Fees:

Mice (BALB/c)                          $3600
Rats (Fischer or Lewis)             $3625
Hamster (Armenian)                $3900

    Three animals will be immunized for four to six months with cDNA encoding a protein antigen and/or using the peptide/protein antigen itself.  For cDNA, at least 6mgs of antigen is required.  For protein or peptides, at least 4mgs of antigen is required.  The Core will test for antibody production using an ELISA assay.  It will be the investigator's responsibility to perform a second assay based on the desired activity of the investigator. 

    If both assays yield positive results, a hemisplenectomy will be performed on the best immune responder.  The B cells from this animal will be fused with SP2/0-IL6 myeloma cells to make hybridomas.  The animal will be kept alive for the further fusion studies if necessary.  Hybridomas will be subcloned to ensure that cell lines are monoclonal.  The investigator may then screen all ELISA-positive clones at Day 21 after fusion (usually about 15 ~ 30 clones), by their secondary assay to determine the hybridoma lines producing the best antibody response for their desired purpose.  Turnaround time is 6-8 months.

Monoclonal Antibody Performance: 

• Animals for monoclonal production will be maintained for 17 weeks, during which 7 immunizations will be performed.  At such time, based on the ELISA response of the mouse sera, along with the client performed other assay, an animal will be selected for the first hemisplenectomy and fusion to be performed. 
• A second fusion is performed only upon failure of the first fusion to produce a single hybridoma cell line secreting an IgG class antibody which is ELISA positive against the directed antigen.  
• The client will be billed for animal boarding costs of all immunized animals until the date of sacrifice.
• The Antibody Core Facility will provide between 10 and 20 hybridoma cell lines, which are shown positive to the stated peptide or protein by ELISA screening. A positive response is an ELISA reading of hybridoma supernatant with a three-fold higher reading over background (using 0.5mg of antigen per well).
• It is the responsibility of the client to screen all ELISA-positive clones by some other acceptable method chosen by the client, in order to determine which hybridoma clones should be eliminated and which to proceed with further investigation.  We ask that the results from any other assay performed by the client to be sent to the Antibody Core Facility for our record keeping.
• The Core will subclone up to 6 cell lines which the client deems acceptable.
• The client is expected to deliver their assay results within 2 weeks, to ensure optimal subcloning time and isolation of monoclonal antibody producing hybridomas.  

Key points about monoclonal antibodies and hybridomas:

• Hybridomas are innately genetically unstable.  A hybridoma cell line thus can “shut off” its production of monoclonal antibody at any time.  To guard against this, the client is strongly advised immediately to culture large quantities of the useful hybridoma cell line, so that stocks of culture supernatant and cells can be frozen away for long-term use.
• Some monoclonal antibodies lose their activity upon freezing.  Freezing the monoclonal antibodies in an environment rich in protein (i.e., culture medium containing high serum) often will help to stabilize the monoclonal antibody’s activity.
• The successful generation of hybridomas, secreting monoclonal antibodies with specific performance traits, is based solely on probability.  The Core’s success is enhanced by our experience.  The monoclonal antibody Core will consider its performance acceptable for a given (single) project when at least one hybridoma cell line secreting an IgG class of antibody directed against the relevant antigen (supplied by the client) reacts by ELISA.   In some cases, the Core may be able to supply a number of hybridomas to the client.
• For more information, please review our Antibody Production Guidelines, speak with Dr. Lai or the Antibody Core Facility staff.

How to proceed:

1. Fill out the order form, which may be downloaded as an Adobe PDF file here. The order form must be accompanied by an IDR, together with required reagents (see below), to Dr. Wayne Lai (K3.118 or Y4.312D or FAX 214-648-2951).
   
   
   
2. Investigators must file an "application for use of animal subjects" with the Institutional Animal Care and Use Committee (IACUC).  Antibodies may be created using the Antibody Core's protocol.  A short form of the application may be downloaded at this site.  Investigators should consult with Dr. Lai if they require guidance in filling out and submitting this form.
   
   
3. For  cDNA microparticle immunization, provide 900µg  cDNA cloned into PcDNA3.1 . Prepare cDNA using Qiagen endotoxin-free kit and dissolve the cDNA in sterile water or PBS (2µg/ul)
   
   
4. For protein or designed peptide antigens, we require at least mg of protein or protein complex.

Cryopreservation and Storage

Cryopreservation procedure  fee: $20/clonal line (2 vials)

Cryopreservation storage  fee: $50/year/each clone line (2 vials)