Secretory IgE in sputum
The site of SIgE is similar to that of SIgA, and is also produced by plasma cells in the lamina propria of the respiratory tract. It is distributed in mucosal tissues and exocrine fluids. These sites are the site of invasion of allergens and the site of allergic reactions. IgE is a monomer, 8S, 19×104 ku, and its heavy chain is longer than the γ chain. One more functional region (CH4), which can bind to cells (such as mast cells, basophils) and trigger the release of intracellular bioactive substances under certain conditions. Therefore, IgE is the main antibody causing type I allergy. .Basic Information
Specialist classification: Respiratory examination classification: sputum examination
Applicable gender: whether men and women apply fasting: fastingReminder: If the main reagent components are improperly prepared or improperly stored, they are easily degraded and inactivated. Normal value
0.1 to 9 mg/L.Clinical significance
Increase: IgE is monomer, 8S, 19×104ku, and its heavy chain is longer than γ chain. One more functional region (CH4), which can bind to cells (such as mast cells, basophils) and trigger the release of intracellular bioactive substances under certain conditions. Therefore, IgE is the main antibody causing type I allergy. . In patients with bronchial asthma and hypersensitivity pneumonitis, the SIgE content in the sputum can be increased.High results may be diseases: bronchial asthma, allergic pneumonia
The main reagent components in the ELISA, such as antigens, antibodies, enzyme conjugates, substrates, etc., are easily degraded and inactivated if not properly prepared or improperly stored. There are many steps in the ELISA, and if the operation is not standardized, it is difficult to obtain accurate results. Therefore, in order to ensure the effectiveness of the test, quality control must be performed for each test. The positive and negative controls provided in the excellent kits can generally be used as the quality control of the test. According to the instructions, the effectiveness of the test can be judged from the obtained absorbance values. Taking the most commonly used HBsAg test in clinical tests as an example, the negative control in the kit should be HBsAg-negative human serum, and the positive control should indicate HBsAg content (eg 9±2 ng/ml). Three negative controls and two positive controls should be made simultaneously for each batch of tests. The absorbance value obtained by the negative control assay should be less than a certain value (for example, 0.100), and the mean value of the positive control absorbance minus the negative control absorbance should be greater than a certain value (for example, 0.200). These values are specific experimental values for the kit under the specified assay conditions. Therefore, if the test results meet the requirements, it indicates both the effectiveness of the reagents used and the correctness of the operation. Only in this case, the batch test is effective.
Some of the positive and negative controls contained in the kit do not meet the requirements for quality control, or the laboratory's measurement conditions such as colorimeter, etc., and the difference in the kit description, the appropriate quality should be used according to the actual situation. Controls and quality control values from the laboratory derived from the experiment. The quality control serum of the commodity is expensive. Qualitative determination of ELISA quality control products can generally be prepared by themselves. The HBsAg test is still taken as an example. Negative controls can be taken from blood donors who are HBsAg negative with high quality reagents. The positive control can be prepared by adding an appropriate amount of HBsAg-positive serum to the negative control serum, and comparing the mixed serum with a reference standard or a quantitative control to determine the HBsAg content. Then, appropriate dilution is carried out to obtain the HBsAg-positive serum of the desired concentration, and then an appropriate amount of antibacterial preservatives such as gentamicin and salicylic acid are added, and then cryopreserved at a low temperature.Inspection process
ELISA is a multi-reaction, multi-reagent, multi-step test method, which must be carefully and carefully operated according to requirements, otherwise accurate results will not be obtained. The ELISA procedures used to detect different items are slightly different, but include steps such as loading, holding, washing, and colorimetry. The operation points of each step are described below.
1. Reagent preparation: Dilute or prepare the reagents required for the test according to the instructions of the kit. Distilled or deionized water used in ELISA, including for washing, should be fresh and of high quality. The self-contained buffer should be measured with a pH meter. The temperature of the test reagent taken out from the refrigerator should be used after reaching room temperature. In tests using HRP as a marker, containers contaminated with metal are not available.
2. Loading: Prepare a good ELISA plate as required. Serum (skinning fluid) specimens should be freshly collected or properly stored in the refrigerator. Highly hemolyzed or turbid specimens should not be used. Specimens containing sodium azide as a preservative are not available for one-step ELISA for HRP labeling. When adding the sample, add the specimen to the bottom of the hole, avoid adding it to the upper part of the hole wall, and be careful not to splash it, and no air bubbles should appear. The accuracy of the amount of sample added in the quantitative determination should meet the quantitative requirements. In the qualitative measurement, the accuracy of the amount of the sample is sometimes not emphasized, for example, it is prescribed as a drop. At this point, you should use the same diameter dropper and maintain the correct loading position so that the volume of each drop is basically the same. The operation and requirements for the addition of the enzyme conjugate and the addition of the substrate are the same as for the addition of the sample.
3. Insulation: There are generally two antigen-antibody reactions in the ELISA, that is, after the addition of the sample and after the addition of the combination. At this time, the temperature and time of the reaction should be as accurate as required. The insulated container is preferably a water bath, and the bottom of the ELISA plate should be placed in the water to quickly balance the temperature. Each ELISA plate should not be stacked together. To avoid evaporation, the board should be covered. The plate can also be placed flat in a wet box with wet gauze on the bottom. The wet box should be pre-warmed to the specified temperature. For example, the insulation of the incubator is more important.
4, washing: washing in the ELISA process is not a reaction step, but decided to close the key to success. The purpose of washing is to wash away the substances in the reaction solution which are bound to the solid phase antigen or antibody and the interfering substances which are non-specifically adsorbed to the solid phase carrier during the reaction. The adsorption of proteins by plastics such as polystyrene is universal, so non-specific adsorption should be avoided as much as possible during the ELISA assay, and this non-specifically adsorbed interfering substance should be washed away during washing. The addition of Tween to the washing liquid can improve the washing effect. If the washing is not thorough, especially at the last time, if there is non-specific adsorption of the enzyme conjugate, the blank value will be raised. In addition, in the indirect method, if the non-specific IgG in the specimen is adsorbed on the solid phase without being washed, it will also interfere with the enzyme-labeled antibody.
The ELISA plate is generally washed by the following methods: 1 absorbing the reaction solution; 2 filling the plate with the washing solution; 3, placing it for 2 minutes, shaking slightly; 4 absorbing or pouring the liquid in the hole and patting it on the absorbent paper. The number of washings is generally 3 to 4 times, and sometimes even 5 to 6 times. A plate ELISA automatic washer can also be used, but the actual use of the instrument should be checked first. Each pipette of the washer must also be placed close to the bottom of the corresponding hole. In order to ensure the same washing effect of each hole.
5. Color development and colorimetric: The temperature and time of the reaction after adding the substrate in the quantitative determination should still be accurate according to the regulations. However, the reaction can generally be carried out at room temperature in a qualitative assay. If the substrate is OPD, it should be placed away from light. The reaction time does not need to be strictly controlled, and sometimes the stop solution can be added in time according to the coloration of the positive control and the negative control. To ensure the same reaction time for each well, the procedure and speed of adding the stop solution should be the same as when adding the substrate.
Qualitative determination, such as a negative reaction, is light, and sometimes the results can be judged visually. Plate ELISA generally uses an enzyme label reader to read absorbance at a specified wavelength. The automatic label reader should be tested for compatibility with the wells of the ELISA plate used and the repeatability of the results before use.
When colorimetric, first check the zero point with distilled water, read the substrate pores (the pores without any reaction and only add the substrate) and the blank wells (the pores measured by the saline or PBS instead of the specimen for the whole process) to record this time. The reagent status of the test. After that, the blank hole can be used to calibrate the zero point, and the absorbance of the sample hole, the standard hole and the control hole can be read. If the zero point is still corrected by distilled water, the absorbance of the above holes should be subtracted from the absorbance of the blank holes in the calculation.Not suitable for the crowd
Inappropriate people: Generally there are no people who are not suitable.Adverse reactions and risks
No adverse reactions.