Monoclonal Antibodies

AUSTRAL Biologicals Custom Services

AUSTRAL Biologicals offers a custom monoclonal antibody service which involves the development of hybridoma cell lines that secrete monoclonal antibodies specific for a given antigen. 

The company has access to excellent facilities and a very experienced staff of scientists and technicians which have developed more than five thousand hybridoma cell lines to more than two hundred different antigens. 

The hybridoma development program is divided into a series of three steps described below in this brochure. The customer may maintain control of the project by evaluating the progress at each phase. 

1. Immunization
   An amount of 0.5 to 3 mg of the antigen (proteins or peptides) will be required. A greater amount will be required if the antigen is of low purity. It is not advisable to use antigens whose purity is below 50%.
   
   The typical protocol calls for 2-4 mice. Each mouse will receive a primary injection with a mixture of KLH-peptide or "blue carrier"™-peptide conjugates. The conjugates to the individual peptides will be made with either commercially available KLH and/or "blue carrier", and coupled via CDI [1-ethyl-3(dimethyl aminopropyl) carbodimide]. The pH of the reaction will vary from 4-6. The molar ratio used during the conjugation will be 1KLH/100 to each peptide. Of course, it does not mean that the final product will have that ratio.
   
   The subject(s) presenting the highest ELISA titer of serum antibodies will receive a boost; after 3 days its splenic lymphocytes will be isolated to make a somatic fusion. 
   
   This step is considered completed when an appropriate titer of serum antibodies has been obtained. 
   
   If subjects do not develop a sufficient antigen-specific immune response, we will restart the project with a second batch at no additional charge.The duration of this step is usually 4 to 6 weeks, depending on the immunogenicity of the antigen 

2. Fusion and screening
   The splenic lymphocytes from the immune subjects will be fused with cells of the non secreting plasmacytoma cell line BIOS/2, deficient in the enzyme hypoxanthine guanine phosphoribosil transferase (HGPRT). Polyethyleneglycol (PEG) will be used as the fusogen agent. The hybrids will be seeded on 96-well plates in a selection medium containing Hypoxanthine, Aminopterine and Thymidine (HAT). After 10 days selected hybridomas will be screened the by ELISA or by indirect immunofluorescence. The supernatants from all viable hybridoma colonies will be screened by means of direct or indirect ELISA (if user provides a polyclonal serum from another subject different from the original immunized with the antigen of interest) or through indirect immunofluorescence. 

3. Timetable
   After 40 days from the primary injections, an ELISA test is performed on the elected mice to check the immune response. Around day 73 a hybridoma clone is obtained by fusion and expanded to a 24 well plate. At day 87, a new ELISA test checks the expansion on the 24 well plates. Around day 92, several supernatants from a variety of putative clones are sent to you for evaluation. You will indicate to us which clones are suitable for your needs and we will re-clone those hybridomas. 
   
   The investigator needs to have these supernatants analyzed during the next 2-3 days and decide which of up to 6 hybridoma populations should be subcloned. If no positives are identified, a second will be scheduled. 
   
   The duration of this step is usually 4 to 6 weeks.

4. Cloning and Ascites Production
   At this stage, the specific hybridomas selected by the client will be cloned by limiting dilution. This assures the monospecificity of the monoclonal antibody obtained and the homogeneity of the line. The assay of positive clones shall be carried out as in step 2. Selected hybridoma lines will be expanded and frozen in liquid nitrogen. Subsequently, the subclass of the monoclonal antibody shall be determined either by ELISA or by passive agglutination.
   
   Approximately 5 to 10ml of ascites fluid obtained from Balb-c or hybrid subjects will be delivered to the investigator. For large scale ascites production see additional services.
   The duration of this step is usually 4 to 6 weeks.

5. Cost and Payments
   Each antibody will cost a total of US$ 4,800.00 We will offer a 10% discount for 3 or more antibodies ordered at the same time.
   
   During the first round of immunization up to the point of you receiving the supernatants, no payment is required.  After you receive the supernatants for your evaluation, we will expect a payment of US$ 2,400.00.  When you receive the ascitic fluids and the hybridomas cells, we expect to receive a final payment of US$ 2,400.00. When payment is received, you will be the sole owner of the cells, though we will keep a stock in case you need to obtain new ascitic fluids.

6. Ordering Information

Information about the antigen must be supplied when placing an order. Antigen should be shipped to AUSTRAL by courier mail as solution in wet ice or frozen in dry ice.

Click here to complete the Custom Antibodies Antigen Form Online or 

Download the Custom Antibodies Antigen Form

You may also contact AUSTRAL Biologicals at:

Tel: 1-800-433-7105 or 925-543-0018
Fax: 925-275-2136
Email: sales@australbiologicals.com
2410 Camino Ramon, Suite 163
San Ramon, California 94583 USA