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Antibody Development

Antibody     ICT offers monoclonal and polyclonal antibody development, production, purification and conjugation services (under GMP conditions, if necessary). Antibodies can be developed and produced in many species including: mice; goats; chickens; rabbits; and sheep. Antibody production can also proceed in cell culture and hollow fiber systems. Antibody development generally takes 2 to 6 months, costs $4,000 to $30,000, and requires 10 - 170mg of antigen. By working with our preferred partners, ICT can help you develop and select an antibody that’s right for your project.

     Successful development of an immunoassay is highly dependent upon the antibodies. They must be highly specific and possess a high binding affinity to the test analyte. Development of quality antibody components is an absolute first step in the development of any ELISA based assay format. ICT has the experience and technical expertise to provide you with highly functional monoclonal and polyclonal antibodies.

Project Prices and Timelines

     Here are some basic project guidelines to give you an idea of what is needed to develop monoclonal and polyclonal antibodies. Generally, it costs between $4,000 - $30,000 to develop monoclonal and polyclonal antibodies. This includes purification of the analyte and conjugation to carrier proteins (if necessary). This will produce approximately 1gm of monoclonal. The amount of specific polyclonal antibody usually runs between 1%-15% of total serum IgG depending upon the animal's response. All antibodies made become property of the customer. 
     It takes about 6 months to generate antibodies and requires 5-20 mg of purified analyte to generate monoclonal antibodies and 20-70 mg for polyclonal antibodies. Purified antigen is also necessary (50-100mg) to construct affinity columns for antibody purification. If there is not enough purified analyte to make antibodies, ICT can try to source the molecule, or have one of our preferred vendors produce recombinant material in E. coli or Baculovirus.Depending on your specific antibody project, the amount of antigen required may vary, conjugations may be necessary, several fusions may have to be done, the antibody may have to be purified, and the time of the project will vary. To prepare a specific quote for your project, please review the antibody development questionnaire, and then contact ICT at 1-800-829-3194.

What are antibodies?Purification of antibodies.

     Antibodies are glycoprotein immunoglobulins produced by lymphoid cells in animals in response to an antigen stimulus. Antibodies are found in serum and tissue fluids and will bind to the antigen whenever it is encountered. Antibodies are capable of binding with high specificity and affinity to the target molecule (antigen), and are stable for years when frozen at -80° C. These characteristics make them well suited in many areas, such as: immunoassays; purification protocols; flow cytometry; and immunohistochemistry.
     Monoclonal antibodies are a homogenous population of a specific antibody with one paratope. They have defined specificity and affinity toward one epitope on the antigen, are derived from one cell clone, and usually can be produced indefinitely. Polyclonal antibodies constitute a heterogeneous population of antibodies with differing paratopes for an antigen. They are commonly produced in rabbits, goats, chickens, and many other animals.
     Affinity purification of antibodies (and antigens) enhances their reactivity and utility for improved results in many applications. The specificity and reactivity of these antibodies are critical when developing a specific assay. ICT can provide a consistent source of reliable antibodies to add control to the antibody development, selection, and production process.
     When developing the monoclonal and polyclonal antibodies, some large molecular weight target proteins can be injected directly into the animals, and will therefore not need further modification to stimulate an immune response (i.e., conjugation to a carrier protein, visit Conjugation Services). Small molecules may require conjugation to a carrier protein to generate an adequate immune response in the host animal. 

Antibody Development Questionnaire

     In order to serve you better, please review and answer as many of the following questions as best you can and give us a call at 1-800-829-3194 to discuss your project.

  • If you want polyclonal antibodies: From what species (rabbit, goat, etc.)? Do you want it purified by Protein G?
  • If you want monoclonal antibodies: From what species (we assume mouse.)? Do you want it purified by Protein A?
  • In what format do you want the antibodies (ascites, cell culture supernatant, whole serum, lyophilized, etc.)?
  • Do you want it affinity purified, or purified any other way?
  • How much antibody do you need (in mg)?
  • Do you require GMP compliance?
  • What is the specific antigen that you want to make antibodies against?
  • What are the characteristics of the antigen?
  • Is the analyte an infectious biological agent (virus, bacteria)?
  • Are there any general toxicity issues associated with the handling or use of the analyte as an immunogen in animals?
  • Amount of purified antigen available?
  • Molecular weight?
  • If the antigen is a small compound or peptide, what is the structure or amino acid sequence?
  • Isoelectric focusing point (pI)?
  • Extinction coefficient?
  • Is the antigen acid labile?
  • Storage buffer (including pH)?
  • Are there antigen solubility issues that require the addition of guanidine or imidazole to the antigen solution (these components are toxic at high concentrations when injected IP)?
  • Storage conditions?
  • If the antigen is lyophilized, what stabilizers were used?
  • Will it have to be conjugated to a carrier protein?
  • Are there any cross-reacting or interfering substances in the buffer that may affect the generation of antibody (such as imidazole)?
  • Would you like ICT to screen the bleeds for positives?
  • Is there any other information that may affect the antibodies?

 

Monoclonal Antibody (MoAb) Development in 4 Mice Non-GMP-Compliance with Production in Cell Culture

  In general, it may cost between $4,000 - $30,000 to develop and produce monoclonal antibodies. This can include purification of the analyte and conjugation to carrier proteins (if necessary). This will produce approximately 1gm of monoclonal. It takes about 6 months to generate monoclonal antibodies from the time the purified antigen is obtained and immunizations have begun.  Between 5-20 mg of purified analyte is required for this process.

     Development and production of monoclonal antibodies may require 2.5 mg of purified antigen per mouse (10 mg total for 4 mice). If the target antigen is of high molecular weight, it will probably not need further modification to stimulate an immune response, and can be injected directly into the mice with the proper adjuvant. If the antigen has a small molecular weight, it may have to be conjugated to a carrier protein before immunization (visit Conjugation Services). If desired, ICT can quote a price for Cloning & Expression of the antigen if you do not have enough available, or it is not of sufficient purity.
     MoAb production will start after Week 21, using a cell culture system (see Section 2 below). If available, all positive clones can be tested for cross-reactivity with related compounds. This will ensure the selection of a specific clone. More than 1 fusion may be needed to develop the proper specific monoclonal antibody. If so, an additional charge will be added per additional fusion.  Production will continue as antibody is needed. 

Week 1 First Immunization of 4 mice
Week 2 Second Immunization
Week 4 Test Bleed
Weeks 5-6 Third Immunization
Week 8 Fourth Immunization
Week 9 Test Bleed
Week 10 Cell Fusion
Weeks 12-14 Screening
Weeks 15-16 Subcloning
Weeks 17-18 Screening
Weeks 20-21 Cryopreservation of 3 Best Clones

1. Development (Non-GMP Compliance)...............................................$ If additional fusions are required, an additional charge will be added per fusion.
2. Once the MoAb has been developed (after Week 21, see Section 1 above), production will begin using a cell culture system. This system may yield between 0.5 – 1.0 gm antibody per run, 1 run..............................$
3. Protein A purification of MoAb IgG, Up to 100 mL supernatant, after concentration........$ Additional charges are added for each additional 10 mL.
Total, Non-GMP Development and Production in Cell Culture ..................... range of $8,000 - $20,000

Polyclonal Antibody (PoAb) Sample Proposals

     For some projects, we recommend using rabbits as the host animal in which to generate polyclonal antibodies. Rabbits tend to produce a high quality antisera from which the protein specific IgG can be easily purified, and are less expensive to maintain than larger animals, such as goats. In general, it may cost between $4,000 - $15,000 to develop polyclonal antibodies in rabbits (which includes purification of the analyte and conjugation to carrier proteins). The amount of specific antibody usually runs between 1%-15% of total serum IgG depending upon the rabbit response.
     It takes 2-6 months to generate antibodies and requires 20-70 mg of purified analyte for polyclonal antibodies. Purified antigen may also be needed (50-100mg) to construct affinity columns for antibody purification, if necessary (visit Antibody Purification).

PoAb Development and Production in 3 Rabbits with Protein A Purification, GMP

1.  Animal Purchase (3 rabbits)……………………………...………...…$
2. Development and production of polyclonal antibodies in rabbits may require 6 mg of purified antigen per rabbit (18 mg total for 3 rabbits). Starting after Week 8, immunization boosts are given once a month, with production bleeds taken 2 weeks following the immunizations. Rabbit PoAb development and production normally takes 12-14 weeks, with production bleeds generating 20-25 mL of serum per rabbit per bleed.

Week 1 Pre-immunization Test Bleed and First Immunization
Week 3 Second Immunization
Week 5 Third Immunization
Week 7 Fourth Immunization
Week 8 Test Bleed and Screen
Weeks 9-14 Boost and Production

PoAb from 3 rabbits………………………………….........…..........….$
3. Once antibody titers reach the desired level, the on-going immunization process will require an additional 15 mg of antigen (for 3 rabbits). Injections will be given monthly, and production bleeds will be collected 2 weeks later. A minimum of 4 months of production bleeds are recommended for production. The serum can be aliquoted and stored at –80c indefinitely and purified as antibody is needed.............................…...........…..$
4. Total IgG can be purified by Protein A. Minimum charge for Protein A purification (up to 350 mL of serum)……......$500* An additional charge will be added for each additional 1.0 mL

Total PoAb Production and Purification in 3 Rabbits.... $4,000-$9,000

 

All prices are in US dollars.

 

Check out ICT's line of HRP-conjugated AffiPure goat secondary antibodies for human IgG, mouse IgG, and rabbit IgG detection in all ELISA test formats.

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