culture volume and culture time. This approach also leads to earlier intervention if there is significant decrease or increase in antibody production levels.
  • The use of serum-free media decreases possible contamination of purified antibody by bovine immunoglobulin.
  • Conditions are endotoxin low and we can remove residual endotoxin from purified antibody as a final step.
  • Subcloning the cell line before large scale production results in improvement of antibody yields and lower cost.
  • Antibody product is provided in sterile phosphate-buffered saline and analyzed by SDS-PAGE and light scattering. Other solvent options are available.

Advantages

  • Obtain mg to gram amounts of purified antibody
  • Production in culture eliminates mouse ascites serum immunoglobulins
  • Serum-free conditions lowers contamination of bovine immunoglobulin
  • Purified antibody can be formulated in appropriate buffer or lyophilized so you can use immediately
  • Lower endotoxin levels so you can use antibody in vivo

To initiate a production, please contact us for a quote. We will need certification that the hybridoma cells are free of mycoplasma, an estimate of production rate, and any information you have on growth conditions including doubling times, media requirements, and antibody isotype.

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Western Blot

Price on request

Western blotting is a common tool used for detecting proteins in crude protein mixtures such as serum and cell lysate. The proteins in the mixture are separated on the basis of size and charge by gel electrophoresis and then transferred onto nitrocellulose. This provides a solid matrix with immobilized protein. The separated... Show more »

Western blotting is a common tool used for detecting proteins in crude protein mixtures such as serum and cell lysate. The proteins in the mixture are separated on the basis of size and charge by gel electrophoresis and then transferred onto nitrocellulose. This provides a solid matrix with immobilized protein. The separated proteins are then “probed” with specific antibodies. Bound antibodies are detected using a secondary antibody conjugated to an enzyme, similar to an ELISA assay. Chromogenic substrates include those where the products precipitate on the nitrocellulose or chemiluminesce that can be detected using autoradiography.

Uses

  • Antigen detection and quantitation in complex samples
  • Cross-reaction with homologous antigens
  • Binding comparison of different antibodies to single antigens
  • Antibody binding to antigen fragments and chemically modified antigens

Some important points about western blots:

  • Not all monoclonal antibodies can bind proteins following reduction of disulfide bonds and denaturation with sodium dodecylsulfate.
  • Antibodies recognizing linear, or non-conformation dependent epitopes are more likely useful for immunohistochemistry or other assays where antigens are subjected to denaturing conditions.
  • Cellular components can be identified from cell lysates or membrane preparations
  • Proteins can be enriched by immunoprecipitation prior to gel electrophoresis.
  • Antibodies that bind well in western blots do not always bind strongly in ELISA when the antigen is immobilized on plastic.
  • In order to obtain monoclonal antibodies that bind proteins in western blot assays, positive antibodies from ELISA should be screened directly in western blots early in the hybridoma selection process.
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Immunoassays

Price on request

Multiplex Bead-Based Assays

Screening assays for hybridoma antibodies have improved and benefited from advances in technology and the availability of reagents and instrumentation for measuring bimolecular interactions. One method we’ve embraced at Green Mountain Antibodies is fluorescent polystyrene beads with flow... Show more »

Multiplex Bead-Based Assays

Screening assays for hybridoma antibodies have improved and benefited from advances in technology and the availability of reagents and instrumentation for measuring bimolecular interactions. One method we’ve embraced at Green Mountain Antibodies is fluorescent polystyrene beads with flow cytometry.Although our primary screening method is solid-phase enzyme-linked immunosorbent assay (ELISA) in 96-well plates, application of fluorescent bead-based assays in a second round of screening positive monoclonal antibodies, allows for further definition of antibody specificity and clone selection before antibody purification. You can obtain monoclonal antibodies with more targeted specificity using protein fragments, homologous proteins, or chemically modified antigens coupled to different bead sets.

Advantages

  • Uses less antigen and smaller sample volumes than ELISA
  • Allows the use of multiple antigens in a single reaction volume
  • Shorter assay times
  • Signal over several logs of antibody and antigen concentrations
  • Covalent attachment of antigen to the surface
  • Permits chemical modification of attached antigen
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Assay Development

Price on request

ELISA Assay Development

Many immunochemical methods use an indicator molecule attached to a pure antigen or antibody, such as an enzyme attached to an antibody. ELISA is useful for determining serum antibody concentrations; detecting and measuring antigens in a variety of tissues and samples; identifying hybridomas producing... Show more »

ELISA Assay Development

Many immunochemical methods use an indicator molecule attached to a pure antigen or antibody, such as an enzyme attached to an antibody. ELISA is useful for determining serum antibody concentrations; detecting and measuring antigens in a variety of tissues and samples; identifying hybridomas producing antigen-specific antibodies; and measuring idiotype-anti-idiotype interactions.

Types of assays we routinely use:

  • Indirect ELISA: Our standard ELISA method used for screening hybridomas. Antigen is immobilized on to wells of 96-well plates and antibody-antigen binding detected using a secondary antibody conjugated to an enzyme and a chromogenic substrate.
  • Capture ELISA: A secondary antibody (for example, goat anti-mouse) is used to capture murine antibodies. Avidin-peroxidase and a chromogenic substrate detect bound biotin-labeled antigen.
  • Competitive ELISA: Using a labeled protein antigen, single monoclonal antibodies are absorbed onto plastic wells and bind biotin-labeled protein antigen. Antigen-antibody complexes determined using avidin-peroxidase. Inhibition of biotin-protein by unlabeled protein forms the basis for a standard curve for protein quantitation.
  • Solution-phase immunoassays: Useful for antigen-antibody interactions in solution and for quantifying soluble protein antigens. Directly measures free antibody in antigen-antibody mixture.
  • Whole Cell: Cells are immobilized onto 96-well plates. Antibody binding to cell antigens detected using a secondary antibody and chromogenic substrate.
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Hybridoma Screening

Price on request

Having a high number of distinct positive hybridoma antibodies is a good outcome. However, a restricted budget often times means not all of them can be expanded, frozen, and the antibody purified and characterized. We believe an alternative to “brute force” production and characterization of positive hybridomas is the more precise... Show more »

Having a high number of distinct positive hybridoma antibodies is a good outcome. However, a restricted budget often times means not all of them can be expanded, frozen, and the antibody purified and characterized. We believe an alternative to “brute force” production and characterization of positive hybridomas is the more precise selection based on secondary screening assays following an initial selection by solid-phase ELISA. The approach of using additional screening methods, or different antigens, often leads to several fit-for-purpose antibodies from a single fusion.

Advantages

  • Identify different antibody clones earlier in the clone selection part of the process
  • Know the apparent affinities of antibodies before production and purification
  • Select discrete antibodies to linear or conformational epitopes
  • Obtain metal-ion or cofactor dependent antibodies
  • Know relative antibody production levels of individual clones early in clone selection

Possible secondary screening methods include:

  • Blotting following SDS-PAGE and transfer to nitrocellulose, or directly using a dot blot apparatus
  • Quantitative analysis of kinetic rate constants and apparent affinity using Octet
  • Multiplex assays using fluorescent bead-based assays with different antigens bound to distinct bead sets
  • ELISA in the presence of metal ions or EDTA
  • Determination of isotype or IgG concentration to select high producing clones or a specific isotype antibody
  • COMING SOON! Clone selection using variable region sequencing
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Bioconjugation/Chemical Crosslinking

Price on request

Label Antibodies with Biotin and Fluorescent Labels

Labeled antibodies are widely used in different assays. Although labeling methods are often kit-based and simple to perform, we believe your time is better spent running experiments, not preparing reagents. Labeling antibodies with biotin and reactive fluophores are two... Show more »

Label Antibodies with Biotin and Fluorescent Labels

Labeled antibodies are widely used in different assays. Although labeling methods are often kit-based and simple to perform, we believe your time is better spent running experiments, not preparing reagents. Labeling antibodies with biotin and reactive fluophores are two common modifications we can do for you.

Uses

  • Labeled antibody for sandwich ELISA, immunofluorescence, FACS or bead-based assays
  • Increase assay sensitivity and decrease background
  • Signal amplification in ELISA and Western blots
  • Screening hybridomas when antigen is limiting
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Protein Aggregation Analysis

Price on request

Light scattering is a rapid non-destructive method for studying motions of macromolecules in solution and cells in suspension. For proteins such as monoclonal antibodies, dynamic light scattering measures the diffusion and size of the antibodies while static light scattering reports the molecular weight and radius of gyration. By... Show more »

Light scattering is a rapid non-destructive method for studying motions of macromolecules in solution and cells in suspension. For proteins such as monoclonal antibodies, dynamic light scattering measures the diffusion and size of the antibodies while static light scattering reports the molecular weight and radius of gyration. By knowing that your purified antibody is in solution following purification you can eliminate one more problem variable in understanding why a particular antibody “doesn’t work” in a particular assay. And if the antibody does aggregate, light scattering is a tool for determining alternative solution conditions to improve its behavior in solution.

Uses

  • Detect antibody aggregation or precipitation
  • Indication of antibody heterogeneity
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Anti-Idiotype Antibody Production

Price on request

Considerable numbers of human monoclonal antibodies are now approved drugs or in drug pipelines. Similar to other proteins, monoclonal antibodies-with specificity to the antigen-binding regions of the antibody drugs-are useful for determining the drug’s presence and concentration in blood or other tissues.

Our approach is to... Show more »

Considerable numbers of human monoclonal antibodies are now approved drugs or in drug pipelines. Similar to other proteins, monoclonal antibodies-with specificity to the antigen-binding regions of the antibody drugs-are useful for determining the drug’s presence and concentration in blood or other tissues.

Our approach is to immunize with the purified antibody (or Fab fragment) and to screen against the antigen IgG and an irrelevant IgG as a control. Antibodies (anti-ID) with the correct specificity are expanded. Specificity to the variable region is confirmed using the ligand recognized by the antigen antibody.

Applications

  • Neutralize the activity of the target antibody by blocking the antibody’s ligand binding site
  • Quantitate the antibody in serum or expression system supernatants
  • Occasionally anti-ID mAbs will be a mirror image of ligands for the idiotype.
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Hybridoma Development

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Protein Characterization

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Pharmacology & Toxicology

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Biochemistry & Molecular Biology

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Project Management

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Protein Expression Visualization

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Protein Services

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Antibody Services

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Therapeutic Monoclonal Antibody (mAb) Development

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Biology

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