9 Tips For Correct ELISA Operation

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 4-6 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., has no time and position impact, 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°C for a week. If it is an operation with bacteria, it is recommended to be frozen. 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.

Incubation

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°C 30 minutes to 1 hour, and imported ELISA kits are usually 37°C for 1-2 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.

(2) The choice of incubation temperature. In the instructions of some ELISA kits, it is pointed out that there are two incubation temperatures, for example, one is at 37°C for 1 hour, and the other is at 43°C for 45 minutes. From the perspective of the nature of the antigen-antibody reaction in immunoassays, the reaction is most complete at a lower temperature for a longer time. For example, react at 2-8°C for 24 hours. The higher reaction temperature will shorten the reaction time due to the increase in molecular movement. This is no problem for the determination of strong positive samples with more molecules, but there is a possibility of missed detection for weak positive samples with less molecular content. Therefore, we recommend that we try to use conditions with lower incubation temperature and longer reaction time in clinical ELISA assays.

(3) Elimination of “edge effects”. In the previous ELISA assays using 96-well plates, “edge effects” were often found, that is, the outer peripheral holes were deeper than the center holes. The reason for this “edge effect” may be the surface of the 96-well plate and the center hole. Differences in thermodynamic characteristics. However, studies have confirmed that the thermodynamic gradient in incubation may be the root cause. Polystyrene itself is a poor thermal conductor. In routine ELISA determinations in the laboratory, the plate is placed in a 37°C incubator from room temperature (usually around 25°C). When the plate is also warming up, there may be a gap between the outer peripheral hole and the central hole. There is a thermodynamic gradient. Therefore, using a water bath or heating the plate and solution to the incubation temperature (such as 37°C) when the reaction solution is added to the wells can easily eliminate the “edge effect” and improve the repeatability of the measurement.

All in all, in clinical ELISA assays, to ensure good results, the following simple methods can be used to ensure incubation conditions, that is, use a water bath as much as possible, let the microplate float on the water during incubation, or put a sand cloth soaked in water. Put it into a large lunch box into a wet box and place it in the incubator. This will cause the temperature of the reaction solution to be caused by the direct contact of the bottom of the slat hole with 37°C water or wet cloth and the high temperature in the water bath or wet box. Quickly balance with the greenhouse.

Wash the board

The solid-phase immunoassay technique is a heterogeneous immunoassay technique. It is necessary to separate the antigen or antibody specifically bound to the solid phase from the non-specific components adsorbed during the reaction incubation by washing operation to ensure the specificity of the ELISA assay. Sex. Therefore, plate washing is also an extremely critical step for ELISA determination. The washing liquid used in the ELISA kit with HRP as the labeled enzyme is generally neutral PBS containing 0.05% Tween20. Tween20 is a non-ionic detergent that contains both hydrophilic and hydrophobic groups. The mechanism of action in washing is to use its hydrophobic group to form a hydrophobic bond with the hydrophobic group of the protein passively adsorbed on the solid phase of polystyrene through hydrophobic interaction, thereby weakening the adsorption of the protein to the solid phase, and at the same time in its affinity The combination of the water group and the water molecules in the liquid phase promotes the protein to leave the solid phase and enter the liquid phase, so that the purpose of removing non-specific adsorbents can be achieved, but because the coating of antibodies or antigens is usually carried out in alkaline Under the conditions, the hydrophobic interaction with the solid phase will passively adsorb to the solid phase. Therefore, attention should be paid to the use concentration of non-ionic detergent. The concentration of Tween20 in the plate washing solution is higher than 0.2%, which can make the antigen coated on the solid phase. Or antibody desorption affects the lower limit of the test.

In clinical laboratories, there are generally two ways of washing plates for ELISA determination, namely manual and washing machine washing. Manual plate washing means that after each reaction incubation, the reaction solution is sucked out or dried, and then filled with washing solution in the wells of the plate. After 2-3 minutes, the washing solution is sucked out or dried, and then placed on absorbent paper. Pat dry. Repeat the above washing steps 3-4 times, and finally pat dry on absorbent paper to proceed to the next measurement operation. The plate washer is to change the above manual operation to the plate washer. One of the characteristics of the plate washer is that it cannot be patted dry after each washing, so there is more liquid residue, and the amount of liquid residue is small. The number of times required to wash the plates thoroughly is less than that of a plate washer with a large amount of residue. In the plate washer used in a specific clinical laboratory, how many times can be washed to meet the requirements, you can perform the following simple experiment: choose 4×8 HBsAgELISA coated strips, and add the same weak positive to every 2×8 wells. And a negative sample. After adding the enzyme conjugate according to the kit instructions and completing the incubation, wash the wells according to the first row of 4 wells once, the second batch of 4 wells twice, and the third row of 4 wells 3 Times-Wash 8 times until the 4th row of the 8th row, add the substrate to determine the color development, such as washing 3 times, the color development will not change, that is, the colorimetric determination of the wells of the 3 washes and the 4 and 5 washes of the plate If the absorbance of the wells are the same, and the positive/negative values remain the same, it can be determined that the plate washer used in the laboratory can meet the requirements after three washings.

Color rendering

In the current commercial ELISA kits that use HRP as the labeled enzyme, 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 substrate, the kit provides OPD tablets Or powder, prepared before use. The color reaction conditions of general commercial kits are 37°C or room temperature for 15-30 minutes. Theoretically, only 30 minutes at 37°C can complete the catalytic reaction of the HRP substrate, although most of the catalytic reaction can be completed within the first 10 minutes. Therefore, in order to make the weakly positive sample wells have sufficient color development, it is recommended to terminate the reaction colorimetric measurement after reacting at 37°C for 25-30 minutes.

In addition, before adding the substrate to start the color reaction, it is best to check the effectiveness of the substrate solution. You can add one drop of each of the two solutions A and B to a clean empty plate hole or eppendorf tube to observe whether If there is color development, it means that the substrate has deteriorated. With OPD as the substrate, it should be colorless after preparation, otherwise it cannot be used. With TMB as the substrate, the entire color reaction process does not need to be protected from light, while with OPD as the substrate, it needs to be protected from light. For the color reaction characteristics and points of attention of TMB and OPD, please refer to the previous chapters. After the color reaction is completed, add acid to terminate the reaction, and after shaking and mixing, the following colorimetric determination or visual judgment can be carried out.

Color comparison

The colorimetric determination of ELISA is carried out by a microplate reader. Some colleagues may think that since it is carried out by a microplate reader, everything will be fine at this time. In fact, this is not the case, because there are more advanced microplate readers. If the function is used improperly, you will get incomprehensible results. For example, after colorimetric determination with a microplate reader, the absorbance value of many negative measuring wells is negative or the false positive rate is greatly increased, and so on. This is mainly caused by the incorrect understanding and use of the microplate reader. As for how to correctly understand and use the microplate reader, please refer to the following chapters. Here, only the following two points are emphasized:

(1) During colorimetric determination, be sure to pay attention to whether the wavelength of the microplate reader has been adjusted properly or whether the filter used is correct. Because some clinical laboratories use TMB as the substrate and OPD as the substrate when performing ELISA assays. The colorimetric wavelength of the former is 450nm and the latter is 492nm. The filter needs to be available at any time according to requirements. replace. Therefore, the problem of misuse of the filter is prone to occur.

2) The choice of single-wavelength or dual-wavelength colorimetry. Basically all microplate readers above the mid-range have both single-wavelength and dual-wavelength colorimetric functions. The so-called single-wavelength colorimetry is the usual colorimetric determination at the wavelength with the maximum absorption of the color, such as 450nm or 492nm; while the dual-wavelength dual-color microplate reader measures at sensitive wavelengths such as 450nm and non-sensitive wavelengths such as 630nm. Once, the absorbance measurement value above and below the sensitive wave is the sum of the absorbance of the specific color developed by the enzyme reaction of the sample and the absorbance caused by the fingerprints, scratches, dust and other dirt on the plate hole; the measurement at the non-sensitive wavelength is to change the wavelength to a certain value , So that the absorbance value of the specific color developed by 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 value at the sensitive wavelength and the absorbance value at the very sensitive wavelength. Therefore, dual-wavelength colorimetric measurement has the advantage of eliminating the influence of non-specific absorption, fingerprints, scratches, dust, etc. of the microtiter plate itself and the specimen in the plate hole on the absorbance of the specific colorimetric measurement. Generally, there is no need to set a blank hole. If a blank hole is still set when using dual-wavelength colorimetry, it may cause the aforementioned phenomenon that the absorbance of the measuring hole is negative. Because there is a certain degree of uncertainty in the non-specific absorption of a single blank hole in the ELISA measurement, that is to say, the difference in the position of the blank hole in each measurement or the same measurement may result in different absorbance measurement values, so the colorimetric measurement in ELISA When, it is best to use dual-wavelength colorimetry.

Result Judgment

Clinical ELISA assays are divided into two categories, qualitative and quantitative, according to the way they express the results. The qualitative determination is only to make a “yes” or “no” conclusion on whether the sample contains the antigen or antibody to be tested, which is represented by “positive” and “negative” respectively. It can be seen that qualitative determination is usually used for the determination of antigens or antibodies of infectious pathogens to determine the presence or absence of infection by specific pathogens. The quantitative determination is the quantitative determination of the amount of the antigen to be tested in the specimen, expressed in specific numerical values. Quantitative determination is basically used for the determination of non-pathogen antigenic substances, such as hormones, cytokines, tumor markers, small molecule drugs and so on. At present, most of the ELISA kits used clinically in China are used for the qualitative determination of antigens or antibodies of infectious pathogens, and a small number are used for the quantitative determination of αFP, hCG, cytokines, etc. The basis for determining “positive” and “positive” in the qualitative determination of ELISA is the positive judgment value (Cut-off) determined by the kit. The “quantity value” of quantitative determination is based on the dose-response curve (also known as the standard curve) obtained from the simultaneous measurement of the standard products in the kit. The data processing of the qualitative and quantitative determination results of ELISA will be discussed in a special chapter later. The point that this department only wants to emphasize is that the judgment of “negative” and “positive” results of qualitative ELISA can only be based on the Cut-off value determined by the kit itself, and cannot be based on the weak positive test supplied by the Clinical Testing Center of the Ministry of Health. Value quality control serum. Why should this point be emphasized? The main reason is that in the past, many primary laboratories used the weakly positive fixed value quality control serum (previously called “cut-off value” serum) supplied by the department center to determine the absorbance of the result. If it is higher than it is judged to be positive, otherwise it is negative. Regardless of the Cut-off value of the kit. So far, there are still some laboratories insisting on this wrong approach. The cut-off value of the kit is established on the basis of a series of scientific experiments and statistical studies (see below for details), and the weakly positive fixed value quality control serum supplied by the Ministry Center is mainly for clinical laboratories. Used for indoor quality control.

Let me explain some abbreviations commonly used in qualitative determination of ELISA results.

(1) S/CO: where S is the abbreviation of Sample or Specimen, which represents the absorbance value measured by the specimen, and CO is the abbreviation of Cut-off value. In addition to the competitive inhibition method, in other qualitative determination modes of ELISA, when the S/CO value is greater than or equal to 1, the determination of the specimen 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 for Negative (negative control), and P is the abbreviation for Patient (patient). Many earlier kits use S/N or P/N≥2.1 as the positive determination standard, and there are still some kits that use this method. This method is not fundamentally different from the S/CO method, except that the former considers 2.1 times the negative control (N) as the Cut-off value.

Results report and explanation

The report of clinical ELISA test results is relatively simple. For qualitative determination, report negative or positive; for quantitative determination, report the specific value. The interpretation of the results is much more complicated than that, and it requires laboratory technicians to have a more comprehensive knowledge base of the measured items. For example, the interpretation of the “two-and-a-half” result of hepatitis B not only requires the examiner to know the clinical significance of the different result models, but also has a deeper level of the molecular biology of hepatitis B virus, molecular variation and its influence on the phenotype. Understanding. Nowadays, inspection is no longer a simple laboratory measurement, but has become a clinical medicine discipline, namely laboratory medicine. This requires laboratory physicians who are engaged in medical laboratory work to be aware of the measurement items they are doing, as well as their knowledge. Therefore, otherwise, it will inevitably be eliminated by the times.

In summary, although the operation steps of the ELISA assay are very simple, there are many factors that may affect the assay results, which are distributed in each step of the assay 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 common problems and their causes are summarized in the table below.

Possible problems and possible causes in clinical ELISA determination