Editing Antibody Production in China

== Materials ==

*Up to ten 6-8 weeks old female Balb/c&nbsp; 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 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):&nbsp;<br/>Hypoxanthine, aminopterin and thymidine supplement (HAT), hypoxanthine and thymidine supplement (HT)&nbsp;

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

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

For 10 mice:&nbsp;<br/>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<br/>Final concentration: ~0.24 μg/μl, total 1050 μl.

3. Inject 100&nbsp; 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&nbsp;the 2nd immunization, draw blood from tails or eyes of mouse and test titer&nbsp;by ELISA,&nbsp;ELISA titers should be in the range of 1:1000~1:10000.

6. At week 6,&nbsp;immunize mice again as above.

7. Make hybridoma at week 7

== ELISA Protocol (simplified) ==

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

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

2. Follow this protocol for ELISA

== <br/>Hybridoma Protocol (simplified) ==

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

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