The basis of ELISA is the immobilization of antigen or antibody and the enzyme labeling of antigen and antibody. The antigen or antibody bound to the surface of the solid phase carrier still maintains its immunological activity, and the enzyme-labeled antigen or antibody retains both its immunological activity and enzyme activity. During the measurement, the test specimen (the antibody or antigen in the measurement) reacts with the antigen or antibody on the surface of the solid-phase carrier. The antigen-antibody complex formed on the solid phase carrier is separated from other substances in the liquid by washing. After adding enzyme-labeled antigen or antibody, it is also bound to the solid-phase carrier through the reaction. At this time, the amount of enzyme on the solid-phase carrier is in a certain ratio to the amount of the test substance in the specimen. After adding the substrate of the enzyme reaction, the substrate is catalyzed by the enzyme into a colored product, and the amount of the product is directly related to the amount of the tested substance, so qualitative or quantitative analysis can be performed according to the degree of color. Due to the high catalytic efficiency of the enzyme, it indirectly amplifies the result of the immune response and makes the determination method reach a high sensitivity.
There are three basic principles of ELISA:
(1) The antigen or antibody can be physically adsorbed on the surface of the solid carrier, possibly because the hydrophobic part between the protein and the polystyrene surface adsorbs each other and maintains its immunological activity;
(2) Antigens or antibodies can be linked with enzymes through covalent bonds to form enzyme conjugates, and this enzyme conjugate can still maintain its immunological and enzymatic activities;
(3) After the enzyme conjugate is bound to the corresponding antigen or antibody, the color reaction of the added substrate can be used to determine whether there is an immune response, and the depth of the color reaction is directly proportional to the amount of the corresponding antigen or antibody in the specimen. Therefore, the test results can be displayed according to the degree of color development of the substrate.
Antigens are substances that can cause specific immune responses in the body. After the antigen enters the body, it can stimulate the body to produce antibodies and cause cellular immunity.
Antibodies are immunoglobulins (Ig) that can specifically bind to antigens. Ig is divided into five categories, namely IgG, IgA, IgM, IgD and IgE. Ig related to immunoassay is mainly IgG and IgM.
The direct method can only be used to determine the antigen:
①. Connect the antigen to the solid phase carrier and wash to remove unbound antigen and impurities.
②. Add enzyme-labeled antibody and heat the reaction. The antigen on the solid-phase immune complex binds to the enzyme-labeled antibody. Wash the unbound enzyme-labeled antibody thoroughly. At this time, the amount of enzyme on the solid phase is related to the amount of the tested antigen in the specimen.
③. Add substrate reaction. The enzyme on the solid phase catalyzes the substrate to become a colored product. At this time, the degradation amount of the substrate = the amount of antigen.
Double antibody sandwich method, this method is suitable for the determination of bivalent or higher molecules, but not suitable for the detection of hapten antibodies and small molecule monovalent antigen antibodies. This method can measure both antibodies and antigens:
Principle of antigen test:
①. The specific antibody is connected to the solid-phase carrier to form a solid-phase antibody. Wash to remove unbound antibody and impurities.
②. Add the tested specimen and keep warm. The antigen in the specimen combines with the solid-phase antibody to form a solid-phase antigen-antibody complex. Wash to remove other unbound substances.
③. Add enzyme-labeled antibody and heat the reaction. The antigen on the solid-phase immune complex binds to the enzyme-labeled antibody. Wash the unbound enzyme-labeled antibody thoroughly. At this time, the amount of enzyme on the solid phase is related to the amount of the tested antigen in the specimen.
④. Add substrate reaction. The enzyme on the solid phase catalyzes the substrate to become a colored product. Through colorimetry, the amount of antigen in the specimen is measured.
The reaction mode of double-antibody sandwich method is similar to that of antigen detection. Specific antigen is used to coat and prepare enzyme conjugate to detect the corresponding antibody.
Principle: Use enzyme-labeled anti-antibody (anti-human immunoglobulin antibody) to detect the test antibody bound to the solid-phase antigen.
Steps:
①. Link the specific antigen with the solid-phase carrier to form a solid-phase antigen. Wash to remove unbound antigen and impurities.
②. Add diluted test serum to keep the reaction warm. The specific antibody in the serum binds to the solid-phase antigen to form a solid-phase antigen-antibody complex. After washing, only specific antibodies are left on the solid-phase carrier, and other components in the serum are washed away during the washing process.
③. Add enzyme-labeled antibody. The antibody in the solid-phase immune complex binds to the enzyme-labeled anti-antibody, thereby indirectly labeling the enzyme. After washing, the amount of enzyme on the solid-phase carrier is positively correlated with the amount of antibody tested in the specimen.
④. Add substrate to develop color.
Principle: The antibody (antigen) in the specimen competes with a certain amount of enzyme-labeled antibody (antigen) to bind to the solid-phase antigen (antibody). The more the amount of antibody (antigen) in the specimen, the less the enzyme-labeled antibody (antigen) bound to the solid phase, so the positive reaction is lighter than the negative reaction.
Coating antigen: Dilute the antigen appropriately with the coating solution, usually 1-10 micrograms/well, add 200 microliters per well, incubate at 37°C for 1 hour, and place in a refrigerator at 4°C for 16-18 hours.
Washing: Pour out the liquid in the wells of the plate, fill up the washing liquid, let it stand for three minutes, repeat three times, and finally put the reaction plate upside down on absorbent paper to allow the washing liquid in the wells to drain.
Add 200 μl of blocking solution and place at 37°C for one hour.
Washing is the same as 2.
Add the tested serum: use the diluent to make several dilutions of the tested serum, 200 microliters per well. At the same time as a dilution control. Place at 37°C for 2 hours.
Washing is the same as 2.
Add horseradish peroxidase goat anti-rabbit IgG, 200 microliters per well, and place at 37°C for 1 hour.
Washing is the same as 2.
Add substrate: add 200ml o-phenylenediamine solution, 10-15 minutes in the dark at room temperature.
Add stop solution: 50 microliters per well.
Observation results: Record 490nm readings with an enzyme-linked immunosorbent assayer.
In the formal experiment, the experimental conditions should be controlled by the positive control and the negative control respectively, and the samples to be tested should be made in duplicate to ensure the accuracy of the experimental results. Sometimes the background is high, indicating a non-specific reaction, which can be blocked with goat serum, rabbit serum or BSA.
In ELISA, the selection of various experimental conditions is very important, including
(1) Selection of solid phase carrier:
Many substances can be used as solid phase carriers, such as polyvinyl chloride, polystyrene, polyacrylamide and cellulose. The form can be a concave plate, a test tube, etc. At present, 96-well polystyrene concave well plates are commonly used. No matter what kind of carrier, it can be screened before use: Coating with the same amount of antigen, reacting under the same experimental conditions, observing whether the color reaction is uniform, and judging whether its adsorption performance is good.
(2) Selection of coating antibody (or antigen):
When the antibody (or antigen) is adsorbed on the surface of the solid phase carrier, the purity is required to be good, and the pH is generally required to be between 9.0 and 9.6 during adsorption. Adsorption temperature, time and protein content also have a certain influence, generally 4℃ for 18-24 hours. The optimal concentration of protein coating needs to be titrated: that is, after coating with different protein concentrations (0.1, 1.0 and 10μg/ml, etc.), under the same other test conditions, observe the OD value of the positive sample. Choose the concentration with the largest OD value and the smallest amount of protein. For most proteins, it is usually 1-10μg/ml.
(3) Selection of working concentration of enzyme-labeled antibody:
Firstly, a direct ELISA method was used for preliminary titer titration. Then fix other conditions or adopt the “square matrix method” (different dilutions of the coating material, the reference product of the sample to be tested, and the enzyme-labeled antibody) in the formal experimental system to accurately titrate its working concentration.
(4) Selection of enzyme substrate and hydrogen donor:
The selection requirements for hydrogen donors are cheap, safe, and have obvious color reaction, and they are colorless. Some hydrogen donors (such as OPD, etc.) have potential carcinogenic effects and should be protected. Those who have the conditions should use non-carcinogenic and highly sensitive hydrogen donors. For example, TMB and ABTS are currently more satisfactory hydrogen donors. After the substrate has acted for a period of time, a strong acid or a strong base should be added to stop the reaction. Usually the substrate action time is 10-30 minutes. The substrate liquid must be freshly prepared, especially H2O2 is added just before use.
(5) Sample:
Roughly determine the content of the sample in the specimen by checking the literature or preliminary experiments, and then dilute the sample and add the sample.
Feature 1: Sensitivity
The sensitivity of this assay comes from the enzyme as the reporter group. As we all know, enzyme is a kind of organic catalyst, and a small amount of enzyme can induce a large number of catalytic reactions and produce observable color reaction phenomena. Therefore, this system is often referred to as an enzyme amplification system. ELISA realizes the tracking of the antigen or antibody at the cellular or subcellular level, or quantification at the microgram or even nanogram level.
For example, when determining the content of a substance in serum, the sensitivity of chemical colorimetry is at the mg/ml level, and the sensitivity of enzyme reaction assay is about 5-10μg/ml.
Feature 2: Specificity
Its specificity comes from the selectivity of antibodies or antigens. The binding of antigen and antibody essentially only occurs between the epitope of the antigen and the antigen binding site of the antibody. Because the two are complementary in chemical structure and spatial configuration, the antigen-antibody reaction has a high degree of specificity.
How to correctly carry out elisa determination operation steps
Clinical ELISA assays are usually manually operated with microwell plates as the solid phase. The assay operation is very simple, generally involving specimen collection and preservation, reagent preparation, sample addition, incubation, plate washing, color development, In terms of colorimetry, result judgment, and result reporting and interpretation, improper steps in any of these steps will affect the measurement results, especially the steps of sample addition, incubation and plate washing. Now they are described as follows.
Collection and preservation of clinical specimens
The most commonly used clinical specimens for ELISA determination are serum (plasma), and sometimes saliva, cerebrospinal fluid, urine, feces and other specimens are also used for specific testing purposes. At present, the clinically used serum samples to determine the markers generally include antigens and antibodies of infectious pathogens, tumor markers, hormones, special proteins, cytokines, and therapeutic drugs. For the collection of serum samples used for hormone and therapeutic drug determination, it is necessary to pay attention to the collection time or even the body position that may affect the determination results. For example, cortisone will have a peak between 4 and 6 in the morning: growth hormone, luteinizing hormone (LH) and follicle stimulating hormone (FSH) are all released in a paroxysmal manner. Therefore, when measuring these hormones , It is necessary to take several blood samples in closely connected time intervals, and use the median value as the measured value. Another example is when changing from a lying position to a standing position, the renin activity in the serum will increase significantly. Another example is the detection of therapeutic drugs, the best time after taking the drug should be selected according to the pharmacokinetics for blood sampling. The collection of serum specimens used for the detection of antigens and antibodies of infectious pathogens, tumor markers and special proteins, etc., does not affect the time and position, but the following aspects should be considered in the handling and storage.
(1) Pay attention to avoid severe hemolysis. Hemoglobin contains a heme group, which has a peroxide-like activity. Therefore, in the ELISA assay with HRP as the labeling enzyme, if the hemoglobin concentration in the serum sample is higher, it is easy to adsorb during the incubation process. In the solid phase, it reacts with the HRP substrate added later to develop color.
(2) During sample collection and serum separation, attention should be paid to avoid bacterial contamination as much as possible. For the growth of bacteria, some of the secreted enzymes may decompose proteins such as antigens and antibodies; the other is for some bacterial endogenous enzymes such as The β-galactosidase of Escherichia coli itself can cause non-specific interference with the assay method labeled with the corresponding enzyme.
(3) If the serum specimen is separated by aseptic operation, it can be stored at 2～8℃ for one week. If it is a bacteria operation, it is recommended to freeze it. The long-term storage of samples should be below -70°C.
(4) For frozen serum specimens, care must be taken to avoid repeated freezing and thawing caused by power outages. The mechanical shearing force produced by repeated freezing and thawing of the specimen will have a destructive effect on the protein and other molecules in the specimen, thereby causing false negative results. In addition, attention should be paid to the mixing of freeze-thaw specimens. Do not vigorously shake, just invert and mix repeatedly.
(5) If the specimens have turbidity or flocculation caused by bacterial contamination during storage, the supernatant should be centrifuged and the supernatant should be taken for testing.
Reagent preparation
In clinical laboratories, generally little attention is paid to the preparation of reagents. The usual practice is to take the reagents out of the refrigerator and use them during the experiment, ignoring that this method may affect the problem of insufficient incubation time later. The consequence of this is false negatives on some weakly positive specimens. Therefore, the most important thing in the preparation of reagents in the ELISA assay is to take the kit out of the refrigerator before the start of the experiment and place it at room temperature for more than 20 minutes before performing the measurement, so that the kit is at room temperature before use. balance. The purpose of this is mainly to enable the temperature in the reaction micropores to reach the required height quickly in the subsequent incubation reaction step to meet the measurement requirements. Secondly, the washing liquid in the current commercial ELISA kits needs to be diluted and prepared with the provided concentrated solution when used in the laboratory, so the quality of the distilled or deionized water used in the dilution should be guaranteed. In addition, when the kit uses OPD as the substrate, the substrate solution should be prepared temporarily before the reaction is developed.
Add serum samples and reaction reagents
In the current commercial ELISA kits, the addition of serum samples is almost the only step that requires the use of a micropipette to add samples. The key points that must be paid attention to when using the micropipette to add samples are: do not add the sample too fast, avoid adding to the upper part of the hole wall, and do not splash or generate bubbles. The sample addition is too fast, and the accuracy and uniformity of the micro sample addition cannot be guaranteed. The non-coated area added to the upper part of the hole wall can easily lead to non-specific adsorption. Splashes can contaminate adjacent holes. When bubbles appear, there is a difference in the interface of the reaction liquid. The addition of reagents in the domestic kits is basically dripping from the dropper. In addition to the angle of the dripping, the speed of the dripping is also very important. If the dripping is too fast, it is easy to repeat dripping or adding in two. The phenomenon between pores will cause non-specific adsorption in the non-coated area in the pores, which will cause non-specific color development. Therefore, sometimes a specimen is tested positive this time with the same kit, and the next test is negative, which is often caused by the above-mentioned sample addition and reagent errors.
Incubate
Incubation is one of the most critical factors affecting the success or failure of the ELISA assay. ELISA is a solid-phase immunoassay. The antigen-antibody binding reaction is carried out on the solid phase. To make the antigen or antibody in the liquid phase completely bind to the specific antibody or antigen on the solid phase, it must be reacted under certain temperature conditions. time. The time required for incubation is inversely proportional to the temperature, that is, the higher the temperature, the shorter the time required. The most commonly used incubation temperatures are 37°C and room temperature, followed by 43°C and 2-8°C.
The incubation step is the most problematic step in the clinical ELISA assay. Generally, the reaction incubation time of domestic ELISA commercial kits is 37℃ 30 minutes to 1 hour, and imported ELISA kits are usually 37℃ 1 to 2 hours to have a more complete binding. If it is less than 1 hour, it may affect the determination. Lower limit. Therefore, regarding the incubation, the following points must be paid attention to in the actual measurement operation:
(1) Ensure that there is sufficient reaction time at the set temperature. Generally speaking, after adding samples and/or reagents, when the microplate is brought from room temperature to a water bath or incubator, the temperature in the wells will rise from room temperature to 37°C, and it takes a certain amount of time, especially at room temperature In the state of relatively low and non-water bath, this period of heating time may be relatively long. In clinical laboratories, few people pay attention to this problem. Usually, the microplate is placed in the incubator and the timer starts. It is easy to cause the problem that the incubation time is not enough in the actual measurement and the weakly positive samples cannot be detected. A colleague from a blood station in the south posed a question, that is, in the winter of every year, there is always more than one month, in the indoor quality control of the HBsAg determination, the determination of 1 is provided by the Clinical Laboratory Center of the Ministry of Health. When ng/ml is weakly positive, it is always undetectable. I don’t know why? This may be related to the low indoor temperature in the southern winter. At this time, the 37°C incubation time after the microplate was transferred to the incubator was not enough, so that the weakly positive samples were tested as negative. Therefore, in order to ensure sufficient incubation time at 37°C, clinical laboratories can determine by themselves how long the temperature in the wells will reach 37°C after the microplate is brought from room temperature to the incubator in different seasons (at different room temperature) in the laboratory. , So as to appropriately extend the time of placing the slats in the incubator. The specific method is to use a small thermometer to measure and observe in the reaction solution of the wells.
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