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New MAb FAQ

Frequently Asked Questions

  • Do you need monoclonal or polyclonal antibodies?

  • Monoclonal antibodies are worth the significant amount of time and effort it takes to generate them if you need one of the following:

    • a highly specific antibody

    • a “limitless” supply of antibody

    • a clinically relevant reagent


    Polyclonal antibodies are the better choice when all you need is a “quick and dirty” reagent.

    If you want to discuss these two options, please call our facility head, Frances Weis-Garcia at 646-888-2354 or 646-888-2331.




  • What services are offered by the facility?

  • You can choose any combination of the following services:

    CONSULTATION:

    We are here for you before, during, and after the process of making new MAbs helping you make informed choices. This is most important when deciding how to:

    • Present the antigen to the animals, because the source of your antigen greatly influences the spectrum of MAbs the animal will generate.

    • Identify which hybridoma among the thousands generated produces the MAb that will work in your assay, because … what you screen for is ... what you get!!!



    IMMUNIZATION:


    • 5 mice or 4 rats or 4 hamsters

    • 6 months of housing

    • Up to 5 immunizations

    • Pre-immune sera and up to 4 test seras



    Advantage: Choosing this service eliminates the time and hassle involved in getting an IACUC protocol approved.

    FUSION:


    • Final boost of an animal (IV or IP challange with the antigen without adjuvant)

    • Fusion of the splenocytes with an appropriate myeloma

    • Plating and selection of the fusion products in about 25 – 96 wells plates

    • Qualitative analysis of the how many hybridomas were generated

    • One feeding



    Advantage: Although the fusion protocol is relatively simple, highly-efficient fusions require some practice.

    MAINTENANCE OF A PLATED FUSION:


    • Provide conditioned media from each well (culture) as it is ready for you to screen for functional MAb

    • Feed the plates

    • Expand positive wells (cultures) and provide more conditioned media for secondary/tertiary screens



    Advantage: Opting for this service allows those doing the screening to concentrate on finding the antibodies and not the laborious task of keeping the cells alive.

    ELISA SCREENING:


    • Prove that we can perform the ELISA you develop in your lab

    • Perform this triage assay on all wells (cultures) to be screened

    • Analyze the data and identify positive wells (cultures)



    Advantage: Opting for this service allows those with the greatest degree of experience with the antigen and techniques to concentrate on finding the MAbs that possess the desired functionality after they pass the first filter, specifically the ELISA.

    FREEZING POSITIVE CULTURES:


    • Freezing a minimum of 10 positive wells, more if you like

    • Transfer as many positive wells to you for freezing (no additional charge)

    • Transfer 1 vial of the positive culture the facility froze to your lab

    • Store 1 duplicate vial of the positive culture the facility froze in the
      facility as a backup



    CLONING OR SUBCLONING:

    Once a hybridoma culture that produces a MAb with the sought functionality has been identified, the culture MUST be cloned at least once to establish a stable line from which the MAb can be reliably produced.

    If you request this service, we will:


    • Plate the culture in semi-solid media
    • Pick all hybridomas that produce MAb into a 96 well, with the ClonePixFL

    • Provide media from the clones for you to determine if the MAb it produces functions in your assay

    • Expand and freeze up to 4 clones that score positive in your assay

    • Transfer 1 vial of the positive culture the facility froze to your lab

    • Store 1 duplicate vial of the positive culture the facility froze in the facility as a backup




    FREEZE SPLEEN FROM IMMUNIZED ANIMAL:


    • Final antigen boost of an animal(s)

    • Prepare splenocytes and freeze

    • Transfer all the vials to your lab



    Advantage: If you have other good responding animals, you can have their splenocytes frozen for future fusion, allowing you to keep the repertoire of B cells generated in the animal longer than the animal can survive.


  • How long does it generally take to have a hybridoma stock from which functional MAbs can be produced?

  • This is something that is very much dependent upon your antigen and what you need the MAbs to do. But on average it could take about 9 months to have stable clones that are ready for production … and this is why:

    1. To get an animal with a good immune response to your antigen (i.e., able to be titered out to 1:10,000):

    * For a bacterial, frog, or fly antigen: < 2.5 months
    * For highly conserved mammalian antigens: 4 months
    * For peptide antigens: up to 8 months

    2. Once you have an animal with a good titer, you need to wait 1 month before the animal can be boosted one last time and the splenocytes fused.

    3. The fusion, maintenance, and screening requires about 1 month.

    4. Getting a stable clone requires one to two rounds of cloning, so add 1 more month.


  • How should the antigen be prepared for immunization?

  • Besides being in a physiologically relevant buffer, two parameters that influence which option is best for making the immunogen (immunized antigen):


    1. What 3 dimensional conformation will the antigen exist, in your functional assay:

    * Denatured (i.e., western blot)
    * Native, but in the presence of some detergent (i.e., immunoprecipitations)
    * Native (i.e., FACS and in vivo studies)
    * Fixed (i.e., immunoflourescence and immunohistochemistry)
    * Complexed to other proteins


    2. What your lab is capable of doing or contracting out.

    The objective is to present the antigen ...

    * in as similar a conformation to what it will be asked to recognized in the desired assay as possible.
    * as "pure" as reasonably possible, because contaminants that are foreign to the host will only "distract" the immune system from what you really want it to be working on, your antigen.
    * in a non-toxic buffer.

    Two basic examples:

    If you want MAbs that immunoprecipitate a relatively native form of a glycosylated protein, ideally you would overexpress the antigen in a baculoviral or mammalian system to obtain a properly glycosylated antigen.

    If you want MAbs that recognize a cell surface proteins when they in complex with another protein, one could try biasing the immune response to those conformational differences by immunizing with live syngeneic cells.



  • What are the advantages to using the facility versus a company?

  • Given that most companies only screen by ELISA and WB and save up to 10 positives the chances of finding a MAb that works in your assay amongst those ten is limited.

    We encourage you to incorporate your desired functional assay into the screening process to significantly increase your chances of finding as many antibodies as possible.

    In addition, you can save every well (culture) that scores positive.