Step1: Hyridoma cell culture

• Thaw freezing cell line 9E10 which express secreted monoclonal antibody(subtype IgG1) against Myc epitope in RPMI1640 medium containing 10% FBS and appropriate amount of ampicillin and streptomycin, incubate cell at  37degree with 5% CO2.
• Once cell get into log phase growth, cell should be passaged by every one day,  the cell density start at 2x 105/ml. Note: 9E10 and 12CA5 cell are half adhesive , once they are completely confluent, they will immediately start cell death.
• Centrifuge cultured medium at 1000 rpm for 5 minitures, collect supernatant into sterile containers, if necessary add  sodium azide up to 0.2%. for several month storage, keep supernatant at 4 degree, otherwise, freeze them at -20 degree.
• Freeze log phase cells for stock, put at least 2 million cell in 1 ml RPMI1640 medium containing 20% FBS,15% DMSO, keep freezing vials at -70  for no more than one month, then transfer them to nitrogen tank.

 

• Affinity column choosing: protein G for mouse IgG1 and protein A for mouse IgG2a, IgG2b, IgG3
• set appropriate volume of protein G sepharose column according to the common rule: culture supernatant contain 20-50ug/ml antibody, 1ml of wet beads bind approximately 10-20mg antibody, wash column with 100mM Tris-HCL pH 8.0.
• Adjust cell culture supernatant pH by adding 1/10 volume of 1.0M tris-HCL pH8.0, pass it through protein G column at speed of 2ml/min.
• wash column with at least 10  column volume of 100mM Tris-HCL then wash with 10mM Tris-HCL.
• Elute the column with 50 mM glycine(pH3.0), add this buffer stepwise at 1ml per time, collect elute fraction into 1.5 ml eppendorf tube containing 100ul 1M Tris-HCL for immediate neutralization of antibody solution
• During collecting  elute fraction, use bradford solution to monitor eluted protein
• Running 5 ul of each fraction on 11% SDS-PAGE gel to check the purity of antibody
• Combine the fractions containing antibody, determine the antibody concentration by measuring OD280nm(1 OD= 0.75 mg/ml)
• Make series dilution of antibody such as 1:500, 1:1,000, 1:2,000, 1:3,000, 1:4,000, perform western blot to determine the optimal titre of the antibody, when doing titration, the antigen should be Myc (for 9E10) or HA (for 12CA5) tagged protein which was confirmed by other Western bolt.