• Protein : customer should provide 5mg antigen for immunization and 1mg for ELISA test.
• Peptide: design & Synthesis.

Preparation of 4 BALB/C mice, 6-weeks-old

• Day 0: 50ul pre-immune sera and 1st -immunization with CFA
• Day 21: 2nd -immunization with IFA
• Day 35: 3rd-immunization with IFA
• Day 45: serum collected for ELISA test
• We will provide ELISA data by e-mail

Day 00 : Serum collected 2ml (0.5-1ml pre-immune serum)
Day 01 : 1st -immunization, complete Freund’s adjuvant 200μg antigen
Day 15 : 2nd -immunization, complete Freund’s adjuvant 200μg antigen
Day 29 : 3rd -immunization, incomplete Freund’s adjuvant 100μg antigen
Day 35 : Serum collected 2-3ml , ELISA and WB test
Day 36 : 4th -immunization, incomplete Freund’s adjuvant 100μg antigen

• Day 43 : 5th -immunization, incomplete Freund’s adjuvant 100μg antigen. If Elisa test is negative, 2-3ml bleeding for ELISA and WB test
• Day 50 : 6th -immunization, incomplete Freund’s adjuvant 100μg antigen. If test is still negative, we need to discuss about project
• Day 57 : 7th -immunization, incomplete Freund’s adjuvant 100μg antigen
• Day 64 : last bleeding
• Day 65 : serum purification, final tests
• Day 69 : Delivery

• Day 61 1st -subcloning
• Day 71 ELISA test of cell supernants and selection of positive clones
• Day 72 2nd -subcloning
• Day 82 ELISA test of cell supernants and selection of positive clones
• Day 83 3rd-subcloning
• Day 83-day 84 ELISA test of cell supernants and selection of positive clones
• Day 84-90 Choose 1 subclone with the highest ELISA titer for each positive hybrid

• Day 91 for each clone : injecting 2 mice with liquid paraffin
• Day 91-98 culture
• Day99 ELISA test for cell supernants; Identification of all positive clones
• Day92 Injection of the hybridoma to mice intraperitoneally
• Day100-106 Ascites collection
• Day107-114 Antibody purification by protein A/G and ELISA test