polyclonal antibody preparation process
Antibody is immune globulin which be capable of combining with specific antigen. It general falls into two categories: Polyclonal antibody and Monoclonal antibody. Polyclonal antibody can combine with multiple epitopes of antigen. This chapter is mainly talking about the production steps of polyclonal antibody with rabbit source.
Polyclonal antibody general preparation process: Complete antigen preparation→Rabbit immunization→Titer of antibody detection and the final bleed→Antibody affinity purification→Antibody Concentration and Preservation.
Detailed experimental procedure is as below:
(2) Collect blood before immunized (For negative reference use)
a. Put the rabbit carefully in a fixed frame and keep it calm.
b. Shave the rabbit’s hair on its ear to make blood vessels visible(or not shave).
c. If necessary, use a small cotton ball dipped in alcohol to smear blood vessels and make it swell.
d. Draw 10 ml blood from the ear vein with a syringe(approximately 5ml serum).
e. Pull out the needle carefully and control the wound with appropriate pressure. Then disinfect it with alcohol cotton ball.
f. Inactivate the collected blood for 30minutes at 37 oC. Finally allow samples to clot overnight at 4 oC and release the serum.
g. Centrifuge samples 10minuntes at 10000r/min.
h. Collect supernatant, is serum.
（3）Rabbit immunization
a. Antigen volume for two rabbits is 1ml and its buffer solution must be free from chemical reagent which maybe harmful to the rabbit. 400ug antigen is relatively suit for the first immunization. In order to obtain better result, reduce its dosage. Subsequent immune dosage should be 100ug each time.
b. Fully blend 1ml Freund's complete adjuvant and 1ml prepared antigen till it is milk white.
c. Remove the rabbit from its cage carefully and immunize 4 parts(either back and thigh root) of each rabbit with 250ul antigen. Needle inserts subcutaneous 1-2cm at 45-degree angle. In order to prevent antigen outflow, stay for a few seconds after injection.
d. Immunization cycle is 20 days. Collect the blood after 7-10 days(including the midway take blood test and eventually bloodletting). Total immune is 4 to 5 times.
（4）Titer detection
(5)Antibody purification
The preparation of affinity column
a. Weight 1mg CNBr-Sepherose 4B agarose gel and put into 2mM HCl acid and make it fully dissolve and swell overnight at 4℃. Then get 3ml gelatinous media.
b. Transfer the gelatinous media into purification column. Wash this media 3 times with 20ml 2mM HCl.
c. Wash the media with Coupling buffer once. It is better to finish this procedure in minutes as activating group is easy to be hydrolyzed under the Coupling buffer PH.
d. Add 5ml Coupling buffer which dissolved 5-10mg antigen into the gelatinous media.
e. Gently shake mixing for 2-4 hours at room temperature or overnight at 4℃. When finished, aspirate a little dosage of this solution if it needs to test joint efficiency.
f. Wash the media with 20ml Coupling buffer once.
g. Add 1% BSA solution and incubate 2 hours at room temperature or overnight at 4℃.
h. Wash the media with phosphate buffer (PBS) 3 times at least, each time more than 15ml.
i. Once Antigen collected gelatinous media is completed, it can be used in purification.
j. If it’s not been used or it will be used later, it should be sealed up with 20% alcohol.
The purification of anti-serum
a. Dilute the anti-serum 1:1 with PBS. Centrifuge at 5000-10000rpm for 15 minute.
b. Wash antigen affinity column with 10x volume of PBS to balance the column.
c. Add 10ml prepared anti-serum into the balanced column.
d. Gently shake it mixing for 2-4 hours at room temperature or overnight at 4℃.
e. Wash antigen column with 10x volume PBS to remove miscellaneous protein which is combined into the column.
f. Wash the column with 2x volume antibody eluent to get the specific antibody.
g. Balance the column with 10x volume PBS.
h. Column should be resealed up with 20% alcohol and stored at 4℃.
After getting the elution antibody and sucrose or polyethylene glycol concentrating, dialyze and desalt in PBS. Using the UV VIS spectrophotometer, OD value of the antibody could be measured at wavelength 280nm. The concentration of detected antibody is the OD value divide 1.35. It can be stored for a long time at -20℃ after adding 40-50% glycerol.
Polyclonal antibody general preparation process: Complete antigen preparation→Rabbit immunization→Titer of antibody detection and the final bleed→Antibody affinity purification→Antibody Concentration and Preservation.
Detailed experimental procedure is as below:
- Rabbit preparation
(2) Collect blood before immunized (For negative reference use)
a. Put the rabbit carefully in a fixed frame and keep it calm.
b. Shave the rabbit’s hair on its ear to make blood vessels visible(or not shave).
c. If necessary, use a small cotton ball dipped in alcohol to smear blood vessels and make it swell.
d. Draw 10 ml blood from the ear vein with a syringe(approximately 5ml serum).
e. Pull out the needle carefully and control the wound with appropriate pressure. Then disinfect it with alcohol cotton ball.
f. Inactivate the collected blood for 30minutes at 37 oC. Finally allow samples to clot overnight at 4 oC and release the serum.
g. Centrifuge samples 10minuntes at 10000r/min.
h. Collect supernatant, is serum.
（3）Rabbit immunization
a. Antigen volume for two rabbits is 1ml and its buffer solution must be free from chemical reagent which maybe harmful to the rabbit. 400ug antigen is relatively suit for the first immunization. In order to obtain better result, reduce its dosage. Subsequent immune dosage should be 100ug each time.
b. Fully blend 1ml Freund's complete adjuvant and 1ml prepared antigen till it is milk white.
c. Remove the rabbit from its cage carefully and immunize 4 parts(either back and thigh root) of each rabbit with 250ul antigen. Needle inserts subcutaneous 1-2cm at 45-degree angle. In order to prevent antigen outflow, stay for a few seconds after injection.
d. Immunization cycle is 20 days. Collect the blood after 7-10 days(including the midway take blood test and eventually bloodletting). Total immune is 4 to 5 times.
（4）Titer detection
- a. Two references: Serum from not immunized rabbit is as negative control which is all diluted (Blocking buffer) from first antibody initial concentration. Former positive serum is as positive control.
- Add 100ul of 1ug/ml antigen to each well of 96 well microtitre plate. Store overnight at 4℃,or incubate for 2 hour at 37℃.
- Expiry kit liquid of each well, add 200ul of blocking buffer to each well. Store overnight at 4℃,or incubate for 2 hour at 37℃.
- Expiry blocking buffer of each well completely by snapping the plate onto absorbent paper. Totally wash 3 times with Wash Buffer. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. If you wish to store the plate for future use, dry each plate at 37℃ then store in a sealed bag at -20℃.
- Get the pre-coated, ready to use 96-well plate. Add 100ul of negative control in the first well. Add positive control 100ul to the second well and add 1:500 sample to be tested to the third one and continue this 1:1 dilution to the rest wells. Incubate for 1 hour at 37℃.
- Remove the liquid. Wash 3 times with Wash Buffer and remove the remaining liquid by snapping. Add 100ul of HRP conjugated Mouse Anti-rabbit IgG (1:200 dilution) to each well, incubate 45 minute at 37℃.
- Remove the liquid, wash 3 times with Wash Buffer and remove the remaining liquid by snapping. Add 100ul of TMB Substrate(Sigma one-component TMB) and incubate 5-20 minute (Incubation time depends on color development) at 37℃.
- Add 50ul of Stop Solution（2N H2SO4）to each well. Read absorbance values on a ELISA reader using wavelength of 450nm.
(5)Antibody purification
The preparation of affinity column
a. Weight 1mg CNBr-Sepherose 4B agarose gel and put into 2mM HCl acid and make it fully dissolve and swell overnight at 4℃. Then get 3ml gelatinous media.
b. Transfer the gelatinous media into purification column. Wash this media 3 times with 20ml 2mM HCl.
c. Wash the media with Coupling buffer once. It is better to finish this procedure in minutes as activating group is easy to be hydrolyzed under the Coupling buffer PH.
d. Add 5ml Coupling buffer which dissolved 5-10mg antigen into the gelatinous media.
e. Gently shake mixing for 2-4 hours at room temperature or overnight at 4℃. When finished, aspirate a little dosage of this solution if it needs to test joint efficiency.
f. Wash the media with 20ml Coupling buffer once.
g. Add 1% BSA solution and incubate 2 hours at room temperature or overnight at 4℃.
h. Wash the media with phosphate buffer (PBS) 3 times at least, each time more than 15ml.
i. Once Antigen collected gelatinous media is completed, it can be used in purification.
j. If it’s not been used or it will be used later, it should be sealed up with 20% alcohol.
The purification of anti-serum
a. Dilute the anti-serum 1:1 with PBS. Centrifuge at 5000-10000rpm for 15 minute.
b. Wash antigen affinity column with 10x volume of PBS to balance the column.
c. Add 10ml prepared anti-serum into the balanced column.
d. Gently shake it mixing for 2-4 hours at room temperature or overnight at 4℃.
e. Wash antigen column with 10x volume PBS to remove miscellaneous protein which is combined into the column.
f. Wash the column with 2x volume antibody eluent to get the specific antibody.
g. Balance the column with 10x volume PBS.
h. Column should be resealed up with 20% alcohol and stored at 4℃.
After getting the elution antibody and sucrose or polyethylene glycol concentrating, dialyze and desalt in PBS. Using the UV VIS spectrophotometer, OD value of the antibody could be measured at wavelength 280nm. The concentration of detected antibody is the OD value divide 1.35. It can be stored for a long time at -20℃ after adding 40-50% glycerol.
