Hybridoma screening FAQ
1. The clone was originally positive, but why did it turn negative?
Answer: The first thing to note is that under normal circumstances, hybridomas are not absolutely stable, and chromosome loss is indeed prone to occur, especially when cells are moved to a new environment for growth, such as expanding from small pores to large pores, or during recovery operations. , It is more likely to change from positive to negative. But there are also some ways to minimize the number of positives becoming negatives. Generally, attention can be paid from these aspects:
(1) Always ensure that the cells are in the best growth environment. Especially the medium cannot be changed for a long time. Generally speaking, when the cells grow to a certain density, the medium will start to change color. At this time, it is necessary to change the medium. In addition to changing the medium, the cells should be controlled well. Density, can discard too many cells, so that the newly added medium will not be quickly consumed.
(2) For important cell lines, keep the positive cells in seed at every step.
(3) Do not operate the cells too frequently. When the cell growth density is not very high, do not operate frequently, otherwise the cells are prone to death or obvious changes in growth morphology.
(4) For cell lines with a large number of passages, frequent subcloning is required, and each subcloned strain should be frozen for seed preservation.
(5) When the cells are contaminated by microorganisms such as mycoplasma, it will also turn from positive to negative.
2. The reason why no positive clones can be obtained after hybridoma cell fusion?
A: This is a monoclonal antibody preparation equipment experiment most vexing problems, possible causes are:
(1) non-immunized animals were fused success.
(2) There are fewer clones after fusion, so the probability of getting positive clones is also smaller.
(3) The method of screening clones is incorrect. For example, some small molecules cannot be directly coated on the ELISA plate, and there are many factors such as the use of inappropriate secondary antibodies. Some people directly do not use ELISA as a screening method, and the success rate remains to be determined.
(4) The antigen component is the same or similar to the substance in the cell culture condition, or it has a competitive reaction with the substance with the same or similar antigen structure in the screening process. For example, if it is necessary to prepare anti-BSA monoclonal antibodies, when performing ELISA screening test, do not use the sample diluent containing BSA for blocking or diluting the secondary antibody operation, otherwise the BSA in the bovine serum will directly bind to the antibody, and finally perform ELISA screening No positive results can be obtained at the time. The solution is to use the serum of other animals or use serum-free medium. For another example, if it is necessary to prepare a casein monoclonal antibody, skimmed milk powder cannot be used as the diluent of the secondary antibody during ELISA screening.
(5) All other factors that can affect related screening methods such as ELISA.
3. Why can't my hybridoma cells prepare ascites or the prepared ascites has no antibody or ascites has no titer?
Answer: Most of the reasons for the failure to prepare ascites are man-made:
(1) Incorrect sensitization, such as using incorrect sensitizers, or the sensitization time is too long from the time of hybridoma inoculation.
(2) The inoculation position of the hybridoma was not correct, and it did not hit the abdominal cavity, but hit the subcutaneously.
(3) The number of hybridoma cells inoculated is too small or the cell condition is not good. Generally, it should not be less than 100,000 cells, and it is recommended that it be around 1 million cells.
(4) The strains of mice with ascites fluid are too far away from the animal strains of the cell sources involved in the fusion, so that the mice with ascites have a strong immune response to hybridomas and kill them. It is recommended to replace the mouse strains and re- Vaccination.
There is no titer for ascites. Possible reasons:
(1) The hybridoma for ascites is extremely unstable. It loses the ability to secrete antibodies before hitting mice or loses secretion ability when hitting the abdominal cavity.
(2) The wrong sensitizer is used when sensitizing the mice. For example, if Freund's complete adjuvant is used, the mice will have ascites even if the hybridoma is not used.
(3) It is extremely rare that the antibody produced by this hybridoma can interact with the molecules in the mouse body, so the antibody produced by the hybridoma is neutralized by the relevant protein of the mouse. In this case, the body of the mouse may be neutralized. There will be obvious abnormalities.
(4) There is a problem with the experimental protocol for detecting ascites titer, or the ascites dilution ratio is too large.
4. How to solve the reason why the titer is low or even there is no titer after immunization?
Answer: You can think from the following directions:
(1) The designed antigen has extremely high homology with the endogenous protein of the immunized animal or the antigen is a small molecule substance with extremely poor immunogenicity. In the former case, the antigen should be redesigned. When designing the antigen, try to select the specific sequence of the target protein. It can be designed as a short peptide and then coupled with the carrier protein for immunization; in the latter case, first confirm whether the small molecule substance has been Correctly couple with the carrier protein. If there is no problem with the coupling, you can replace other carrier proteins. Generally speaking, the BSA-coupled small molecule is used as the immunogen, and the OVA is coupled as the test source.
(2) The immunization cycle is incorrect. If the immunization cycle is too long or too short, and the interval between each immunization step is too long or too short, it may affect the immune effect. The immunization interval is generally 2 to 3 weeks.
(3) Immunity adjuvant is inappropriate. Certain adjuvants may not be effective for certain antigens during immunization, and Freund’s adjuvant cannot be guaranteed to be completely effective. At this time, you can consider changing the adjuvant.
(4) Immunization dose is unreasonable. Too large immunization dose may lead to immune tolerance, and too small immunization dose may not activate immune response. Generally speaking, the actual immunization dose should not differ by more than two times from the standard dose.
(5) The immunization route is unreasonable. For some weakly immunogenic antigens, intrasplenic immunization can be considered.
(6) There are errors in the process of detecting potency, especially considering whether the coating is successful or whether the secondary antibody is used correctly. At the same time, the dilution gradient of the primary antibody (ie serum) should be relaxed as much as possible. Generally, it is recommended to start the gradient dilution from 1:500 or 1:1000. For small molecule substances, a titer of 1:2000 can still be regarded as successful immunity.
5. Why do cells fail to grow after fusion or there are few clones after fusion?
Answer: This question is something we should pay attention to in the monoclonal antibody preparation experiment. It may be caused by the following reasons, which can be investigated one by one.
(1) The animal strain used for immunization is incorrect or impure. The animal used for immunization should generally be of the same strain as the animal from which the myeloma is derived. For example, when using SP2/0 myeloma cells, Balb/C mice should be used, and mice of pure strain must be used.
(2) If too high a concentration of HAT is added to the medium, or only the concentration of A is too high or the concentration of HT is too low, it is recommended to use pure myeloma and existing hybridomas for HAT concentration screening.
(3) Incorrect culture medium or incorrect serum concentration or poor quality serum.
(4) Feeder cells were not prepared correctly.
(5) After fusion, cells were not transferred or diluted in time. After fusion, some people like to put the fused cells in a culture flask and then drop them onto a 96-well plate. If the culture time is too long in the culture flask, the cloning rate may be low.
(7) The cells are contaminated. Observe carefully under the microscope whether there is obvious microbial contamination. Even if there are no obvious microorganisms, you should consider whether there is mycoplasma contamination. If possible, you can do mycoplasma detection.
(8) The cell culture conditions are incorrect. Ensure that the cells are cultured in a constant 37°C humid environment, while ensuring that the CO2 concentration is around 4-5%.