A standard ELISA operation points <br> <br> quality reagents, good equipment and proper operation is essential to ensure accurate and reliable ELISA test results. Distilled or deionized water used in the ELISA, including those used for washing, should be fresh and of high quality. The purified water required by our company has a conductivity of less than 1.5 μs/cm.
1. The samples taken and stored <br> <br> most are serum ELISA test for the specimens. Serum samples can be collected by routine methods. Care should be taken to avoid hemolysis. Red cell lysates can release substances with peroxidase activity. In HRP-labeled ELISA assays, hemolysis specimens may increase nonspecific color development. Serum samples should be tested when fresh. If bacteria are contaminated, the cells may contain endogenous HRP and false positive reactions may also occur. In general, serum samples measured within 5 days can be stored at 4°C. Excessive preservation of specimens in the refrigerator leads to aggregation of serum IgG and deepens the indirect reagent background. More than one week to determine the need for -20 °C preservation. After the frozen serum is thawed, the protein is partially concentrated and unevenly distributed. Mix well and avoid bubbles. The turbid or precipitated serum samples should be centrifuged or filtered before they are clarified. Repeated freezing and thawing may cause the antibody titer to fall. Therefore, if serum samples for measuring antibodies need to be preserved for multiple tests, a small amount of ice should be stored. Preservation of serum should be performed with care aseptically, and appropriate preservatives may also be added.
Specimens with incomplete anticoagulation result in false positives due to interference with fibrinogen. It is recommended that anticoagulants, especially heparin anticoagulants, be avoided as much as possible.
2. Adding Samples Add the sample to the bottom of the well of the ELISA plate to avoid adding to the upper part of the well.
3. When the reaction kinetics <br> <br> incubated in an ELISA method for establishing, experiments show that the two antigen-antibody reaction is typically over 1-2 hours, up to 37 ℃ generating product peak. In order to avoid evaporation, the board should be covered, plastic cover paper or plastic wrap can also be used to cover the board hole, the reaction board should not be stacked, in order to ensure that the temperature of each board can be quickly balanced. It should be noted that the temperature and time of incubation should be as precise as required. Because the company's kit incubation is done in an air bath, using a water bath can result in a high value or flower plate. In addition, there are edge effects in the incubation, and the value on the edge will be high. It is suggested that the quality control should be placed at the non-edge position for objective judgment results.
4. Although it is washed with a washing <br> <br> reaction step ELISA procedure, but determine the success of the experiment. ELSIA is washed to achieve separation of free and bound enzyme labels. By washing to remove substances that remain in the plate wells from being unable to bind to the solid-phase antigen or antibody, and interference substances that are non-specifically adsorbed to the solid-phase carrier during the reaction. Adsorption of proteins by plastics, such as polystyrene, is common, and the non-specifically adsorbed interfering substances should be washed off during washing. It can be said that in the ELISA operation, washing is the most important key technology and should be highly valued by the operator. The operator should wash it strictly according to the requirements, and should not be sloppy.
Most of the wash solution is a neutral buffer containing a non-ionic detergent. The binding of the polystyrene carrier to the protein is hydrophobic. The non-ionic detergent contains both hydrophobic and hydrophilic groups. The hydrophobic group binds to the hydrophobic group of the protein by hydrophobic bonds, weakening the protein and The combination of the solid-phase carrier and the combined action of the hydrophilic group and the water molecule cause the protein to return to the aqueous solution state, thereby escaping the solid-phase carrier. The non-ionic detergent in the washing liquid is generally Tween 20, and its concentration can be between 0.05% and 0.2%. Above 0.2%, the antigen or antibody coated on the solid phase can be desorbed to reduce the test. The sensitivity.
When washing the plate, be careful not to mix the lotions of the various kits. If the lotion needs to be diluted, it should be diluted as required. The conductivity of the water used should be less than 1.5us/cm. If the lotion should be thawed, it should be prepared after melting. The soaking time of the washing plate is guaranteed to be about 40 seconds. The better the liquid in the hole is absorbed by the plate washer, the better the washing effect is. The manual washing of the plate prevents the lotion from forming bubbles in the hole.
5. Color development and colorimetric TMB After HRP treatment, the color development peaked about 40 minutes, then gradually weakened, and completely disappeared to colorlessness after 2 hours. There are several kinds of stop solutions for TMB, and the reaction can be terminated by enzyme inhibitors such as sodium azide and sodium dodecyl sulfate (SDS). This type of terminator can maintain blue color for a long time (12-24 hours) and is a good terminator for visual judgment. In addition, various types of acidic stop solutions will turn blue into yellow, and absorbance can be measured at a specific wavelength (450 nm). Enzyme-labeled colorimetric apparatus is referred to as microplate reader, and usually refers to a photometer that is designed to measure the absorbance of ELISA results. The main performance indicators of the microplate reader are: reading speed, reading accuracy, repeatability, accuracy and measurable range, linearity and so on. Excellent microplate readers generally have an accurate reading of 0.001, an accuracy of ±1%, and a repeatability of 0.5%. The microplate reader should not be placed in sunlight or under strong light. During operation, the room temperature should be 15~30°C. Before use, the instrument should be warmed up for 15-30 minutes, and the reading result is more stable.
When reading A values, the sensitive absorption peak of the product should be selected, for example, the wavelength of 492nm for OPD. Some microplate readers can be used for dual-wavelength type readings, that is, each well read twice before, the first at the optimal wavelength (W1), the second at the insensitive wavelength (W2), and no movement between two measurements. The position of the board, the final measured A value is the difference between the two (W1-W2). Dual wavelength readings reduce light interference caused by scratches or fingerprints on the container.
Analysis of the causes of the two backgrounds and false positives 1. Differences between genetically engineered antigens and synthetic peptide antigens 1.1 Genetically engineered antigens are antigens for prokaryotic or eukaryotic expression of antigenic genes in plasmid vectors, mostly expressed in E. coli or yeast. system. Compared with synthetic peptides, these antigens have the following characteristics:
a. Large molecular weight. Synthetic peptides are prepared by chemical methods. Due to the limitations of the process, the number of synthesis is limited and can only reach several hundred amino acids. The antigens prepared by genetic engineering have larger molecular weights.
b. Good stability. The stability of the coated antigen can ensure the validity of the kit. The effective period of the kit that uses the synthetic peptide as the coating antigen is only 3-4 months, and the use of the genetically engineered antigen is greatly prolonged.
c. Genetically Engineered Antigens Fusion-specific expression of specific antigenic determinants. The expression product contains more antigenic determinants, which can increase the sensitivity of the kit and increase the detection rate.
d. Purification is difficult. The purification of genetically engineered antigens is difficult.
1.2 Synthetic polypeptide antigens are polypeptide fragments that are artificially synthesized based on the amino acid sequence of an antigenic determinant of a protein antigen molecule. Synthetic peptide antigens have the following characteristics:
a. The molecular weight is too small b. Generally only contains one antigenic determinant c. High purity d. Poor stability Due to the superiority of genetically engineered antigens over synthetic peptide antigens, ELISA diagnostic reagents have gone from synthetic peptides to genetic engineering. The antigen transition. As for the HCV ELISA kit, the first generation products were synthetic peptide antigens, mainly peptide fragments of HCV-specific antigenic determinants; the second-generation products coated antigens were both genetically engineered antigens and synthetic peptides, but were the genes of the time. The engineering antigens are incomplete, including only the core fragment of HCV; the third generation products basically use genetic engineering antigens, and these antigens include more, more stable, and higher purity HCV-specific antigens. The sensitivity of the third generation reagents has greatly increased.
Due to historical reasons, people often measure the ELISA reaction kit based on the quality of the reaction background. Therefore, in order to maintain a good background, some manufacturers use a single-segment genetic engineering antigen and synthetic peptide coating, and the popularity of such kits. The sensitivity to disease is not enough and stability is also a problem. What is gratifying is that there are also manufacturers that stick to the high epidemiological sensitivity of the kits, and scientifically treat the results.
2. Causes of false-positive background 2.1 Effect of antigenic factors 2.1.1 Effects of fusion proteins on the specificity of genetically engineered antigens. Taking the hepatitis C diagnosis kit as an example, since the coated genetic engineering antigen is a fusion protein and contains some sequences from the expression vector, it can react with anti-e. coli factors in the serum to generate a suspicious sample.
1. The samples taken and stored <br> <br> most are serum ELISA test for the specimens. Serum samples can be collected by routine methods. Care should be taken to avoid hemolysis. Red cell lysates can release substances with peroxidase activity. In HRP-labeled ELISA assays, hemolysis specimens may increase nonspecific color development. Serum samples should be tested when fresh. If bacteria are contaminated, the cells may contain endogenous HRP and false positive reactions may also occur. In general, serum samples measured within 5 days can be stored at 4°C. Excessive preservation of specimens in the refrigerator leads to aggregation of serum IgG and deepens the indirect reagent background. More than one week to determine the need for -20 °C preservation. After the frozen serum is thawed, the protein is partially concentrated and unevenly distributed. Mix well and avoid bubbles. The turbid or precipitated serum samples should be centrifuged or filtered before they are clarified. Repeated freezing and thawing may cause the antibody titer to fall. Therefore, if serum samples for measuring antibodies need to be preserved for multiple tests, a small amount of ice should be stored. Preservation of serum should be performed with care aseptically, and appropriate preservatives may also be added.
Specimens with incomplete anticoagulation result in false positives due to interference with fibrinogen. It is recommended that anticoagulants, especially heparin anticoagulants, be avoided as much as possible.
2. Adding Samples Add the sample to the bottom of the well of the ELISA plate to avoid adding to the upper part of the well.
3. When the reaction kinetics <br> <br> incubated in an ELISA method for establishing, experiments show that the two antigen-antibody reaction is typically over 1-2 hours, up to 37 ℃ generating product peak. In order to avoid evaporation, the board should be covered, plastic cover paper or plastic wrap can also be used to cover the board hole, the reaction board should not be stacked, in order to ensure that the temperature of each board can be quickly balanced. It should be noted that the temperature and time of incubation should be as precise as required. Because the company's kit incubation is done in an air bath, using a water bath can result in a high value or flower plate. In addition, there are edge effects in the incubation, and the value on the edge will be high. It is suggested that the quality control should be placed at the non-edge position for objective judgment results.
4. Although it is washed with a washing <br> <br> reaction step ELISA procedure, but determine the success of the experiment. ELSIA is washed to achieve separation of free and bound enzyme labels. By washing to remove substances that remain in the plate wells from being unable to bind to the solid-phase antigen or antibody, and interference substances that are non-specifically adsorbed to the solid-phase carrier during the reaction. Adsorption of proteins by plastics, such as polystyrene, is common, and the non-specifically adsorbed interfering substances should be washed off during washing. It can be said that in the ELISA operation, washing is the most important key technology and should be highly valued by the operator. The operator should wash it strictly according to the requirements, and should not be sloppy.
Most of the wash solution is a neutral buffer containing a non-ionic detergent. The binding of the polystyrene carrier to the protein is hydrophobic. The non-ionic detergent contains both hydrophobic and hydrophilic groups. The hydrophobic group binds to the hydrophobic group of the protein by hydrophobic bonds, weakening the protein and The combination of the solid-phase carrier and the combined action of the hydrophilic group and the water molecule cause the protein to return to the aqueous solution state, thereby escaping the solid-phase carrier. The non-ionic detergent in the washing liquid is generally Tween 20, and its concentration can be between 0.05% and 0.2%. Above 0.2%, the antigen or antibody coated on the solid phase can be desorbed to reduce the test. The sensitivity.
When washing the plate, be careful not to mix the lotions of the various kits. If the lotion needs to be diluted, it should be diluted as required. The conductivity of the water used should be less than 1.5us/cm. If the lotion should be thawed, it should be prepared after melting. The soaking time of the washing plate is guaranteed to be about 40 seconds. The better the liquid in the hole is absorbed by the plate washer, the better the washing effect is. The manual washing of the plate prevents the lotion from forming bubbles in the hole.
5. Color development and colorimetric TMB After HRP treatment, the color development peaked about 40 minutes, then gradually weakened, and completely disappeared to colorlessness after 2 hours. There are several kinds of stop solutions for TMB, and the reaction can be terminated by enzyme inhibitors such as sodium azide and sodium dodecyl sulfate (SDS). This type of terminator can maintain blue color for a long time (12-24 hours) and is a good terminator for visual judgment. In addition, various types of acidic stop solutions will turn blue into yellow, and absorbance can be measured at a specific wavelength (450 nm). Enzyme-labeled colorimetric apparatus is referred to as microplate reader, and usually refers to a photometer that is designed to measure the absorbance of ELISA results. The main performance indicators of the microplate reader are: reading speed, reading accuracy, repeatability, accuracy and measurable range, linearity and so on. Excellent microplate readers generally have an accurate reading of 0.001, an accuracy of ±1%, and a repeatability of 0.5%. The microplate reader should not be placed in sunlight or under strong light. During operation, the room temperature should be 15~30°C. Before use, the instrument should be warmed up for 15-30 minutes, and the reading result is more stable.
When reading A values, the sensitive absorption peak of the product should be selected, for example, the wavelength of 492nm for OPD. Some microplate readers can be used for dual-wavelength type readings, that is, each well read twice before, the first at the optimal wavelength (W1), the second at the insensitive wavelength (W2), and no movement between two measurements. The position of the board, the final measured A value is the difference between the two (W1-W2). Dual wavelength readings reduce light interference caused by scratches or fingerprints on the container.
Analysis of the causes of the two backgrounds and false positives 1. Differences between genetically engineered antigens and synthetic peptide antigens 1.1 Genetically engineered antigens are antigens for prokaryotic or eukaryotic expression of antigenic genes in plasmid vectors, mostly expressed in E. coli or yeast. system. Compared with synthetic peptides, these antigens have the following characteristics:
a. Large molecular weight. Synthetic peptides are prepared by chemical methods. Due to the limitations of the process, the number of synthesis is limited and can only reach several hundred amino acids. The antigens prepared by genetic engineering have larger molecular weights.
b. Good stability. The stability of the coated antigen can ensure the validity of the kit. The effective period of the kit that uses the synthetic peptide as the coating antigen is only 3-4 months, and the use of the genetically engineered antigen is greatly prolonged.
c. Genetically Engineered Antigens Fusion-specific expression of specific antigenic determinants. The expression product contains more antigenic determinants, which can increase the sensitivity of the kit and increase the detection rate.
d. Purification is difficult. The purification of genetically engineered antigens is difficult.
1.2 Synthetic polypeptide antigens are polypeptide fragments that are artificially synthesized based on the amino acid sequence of an antigenic determinant of a protein antigen molecule. Synthetic peptide antigens have the following characteristics:
a. The molecular weight is too small b. Generally only contains one antigenic determinant c. High purity d. Poor stability Due to the superiority of genetically engineered antigens over synthetic peptide antigens, ELISA diagnostic reagents have gone from synthetic peptides to genetic engineering. The antigen transition. As for the HCV ELISA kit, the first generation products were synthetic peptide antigens, mainly peptide fragments of HCV-specific antigenic determinants; the second-generation products coated antigens were both genetically engineered antigens and synthetic peptides, but were the genes of the time. The engineering antigens are incomplete, including only the core fragment of HCV; the third generation products basically use genetic engineering antigens, and these antigens include more, more stable, and higher purity HCV-specific antigens. The sensitivity of the third generation reagents has greatly increased.
Due to historical reasons, people often measure the ELISA reaction kit based on the quality of the reaction background. Therefore, in order to maintain a good background, some manufacturers use a single-segment genetic engineering antigen and synthetic peptide coating, and the popularity of such kits. The sensitivity to disease is not enough and stability is also a problem. What is gratifying is that there are also manufacturers that stick to the high epidemiological sensitivity of the kits, and scientifically treat the results.
2. Causes of false-positive background 2.1 Effect of antigenic factors 2.1.1 Effects of fusion proteins on the specificity of genetically engineered antigens. Taking the hepatitis C diagnosis kit as an example, since the coated genetic engineering antigen is a fusion protein and contains some sequences from the expression vector, it can react with anti-e. coli factors in the serum to generate a suspicious sample.
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