High-affinity monoclonal antibody preparation problems and countermeasures
1. Monoclonal Antibody Preparation - Problems and Countermeasures in the Process of Myeloma Cell SP2/0 Culture
1, slow cell growth or cell death
Under normal growth conditions, the cells are passaged once a day, the better the growth, the more the adherence measures: 1 the medium formula is wrong; 2 the seeding density is too low, less than 10 to the 4th power; 3 contaminated mycoplasma or other unknown bacteria, mycoplasma The current situation is the emergence of small black spots; bacterial contamination is turbid; mold contamination is the appearance of white visible colonies; unknown bacterial contamination appears in a sand-like tiled background, the field of view is dark. 4 cell bottle is not cleaned
2, abnormal cell morphology
Under normal growth conditions, the cells are round, translucent, and grow in pairs, the better the growth, the more adherence, the less suspension: 1 medium formula is wrong; 2 the seeding density is too low, less than 10 4th power; 3 pollution Mycoplasma or other unknown bacteria, the status quo of mycoplasma is the appearance of small black spots; bacterial contamination is turbid; mold contamination is the appearance of white visible colonies; unknown bacterial pollution appears in the sand-like tiled background, the field of view is dark. 4 cell bottle is not cleaned
Monoclonal Antibody Preparation - Problems and Countermeasures in Cell Fusion Screening
1, the fusion rate is low, the number of positive holes is small
1 immune problem. Due to weak immunogenicity or improper immunization pathway, the immunity is weak and the titer is low.
2 temperature, time, PEG molecular weight, time of action, etc. during the fusion process.
3 Whether the spleen cells have taken out enough cells to interfere with tissue debris.
4 The pre-fusion myeloma cells are in good condition, and the cells are round, translucent and logarithmically grown.
2, the overall affinity of the monoclonal antibody is low
1 immune problem. Improper immune pathways or immunogenic problems.
2 The cloning cell screening process is missing, the screening method is improper or the detection method is improper, resulting in no fused to high affinity fusion cells.
3 Due to the inappropriate growth environment of the subcloned cells, the chromosomes of the fusion cells are lost, the secretion characteristics are changed, and the affinity changes from high to low. At this time, the culture solution needs to be changed in time. When the cells grow to a certain density, the medium begins to change color. It affects the growth of fused cells, causing chromosome loss; controlling the density of cells, the density is too large, so that the newly added medium will not be consumed quickly, affecting the growth of fused cells, causing chromosome loss; do not operate cells too frequently, when cells When the growth density is not very large, do not operate frequently, otherwise the cells are prone to death or the growth pattern changes significantly.
The operation is meticulous to prevent cells from being contaminated by microorganisms such as mycoplasma.
3, antibody affinity changes from high to low
1 The culture medium is not changed in time. When the cells grow to a certain density, the medium begins to change color, affecting the growth of the fused cells, causing chromosome loss or trait changes.
2 Control the density of the cells. If the density is too large, the newly added medium will not be consumed quickly, affecting the growth of the fused cells, causing chromosome loss or trait changes.
3 Operate the cells too frequently. When the cell growth density is not very large, do not operate frequently, otherwise the cells are prone to death or the growth pattern changes significantly.
4 cells are contaminated by microorganisms such as mycoplasma.
5 subcloning more than 5 times, resulting in a frequent change in the cell growth environment, weak positive hybridoma cells with chromosome loss or trait changes.
Third, the problems encountered in the preparation of monoclonal antibody ascites and countermeasures
1, less ascites or no ascites
1 sensitization is incorrect, incorrect use of sensitizer, or sensitization time and time to inoculate hybridoma are too long, usually 7-14 days, depending on the abdominal condition of the mouse.
2 The hybridoma cells or sensitizers were inoculated incorrectly, and they did not hit the abdominal cavity, but hit the skin.
3 The number of hybridoma cells inoculated is too small, too much or the cell state is not good. Generally, it is not suitable for about 500,000 cells.
4 It is recommended to use the mother or the mother who has given birth to prepare ascites. Â
2, ascites has no potency
1 The hybridoma for the preparation of ascites is extremely unstable. Before it hits the mouse, it loses its ability to secrete antibodies or loses its secretion capacity when it hits the abdominal cavity.
2 The sensitizer was used in sensitized mice.
3 The number of hybridoma cells inoculated is too small, or the cell state is not good.
4 Solid tumors are formed due to incorrect injection methods and locations.
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