Antibody Development

How do the Rapid Prime Method and Standard Method of Producing Mouse Monoclonals Compare?

Rapid Prime vs Standard Immunization Method

Advantages Disadvantages
Standard Method- 4 female BALB/c mice are immunized intraperitoneally. Best responding 2 mice are used for fusionAntigen amount required:0.5mg minimum for immunizing (25µg/mouse approach)
1mg minimum for screening
  1. Gold standard – well characterized
  2. Theoretically produces higher affinity antibodies (longer immunization schedule allows for more affinity maturation and can produce high affinity antibodies)
  3. Test bleed done after 2nd boost to ensure appropriate  immune response is occurring
  4. Smaller amount of immunizing antigen required
  5. Back-up mice can be used for a repeat fusion if necessary
  6. Method available in rats
  1. Uses Freund’s Adjuvant (category of invasiveness “D” according to CCAC*)
  2. Slower than RP Approximately 115 days to identify positive clones
  3. Intraperitoneal injections considered more invasive (mice can  have more health issues)
  4. Final pre-fusion boost is intravenous and can cause anaphylaxis
  5. Final pre-fusion boost cannot be IV for whole bacteria antigen

*CCAC = Canadian Council on Animal Care

Rapid Prime – 4 female BALB/c mice are immunized by a proprietary method. All 4 mice are used for fusionAntigen amount required:2mg minimum for immunizing, 1mg minimum for screening
  1. Proprietary method developed in-house at IPA and used successfully for last the 15 years
  2. Does not use Freund’s adjuvant (considered more mouse friendly approach)
  3. Fast – approximately 65      days to identify positive clones
  4. Statistically creates more IgG antibodies
  5. Produces antibodies to conformational epitopes
  6. Works well for conserved and small proteins
  1. Could theoretically result in lower affinity antibodies
  2. Test bleed is not done prior to fusion. Localized immune response is created therefore immune sera may or may not be of high titer
  3. Larger amount of immunizing antigen required
  4. No back-up mice therefore no repeat fusion possible
  5. Used in mice only. Not available in rats.
  6. How is immune response evaluated in a Polyclonal Project?

How is immune response evaluated in a Polyclonal Project?

The immune serum after the second boost is titrated by indirect ELISA to assess immune response. This sera can also be shipped to client for evaluation in the client’s specific assays before terminating the rabbit.

How is immune response evaluated in a Monoclonal Project?

The immune serum after the second boost is titrated by indirect ELISA to assess immune response before fusing mice in a Standard Method Project. Mice are titrated separately to assess individual responses. These sera can also be shipped to client for evaluation in the client’s specific assays before proceeding to fuse the mice. Top 2 responding mice are fused.

Does a high IgG titer in the serum guarantee IgG hybridomas?

It is highly likely that IgG secreting hybridomas will be made with a high IgG sera titer but it is not a guarantee. A high IgG titer to antigen does not always correspond to producing IgG mAbs. The Standard Method allows for a test bleed stage where titer is assessed after the second boost. The IgG and IgM titers are assessed individually. Mice that only have a strong IgM titer most likely will not produce IgG hybridomas.

How are hybridomas made at ImmunoPrecise?

We essentially use the same method that was invented by Cesar Milstein and Georges J. F. Köhler in 1975 with a few variations. We use SP2/0 murine myeloma for making mouse hybridomas and P3X63Ag8 murine myeloma for making rat hybridomas. HAT (hypoxanthine-aminopterin-thymidine) selection is used to generate the hybridomas with a Single-Step cloning method.

What is Single-Step cloning?

IPA uses a proprietary semi-solid HAT selective media to grow mouse and rat hybridomas post fusion. This special media allows for the support of single cell colonies immediately post-fusion and hybridomas are essentially monoclonal from the start. No serial dilution (limiting dilution) cloning is required. All hybridomas that grow from the fusion are able to be tested for specific antibody within 15 days post-fusion. This quick detection of antibody specific clones allows for immediate subcloning and cryopreservation of valuable cultures.

How are the hybridomas screened post-fusion?

Our standard method for screening hybridomas is indirect ELISA on antigen probing for IgG and IgM antibody. Indirect ELISA allows for identification of all antibodies that bind to antigen. Further testing of the identified positive clones is required to determine their specificity, isotype and antibody secretion levels.

Can ImmunoPrecise test antibodies by other assays than ELISA?

Yes, IPA can test antibodies by W. blot, dot blot, BIAcore, FACS and specialized custom ELISAs (competitive, inhibition, sandwich). Please contact us to discuss your exact testing needs.

Do some hybridomas stop making antibody?

Yes, hybridomas are unnatural cell lines that would never be found in nature. Hybridoma stability issues include mutations and chromosome losses which have potential effects on the yield and quality of the antibody product. Mouse hybridomas can be unstable in the early stages after fusion but subcloning generally produces stable clones.The creation of a successful monoclonal antibody requires the production of a stable hybridoma cell line that continues to secrete the antibody of interest. Many of the factors surrounding the success of a project are controllable, such as the timing of injections, sterility of cell culture and proficiency of workers. However even when all is done correctly cell lines can be lost with the leading cause due to immunoglobulin chain loss.