1) How long does it take to develop an antibody?
We perform two types of immunizations: a) foot pad injections and/or b) subcutaneous injection.
- The total procedure for footpad will take 5 weeks and for subcutaneous 8 weeks.
- On those times and after our first screening performed by ELISA, we provide the user with supernatants of the positive selected clones (2 ml each) for their own screening based on the desired application.
- The users have time to perform their screening and select, with our advise, the best clones that match their desired application for the Core to subclone and purify.
- If the users do not have in hand the functional or other screening ready to assay the clones, this is a bottle neck. Don't worry, we are here to help you.
2) What type and how much antigen do I have to provide?
- We immunize with peptides, proteins, cells expressing the protein and DNA.
- Antigen peptide or protein: 500 ug of antigen is required, at least 0.8 mg/mL concetration for footpad immunization, because the volume to be injected is small.
- Subcutaneous immunization has less volume restrictions.
- This amount includes the needs for screening.
- Antigen cells expressing the protein: cells can be provided in a flask. The Core will maintain the cells.
- Antigen DNA: upon discussion.
- Every project is different and peptides, proteins or parental cells will be required as negative controls.
3) What determines the route of immunization that will be done?
- The mice will be immunized preferable at the footpad if the primary or transfected cell lines are the immunogen.
- Peptides or purified proteins can be used for both footpad or subcutaneous immunization, depending on the concentration.
4) What type of mice does the Core use?
The Core uses:
- Regular Balb-C mice
- KO mice
- Special strain mice for cases in which KO are not available
- Other strains as required for each project and discussed during project development
5) What type of screening does the Core perform?
- Primary screening will be done by ELISA, either on peptides, proteins or cell-based ELISA.
- When the antigen provided is a protein, special plates could be used for particular applications, such as generation of blocking or agonist antibodies, allowing the protein coated on the ELISA plate to maintain a "native-like" conformation.
- We advise for development of your functional assays, in particular when the antibodies are intended for therapeutic use (ADCC, CDC, apoptosis, etc).
5) Do you perform FACS?
- Yes, only for secondary screenings and at an additional cost.
- With the 2 ml supernatant volume provided as described above, users can run their own FACS to determine antibody binding to antigen expressed on cells.
6) Do you perform western-blot (WB) for screening?
No, but we can provide protocols on how to proceed for screening with more than 100 samples if WB is your goal.
7) Do you generate rat antibodies?
8) Do you generate rabbit antibodies?
Under development: we will soon perform rabbit monoclonal antibodies.
9) How many clones may I receive?
- All the clones that show strong binding by ELISA, which can be in the range of 100 to 2,000.
- There are different price levels for different amount of clones desired.
10) I don't have a purified protein or peptide for immunization; can I provide cells expressing the recombinant protein?
- Yes, if it is a surface molecule.
- The extracellular domain needs to be expressed on cell surface.
- This is the preferred antigen for applications such as functional blocking or agonist antibodies.
11) Which are the best cells to express my protein?
It depends on your project
- Mouse fibroblast L cells
- CT39 T cell line
- Rat fibroblast
12) I don't have the mouse or rat cells that you suggest to express my protein; can you provide me with them?
Yes, at no extra cost.
13) Does the Core test mycoplasma on the cells we provide?
If you cannot provide recent proof of mycoplasma screening, we can do it for you at an additional cost.
14) What minimal length of peptide we need to provide?
8 meers. If the peptide is less than 30 amino acids, it needs to be conjugated with KLH (or other carrier protein). KLH is a marine organism molecule that trigger the immune response in case of small peptides. Companies that synthezise the peptides also provide the KLH conjugation.