1. The basic principle of ELISA ELISA is based on immunological reaction, combining the specific reaction of antigen and antibody with the efficient catalytic action of enzyme on substrate, and has almost high specificity and sensitivity. The soluble antigen-antibody system can be used for detection, and its minimum measurable value is ng or even pg level. The basic principle of the method is as follows: (1) The antigen or antibody is bound to the surface of a solid phase carrier (currently the most commonly used polystyrene microtiter plate) and maintains its immunological activity.
This step is referred to as "coating of the solid phase carrier" and has been completed in commercial kits. (2) further linking the antigen or antibody to an enzyme (such as horseradish peroxidase, alkaline phosphatase, etc.) to form an enzyme-labeled antigen or an enzyme-labeled antibody, and the enzyme-labeled antigen or antibody retains its immunological activity. The activity of the enzyme is also retained. At the time of detection, the specimen to be examined (the antibody or antigen to be assayed therein) and the enzyme-labeled antigen or the enzyme-labeled antibody are added in the corresponding order to react with the antigen or antibody on the solid phase carrier. The excess unbound material in each step is washed away by washing, and the amount of the enzyme bound to the solid phase carrier is proportional to the amount of the test substance in the sample. (3) Finally, the substrate of the enzyme reaction is added, and the substrate is catalyzed by the enzyme to form a colored product, and the amount of the colored product is directly related to the amount of the test substance in the sample, so that the quantitative analysis can be performed according to the depth of the color reaction. This color reaction can be quantitatively determined by a microplate reader, thus combining the sensitivity of the enzyme chemical reaction with the specificity of the antigen-antibody reaction, making the ELISA method a both specific and sensitive detection method.
2. The application of ELISA technology in clinical medical research is complicated in ELISA steps and difficult to prepare reagents. Only satisfactory reagents and standardized operations can be used to obtain satisfactory results. Currently, a wide range of test items are generally sold in kits. The complete ELISA kit should contain a solid substrate, an enzyme conjugate, a substrate, and various concentrated diluents, buffers, and the like.
In medical research, the items tested by ELISA reagents can be mainly divided into the following categories: (1) detection of various pathogens and their antibodies: such as hepatitis virus, cytomegalovirus, rubella virus, herpes virus, HIV, etc.; Protein: tumor markers such as alpha-fetoprotein, carcinoembryonic antigen, etc.; hormones such as HCG, FSH, TSH, etc.; cytokines such as TNF. 0l, VEGF, NFKB, etc.; apolipoproteins such as Apo A-I, ApoE, etc.; (3) non-peptide hormones: such as T3, rr4, estradiol, cortisol, etc.; (4) detection of drug concentration in the blood: Such as the treatment of heart disease drugs digoxin, antiepileptic drugs theophylline, antibiotics gentamicin and so on.
Specimens used in basic research come from a variety of sources, such as clinical blood samples, blood samples from experimental animals, tissue samples from experimental animals, cultured cells, and cell culture supernatants, etc., which can be used as test samples to detect some of them. The content of the substance. The specific sample should be selected for the detection of specific indicators, depending on the purpose of the experimental study and the expression characteristics of the substance to be tested.
3. Application characteristics of ELISA in basic medical research The application of ELISA method in basic research is very different from that in clinical laboratory work. The differences are mainly as follows: (1) The specimens used in ELISA testing in basic research are widely used. Human, rat, mouse, rabbit, pig, dog and other common experimental animal species of blood, tissue, cell and cell culture supernatant, etc., while the clinical test specimens are human blood (serum or plasma) And urine, etc.; (2) basic research is exploratory, and the results are also uncertain. Without the normal range of values, it is necessary to judge the significance of the measured values ​​of samples between different experimental groups according to the specific values ​​of the ELISA test indicators; The clinically tested ELISA test results have a positive or negative definition, or there is a normal range of values ​​as a criterion for interpreting the test results. (3) For clinical test specimens such as serum and plasma, the ELISA test kits for various indicators will give the optimal dilution factor for the sample to be tested; in the basic research, especially when the measured index is in the tissue When expressing, that is, when the sample to be tested is derived from tissue, the ELISA test kit usually does not recommend the dilution factor of the sample to be tested; at this time, the researcher must first perform an optimal dilution factor through the preliminary experiment before testing. The detection steps for tissue samples are complex and cumbersome.
4. Classification of ELISA and main operational procedures ELISA methods can be used for both antigen determination and antibody determination. Depending on the type and nature of the substance to be tested in the sample, various types of detection methods can be designed. The ELISA methods used in clinical laboratory work are mainly divided into the following types: (1) for detecting antigens: double antibody sandwich method and competition method; (2) for detecting antibodies: double antigen sandwich method, indirect method , competition law and capture package method. However, in the basic medical research, most of the detection indexes involved are antigenic substances. Therefore, only the methods for measuring antigenic substances by the double antibody sandwich method and the competition method will be described in detail below.
4.1 Double-antibody sandwich assay for antigenic studies showed that patients with congestive heart failure had elevated levels of interleukin-6 (IL-6) in the circulation and tissues, and excessive IL-6 production would destroy cells. Factor network may lead to progression of myocardial injury and dysfunction, therefore, circulating IL-6 levels are closely related to the severity of left ventricular dysfunction. Here, the ELISA procedure and main points of the double antibody sandwich method are described by taking rat plasma IL-6 as an example.
4.1.1 Operation procedure (1) Take out the kit and equilibrate at room temperature for 30 min; (2) Prepare various working fluids according to the instructions: lotion, standard dilution and sample dilution; (3) Recommended according to the instructions The sample dilution is diluted with the sample to be tested, and the standards of each concentration are prepared according to the specifications (8 concentration gradients from blank control to low concentration up to high concentration); (4) Standards and samples are added: Standard wells and sample wells were added to the standard and sample 50 L/well, and the sealing film was attached and incubated for 2 h at room temperature. (5) Washing the plate: discard the liquid in the well and pat the liquid on the absorbent paper. Dry, add 3001~L per well, soak for 1 min each time, and repeat the washing 4 times; (6) Add enzyme-labeled antibody: add 100IzL of enzyme-labeled antibody per well, incubate for 2h at room temperature; Open the microplate reader and pre-establish the corresponding detection procedure; (7) Wash the plate: repeat the steps in (5); (8) Add the enzyme substrate: add lOOp per well, L enzyme substrate solution, incubate at room temperature in the dark Within 30 min (30 min, visual observation showed that the standard wells gradually faded from low to high concentrations of light blue Deep, the highest concentration of 34 wells showed a clear gradient of blue, while the low concentration of 3 to 4 wells was not obvious, it could be terminated); (9) Termination reaction: 100 mL of stop solution per well, immediately blue Turned to yellow; (10) Reading: The optical density value of each well was measured at a wavelength of 450 nm of the microplate reader within 10-15 minutes after the addition of the stop solution (using the previously established detection procedure in step (6)).
4.1.2 Standard curve for detection of plasma IL-6 in rat by double antibody sandwich method In the detection procedure setting procedure of multi-function microplate reader (instrument: TECAN, GENios plus), the standard curve can be set in detail. The concentration value of each point and the dilution factor of the sample. Therefore, when the detection data is finally output, the program software can automatically convert the OD value of the sample to the corresponding concentration according to the OD value of each point on the standard curve and its corresponding concentration. The detection is convenient, intuitive and accurate.
4.2 Competition method for detecting antigenic substances The competition method for detecting antigens is similar to the double antibody method. The difference is as follows: when the double antibody sandwich method is used to detect antigen, the sample to be tested is first added to be coated with the solid phase carrier. The antibody is reacted, the unbound antigen is washed away after the specific binding reaction is completed, and then the enzyme-labeled secondary antibody is added to continue reacting with the antigen on the solid phase carrier to form a sandwich complex on the sandwich complex. The label is measured to obtain the content of the antigen in the sample to be tested; and in the process of detecting the antigen by the competition method, the sample to be tested and a certain amount of the labeled antigen are simultaneously added, so that the surface of the solid phase carrier is limited. The antibody undergoes a competitive reaction, and after the reaction, the free antigen is washed away for a period of time. As a result of the competitive reaction, the amount of the label bound to the solid phase carrier is inversely proportional to the amount of the antigen in the sample, thereby determining the antigen in the sample. content.
Apo A-I accounts for 67% of high-density lipoprotein (HDL) and promotes the maturation of high-density lipoprotein.
4.3 The main difference between the double antibody sandwich method and the competition method for detecting antigens (1) The double antibody sandwich method is suitable for detecting macromolecular antigens such as proteins. Generally, two monoclonal antibodies against different determinants on the antigen are selected for coating. A solid phase carrier and an enzyme-labeled antibody. However, the lack of small-molecule antigens or semi-antibiotics can be used as two or more sites for the sandwich method, and the two-point sandwich cannot be formed. Therefore, the competition method mode is usually selected. (2) The characteristics of the standard curve are different. In the double antibody sandwich method, the amount of enzyme bound to the solid phase carrier is proportional to the amount of the antigen (or antibody) to be tested in the sample. Therefore, the absorbance value (OD value) of the sample is proportional to the concentration, and the standard curve is shown in the figure. 3 is shown. However, in the competition method, the antigen in the specimen and a certain amount of the enzyme-labeled antigen compete with the solid-phase antibody, that is, the more the antigen content in the specimen, the less the enzyme-labeled antigen bound to the solid phase, and finally The color is also lighter. Therefore, the absorbance value (OD value) of the sample is inversely proportional to the concentration.
5. Precautions for ELISA test
5.1 How to order the kit The EL1SA kit currently used in basic research is different from the kit used in clinical testing and cannot be used for medical diagnosis, but only for research. After determining the indicator to be tested, it is necessary to purchase a kit of the corresponding kind according to the species source of the sample.
Then, the number of required kits is calculated based on the number of samples to be tested. Taking the most commonly used 96-well microplate as an example, each plate needs to be a standard curve. In general, the standard curve is set to 6 to 8 different concentration values. If 8 points are calculated, each sample needs to be duplicated. , the maximum number of samples that can be detected per board is 40.
Dividing the number of samples to be tested by 40 is the number of kits you need to purchase. One more thing to note before ordering an ELISA kit: Some kit instructions require that the pre-treatment of the sample must use a specific kit of reagents produced by the same company, so if you measure the amount of an indicator in your tissue, you need to use the appropriate Tissue protein extraction reagent and protein concentration detection reagent. In this case, all relevant reagents should be purchased in advance in accordance with the instructions, so as not to delay the experiment.
5.2 The kit needs to be taken out of the refrigerator before use and equilibrated for about 30 minutes at room temperature for testing.
5.3 Collection and preservation of specimens When collecting serum samples, the whole blood specimens should be placed at room temperature for 2 hours or 4 °C overnight, and the supernatant can be separated by centrifugation, or the supernatant can be stored at _20~C or below. Take a backup. In order to avoid the degradation of the test substance in the serum caused by repeated freezing and thawing, it is necessary to pack the serum in an appropriate amount and then freeze it. If a plasma sample is required for ELISA, an anticoagulant (such as EDTA, heparin or sodium citrate) may be added to the blood sample, and the supernatant may be centrifuged for 30 minutes or frozen for storage. For the cell culture supernatant, it was centrifuged at 1000 rpm for 5 min, and the supernatant was taken for detection in order to remove floating cells suspended therein. Before performing the ELISA assay, the sample needs to be diluted and used for testing according to the requirements of the corresponding instructions.
5.4 Dilution factor of sample When the sample to be tested is serum or plasma, the ELISA test reagent instructions usually give the recommended dilution factor for easy operation. A more complicated and cumbersome situation is the detection of a quantitative expression of a substance in a tissue sample. No ELISA reagent specification will clarify a key question: After extracting the protein from the tissue sample, how many times the dilution of the sample is most suitable for testing?
Because the abundance of the substance to be tested may be different in different tissues; at the same time, for different researchers, even if the same tissue is used for detection, if the degree of homogenization or lysis of the tissue is different, the extracted The protein concentration is naturally different, so there will be differences in the dilution factor for the sample. In this case, the investigator must first extract the tissue protein supernatant and determine the protein concentration, and then combine the concentration of the test substance in the tissue reported in the literature (generally the substance to be tested per gram of tissue protein). Quality), estimate the dilution factor of the protein supernatant. Then, based on the estimated dilution factor, it is necessary to set the dilution factor as the center, and set another 2-3 dilutions, and then try to find the optimal dilution factor through the preliminary experiment, and finally carry out the formal experiment.
5.5 Selection of anticoagulants For some special detection indicators, in order to make the measurement results accurate and reliable, you need to know the relevant knowledge. For example, when performing lipoprotein analysis with plasma samples, attention needs to be paid to the choice of anticoagulant. Due to the hypotonic effect of low molecular weight anticoagulants such as sodium citrate and sodium fluoride, a large amount of water molecules enter the plasma to cause dilution of plasma components. The divalent cation chelating agent EDTA, due to its large molecular weight, has a low hypotonic effect and has little effect on plasma protein concentration, only 3% or 4%. Heparin has a larger molecular weight than EDTA, which produces anticoagulation. The dilution effect on plasma was not detected at the concentration of action. Therefore, both EDTA and heparin can be used as anticoagulants for lipoprotein analysis studies. Meanwhile, since EDTA can inhibit the oxidation reaction and enzyme component changes during plasma cryopreservation, ED-TA is in practice. The most commonly used anticoagulant. The ELISA kit for basic research generally recommends the anticoagulant to be used in the test instructions. Please pay attention to the blood sample before taking it.
5.6 Other instruments and consumables that need to be prepared In addition to the microplate reader, the instruments used are the special temperature control shaker, 37~C incubator and vortex mixer, which need to be operated according to different kits. Choose to use it. In addition, pipettes, disposable tips, absorbent paper, latex gloves, beakers and measuring cylinders, deionized water, ice boxes, test tubes and centrifuge tubes of various ranges. All the above items should be prepared at hand before the test starts to make the inspection process go smoothly.
5.7 ELISA operation points The following steps are more important in the ELISA operation: (1) Loading: The sample should be accurate, the reagent should be added vertically, and the reagent should not be tilted or artificially increased.
Use disposable tips to prevent cross-contamination. Calibrate the sampler regularly. Specimens should be added to the bottom of the micropores to avoid being added to the upper part of the hole wall, and be careful not to spill and create bubbles. (2) Incubation: The 96-well microtiter plate has a special structure, which is easy to produce edge effect. The antigen-antibody binding and enzymatic reaction have strict temperature requirements. The temperature of the pores around the microplate and the internal pores are different, which causes the surrounding and internal. There are differences in hole results. Therefore, the whole plate should be closed with a sealing film during the incubation process to avoid edge effects. The temperature of the incubator must be strictly controlled, and the incubation time should be as accurate as specified in the instructions. (3) Washing: Washing is an important part of ELISA operation. The consistency of hand washing conditions is poor, which has a great impact on the results. Improper use of semi-automatic and fully automatic washing machines will also affect the results. Make sure that the washing liquid fills each plate hole during washing, but do not overflow the plate hole. After the liquid is discarded, the liquid in the hole is patted dry on the absorbent paper. In order to better the washing effect, the laboratory can adopt a combination of machine and manual washing, and then manually wash the plate 1-2 times after the machine is washed, or increase the number of times of washing.
(4) Color termination: The color development time should be strictly in accordance with the instructions. The microplate reader should be read within 15 min of the color termination.
5.8 Establishing the test procedure using the fully automatic multi-function microplate reader For the specific test indicators, the concentration of the standard sample, the number of samples, the dilution factor of the sample, the sample arrangement and the data are set using the microplate reader and its related software. Conditions such as the output format, the detection procedure is established in advance before the color reaction is terminated. After the coloration is terminated, the reaction plate can be placed in the microplate reader, the detection program can be opened for reading, and then the result can be printed immediately by the connected printer, which is simple and quick.
In summary, although the ELISA method is simple and convenient, there are many factors affecting the measurement results. Therefore, in the actual operation process, it is necessary to strengthen the quality management and strictly carry out the experimental operation according to the kit instructions. The application of automatic microplate reader can realize ELISA standardized detection to avoid or reduce the influence of relevant factors, and strive to achieve accurate results and provide a reliable basis for medical scientific research.
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