Custom Monoclonal Antibodies

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Proteintech offers mouse hybridoma-based production of custom monoclonal antibodies made from synthesized peptides or recombinant proteins.

Proteintech’s monoclonal antibody production protocol

1) Immunization
3 to 5 adult female Balb/C mice are chosen for immunization following the standard protocol.
Day 0-Primary immunization
Day 22-Boost immunization
Day 37-2nd Boost immunization


2) Titer determination and shock immunization
The titer of the antiserum of each immunized mice is determined by indirect ELISA against the immunogen or the free peptide if the conjugated peptide was used as the immunogen.The highest titer mouse is then shock immunized to increase the number of its plasma cells


3) Cell fusion and screening
The shock immunized mouse is sacrificed and its splenocytes are fused with myeloma cell line SP2/0. The fused cells are then cultured in plates with HAT selection medium.
After two rounds of renewal of culture medium, the supernatant from each well of the culture plates are subjected to indirect ELISA. Hybridomas from each positive well are then picked into new plates. After culturing for two days, the second round of ELISA is performed to comfirm the positive hybridomas.


4) Subcloning
The positive hybridomas are subjected to two rounds of subcloning by limiting dilution. After each round of subcloning, ELISA is performed to verify each subclone.


5) Expansion and cryopreservation
Stable positive subcloned hybridomas are enlarged to 24-well plates and then cryopreserved in nitrogen vapor phase. 5-10ml supernatant for each hybridoma is collected and send to the customer for further testing. The customer can choose 1-3 hybridomas to generate ascites and purify monoclonal antibodies.


6) Ascites generation and purification
The chosen hybridomas are used to generate ascites. Isotypes and titers are determined by indirect ELISA after harvesting the ascites. Monoclonal antibodies will then be purified from the ascites according to the isotypes of the antibodies. 1-2mg purified monoclonal antibodies for each hybridoma is sent to you. Frozen hybridomas will also be sent to the customer.

Timescales

Day 0-52 Immunization
Day 53 Titer determination
Day 54 Shock immunization
Day 57 Cell fusion
Day 67 Primary screening
Day 71 First subcloning
Day 86 Second subcloning
Day 99 Enlargement
Day 110-120 Supernatant shipped
Ascites generation and purification will be done in 40-50 days after the customer feedback regarding hybridomas.

 

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