Monoclonal Antibody research and development procedure
I. Antigen Preparation
Protein antigens can be directly used as an immunogen while it is necessary to reorganize chemical structure of polypeptide hapten (or small molecule compound) .
1. mcKLH conjugation with sulfo-SMCC
Add 2ml ultrapure water into 10mg mcKLH so that we get 5mg/ml solution of the mcKLH. And then prepare 2.5mg/ml solution with
5mg Sulfo-SMCC dissolved in ultrapure water.Mix the two solutions together for equivalent volume and stirred at room temperature for 2 hours. Finally, in order to remove the excess Sulfo-SMCC, dialyze the complexs in PBS.
2. Polypeptide(or small molecule compound) hapten conjugation
Weigh 10mg polypeptide and dissolve it in 5mL conjugate buffer. Then mix polypeptide with the activated carrier protein and stirred at room temperature for 2 hours. Finally, dialyze the complexs in PBS.
II. Mouse immune
1. Preparation
Select healthy, six weeks, Balb/c female mice.
2. Antigen injection
a. The immunizing dose is 100μg/0.2ml for each mouse, 100μg is appropriate for per mouse for the first time, or less dose will get better results.
b. Emulsify 0.1ml Freund’s complete adjuvant and 0.1ml antigen fully.
c. Immune period is 2-3 weeks. Total times of immunization is 4-5 times.
Immunization program:
3. ELISA immunizing potency test
Detect antibody titers in the immune serum by indirect ELISA method.
a. two reference: Negative reference is not been immuned serum of animals while positive serum or cell supernatant is as the positive reference. Dilute both according to first antibody initial concentration.
b. Add 2ug/ml coated antigen into each well of 96-well plate, stay overnight at 4℃ or incubate 2 hours in 37℃.
c. Discard the liquid, add 200ul blocking solution into each well, stay overnight at 4℃ or incubate 2 hours in 37℃.
d. Throw away the blocking solution, wash 3 times with the washing buffer and air the plate or 37℃ drying. Seal it with the hermetic bag and store at -20℃.
e. Add 100ul primary antibody to each well. If it is hybridoma cell, the primary antibody is cell supernatant. If it is the immunized animal’s serum, it needs a series of dilution(diluted in blocking buffer). Usually, the initial dilution ratio is 1:500. Then do the double dilution and incubate 1 hour at 37℃.
f. Wash the plate 3 times. Same operation as above.
g. Add appropriate HRP labeled anti-mouse antibody, incubate 45 minutes at 37℃.
h. Wash the plate 3 times. Same operation as above.
i. Add 90ul TMB substrate solution into each well, incubate 15-20 minutes at 37℃.
j. Add 50ul stop solution into each well.
k. Read at 450nm immediately.
III. Fusion of hybridoma cell
1. Preparation
a. Culture of SP2/0. About two weeks before fusion, recover the cells and culture them till they are in logarithmic growth phase. Myeloma cell suspension preparation is as below:
b. 48-36 hours before fusion, amplification culture of the myeloma cells.
c. on the day of fusion, blow the cells gently with tips and collect the cells in a 50ml centrifuge tube .
d. 1000r/min centrifugation for 5-10 minutes and discard the supernatant.
e. Add 30ml incomplete culture medium, wash once with the same method of centrifugation. Then resuspend the cells in 10ml incomplete culture medium and mix until it is well-distributed
f. Take the myeloma cell suspension and use 1mg/ml trypan blue dye for living cells count. While the cells is counted, 0.5ml cell suspension is added into 0.5ml trypan blue dye, mix them and count with hemocytometer. The number of cells is calculated as follows: the number of cells per milliliter = 4 large squares cells ×104/4×2.
2. Keep ultraviolet irradiating room overnight.
3. Preparation of feeder cells. Normally，it needs to be prepared the day before. Take normal BALB/c mouse, remove its eyeballs and collect the blood. Then separate serum from the blood as negative reference .Kill the mouse by cervical dislocation and dip into 75% alcohol for 5 minutes. Fix it on dissecting table, dissect with the abdominal skin on the left side. Spleen can be seen. Take out the spleen and place it in plate and carefully strip the surrounding connective tissue. Grind the spleen with a plunger to disperse the cells. Wash the cells many times with 10ml incomplete culture medium and made it into a single cell suspension. Transfer it into 50ml centrifuge tube, 1000r / min centrifugation for 5-10 minutes. Wash 1-2 times with incomplete culture medium and resuspend it in 10ml incomplete culture medium. Then take the above 0.5ml cell suspension, add 0.5mltrypan blue dye as living cell count. Usually, each mouse can obtain 1×108-2.5×108 spleen cells. Add the above cell suspension into 96 wells, each well 0.1ml and set it in 37℃ 4% CO2 incubater overnight and reserve for next step.
4. B lymphocytes preparation. Take a immunized BALB/c mouse, remove its eyeballs and collect the blood. Separate serum from the blood as positive reference. Kill the mouse by cervical dislocation and soaked in 75% alcohol for 5 minutes. The method of obtaining spleen and preparation of feeder cells are the same.
5. cell fusion and Selective culture of hybridoma cells.
a. Mix 1×108 spleen cells with 2×107-5×107 SP2/0 myeloma cell in a 50ml tube, supplement the incomplete culture medium to 30ml and mix well.
b. 1000r / min centrifugation for 10 minutes. Absorb the supernatant fully.
c. Tap bottom of the tube to make the cells loose and homogeneous.
d. Gently turn the centrifuge tube while add 1ml pre-heat PEG within one minute (the best time is 45 seconds).
e. Add 10ml incomplete culture medium which has been heated to 37℃ within 90 seconds，then place quietly 10 minutes in 37℃
f. 1000r/min centrifugation for 5 min and remove the supernatant.
g. Tap the bottom of the tube to make the cells loose. Rotate the centrifuge tube while adding 50ml 37℃ preheated HAT medium. Then transfer to big bottle to make the volume reach 100ml.
h. Add it to prepared 96 wells feeder cell culture plate, each well 0.10-0.15ml and culture it in 37 ℃ 4% CO2 incubator.
i. 5 days later, use HAT medium to exchange half of the medium.
j. 7-10 days later, the HAT medium was replaced with HT medium.
k. Observe the growth of hybridoma frequently, aspirate the supernatant for antibody detection when it grows up to 1/10 of the bottom of the plate.
IV. Screening
Primarily screened by ELISA method and its screening programs based on different projects.
V. Hybridoma cloning
The positive hybridoma cells obtained from the original wells and it might be derived from two or more hybridoma cells, so their secreted antibodies are not aiming at the same antigen epitope. In order to obtain completely the same homogeneous monoclonal antibody, hybridoma cells have to be cloned. There are many cloning methods and we mainly use limiting dilution method.
1. Materials:
a. 96 wells cell culture plate,etc;
b. HT medium;
c. Strong vitality hybridoma cells;
d. Spleen cells from normal mouse.
2. Method:
a. Preparation of feeder layer cells. Method is same as previous.
b. Prepare the cloned hybridoma cells suspension. Dilute the cells suspension to 10 cells per ml with containing 20% serum HT medium, 100 cells per 10ml HT medium. Drop them into well prepared feeder layer cells.
c. 37 ℃, 4% CO2 cultured for 7-10 days．Detect antibody once there appears visible clone. Observe them under an inverted microscope and mark with the hole of single clone and take the supernatant for antibody detection.
d. If need be, we can also select some clone to do subcloning.
VI. Identification of physicochemical characteristics of monoclonal hybridoma
VII. Production of monoclonal antibody．
After obtaining a stable hybridoma cell line, we can produce monoclonal antibody in bulk for different purposes.
1. Ascites produce method
Take the same batch adult Balb/c mouse, about 20g or more.
Method is as below:
a. Liquid paraffin intraperitoneal inoculation, each mouse 0.3-0.5ml.
b. After 7-10 days, intraperitoneal inoculation with PBS or serum -free medium hybridoma cells, each mouse 3×105/0.5ml.
c. 5 days later, observe the mouse daily. If its abdominal swell is obvious and skin is tension when touching, ascites can be collected with a needle. Normally it can be collected 2-3 times, 5-10ml per mouse every time; Or collected one time and kill it.
d. Centrifuge the ascites 3000r/min for10minutes. Remove cellular components and other precipitates, then collect the supernatant to test antibody titers. On top of，separate the monoclonal antibody from supernatant. Finally, split charging and store it -70℃ or lyophilized for further use.
VIII. Preservation of antibody
Add 40-50% glycerol to antibody and stored -20℃.
Protein antigens can be directly used as an immunogen while it is necessary to reorganize chemical structure of polypeptide hapten (or small molecule compound) .
1. mcKLH conjugation with sulfo-SMCC
Add 2ml ultrapure water into 10mg mcKLH so that we get 5mg/ml solution of the mcKLH. And then prepare 2.5mg/ml solution with
5mg Sulfo-SMCC dissolved in ultrapure water.Mix the two solutions together for equivalent volume and stirred at room temperature for 2 hours. Finally, in order to remove the excess Sulfo-SMCC, dialyze the complexs in PBS.
2. Polypeptide(or small molecule compound) hapten conjugation
Weigh 10mg polypeptide and dissolve it in 5mL conjugate buffer. Then mix polypeptide with the activated carrier protein and stirred at room temperature for 2 hours. Finally, dialyze the complexs in PBS.
II. Mouse immune
1. Preparation
Select healthy, six weeks, Balb/c female mice.
2. Antigen injection
a. The immunizing dose is 100μg/0.2ml for each mouse, 100μg is appropriate for per mouse for the first time, or less dose will get better results.
b. Emulsify 0.1ml Freund’s complete adjuvant and 0.1ml antigen fully.
c. Immune period is 2-3 weeks. Total times of immunization is 4-5 times.
Immunization program:
| 0 day | 1st immunization |
| 28 day | 1st enhanced immunization |
| 42 day | 2nd enhanced immunization |
| 49 day | Blood and test |
| 56 day | 3rd enhanced immunization or tail vein injection |
Detect antibody titers in the immune serum by indirect ELISA method.
a. two reference: Negative reference is not been immuned serum of animals while positive serum or cell supernatant is as the positive reference. Dilute both according to first antibody initial concentration.
b. Add 2ug/ml coated antigen into each well of 96-well plate, stay overnight at 4℃ or incubate 2 hours in 37℃.
c. Discard the liquid, add 200ul blocking solution into each well, stay overnight at 4℃ or incubate 2 hours in 37℃.
d. Throw away the blocking solution, wash 3 times with the washing buffer and air the plate or 37℃ drying. Seal it with the hermetic bag and store at -20℃.
e. Add 100ul primary antibody to each well. If it is hybridoma cell, the primary antibody is cell supernatant. If it is the immunized animal’s serum, it needs a series of dilution(diluted in blocking buffer). Usually, the initial dilution ratio is 1:500. Then do the double dilution and incubate 1 hour at 37℃.
f. Wash the plate 3 times. Same operation as above.
g. Add appropriate HRP labeled anti-mouse antibody, incubate 45 minutes at 37℃.
h. Wash the plate 3 times. Same operation as above.
i. Add 90ul TMB substrate solution into each well, incubate 15-20 minutes at 37℃.
j. Add 50ul stop solution into each well.
k. Read at 450nm immediately.
III. Fusion of hybridoma cell
1. Preparation
a. Culture of SP2/0. About two weeks before fusion, recover the cells and culture them till they are in logarithmic growth phase. Myeloma cell suspension preparation is as below:
b. 48-36 hours before fusion, amplification culture of the myeloma cells.
c. on the day of fusion, blow the cells gently with tips and collect the cells in a 50ml centrifuge tube .
d. 1000r/min centrifugation for 5-10 minutes and discard the supernatant.
e. Add 30ml incomplete culture medium, wash once with the same method of centrifugation. Then resuspend the cells in 10ml incomplete culture medium and mix until it is well-distributed
f. Take the myeloma cell suspension and use 1mg/ml trypan blue dye for living cells count. While the cells is counted, 0.5ml cell suspension is added into 0.5ml trypan blue dye, mix them and count with hemocytometer. The number of cells is calculated as follows: the number of cells per milliliter = 4 large squares cells ×104/4×2.
2. Keep ultraviolet irradiating room overnight.
3. Preparation of feeder cells. Normally，it needs to be prepared the day before. Take normal BALB/c mouse, remove its eyeballs and collect the blood. Then separate serum from the blood as negative reference .Kill the mouse by cervical dislocation and dip into 75% alcohol for 5 minutes. Fix it on dissecting table, dissect with the abdominal skin on the left side. Spleen can be seen. Take out the spleen and place it in plate and carefully strip the surrounding connective tissue. Grind the spleen with a plunger to disperse the cells. Wash the cells many times with 10ml incomplete culture medium and made it into a single cell suspension. Transfer it into 50ml centrifuge tube, 1000r / min centrifugation for 5-10 minutes. Wash 1-2 times with incomplete culture medium and resuspend it in 10ml incomplete culture medium. Then take the above 0.5ml cell suspension, add 0.5mltrypan blue dye as living cell count. Usually, each mouse can obtain 1×108-2.5×108 spleen cells. Add the above cell suspension into 96 wells, each well 0.1ml and set it in 37℃ 4% CO2 incubater overnight and reserve for next step.
4. B lymphocytes preparation. Take a immunized BALB/c mouse, remove its eyeballs and collect the blood. Separate serum from the blood as positive reference. Kill the mouse by cervical dislocation and soaked in 75% alcohol for 5 minutes. The method of obtaining spleen and preparation of feeder cells are the same.
5. cell fusion and Selective culture of hybridoma cells.
a. Mix 1×108 spleen cells with 2×107-5×107 SP2/0 myeloma cell in a 50ml tube, supplement the incomplete culture medium to 30ml and mix well.
b. 1000r / min centrifugation for 10 minutes. Absorb the supernatant fully.
c. Tap bottom of the tube to make the cells loose and homogeneous.
d. Gently turn the centrifuge tube while add 1ml pre-heat PEG within one minute (the best time is 45 seconds).
e. Add 10ml incomplete culture medium which has been heated to 37℃ within 90 seconds，then place quietly 10 minutes in 37℃
f. 1000r/min centrifugation for 5 min and remove the supernatant.
g. Tap the bottom of the tube to make the cells loose. Rotate the centrifuge tube while adding 50ml 37℃ preheated HAT medium. Then transfer to big bottle to make the volume reach 100ml.
h. Add it to prepared 96 wells feeder cell culture plate, each well 0.10-0.15ml and culture it in 37 ℃ 4% CO2 incubator.
i. 5 days later, use HAT medium to exchange half of the medium.
j. 7-10 days later, the HAT medium was replaced with HT medium.
k. Observe the growth of hybridoma frequently, aspirate the supernatant for antibody detection when it grows up to 1/10 of the bottom of the plate.
IV. Screening
Primarily screened by ELISA method and its screening programs based on different projects.
V. Hybridoma cloning
The positive hybridoma cells obtained from the original wells and it might be derived from two or more hybridoma cells, so their secreted antibodies are not aiming at the same antigen epitope. In order to obtain completely the same homogeneous monoclonal antibody, hybridoma cells have to be cloned. There are many cloning methods and we mainly use limiting dilution method.
1. Materials:
a. 96 wells cell culture plate,etc;
b. HT medium;
c. Strong vitality hybridoma cells;
d. Spleen cells from normal mouse.
2. Method:
a. Preparation of feeder layer cells. Method is same as previous.
b. Prepare the cloned hybridoma cells suspension. Dilute the cells suspension to 10 cells per ml with containing 20% serum HT medium, 100 cells per 10ml HT medium. Drop them into well prepared feeder layer cells.
c. 37 ℃, 4% CO2 cultured for 7-10 days．Detect antibody once there appears visible clone. Observe them under an inverted microscope and mark with the hole of single clone and take the supernatant for antibody detection.
d. If need be, we can also select some clone to do subcloning.
VI. Identification of physicochemical characteristics of monoclonal hybridoma
VII. Production of monoclonal antibody．
After obtaining a stable hybridoma cell line, we can produce monoclonal antibody in bulk for different purposes.
1. Ascites produce method
Take the same batch adult Balb/c mouse, about 20g or more.
Method is as below:
a. Liquid paraffin intraperitoneal inoculation, each mouse 0.3-0.5ml.
b. After 7-10 days, intraperitoneal inoculation with PBS or serum -free medium hybridoma cells, each mouse 3×105/0.5ml.
c. 5 days later, observe the mouse daily. If its abdominal swell is obvious and skin is tension when touching, ascites can be collected with a needle. Normally it can be collected 2-3 times, 5-10ml per mouse every time; Or collected one time and kill it.
d. Centrifuge the ascites 3000r/min for10minutes. Remove cellular components and other precipitates, then collect the supernatant to test antibody titers. On top of，separate the monoclonal antibody from supernatant. Finally, split charging and store it -70℃ or lyophilized for further use.
VIII. Preservation of antibody
Add 40-50% glycerol to antibody and stored -20℃.
