Development and Quality Control of ELISA Measurement Technology
ELISA test is an experimental diagnostic method with high sensitivity, strong specificity and good repeatability. Due to its stability, easy storage, easy operation, and objective judgment of results, it has been widely used in various fields of immunological testing. However, there are many factors that affect the results of the ELISA test, so it is necessary to strengthen the quality assurance of each link to give full play to its methodological advantages. 1. Development of ELISA kit measurement technology The development of clinical measurement technology mainly lies in the development of methodology, and the development of methodology relies on the continuous improvement of reagent production technology and the application of type markers. At present, molecular biology is and will definitely give us a comprehensive and thorough understanding of the entire life sciences. Its impact on the development of immunoassay technology is also direct and effective. It makes us have some biological activities that were difficult to detect before. The determination of substances becomes a breeze, and the sensitivity and specificity of the detection are greatly improved. 1. The influence of genetic engineering reagents on the development of immunoassay technology The basis of immunoassay technology is the specific binding reaction between antigens and antibodies, so any high-quality diagnostic reagents are inseparable from high-quality raw materials, such as antigens, antibodies, enzymes and so on. In the past, the antigens used for immunoassay were usually various purified antigens, while the antibodies were polyclonal antibodies obtained after immunizing animals with purified antigens and monoclonal antibodies obtained using hybridoma technology, and antigens or antibody labels such as enzyme labels were combined The substance is prepared by each general chemical synthesis method. In recent years, with the development of molecular biology, the use of genetic engineering methods to prepare various special antigens or antibodies and their enzyme conjugates and other immunoassay reagents has become a new generation of reagents, and various new types of measurement methods have also appeared . Characteristics of HBsAg reagent (detection mode: clinical antibody sandwich method): The coating antibody is goat polyclonal antibody, which has high affinity and reactivity to various epitopes of the antigen. The enzyme epitope antibody is a mouse compound unit with different binding sites to HBsAg. The HBsAg variant samples can be measured. Improve detection specificity (99.98%) To improve the detection sensitivity, apply Parl Ehrich theory (PEI) HBsAg standard, the sensitivity to ad standard is 0.05ng / ml and the sensitivity to ay standard is 0.025ng / ml 2. Genetic engineering The early genetically engineered antigens used in immunoassays are HBcAg and HBsAg. The emergence of these two genetically engineered antigens has solved the problem of anti-HBc and HBe determination. Because it is difficult to obtain these two pure antigens in large quantities from the virus itself, and this is common to pathogenic microorganisms that are difficult to culture, such as HIV, HCV and Treponema pallidum. 2.1 HCV-ELISA reagent HCV cannot be cultured in vitro at present, and can only use genetic engineering or chemical synthesis methods to obtain various antigen fragments of HCV structure and non-structural regions. It is known that the HCV genome consists of 7 functional regions: core, E1, E2 / NS1 NS2 NS3 NS4 The NS5 (there are divided into NS5a and NS5b) regions, the advantages of synthetic antigens are easy purification, good specificity, and strong antigen-antibody reaction. 2.1.1 The first generation anti-HCV-ELISA kit: -Several fragments of C1003 antigen cloned by American ortho company are combined with human superoxide dismutase (SOD). Yeast is used to express from the non-binding region of the viral genome, expressed as a fusion protein, and also serves as a coating solid phase carrier. -Antigen combination: two gene fragments of NS4 region are NS 5-1-1 and C1003 -Characteristics: IgG anti-C100 is detected only a few months after the onset, so it is not suitable for early diagnosis. -About 20% of patients do not develop antibodies. The detection rate of IgM anti-C100 in the acute phase was only 64%. -The antigenicity of C100 is weak, and it cannot fully expose the host's immune system during HCV replication. -Poor specificity and sensitivity. 2.1.2 Second-generation HCV-ELISA kit -Coating the solid-phase carrier with genetically recombined HCV structural proteins and non-structural protein antigens. -Antigen combination: core region (C region) polypeptide C22 NS region polypeptide C33 C1003 and NS 5-1-1 -After adding C22 and C33, antibodies appear early, which helps early diagnosis and improves the positive rate. Favorable for early diagnosis. -C22 is early prone to viremia 2.1.3 The third generation HCV-ELISA kit Synthetic peptide antigen, consisting of 36 amino acids Cp9 (inner shell protein) and 19 amino acids Cp10 mixed coated solid phase carrier. Antigen combination: In addition to C 100-3 C22 C33, core and NS3 NS4 NS5 are added Features: * core NS3 NS5 is recombinantly expressed by Baculovirus, there is no non-specific carrier, and the reagent has high specificity. The recombinant protein is fused to form a macromolecular antigen, which is conducive to improving the detection sensitivity. * Added amino acid fragments in NS3 region (proportion is very important). Improved sensitivity of reflection. * NS5 is optimized to remove the non-specific region (GDD) NS3 NS5 is the earliest antibody measured for hematopoiesis, which improves the accuracy of diagnosis. * NS4 is a synthetic peptide of two fragments, which removes the non-specific reaction area and enhances the sensitivity and specificity of the reagent. 2.1.4 Fourth-generation HCV-ELISA kit -The United States recently introduced a single fusion protein that includes all immunodeterminants of the seven functional regions of the HCV genome, called multi-determinant fusion antigens, which uses surfactants to treat the demarcation virus surface complex, release HCV core antigen (core) and use ELISA Method, the sensitivity can reach 500 copies / ml, which is close to the detection level of nucleic acid amplification. -Antigen combination: use Core and NS3 as the main detection fragments (the ratio is very important) plus synthetic peptide NS4 -Features: Direct measurement of Core antigen Not easy to mutate Anti-IgM and IgG detection rate can reach 100% 99.8% specificity and 100% sensitivity Makeup Brush Full Set,Full Makeup Brush Set,All Makeup Brushes,20Pcs Brush Set DONGGUAN YACAI COSMETICS CO.,LTD. , https://www.yacaicosmetic.com