Classification summary of IVD core raw materials!
1. The raw materials of in vitro diagnostic reagents can be divided into two categories:
One type is bioactive macromolecule raw materials that can specifically recognize the target molecule to be tested or catalyze its reaction. The other category is non-biologically active raw materials of various organic and inorganic small molecule substances that do not directly participate in the reaction.
In vitro diagnostic reagent raw materials refer to the reaction system raw materials used for biochemical, immunological or molecular diagnostic reagents, mainly referring to the core reaction system. According to different properties, in vitro diagnostic reagent raw materials can be divided into: antigens and antibodies, enzymes and coenzymes, and other raw materials. Upstream raw materials determine the detection quality of in vitro diagnostic reagents. It can be said that the selection and development of these key raw materials affect the performance levels (such as sensitivity, stability) and application value of in vitro diagnostic reagents. Screening out suitable raw materials is the key to R&D. the primary link. It is also the most important part of the cost of in vitro diagnostic reagents.
2. In vitro diagnostic reagent raw material classification system
So what are the main bioactive raw materials?
The first category is antigens: including proteins, polysaccharides, polypeptides, lipids, small molecule compounds, etc., including natural antigens and artificial antigens. Those obtained directly from biological tissues or cells are natural antigens, and antigens prepared through genetic engineering, chemical synthesis, etc. For artificial antigens.
The second category is antibodies, which mainly include monoclonal antibodies and polyclonal antibodies.
The third category is enzymes, which are collectively called tool enzymes in in vitro diagnostic reagent raw materials.
Other raw materials
Other raw materials include colloidal gold, enzyme substrate systems, luminescent substances and other signal systems, NC membranes, enzyme plate, magnetic beads and other reaction system carriers, as well as various biologically active materials and fine chemical raw materials, etc., which help to develop colors and provide Carrier location or improving the performance of raw materials to ensure stable and smooth diagnostic reactions.
3. Basic principles for the research and development of bioactive raw materials
The research and development of antigens, whether they are natural antigens or recombinant antigens, are the raw materials for preparing antigen in vitro diagnostic reagents, including prokaryotic/eukaryotic recombinant antigens, natural antigens, etc., through genetic engineering recombinant technology, protein purification technology, cell biology technology and chemistry It is prepared by synthetic technology and other methods and is suitable for diagnosis of infectious diseases, food safety diagnosis, diagnosis of digestive tract diseases, diagnosis of immune system diseases, etc. They are all obtained through purification, and the more important antigen molecules for the recombinant antigen are the design, expression vector and host selection.
The research and development of antibodies includes polyclonal antibodies and monoclonal antibodies, which are stored through immunological technology, hybridoma cell technology, cell fermentation technology and other methods, and are used for cardiovascular disease diagnosis, tumor diagnosis, infectious disease diagnosis, etc. Polyclonal antibodies are mainly optimized through immunogens and immunized animals, as well as immunization doses and immunization procedures. Monoclonal antibodies are relatively complex and require later hybridization technology, cell culture, cell screening, and re-immunization to obtain antibodies that recognize specific epitopes. Of course, many antibodies can be sequenced and obtained on a large scale through genetic recombination.
Enzymes and coenzymes are raw materials for preparing molecular diagnostic reagents, immunodiagnostic reagents and biochemical diagnostic reagents such as fluorescence quantitative PCR and genetic diagnosis, including reverse transcriptase, DNA polymerase, alkaline phosphatase, etc., and are widely used in genetic testing, tumor diagnosis, Infectious disease diagnosis, coagulation diagnosis, liver and kidney function biochemical diagnosis and other testing fields. Enzymes are also obtained mainly through purification. Therefore, protein purification technology and antibody preparation technology, as two key core technologies, are the key to the development of bioactive raw materials. It will involve biochemistry, molecular biology, immunology, microbiology, genetic engineering, protein engineering, enzyme engineering and other disciplines.
4. Large-scale preparation of bioactive raw materials
Including raw material preparation, pretreatment, primary purity and refinement.
Raw material preparation can directly select tissues or cells from animals and plants, or engineered cells and bacteria obtained through genetic engineering.
Raw material pretreatment can generally use mechanical methods such as high-speed homogenization, liquid nitrogen grinding or ultrasound to disrupt tissues and cells, or non-mechanical methods such as hypotonicity, repeated freezing and thawing, enzymatic digestion or surfactant treatment, but What should be noted is the crushing strength. If it is too small, the release will not be sufficient. If it is too large, it will cause the denaturation of the bioactive raw materials. The crushed raw materials will appear turbid and can be removed by high-speed centrifugation, membrane filtration, etc. or by precipitation with saturated ammonium sulfate.
The initial purity stage mainly uses precipitation and centrifugal filtration. Commonly used methods include ammonium sulfate precipitation, euglobulin precipitation, isoelectric precipitation and other non-denaturing precipitation methods, or denaturing precipitation methods using organic solvents, acids and alkalis, and heat. .
Purification is mainly based on various chromatographic separations based on molecular size, shape, surface charge distribution, hydrophobicity and specific binding properties, including ion exchange chromatography, molecular sieve exchange chromatography, affinity chromatography, hydrophobic chromatography and Separation by techniques such as electrophoresis.
5. Preservation of biologically active raw materials
Factors that affect the maintenance of biologically active raw material solutions include pH, ionic strength, contamination or residual proteases, storage temperature and the number of repeated freezing and thawing. The stability of raw materials can be maintained by adding a series of buffer systems, cryogenic storage, surfactants, protease inhibitors, preservatives, reducing agents, etc. The most commonly used technology here is freeze-drying.
6. Quality control of bioactive raw materials
The most critical aspect of quality control is to identify the concentration and purity of each batch of purified raw materials through Elisa, protein quantification, SDS-PAGE and other methods. The bioactive raw materials of the final in vitro diagnostic reagent should be of high purity, high activity and state. A uniform finished product should ensure appearance, concentration, purity, uniformity and biological activity.
7. Reasons affecting stability
Among the raw materials used in in vitro diagnostic reagents, proteins such as antibodies, antigens, and enzymes are the most important key raw materials. Its specific binding and catalytic reaction functions are very sensitive to potential harmful effects during pretreatment, storage, use and processing. Storage in a sealed, dry environment will help ensure long-term stability, but unless the protein is properly protected, the drying process may also cause serious denaturation of the protein. When stored in solution, potential harmful factors will increase. For example, in a very simple aspect, changes in pH value or buffer solution can make the stability of the protein in the solution significantly different. A method that stabilizes one solution may not necessarily be effective for another. Other factors in the production process may also affect product performance. These factors include raw material sources, dilution and timing errors, inappropriate temperature and humidity, exposure and container materials, etc. To ensure the validity and accuracy of diagnostic tests, these raw proteins must remain stable and effective. Therefore, an appropriate amount of protein protective agent must be added to the diagnostic reagent.
8. Dry state stability
Although most proteins function in their native conformation in solution, dry or frozen states are still preferred for stable storage. In these relatively stable states, frequent collisions between negative solvents such as proteases and oxidants and proteins will be minimized. However, removing the solvent from the protein molecules through drying or other phase change methods, such as precipitation and freezing, may damage the functional structure of the protein. These phase changes may expose hydrophobic amino acids that are usually folded inside the molecule to the surface of the molecule, causing the protein molecules to bind to other molecules through the hydrophobic surface, leading to the denaturation of the protein molecules. Drying methods include natural air drying or freeze drying, such as drying of conjugates in reagent bottles, and drying of antibodies coated on plastic plates or membrane materials.
Adding appropriate solutes to the solution to be dried to prevent protein denaturation is a very effective way to keep proteins stable. These solutes are characterized by having hydrophilic groups, which stabilize the functional structure of proteins by masking hydrophobic amino acids. Therefore, since the removal of solvent during drying will promote the interaction between protein and protein, especially protein and solid surface, it is very important to add a protective agent before drying.
Whether it is drying methods or storage conditions, there are very strict requirements to maintain stability. When proteins are applied to membranes, very rapid drying or lyophilization is often required to maintain optimal protein performance and viability. For protein coating of plates, test tubes or microbeads, it is more important to ensure thorough drying of the components than rapid drying. For maximum shelf life, all dried products need to be stored in airtight containers with desiccant.
9. Solution state stability
Although antibodies, antigens, enzymes, and other diagnostic proteins are in their native functional state in aqueous solution, this is not their most stable state. In solution, there are many factors that affect the active function of proteins:
Protein molecules become more flexible and prone to changing conformations.
The frequency of collisions of protein molecules with other molecules and with the walls of the container increases.
The possibility of microbial contamination is increased.
More sensitive to the effects of increased temperature and decreased protein concentration.
Proteins are more susceptible to oxidation.
Generally speaking, dissolved protein molecules are most stable at lower temperatures and higher protein concentrations. At just above freezing, protein molecules possess less kinetic energy, which is necessary for the protein to "escape" from its lowest-energy functional conformation. In the solution, part of the loss of protein activity is caused by adsorption on the container wall, depolymerization of oligomerase, and inactivation of protein by trace contaminants such as oxidants, hydrolases, and microorganisms. At higher concentrations (mg per milliliter range or higher), the proportion of lost protein activity to total protein activity is lower.
10. Other factors affecting stability
The protein source, purification method, and chemical coupling method can all affect the activity and stability of the protein reagent. For example, the source of alkaline phosphatase has a significant impact on the stability of the complex; in terms of stable storage of the solution, the required protein and enzymes (such as hydrolases and oxidases) that can damage the protein are removed from the raw materials. Separation is very important. To ensure solution stability, these harmful enzymes and the microorganisms that produce these enzymes must be removed or inactivated. When synthesizing complexes, a number of chemical reaction steps have been shown to enable the formation of covalent bonds between enzymes and antibody molecules. Some of these methods have less negative impact on the stability of certain enzyme-antibody complexes. If an enzyme-linked complex loses enzyme-linked detection activity but still retains enzyme activity, then choosing an alternative chemical coupling method may solve this stability problem.
Sometimes, what is thought to be a stability issue is actually a lack of protein caused by other variables. Maybe it's just an obvious dilution error. Manufacturers must discard container materials that may absorb proteins or inhibit protein activity, such as untreated polystyrene, polysulfone, polycarbonate, or glass. Polyethylene and polypropylene are preferred container materials. Colored enzymes and other protein solutions containing oxidized metal ions cannot be exposed to sunlight.
Types of protein protectants
The factors that affect protein activity mainly include the pH value of the solution, the concentration of salt and sugar, the action of microorganisms and the oxidation of air. In response to market needs, there are currently many commercialized protein protectants on the market that can be used in large quantities by clinical and scientific research manufacturers. They are mainly used for sealing and stabilizing microplates, antibody diluents, protein diluents, enzyme stabilizing solutions, etc. Choosing these commercial reagents will solve many problems for some companies in the early stages of research and development and save research and development time. Some companies, considering the cost, will also start to develop their own protein protection solution formula to reduce costs and obtain better results. At present, protein protective agents mainly include: polyhydroxy compounds, sugars, amino acids, polymers, proteins, etc.
Raw materials are the core of diagnostic reagents. Today, when high-quality raw materials and even top-notch raw materials are readily available for most projects, and the sources of raw materials are comparable to those of major international manufacturers, China's in-vitro diagnostic industry can focus more energy and wisdom on improving and optimizing processes. Create internationally renowned products.
