Frequently Asked Questions
What forms of MAb are available from the facility?
MAbs can currently be ordered either as:
This highly-conditioned media (HSFM and 0.5% FBS, prescreened for ultra-low levels of bovine Ig) contains from 0.2 to 3.0 mg MAb/ml that range in purity from 20 to 80%. Most aliquots contain 10 mg, but some smaller ones are usually available. The aliquots are sterile and do NOT contain azide.
Following purification, all MAbs are transferred into PBS without Ca+2 or Mg+2. They are usually greater than 95% pure and available in 1 mg aliquots. As with the bioreactor supernatant aliquots, the purified MAbs are also sterile and do NOT contain azide.
- In what applications can MAbs as bioreactor supernatant be used and when do you need purified MAb?Bioreactor supernatants can be used in most assays:
* Western blot
* Flow cytrometry
* In vivo (i.e., depletion)
Purified MAb may be preferable when:
* Cellular debris from and/or factors secreted by the hybridoma could interfere with your assay (i.e., gel shifts and growth assays)
* The MAb will be biochemically manipulated (i.e., conjugation to a fluorophore or resin)
- In which assay does a particular MAb function?Due to the broad spectrum of MAbs carried by the facility, it is not feasible to functionally test every MAb produced/purified/modified. To ensure that a particular MAb is functional:
* The submitter is asked to confirm functionality when the MAb is initially produced or purified.
* The facility can ask other groups who have received the MAb previously, in which assay they successfully used it.
* You can always evaluate a small aliquot in your assay before you take and are charged for your entire order.
- Does the facility currently carry the MAb I need?To see the MAb inventory:
* Under "Established MAbs" in the navigation bar
* Choose the "Shop for a MAb"
* Either click search, or narrow your results by selecting from the options within the drop-downs
Log in is required.
Many MAbs are available for delivery in one business day as bioreactor supernatant (BRS) and/or purified. This is shown under "Current MAb Stock". If there is no MAb in stock but the website indicates that the hybridoma is ready for production, simply proceed with the order and the ABCF will begin producing/purifing the MAb as soon as possible. For estimates on how long it should take, please call the facility at 646-888-2331 or 212-327-7030.
- What if the facility does not have the MAb I need?When requesting the production of a MAb not currently carried by the facility, you need to request that a hybridoma be added to our inventory, by (1) filling out a New Submission Form and (2) filling out an order online.
If the ABCF cannot purchase the hybridoma from a cell repository (i.e., ATCC), you will have to obtain the hybridoma from another lab, grow it up and submit 500 µl of antibiotic and cell-free media that is over conditioned by the hybridoma for mycoplasma testing (no extra cost). If the mycoplasma test is negative, you need to bring us the live culture of cells within two business days. If it is not, the facility can provide protocols by which the mycoplasma can be eliminated.
Irrespective of where the hybridoma originates, you need to be aware of the following:
* The process of submitting a hybridoma and preparing it for in vitro production takes about 1 month.
*Once the cell line has been adapted for production, you are required to test conditioned supernatant for functional MAb because the ABCF tests for the presence of the MAb.
*Approximately 1 out of every 20 adapted hybridoma lines that have not been recently cloned FAIL to produce enough MAb in the in vitro production systems (bioreactors). The solutions are:
1. Subcloning the adapted line to clone out a sub-population that still produces MAb.
2. Making ascites is an option, but only after subclones have failed to produce in the bioreactors. This venue almost always solves the problem. For further explanation, call the facility head at 646-888-2354.
* You need to instruct the ABCF on how the MAb (not the hybridoma) can be distributed. The options are:
1. General Distribution (any academic lab can obtain the MAb).
2. General/MTA (any academic lab can obtain the MAb, but if one is NOT from the institution that legally owns the MAb, an MTA needs to be fully executed).
3. Institutional Distribution (only labs from the designated institution can obtain the MAb).
4. Exclusive Distribution (only the submitting lab and anyone the submitting lab head designates in writing can obtain the MAb; there is a minimum order for this type of distribution).
- Are there minimum orders?Yes.
They are active under the following circumstances:
- Non MSKCC or RU laboratories must order at least 10 mg BRS of most antibodies; 1 mg Purified
- HtzGFP mAbs for the bacTRAP protocol: 5 mg of BRS or 1 mg Purified or next larger aliquot size available.
First time order of a mAb or mAbs Exclusive to your lab (not available to other requestors) OR orders when there is none in stock must be one of the following:
- 50 mg BRS; OR
- 25 mg purified; OR
- Combination of BRS and purified (i.e. 20 mg BRS and 10 mg purified)
- Can the MAbs produced by the facility be used in vivo?Yes ... BUT ... before they can be administered to animals, they need to be tested for the presence of viruses that could infect the animal and the rest of the colony. Many, but not all, of the MAbs carried by the ABCF have been found negative for all tested pathogens. If they passed MAP testing, it is indicated on within this website. Simply click "Shop for a MAb" under "Established MAbs in the navigation bar and then choose "Additional Information", which is on the right of the screen. If it has not been tested (i.e. nothing after the field title "MAP:"), then request testing by emailing the facility at email@example.com. The screen results are usually back in about 2 weeks from when the sample is submitted.
- Can the facility produce GMP/GLP quality MAbs?No ... MAbs are for research purposes only.
Although the facility follows standard operating procedures, it has not been certified as a GLP, much less a GMP facility, and as a result, the MAbs cannot be administered to humans.
If you need GMP grade MAbs, please call the facility manager (646-888-2331) or send an email to firstname.lastname@example.org.
- Does the facility determine the endotoxin levels of all MAb it distributes?No, because for most applications the endotoxin load is inconsequential. If it becomes a common request, this will be considered.
- Reconstitution of MAbs lyophilized by the facilityMAbs lyophilized by the facility should be reconstituted with sterile dH20 to original volume as noted on the tube.
- General MAb Storage GuidelinesStorage Guidelines
- Avoid freeze-thaw cycles
- Most antibodies can be kept at 4ºC for use within 1-2 months and for long-term storage.
- For long-term storage, keep at -20ºC / -80ºC (not frost free)
When First Thawed
- Vortex to ensure a homogeneous sample
- Centrifuge to bring liquid off the top of the tube
- Aliquot into volumes anticipated to be consumed within 1 – 2 months
- NOTE: Upon thawing BRS (pink solution), a precipitate may have formed. Do not be alarmed; the precipitate is simply an aggregate of other protein present in the BRS. Simply centrifuge it out and process as outlined above.
0.02% Sodium Azide Recommendations:
- Adding 0.02% (w/v) sodium azide to stocks kept at 4ºC to minimize contamination risks is only recommended if:
- The antibody will not be used in vivo or in cell culture;
- Will be conjugated using an amine-based protocol; or
- Will be used in any experiment in which sodium azide is problematic
- Sodium azide can be dialyzed away if necessary.
- An acceptable alternative is 0.01% thimerosal (merthiolate), which does not have a primary amine.
MAb Storage Concentration:
- Do not store at less then 1 mg mAb/mL.
- If a working concentration is preferred, add additional proteins to bring up to >= 1mg total protein / mL and to ensure stable conditions.
- Bovine serum albumin is a common choice.
- Do not add other proteins if the MAb is likely to be conjugated because the other protein will be conjugated.