Monoclonal Antibody Technology Operation Process---Animal Immunity

Introduction

Monoclonal antibody technique Invented by British scientists Milstein and Kohler in 1975 and won the 1984 Nobel Prize in Medicine. 1984 German G. J. F. Kohler, Argentine C. Milstein and Danish scientist N. K. Jerne shared the Nobel Prize in Physiology and Medicine for the development of monoclonal antibody technology and improved detection technology for very small amounts of protein. The principle is: B lymphocytes can produce antibodies, but incapable of infinite division in vitro; while tumor cells can be infinitely passaged in vitro, but can not produce antibodies. The hybridoma cells obtained by fusing these two cells have the characteristics of two parental cells. The immune response is an important factor in human resistance to disease. An antibody can be produced when the animal is stimulated by an antigen. The specificity of an antibody depends on the determinant of the antigen molecule, and various antigen molecules have many antigenic determinants. Therefore, the antibody produced by the immunized animal is a mixture of various antibodies. The preparation of antibodies by this conventional method is inefficient, limited in yield, and the injection of animal antibodies into the human body can produce severe allergic reactions. In addition, it is extremely difficult to separate these different antibodies. In recent years, the emergence of monoclonal antibody technology is a major breakthrough in the field of immunology.

Animal immunity

Preparation of antigen

The immunological antigen for preparing a monoclonal antibody sequence database is not required to be high in purity, but the high-purity antigen increases the chance of obtaining the desired monoclonal antibody and at the same time reduces the workload of screening. Therefore, the purer the immune antigen, the better, and should be determined based on the antigen being studied and laboratory conditions. In general, the source of the antigen is limited, or the nature is unstable, it is apt to be denatured during purification, or its immunogenicity is strong, or the desired monoclonal antibody is used for purification or analysis of different components of the antigen, and the antigen for immunization is only Initial purification is not required, but there are many impurities in the antigen, especially if the immunogenicity of these hybrids is strong, the antigen must be purified. The antigen for detection may be of the same purity as the immunizing antigen, or may be of different purity depending on the type of screening method used and its specificity and sensitivity.

The choice of immune animals

Mice and rats can be used as immunized animals depending on the myeloma cells used. Because all mouse myeloma cell lines for hybridoma technology are derived from BALB/c mice, all rat myeloma cells are derived from LOU/c rats, so the general hybridoma production is used. These two pure animals are used as immunized animals. However, sometimes interspecific hybridization is required for special purposes, and other animals can be immunized. Interspecies hybridomas generally have an unstable ability to secrete antibodies because chromosomes are easily lost. In the case of mice, it is preferred to use 8-12 weeks of age for the first immunization, and female rats are more convenient to handle.

Determination of the immunization program

Immunization is one of the important steps in the preparation of monoclonal antibodies. The purpose is to differentiate and proliferate B lymphocytes under the stimulation of specific antigens, so as to facilitate cell fusion to form hybrid cells and increase the chance of obtaining hybridomas secreting specific antibodies. Therefore, in designing the immunization program, the nature and purity of the antigen, the amount of antigen, the route of immunization, the number and timing of immunizations, the application of the adjuvant, and the ability of the animal to respond to the antigen should be considered. No immunological procedure can be applied to a variety of antigens. Most of the current immunization procedures are based on methods for preparing conventional polyclonal antibodies. Table 6-1 lists the currently used immunization procedures. Immunization pathways commonly used in vivo immunoassays include subcutaneous injections, intraperitoneal or intravenous injections, as well as footpads, intradermal, nasal drops or eye drops. The last booster is usually administered intraperitoneally or intravenously, and the latter is especially recommended because it allows the antigen to act more rapidly and fully on spleen cells. Spleen fusion was good on the third day after the last booster immunization. The results of many laboratories showed that in the primary immunization and re-immune response, the spleen cells were fused with myeloma cells, and the peaks of specific hybridomas were respectively On days 4 and 22, the hybridomas obtained at the time of the primary immune response mainly secreted IgM antibodies, and the hybridomas obtained in the re-immunization response mainly secreted de novo igg sequencing. The authors realized that there was no significant parallel relationship between the peak of positive hybridoma and the titer of serum antibody in mice, and it was mostly before the peak of serum antibody. Therefore, in order to achieve the highest rate of hybridoma formation, it is necessary to have as many plasmablasts as possible, which is preferable to spleen for fusion on the third day after the last booster immunization. It has been reported that intrasplenic immunization can increase the immunoreactivity of mice to antigens and save time. Generally, it can be fused after 3 days of immunization.

Monoclonal antibody drugs have become the most important class in biopharmaceuticals, and monoclonal antibodies will also be an important way for the future development of the biopharmaceutical industry.