How to operate ELISA correctly

At present, the clinical ELISA detection is usually a manual detection mode with microplate strip as solid phase, and the detection operation is very simple, which generally involves sample collection and preservation, reagent preparation, sample addition, incubation, plate washing, color development, colorimetric analysis, result judgment, result report and interpretation. Improper steps will affect the test results, especially the steps of adding samples, incubating and washing plates. Now described as follows. 1. Collection and preservation of clinical specimens Serum (plasma) is the most commonly used clinical specimen for ELISA, and sometimes saliva, cerebrospinal fluid, urine, feces and other specimens are also used for specific detection purposes. At present, the markers determined by serum samples generally include antigens and antibodies of infectious pathogens, tumor markers, hormones, special proteins, cytokines, therapeutic drugs and so on. For the collection of serum samples for the determination of hormones and therapeutic drugs, it should be noted that the collection time and even body position may have an impact on the determination results. For example, cortisone has a peak between 4-6 am: growth hormone, luteinizing hormone (LH) and follicle stimulating hormone (FSH) are all released in a paroxysmal manner. Therefore, it is necessary to take several blood samples at closely connected time intervals, and take the median value as the measured value. For another example, when changing from lying position to standing position, the activity of renin in serum will increase obviously. Another example is the detection of therapeutic drugs, and the best blood test time should be selected according to pharmacokinetics. The collection of serum samples for detecting infectious pathogen antigens and antibodies, tumor markers and special proteins has no influence on time and body position, but the following aspects should be considered when handling and storing: (1) Attention should be paid to avoiding severe hemolysis. Hemoglobin contains heme group, which is similar to peroxide activity. Therefore, in the ELISA with HRP as the marker enzyme, if the concentration of hemoglobin in the serum sample is high, it will be easily adsorbed on the solid phase during incubation, thus reacting with the HRP substrate added later to develop color. (2) In the process of sample collection and serum separation, attention should be paid to avoiding bacterial contamination as much as possible, because some enzymes secreted by bacteria may decompose protein such as antigens and antibodies; Secondly, endogenous enzymes of some bacteria, such as β -galactosidase of Escherichia coli, will cause non-specific interference to the determination methods of corresponding enzyme markers. (3) Serum samples can be stored at 2 ~ 8℃ for one week if they are separated by aseptic operation, and frozen storage is recommended if they are processed by bacteria. The long-term storage of samples should be below -70℃. (4) Pay attention to avoid repeated freezing and thawing of frozen serum samples due to power failure. The mechanical shear force caused by repeated freezing and thawing will destroy protein and other molecules in the specimen, thus causing false negative results. In addition, we should also pay attention to the mixing of frozen-thawed specimens, do not violently oscillate, and mix them repeatedly. (5) In case of turbidity or floc caused by bacterial contamination during storage, the supernatant should be taken for detection after centrifugal precipitation. Second, reagent preparation in clinical laboratories, reagent preparation is generally not taken seriously. The usual practice is to take the reagent out of the refrigerator and use it in the experiment, while ignoring the problem that this practice may affect the lack of incubation time in the later stage. The direct consequence is that some weakly positive samples are detected by false negatives. Therefore, in the reagent preparation of ELISA, the most important thing is to take the kit out of the refrigerator before the experiment starts, leave it at room temperature for more than 20 minutes, and then measure it, so that the kit can reach the balance with room temperature before use. The main purpose of this is to make the temperature in the reaction micropore reach the required height quickly in the subsequent incubation reaction step to meet the determination requirements. Secondly, the plate washing solution in the enzyme-linked immunosorbent assay (ELISA) kit currently on the market needs to be diluted and prepared with the provided concentrated solution, so the quality of distilled water or deionized water for dilution should be guaranteed. In addition, when OPD is used as the substrate in the kit, the substrate solution should be prepared temporarily before the reaction color development. Third, adding serum samples and reaction reagents In the current commercial ELISA kits, the addition of serum samples is almost the only step to add samples by using micro-sampler. The key points that must be paid attention to when using the micro sampler are: don't add the sample too quickly, avoid adding it to the upper part of the hole wall, and don't splash and generate bubbles. It is too fast to ensure the accuracy and uniformity of micro-addition. The uncoated area added to the upper part of the hole wall is easy to cause non-specific adsorption. Splash will contaminate adjacent holes. When bubbles appear, the interface of reaction liquid is different. In domestic kits, reagents are basically dripped out of dropping bottles. Besides the angle of falling, the speed of falling is also very important. If dropping is too fast, it is easy to repeat dropping or adding between two holes, which will lead to non-specific adsorption in the uncoated area of the hole, thus causing non-specific color development. So sometimes a sample of the same kit is positive this time and negative next time, which is often caused by the above mistakes in adding samples and reagents. 4. Incubation is the most critical factor affecting the success or failure of ELISA. ELISA is a solid-phase immunoassay method, and the binding reaction between antigen and antibody is carried out on the solid phase. In order to make the antigen or antibody in the liquid phase completely combine with the specific antibody or antigen on the solid phase, it is necessary to react at a certain temperature for a certain time. The incubation time is inversely proportional to the temperature, that is, the higher the temperature, the shorter the incubation time. The most commonly used incubation temperatures are 37℃ and room temperature, followed by 43℃ and 2 ~ 8℃. Incubation is the most common problem in clinical ELISA. Usually, the reaction incubation time of domestic commercial ELISA kits is 37℃ for 30 minutes ~ 1 hour, while that of imported ELISA kits is usually 37℃ 1 ~ 2 hours, which may affect the lower limit of determination. Therefore, regarding incubation, the following points must be paid attention to in the actual determination operation: (1) Ensure sufficient reaction time at the set temperature. Generally speaking, after adding samples and/or reagents, when the microplate is taken from room temperature to a water bath box or incubator, it takes a certain time for the temperature in the well to rise from room temperature to 37℃, especially when the room temperature is relatively low and it is not in a water bath state. However, in the clinical laboratory, few people pay attention to this problem. Usually, the microplate is timed as soon as it is put into the incubator, which easily leads to the actually measured incubation time. A colleague from a blood bank in the south once raised a question, that is, there is always more than one month in winter every year. In the indoor quality control of HBsAg determination, 1 ng/ml weakly positive samples provided by the clinical laboratory center of the Ministry of Health are always undetectable. I don't know why. This may be related to the low indoor temperature in winter in southern China. At this time, the incubation time at 37℃ was not enough after the microplate was transferred to the incubator, which made the weak positive sample negative. Therefore, in order to ensure sufficient incubation time at 37℃, the clinical laboratory can determine by itself how long it will take for the temperature in the well to reach 37℃ after the microplate is taken to the incubator from room temperature in different seasons (at different room temperatures) in our laboratory, so as to appropriately extend the placing time of the slats in the incubator. Specifically, a small thermometer is placed in the plate hole reaction solution for measurement and observation. (2) Selection of culture temperature. In the instructions of some ELISA kits, it is pointed out that there are two incubation temperatures, such as 37℃ 1 hour and 43℃ for 45 minutes. Judging from the nature of antigen-antibody reaction in immunoassay, the reaction is most complete at low temperature for a long time. E.g. 2-8 C for 24 hours. At higher reaction temperature, due to the regret of molecular movement, the reaction time is shortened, and there is no problem in the determination of strong positive samples with more molecules, but it is possible to miss the detection of weak positive samples with less molecules. Therefore, we suggest that the conditions of lower incubation temperature and longer reaction time should be used as much as possible in clinical ELISA determination. (3) Eliminate the "edge effect". In the past, in the ELISA determination using 96-well plate, it was often found that there was a "edge effect", that is, the color of the peripheral hole was darker than that of the central hole. The reason for this "edge effect" may be the difference in surface or thermodynamic characteristics between the peripheral hole and the central hole of the 96-well plate. However, some studies have confirmed that the thermodynamic gradient in hatching may be the fundamental reason. Polystyrene itself is a poor thermal conductor. In the routine ELISA in the laboratory, when the plate is put into an incubator at 37℃ from room temperature (usually around 25℃) and the plate is also heated, there may be a thermodynamic gradient between the peripheral hole and the central hole. Therefore, when using a water bath or adding a reaction solution to the well of the plate, heating the plate and the solution to the incubation temperature (such as 37℃) can easily eliminate the "edge effect" and improve the repeatability of the determination. To sum up, in clinical ELISA determination, in order to ensure a good determination effect, the following simple methods can be used to ensure the incubation conditions, that is, to use water bath as much as possible, so that the microplate floats on the water surface during incubation, or to put the emery cloth soaked in water into a wet box in a large lunch box, and put it into a warm box. Because the bottom of the slat hole is in direct contact with water or wet cloth, the temperature of the reaction solution can change rapidly with the greenhouse at 37℃ and the high temperature of the water bath box or wet box. 5. Wash-plate solid-phase immunoassay is a heterogeneous immunoassay technology, which requires washing operation to separate the antigen or antibody specifically bound to the solid phase from the nonspecific components adsorbed in the reaction incubation process to ensure the specificity of ELISA determination. Therefore, washing the plate is also an extremely critical step in ELISA determination. The washing liquid used in ELISA kit with HRP as the marker enzyme is generally neutral PBS containing 0.05%Tween20, which is a nonionic detergent containing both hydrophilic and hydrophobic groups. The mechanism of its action in washing is that its hydrophobic groups form hydrophobic bonds with the hydrophobic groups of protein passively adsorbed on polystyrene solid phase through hydrophobic interaction, thus weakening the adsorption of protein and solid phase. At the same time, under the joint action of hydrophilic groups and water molecules in the liquid phase, protein can be separated from the solid phase and enter the liquid phase, thus achieving the purpose of removing nonspecific adsorbents. However, because the coating of antibody or antigen is usually passively adsorbed on the solid phase through hydrophobic interaction with the solid phase under alkaline conditions, attention should be paid to the concentration of nonionic detergent. When the concentration of Tween20 in the washing solution is higher than 0.2%, the antigen or antibody coated on the solid phase will be desorbed, which will affect the detection limit. In clinical laboratories, there are generally two ways to wash plates by ELISA, namely, manual and plate washing. Manual plate washing means that after each reaction incubation, the reaction liquid is sucked out or spun dry, then the plate holes are filled with washing liquid, and after standing for 2 ~ 3 minutes, the washing liquid is sucked out or spun dry, and then it is patted dry on absorbent paper. Repeat the above washing steps for 3 ~ 4 times, and finally pat dry on absorbent paper, and then the next determination operation can be carried out. Washing the board with a washing machine is to change the above manual operation into washing the board with a washing machine. One of the characteristics of washing dishes with washing machine is that it can't be dried every time it is washed, so there is more liquid residue. The washing machine with less liquid residue needs less times to thoroughly clean the plate than the washing machine with more liquid residue. The washing machines used in specific clinical laboratories can meet the requirements after washing in China. The following simple experiments can be carried out: select 4×8 HBsAgELISA coated slats, add the same weak positive samples and negative samples every 2×8 holes, and add enzyme conjugates according to the instructions of the kit to complete the incubation. When washing plate holes, the first row is washed 1 time, the second row is washed twice, and the third row is washed three times-until the eighth row is washed eight times, and the substrate is added for color determination. If washed for three times, the color will not change, that is, the colorimetric determination of the plate hole washed for three times is the same as the absorbance of the plate hole washed for four times and five times, and the positive/negative value keeps the maximum. Six, color development In the ELISA kit with HRP as the marker enzyme currently on the market, if TMB is used as the substrate, the substrates provided are two bottles of application solutions A and B; If OPD is used as the matrix, the kit provides OPD tablets or powders prepared before use. Generally, the color reaction conditions of commercial kits are 37℃ or room temperature 15 ~ 30 minutes. Theoretically, the substrate catalytic reaction of HRP can be completed within 30 minutes at 37℃, although most of the catalytic reactions can be completed within the first 10 minutes. Therefore, in order to make the holes of weakly positive samples fully develop color, it is suggested that the reaction colorimetric determination should be terminated after 25 ~ 30 minutes at 37℃. In addition, before adding the substrate to start the color reaction, it is best to check the effectiveness of the substrate solution, that is, add a drop of solution A and solution B into a clean empty plate hole or eppendorf tube to see if there is color development. If there is, it means that the substrate has deteriorated. Using OPD as substrate, it should be colorless after preparation, otherwise it cannot be used. Using TMB as substrate, the whole color reaction process does not need to avoid light, while using OPD as substrate, it needs to avoid light. For the color reaction characteristics and precautions of TMB and OPD, please refer to the previous chapter. After the color reaction is completed, add acid to stop the reaction, shake and mix well, and then carry out the following colorimetric determination or visual judgment. Seven, colorimetric ELISA enzyme-linked immunosorbent assay colorimetric determination Some colleagues may think that since the enzyme-linked immunosorbent assay is used, then everything can be done at this time, but it is not, because the more advanced enzyme-linked instruments and meters have more functions, improper use will lead to incomprehensible results. For example, after colorimetric determination with enzyme-labeled instrument, the absorbance values of many negative determination holes are negative or the false positive rate of determination is greatly increased. This is mainly due to the lack of correct understanding and use of enzyme-labeled instruments. As for how to correctly understand and use the microplate reader, please refer to the relevant chapters in the back. Here, only the following two points are emphasized: (1) During colorimetric determination, we must pay attention to whether the wavelength of the microplate reader is adjusted or whether the filter used is correct. In some clinical laboratories, enzyme-linked immunosorbent assay (ELISA) uses both TMB kit and OPD kit, while the colorimetric wavelength of the former is 450nm and the latter is 492nm, so it is necessary to change the filter at any time as needed. Therefore, the problem of misuse of optical filters is easy to occur. (2) The problem of single-wavelength or dual-wavelength colorimetric selection. Mid-range and above enzyme-labeled instruments basically have both single-wavelength and dual-wavelength colorimetric functions. The so-called single-wavelength colorimetric determination is usually carried out at the wavelength with the greatest absorption of color development, such as 450nm or 492nm, while the dual-wavelength two-color enzyme-labeled instrument measures once at the sensitive wavelength, such as 450nm and the insensitive wavelength, such as 630nm. The absorbance measured above and below the sensitive wave is the sum of the absorbance of the specific color development of the enzyme reaction measured by the sample and the absorbance caused by fingerprints, scratches, dust and other dirt on the plate hole. Determination at non-sensitive wavelength refers to changing the wavelength to a certain value, so that the absorbance value of the specific color development of the enzyme reaction of the sample is zero, and the absorbance measured at this time is the absorbance value of the dirt. Finally, the value given by the microplate reader is the difference between the absorbance at the sensitive wavelength and the absorbance at the insensitive wavelength. Therefore, dual-wavelength colorimetric determination has the advantage of eliminating the effects of non-specific absorption, fingerprints, scratches, dust and so on. It is generally unnecessary to set a blank hole for measuring the absorbance of the specific color of the drip plate itself and the sample on the hole of the plate. If the blank hole is still set when the dual-wavelength colorimetry is used, it may cause the phenomenon that the absorbance of the above measuring hole is negative. Because there is a certain degree of uncertainty in the non-specific absorption of a single blank hole in ELISA, that is to say, different absorbance values may be obtained each time or at the same time, so it is best to use dual-wavelength colorimetry for ELISA colorimetric determination. Eight. Results Clinical ELISA assay can be divided into qualitative assay and quantitative assay according to the way of expressing the assay results. Qualitative determination is only to make a "yes" or "no" conclusion on whether the specimen contains the antigen or antibody to be detected, which is represented by "positive" and "negative" respectively. Visible qualitative determination is usually used to determine the antigen or antibody of infectious pathogens to judge the existence of specific pathogen infection. Quantitative determination is the quantitative determination of China of the antigen to be detected in the specimen, which is expressed by a specific value. Quantitative determination is basically used for the determination of non-pathogen antigen substances, such as hormones, cytokines, tumor markers, small molecular drugs and so on. At present, most of the ELISA kits used clinically in China are used for qualitative determination of antigens or antibodies of infectious pathogens, and a few are used for quantitative determination of αFP, hCG and cytokines. The determination of "positive" and "positive" in ELISA qualitative determination is based on the positive determination value (critical value) determined by the kit. The "value" of quantitative determination is based on the dose-response curve (also called standard curve) obtained by simultaneously determining the standard substance in the kit. The data processing of qualitative and quantitative results of ELISA will be discussed in the following special chapters. What I just want to emphasize here is that the "negative" and "positive" results of ELISA qualitative determination can only be judged according to the critical value of the kit itself, and the weak positive fixed-value quality control serum provided by the Clinical Inspection Center of the Ministry of Health cannot be used. Why should we emphasize this point? This is mainly because many grass-roots laboratories use the absorbance of weak positive fixed-value quality control serum (also called "critical serum" in the past) supplied by the Ministry Center to judge the results. If it is higher than it, it is judged as positive, otherwise it is negative, regardless of the critical value of the kit. Up to now, some laboratories still insist on this wrong practice. The establishment of the critical value of the kit is based on a series of scientific experiments and statistical studies (see below). The weakly positive fixed-value quality control serum provided by the Ministry Center is mainly used for indoor quality control in clinical laboratories. Next, we will explain some abbreviations commonly used in determining the qualitative results of ELISA. (1)S/CO: where s is the abbreviation of the sample or specimen, indicating the absorbance value determined by the specimen, and CO is the abbreviation of the cutoff value. Except for the competitive inhibition method, in other qualitative determination modes of ELISA, when the S/CO value is greater than or equal to 1, the sample is positive, and when it is less than 1, it is negative. (2)S/N or P/N: where S is the same as (1), N is the abbreviation of negative control, and P is the abbreviation of patient. Many early kits used S/N or P/N≥2. 1 as the positive standard, and some kits still use this method. There is no essential difference between this method and S/CO method, except that the former takes 2. 1 times of the negative control (n) as the critical value. Nine, the results report and the report explaining the results of clinical ELISA are relatively simple. Qualitative determination can be negative or positive; Quantitative determination reports specific values. The interpretation of the results is much more complicated, which requires laboratory technicians to have a comprehensive knowledge base of the measured items. For example, the interpretation of the "two and a half" results of hepatitis B requires not only the examiners to know the clinical significance of different results patterns, but also a deeper understanding of the molecular biology, molecular variation and its influence on the phenotype of hepatitis B virus. At present, inspection is no longer a simple laboratory measurement, but has become a clinical medicine discipline, that is, laboratory medicine, which requires laboratory doctors engaged in medical inspection not only to know what they are doing, but also to know why they are doing it, otherwise they will be eliminated by the times. To sum up, although the operation steps of ELISA determination are simple, there are many factors that may affect the determination results, which are distributed in all steps of the determination operation, especially sample addition, incubation and plate washing. In order to help you analyze and find the possible causes of problems in the determination, the following table summarizes the common problems and their causes. Possible problems and reasons in clinical ELISA detection. Possible reasons (not the package itself) 1. Weak positive quality control samples cannot detect incubation time or insufficient temperature; The color reaction time is too short; There is something wrong with the distilled water used to prepare the buffer. 2. The repeatability of the determination is poor (the results of two determinations of the same sample are inconsistent). This is a typical problem caused by the determination operation, including (1) sample addition and inaccurate reagent dosage; Inconsistency between holes; (2) The sample is added too quickly, resulting in pollution between holes; (3) adding the wrong sample; (4) When samples and reagents are added, they are added to the uncoated area on the upper part of the hole wall; (5) mixing the components in the kit with different batch numbers; (6) The incubation time, plate washing and color development time are inconsistent; (7) Contaminated sundries in the hole; (8) The filter of the microplate reader is incorrect; (9) When the serum sample is added before complete coagulation, fibrin clot or residual blood cells appear in the reaction hole, which is prone to false positive reaction. 3. White board (positive control does not develop color) (1) missed adding enzyme conjugate; (2) There is something wrong with the preparation of washing liquid, such as the measuring cylinder is not clean and contains enzyme inhibitors (such as sodium azide). (3) omit the developer A or B; (4) Terminating agent is used as developer. 4. All plate holes are colored (1), and the plate is not clean; (2) The chromogenic solution deteriorates; (3) The absorption of added substrate is polluted by enzyme; (4) The washing liquid is polluted by enzyme.