Antibody Production in China

Materials

• Up to ten 6-8 weeks old female Balb/c  mice from Wushi Animal Laboratory (Shanghai, China).
• QuickAntibody-Mouse2W（2周快速鼠多抗制备佐剂), 3 ml, KX0210043, 600Yuan/ml) from 北京博奥龙免疫技术有限公司http://en.biodragon.cn/m/list.php?tid=27
• SP2/0 myeloma cells
• Nunc MaxiSorp™ flat-bottom from ThermoFisher
• HRP-labeled goat anti-mouse IgG
• HRP substrate
• 2 M H2SO4
• Microplate Reader with 450 nm

Sigma-Aldrich (Shanghai, China): 
Hypoxanthine, aminopterin and thymidine supplement (HAT), hypoxanthine and thymidine supplement (HT) 

Thermo Fisher Scientific (Shanghai, China): RPMI 1640 medium powder,  fetal bovine serum (FBS)

2. Dilute the recombinant protein in  0.9% saline solution or sterilize PBS to make a final concentration of 0.5 μg/μl

For 10 mice: 
Original protein concentration is 2 mg/ml, i.e., 2 μg/μl, take 125 μl, add 375 μl 0.9% saline or PBS solution. Mix. This is 0.5 μg/μl diluted antigen

Vortex Quickantibody adjuvant, add 550 μl Quickantibody adjuvant to the above 500 μl diluted antigen
Final concentration: ~0.24 μg/μl, total 1050 μl.

3. Inject 100  μl antigen intramuscularly in hind legs of each mouse, 50 μl to each leg.

It is normal to see precipitation in the antigen/adjuvant mixture. Mix well before drawing into a syringe and inject it as quick as possible after drawing into the syringe.

4. 3 weeks later, immunize mice again as above

5. 1 week after the 2nd immunization, draw blood from tails or eyes of mouse and test titer by ELISA, ELISA titers should be in the range of 1:1000~1:10000.

6. At week 6, immunize mice again as above.

7. Make hybridoma at week 7

ELISA Protocol (simplified)

Nunc MaxiSorp™ flat-bottom from ThermoFisher
HRP-labeled goat anti-mouse IgG
HRP substrate
2 M H2SO4
microplate Reader with 450 nm

1. Dilute recombinant protein in PBS to 2 μg/ml
Original concentration: 2000μg/ml, take 10 μl, add 10 ml PBS. Store at 4 oC
For 1 plate:
each well: 100 μl
96 wells: 10 ml.

2. Follow this protocol for ELISA

Hybridoma Protocol (simplified)

1, Mice with high antibodies titer are further immunized with 20 μg antigen in 100 μL 0.9% saline solution/Quickantibody mix. 
2, 3 days later, kill mice with CO2 (dry-ice) and cut out spleens. 
3. Fuse splenocytes with SP2/0 cells at a ratio of 10: 1  with PEG1450. 
4. Transfer fused cells  into ten 96-well microplates
5. Culture cells in RPMI 1640 with 20% FBS/HAT medium with antibiotics.
6. Five days later, substitute half medium with fresh medium. 
7. Ten days later, screen positive hybridoma cells by ELISA. 
8. Select positive clones with high titer  for further sub-cloning (serial dilution)

Alternatively, isolate total or mRNA from spleens, make an antibody library and screen positive clone by phage display.