What is used to fuse hybridoma cells?
Hybridoma technology is one of the core methods for preparing monoclonal antibodies. The key is to fuse immune B cells with myeloma cells to form hybridoma cells. This article will conduct a structured analysis around the fusion methods, key reagents and operating procedures of hybridoma cells, and attach relevant data comparisons.
1. Core methods of hybridoma cell fusion
| 融合方法 | 原理 | Advantages | limitations |
|---|---|---|---|
| PEG-mediated fusion | Polyethylene glycol destabilizes cell membranes | 成本低、操作简单 | The fusion rate is low (about 1×10⁻⁵) |
| 电融合法 | High-voltage pulses perforate cell membranes | High fusion efficiency (up to 80%) | Equipment is expensive and technical requirements are high |
| 病毒介导融合 | Sendai virus induces membrane fusion | 自然融合过程 | Biosafety risks, poor reproducibility |
2. Performance comparison of commonly used reagents
| 试剂类型 | Typical products | concentration | Action time |
|---|---|---|---|
| PEG solution | PEG1500 | 50%（w/v） | 1-2 minutes |
| 电融合缓冲液 | 甘露醇溶液 | 0.3M | Pulse time 10μs |
| Screening media | HAT medium | 1× | 持续培养 |
3. Key steps of the operation process
1.Cell pretreatment: Immune spleen cells and myeloma cells are mixed at a ratio of 5:1 and centrifuged to remove serum proteins.
2.fusion reaction: Use gradient centrifugation method to gradually add PEG solution, and accurately control the reaction time in a 37°C water bath.
3.selective culture: Use HAT medium (containing hypoxanthine-aminopterin-thymidine) to screen hybridoma cells.
4.clonal culture: Obtain monoclonal cell lines through limiting dilution method, and detect antibody secretion by ELISA.
4. Technical optimization direction
| Optimize dimensions | Specific measures | expected effect |
|---|---|---|
| Fusion efficiency | Optimization of pulsed electric field parameters | Increased to over 85% |
| cell activity | Add antioxidants | Survival rate increased by 30% |
| Screening speed | Automated clone picking | Reduce working hours by 50% |
5. Latest trends in the industry (hot spots in the past 10 days)
1. The latest report in "Nature Protocols" shows that the hybridoma formation efficiency can exceed 90% using microfluidic chip fusion technology.
2. A Shanghai biological company released a new type of electrofusion instrument. Its patented waveform design controls cell death rate below 5%.
3. CRISPR gene editing technology has begun to be applied to the transformation of myeloma cell lines, significantly improving the stability of antibody secretion.
In summary, hybridoma cell fusion technology is developing towards high efficiency and standardization. Researchers need to make a reasonable choice between the traditional PEG method and emerging electrofusion technology based on experimental needs and equipment conditions.
check the details
check the details
en
