1. What is Monoclonality Identification of Cell Lines
The monoclonality of cell lines refers to the tracing of the produced antibody to determine that it is a highly uniform antibody produced by single-cell cloning.
2. Purpose ofMonoclonality Identification
In recent years, recombinant protein drugs have found significant applications in the treatment of diseases, especially neoplastic disease, and most recombinant protein drugs are produced by the expression of mammalian CHO cells. Drug evaluation units require research and development enterprises to issue the monoclonality of the cells used in the drug, so the monoclonality identification of cell lines is an important means to ensure the consistency of protein drugs.
3. Monoclonal Acquisition Methods
Clonality is considered to reduce the heterogeneity of the cell pool, commonly used monoclonal acquisition methods, such as: limited dilution method, ClonePix, flow cytometric fluorescence sorting technology, and new monoclonal selection technology, etc.
3.1 Limiting Dilution Cloning (LDC)
Limited dilution cloning is a common cloning method, requiring seeding density of less than 0.5cell/well.The method has the advantages of simple operation and low requirement on equipment. The disadvantages are long time consumption, low efficiency, and high dependence on manpower. It is easily affected by experimental operators, experimental environment, conditions and other factors to produce different results.
3.2 ClonePix Method
ClonePix is a high-throughput monoclonal screening equipment. This method has the advantages of less manual intervention, high flux, high efficiency and high screening accuracy. However, compared with the limited dilution method, the difference between the monoclonal medium screened by ClonePix method and the later generation process medium is larger, and the performance of clones by limited dilution method in the plate is closer to the state of production.
3.3 Fluorescence-Activated Cell Sorting (FACS)
Fluorescence-activated cell sorting (FACS) is used to select the desired single cells based on factors such as cell size, fluorescent dye and cell surface marker. The method has the advantages of high monoclonal formation rate, and the disadvantages that the selected cells are in one-to-one correspondence with the wells of the well plate through condition optimization, and flow cytometry and cell morphology and state influence the separation effect.
3.4 New Techniques for Monoclonal Selection
There are a variety of monoclonal screening equipment available on the market, such as single cell printing, Solentim VIPS, etc., which can improve efficiency and reduce the reliance on manual. At present, the monoclonal antibody obtained is basically the repeated combination and use of the above methods.
4.Monoclonality Identification by Fluorescence In Situ Hybridization.
The use of fluorescence in situ hybridization technology to detect whether the cells are of single origin, the results are visualized and convincing.
Fluorescence in situ hybridization is a technique for the qualitative, quantitative or relative positioning analysis of DNA using a fluorescence detection system. The basic principle is to hybridise a known fluorescein, biotin or digoxigenin-labelled nucleic acid probe with the DNA of the sample to be tested, based on the complementarity of the DNA sequence, to form a detectable double-stranded nucleic acid hybridization.
5. Advantages of Fluorescence In Situ Hybridization Technology
Fluorescence in situ hybridization has advantages over other hybridization methods:
(1) FISH is non-radioactive element labelling and is safer;
(2) The experimental cycle of FISH is short and multiple sequences can be detected simultaneously;
(3) The sensitivity of FISH is comparable to that of radioactive probes because the hybridization signal is enhanced by multiple immunochemical reactions.
6. KMD Bioscience can Provide Customers with High-Quality Monoclonality Identification Services
KMD Bioscience has been dedicated to cell biology research for many years. We have a team of experienced scientific experts, a skilled laboratory team, a complete cell culture platform and complete experimental equipment and conditions, and have built a complete fluorescence in situ hybridization technology service platform and accumulated a lot of successful experience. With our solid expertise, we can provide a full range of services from probe design to monoclonality identification of cells in a customized manner, aiming to solve problems for our customers in an efficient and cost-effective manner.
7. Introduction of Fluorescence In Situ Hybridization Service Process
KMD Bioscience can provide fluorescence in situ hybridization services, the main technical processes are as follows: probe design and synthesis, sample processing, in situ hybridization experiments, imaging and photography, result analysis and project reporting.
7.1 Probe Design and Synthesis
7.1.1 Ready-to-use probes can be hybridised directly, while non-ready-to-use probes need to be diluted with the hybridization solution at a ratio of 1:1:1:50, denatured at 85°C for 3 minutes and equilibrated at 37°C for 5 minutes.
7.2 Sample Processing
7.2.1 Dewaxing and rehydration, paraffin sections immersed in xylene dewaxing , 2 times for 10 minutes each;
7.2.2 Slices are sequentially passed through 100%, 85% and 75% ethanol for 3 minutes each and PBS for 3 minutes;
7.2.3 Slides are digested with pepsin digestive solution at 37°C for 15-30 minutes, and washed with PBStwice for 3 minutes each time;
7.2.4 Pre-hybridization: remove the hybridization solution from the refrigerator 30 minutes in advance to restore it to room temperature, and the hybridization solution was added dropwise for incubation at 55 C for 1 hour..
7.3 In Situ Hybridization Experiment
7.3.1 Taking out the pre-hybridized slices, removing excess liquid, adding the probe dropwise to the slices, cover the tissue completely and place them in the hybridiser overnight at 37°C;
7.3.2 Washing the slices 3 times for 5 min each in 2 x SSC (0.1% NP-40);
7.3.3 Adding 20ul of DAPI-Antifade Solution dropwise to seal the slides and incubate for 20min away from light.
7.4 Imaging and photography
Photographs of the results of the fluorescence in situ hybridization experiments for different samples of KMD Bioscience:
7.5 Results Analysis and Project Report
KMD Bioscience delivers: the remaining probes, sections and samples, original hybridization imaging photographs, and a detailed report (including a full procedure and analysis of the results).
In conclusion, monoclonality identification is the premise and guarantee of the quality consistency of recombinant protein drugs, and the lack of evidence of monoclonality identification at the time of drug declaration will lead to failure. KMD Bioscience has been involved in the monoclonality identification for many years and can provide key data for the drug reporting process using fluorescence in situ hybridization. KMD Bioscience can detect 100-200 cells, and the service period is short, which is helpful for the enterprise to declare drugs.
