Monoclonal Antibody from Hybridoma Culture Supernatant
Step1: Hyridoma cell culture
Step2: Affinity purification of antibody
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
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