1. What is a neutralizing antibody?

A: When foreign matter enters the body, the immune system in the body will work quickly to produce corresponding antibodies, which will combine with foreign matter to protect the body from interference from foreign matter. Neutralizing antibodies are produced when foreign matter invades the body and can bind to antigens on the surface of foreign matter. Through binding, they prevent foreign viruses from recognizing the body's receptors, thereby blocking the virus from invading cells. Some scientists also call a class of antibodies that can bind to a specific protein and interfere with its function neutralizing antibodies. When pathogens invade the body, they need to recognize the ligands on the body through the specific structure of the pathogen itself, so that the pathogen can infect the body and cause the body to become ill. Neutralizing antibodies are a water-soluble protein produced by the body's own cells in the process of adapting to immunity. When a pathogen invades the body, the body's immune cells are stimulated and secrete neutralizing proteins, and then the neutralizing antibodies are transported to various parts of the body through blood transportation, so that they can bind to the pathogen, prevent the virus from binding to the body, and neutralize the virus.

Production of neutralizing monoclonal antibodies

2. In the development of neutralizing monoclonal antibodies, we may encounter the situation that there is no titer or low titer after immunizing animals. How do we solve it?

A: In the development of neutralizing monoclonal antibodies, we select healthy animals that do not carry pathogens as immunogens, but we may encounter the situation that the molecular weight of the selected antigen is not suitable or the immunogenicity of the antigen is not suitable, which leads to no titer or the titer of Elisa test after animal immunization does not reach the expected target. At the same time, the choice of immune adjuvant, different immunization procedures, the amount of immunogen dose, and whether the immunization method is oral or intramuscular or subcutaneous will also affect the titer of neutralizing antibodies in our serum. If we encounter similar situations as mentioned above, we can first optimize our antigen. If the antigen is a small molecule, we can couple it with a large molecule to increase the immunogenicity of our antigen and ensure that the molecular weight of our antigen is not less than 25kDa. At the same time, we can also replace different immune adjuvants. In addition to the frequently used Freund's complete adjuvant and incomplete adjuvant, we can also use other new adjuvants to try to increase immunogenicity. At the same time, our immunization program should be redesigned according to the different immunogens and adjuvants. It cannot be the same for everyone. If the titer has not reached the expected target after three immunizations, we can conduct a booster immunization to increase the titer. At the same time, we can adopt multi-site immunization such as multi-point back injection, intraperitoneal injection, and intramuscular injection to increase the antibody titer. TechBio has been committed to antibody production research for many years and can provide customers with quality-assured antibodies. TechBio can prepare neutralizing antibodies with high titer, strong specificity and good stability.

3. What is the working principle of neutralizing antibodies?

A: Neutralizing antibodies use the method of preventing pathogens from invading the human body by preventing receptors and cells on the surface of pathogens from recognizing and then entering cells. When the virus has an envelope, neutralizing antibodies can prevent the virus from adsorbing on cells and entering cells. When the virus has no envelope, neutralizing antibodies can bind to the capsid protein of the virus, which is the protein shell of the virus and can protect the genetic material of the virus. Neutralizing antibodies can also change the structure and shape of pathogens, and then prevent the virus from entering cells to replicate and infect the body. In microbial infections, neutralizing antibodies can quickly eliminate the effects of toxins. Once the virus and neutralizing antibodies combine, the pathogen will be engulfed by white blood cells, and the spleen of the body can automatically filter the pathogen, so that the metabolites of the pathogen are excreted from the body through the urinary system. Neutralizing antibodies are generally used to treat diseases in the body through passive immunity. TechBio has been committed to antibody production research for many years, and can provide customers with quality-assured antibodies and recombinant protein products and services, and can prepare antibodies with high titer, strong specificity and good stability. Based on the construction of a complete platform system such as antibody platform and protein platform, TechBio covers the upstream and downstream services of antibody production, and can realize technical services such as antibody preparation, antibody affinity purification and antibody separation and purification, and antibody sequencing.

4. In the neutralizing monoclonal antibody development service, there is no hybridoma cell after cell fusion and the cell does not grow. How do we solve it?

A: In the preparation service of neutralizing monoclonal antibodies, the most important step is to complete the fusion of SP20 cells and spleen cells. After the fusion of the two, the hybridoma cells produced can continuously produce neutralizing antibodies. After the fusion of SP20 cells and spleen cells, we may encounter the situation that the hybridoma cells do not grow or grow very little after fusion. The reason may be that the culture medium and serum we use when culturing hybridoma cells are not suitable for growth, or the fusion reagent PEG we use during fusion is of poor quality or insufficient concentration, resulting in the failure of cell fusion. At the same time, the selection of mouse strains for preparing monoclonal antibodies is also one of the key factors. When hybridoma cells grow, the inappropriate time and concentration of HAT and HT we add will also lead to fusion failure. For the above reasons, how do we solve the situation that hybridoma cells do not grow or grow very little after fusion? TechBio selects suitable culture medium and serum, selects PEG with quality assurance, and TechBio has rich experience in neutralizing antibody projects, which can help customers better complete the preparation of neutralizing antibodies. TechBio selects pure strains of balb/c mice as immune animals, selects appropriate HAT and HT concentrations, and promotes the growth of hybridoma cells. At the same time, TechBio has its own biological cell room.

5. In the neutralizing monoclonal antibody development service, how to screen and identify antibodies, and how to purify antibodies?

A: In the neutralizing monoclonal antibody development service, after the cells are fused and grown to occupy two-thirds of the bottom of the well, we perform Elisa detection on the supernatant of the fused hybridoma cells, and then select the positive clone wells to continue subcloning, but it may happen that no positive clones are detected after fusion or all positive clones are detected. At this time, we can first think about whether there are errors in our Elisa steps, set up PBS, negative control and positive control groups to make sure that there are no errors in our experimental operations, and then we can add blank culture medium to detect whether the serum affects our experimental results. Finally, if the hybridoma cells are too small and the secretion of insufficient antibodies may also lead to insufficient titer, we can wait for the fused cells to grow again for a period of time. Depending on the source and the method of monoclonal antibody preparation, monoclonal antibody samples are divided into different types, such as ascites, hybridoma supernatant, etc. The sample obtained by injecting hybridoma cells into the abdominal cavity of animals to produce antibodies is called ascites sample. The concentration of monoclonal antibodies accumulated in ascites is usually high, which is suitable for large-scale antibody production and purification. Antibody purification methods include Protein A/G purification, ion exchange chromatography, gel filtration, precipitation, and hydrophobic interaction chromatography.