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 |
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| * Polyclonal antibody production |
| 1) primary injection (antigen 500 ㎍
inject per 1 rabbit) |
| i) |
Protein antigen (500 ㎍ / 0.5 ㎖ PBS buffer) is mixed with an equal
volume of the CFA (Complete Freund’s adjuvant)
|
| ii) |
Rabbits normally are given multiple subcutaneous
injections on the back. A normal subcutaneous injection for a rabbit is
approximately 400 ㎕ . |
| iii) |
Up to 10 sites per animal are common . |
|
|
| 2) booster injection (antigen 200 ㎍
inject per 1 rabbit) |
| i) |
Protein antigen (200 ㎍ / 0.5 ㎖ PBS buffer) is mixed with an equal
volume of the IFA (Incomplete Freund’s adjuvant) and an emulsion is formed . |
| ii) |
Rabbits normally are given multiple subcutaneous
injections on the back. A normal sc injection for a rabbit is approximately 400
㎕ . |
| iii) |
Up to 10 sites per animal are common. |
|
|
 |
| * Monoclonal antibody production |
| 1) Immunization |
| i) |
Injection (for a single mouse) |
|
| ①
|
Protein antigen (100 ug/ 0.2ml PBS buffer), are mixed with an equal volume of
the CFA . Inject the emulsion to 6-week female BALB/c mouse intraperitoneally. |
| ②
|
After 4 weeks, Inject the emulsion of 100 ㎍ of antigen per mouse and incomplete
Freund's adjuvant intraperitoneally. |
| ③
|
After 2 week, blood samples are obtained from mice. Serum antibody titer is
determined with ELISA test. If the OD is higher than 1.0 when the dilution fold
is 1/1000, cell fusion can be performed. |
| ④
|
At two weeks after the second immunologic treatment, inject the same amount of
antigen diluted with PBS intraperitoneally. Perform fusion three days later. |
|
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|
| - |
Each serum bleeding has to be performed after 2 weeks from the time of
each injection |
|
| ① |
Dilution fold |
| |
1:100 |
| |
1:1,000 |
| |
1:5,000 |
| |
1:10,000 |
| |
1:50,000 |
| |
1:100,000 |
|
|
| 2) Fusion |
| ①
|
Dissect the spleen out from a mouse. Meticulously
separate the fat tissue and other organs. Be sure to avoid excessive bleeding. |
| ②
|
After rinsing with a small amount of media, transfer
the spleen to a Petri dish filled with 10 ㎖ of DMEM and squash down. |
| ③
|
Centrifuge at 1000rpm for 10 minutes and decant the
supernatant. |
| ④
|
Suspend the pellet in RBC lysis buffer. |
| ⑤
|
Centrifuge at 1000rpm for 10 minutes and decant the
supernatant. |
| ⑥
|
After suspending in DMEM, perform cell counting. |
| ⑦
|
Mix splenocytes with myeloma cells. |
| ⑧
|
Centrifuge at 1000rpm for 10 minutes and decant the supernatant. |
| ⑨
|
Carry out fusion using polyethylene glycol (PEG). |
| ⑩
|
Centrifuge at 800rpm for 5 minutes. |
| ⑪
|
Gently suspend the pellet in 1 x HAT media and
dispense 200 ㎕ to each well of 96 well plate. |
|
| |
| 3) Cloning |
| ① |
Transfer the cells from ELISA-positive well to 24-well
plate and perform cell counting. |
| ② |
Dilute with 20 ㎖ of 1x HT DMEM media (HT supplement,
10% FBS, 1% penicillin/streptomycin) to get 100-200cells/20 ㎖ suspension and
dispense 200 ㎕ to each well of 96 well plate. |
| ③ |
Incubate at 37℃ for 7 to 10 days in a CO₂ incubator.
Feed once in the middle of the period
(Day 5 - 7). |
| ④ |
Repeat cloning until the final clone is confirmed with
ELISA screening. |
| ⑤ |
It is possible to conduct screening with any methods
available other than ELISA. |
|
