Enzyme-linked immunosorbent assay (ELISA) operation points

1 Specimen adoption and preservation

Specimens that can be used for ELISA assays are extensive, and body fluids (such as serum), secretions (saliva), and excreta (such as urine, feces) can be used as specimens to determine an antibody or antigen component. Some specimens can be directly measured (such as serum, urine), while others require pretreatment (such as feces and certain secretions). Most ELISA tests use serum as a specimen. In addition to fibrinogen and anticoagulant, the plasma is equivalent to serum. Preparation of plasma specimens requires the use of anticoagulants, and serum specimens can be obtained as long as the serum naturally solidifies and the blood clot shrinks. In addition to special circumstances, serum was used as a test specimen in medical tests. Plasma and serum can be equally applied in ELISA. Serum samples can be collected according to conventional methods. Care should be taken to avoid hemolysis. When red blood cells are dissolved, substances with peroxidase activity are released. In ELISA assays marked with HRP, hemolyzed specimens may increase non-specific coloration.

Serum samples should be tested when fresh. If there is bacterial contamination, the bacteria may contain endogenous HRP, which may also produce false positive reactions. If stored in the refrigerator for a long time, the polymerization can occur, and the background can be deepened in the indirect ELISA. In general, serum samples measured within 5 days can be placed at 4 ° C, and cryopreservation is required for more than one week. After the frozen serum is melted, the protein is partially concentrated and unevenly distributed. It should be mixed well and gently, avoiding air bubbles, and mixing upside down, do not oscillate strongly on the mixer. Bloody or precipitated serum samples should be centrifuged or filtered before clarification. Repeated freezing and thawing will cause the antibody titer to fall. Therefore, if the serum sample of the antibody is to be stored for multiple tests, it should be stored in small amounts. Preservation of serum should be performed aseptically, and appropriate preservatives may be added.

2 reagent preparation

Prepare the reagents required for the experiment as required by the kit instructions. Distilled or deionized water used in ELISA, including for washing, should be fresh and of high quality. The self-contained buffer is corrected using a pH meter. The test reagent taken out of the refrigerator should be used after the temperature and room temperature are balanced. The parts of the kit that are not needed for this test should be returned to the refrigerator in time for storage.

3 loading

In the ELISA, there are generally three steps of adding the sample, that is, adding the sample, adding the enzyme conjugate, and adding the substrate. When adding the sample, add the added material to the bottom of the LEISA plate hole, avoid adding it to the upper part of the hole wall, and be careful not to splash it, and do not generate bubbles.

The spiked sample is generally added to the well of the plate in a prescribed amount using a micropipette. The nozzle should be replaced every time the specimen is added to avoid cross-contamination. It can also be loaded with a disposable quantitative plastic tube. In this assay (such as indirect ELISA), diluted serum is used, which can be diluted in a test tube at the specified dilution before loading. Dilutions can also be added to the wells, serum samples are added to them, and then shaken on a micro-vibrator for 1 minute to ensure mixing. When adding the enzyme conjugate application liquid and the substrate application liquid, a quantitative multi-channel liquid applicator can be used to complete the liquid addition process quickly.

4 insulation

There are generally two antigen-antibody reactions in an ELISA, ie, after adding the sample and adding the enzyme conjugate. The completion of the antigen-antibody reaction requires a certain temperature and time. This incubation process is called incubation, which is called incubation and may not be appropriate in ELISA.

ELISA is a solid phase immunoassay in which antigen and antibody binding occurs only on the surface of a solid phase. Taking the sandwich method of antibody coating as an example, the specimen added to the well of the plate does not have an equal opportunity to bind to the solid phase, and only the antigen in the solution closest to the pore wall is directly in contact with the antibody. . This is a process of gradual equilibrium, so it needs to be diffused to reach the end of the reaction. The same is true for the binding of the enzyme-labeled antibody added thereafter to the solid phase antigen. This is why ELISA reactions always require a certain amount of incubation.

Temperatures commonly used for incubation are 43 ° C, 37 ° C, room temperature, and 4 ° C (refrigerator temperature). 37 ° C is the usual incubation temperature in the laboratory, and is also the appropriate temperature for most antigen-antibody binding. When the ELISA method was established as the reaction kinetics study, the experiment showed that the two antigen-antibody reactions generally reached the peak at 37 ° C for 1-2 hours. In order to accelerate the reaction, the temperature of the reaction can be raised. Some tests are carried out at 43 ° C, but higher temperatures are not suitable. The antigen-antibody reaction was more thorough at 4 ° C, and the reaction was mostly allowed to stand overnight in the refrigerator in a radioimmunoassay to form the most precipitate. However, because the time required is too long, it is generally not used in ELISA.

Insulation method In addition to the special ELISA instrument with special electric heating block, the water bath is generally used. The ELISA plate can be placed in the water bath box. The bottom of the ELISA plate should be attached to the water surface to make the temperature balance quickly. In order to avoid evaporation, the board should be covered, or the plate hole can be covered with plastic sealing paper or plastic wrap. At this time, the reaction plate can float on the water surface. If an incubator is used, the ELISA plate should be placed in a wet box. The wet box should be made of a material with good heat transfer such as metal, a wet gauze at the bottom of the box, and finally the ELISA plate should be placed on the wet gauze. The wet box should be pre-warmed in the incubator to the specified temperature, especially at lower temperatures. Whether it is water bath or wet box incubation, the reaction plates should not be stacked to ensure that the temperature of each plate can be quickly balanced. For the reaction at room temperature, the room temperature during the operation should be strictly limited to the specified range. The standard room temperature refers to 20-25 ° C, but the specific conditions can be controlled according to the requirements of the instructions. When incubated at room temperature, the ELISA plate should be placed flat on the console. It should be noted that the temperature and time of incubation should be as accurate as required. In order to ensure this, one person should not measure more than two plates at the same time.

5 washing

Washing is not a reaction step in the ELISA process, but it also determines the success or failure of the experiment. ELSIA relies on washing to achieve the separation of free and bound enzyme markers. The substance remaining in the pores of the plate which is not capable of binding to the solid phase antigen or antibody, and the interfering substance which is non-specifically adsorbed to the solid phase carrier during the reaction are removed by washing. The adsorption of proteins by plastics such as polystyrene is universal, and such non-specifically adsorbed interfering substances should be washed away during washing. It can be said that in the ELISA operation, washing is the most important key technology, which should be highly valued by the operator. The operator should wash it strictly according to requirements, and should not be sloppy.

Washing method In addition to some special ELISA instruments equipped with special automatic washing instruments, manual operation has two types of immersion and running water, the process is as follows:

(1) Immersion type a. Drain or dry the reaction solution in the well; b. Wash once with the washing liquid (after the washing liquid is filled into the plate hole, it is smashed); c. Soak, the washing liquid is filled Hole, place for 1-2 minutes, intermittent shaking, soaking time can not be shortened arbitrarily; d. suck the liquid in the hole. Drain dry thoroughly, pump with a water pump or vacuum pump, or pat dry on a clean towel or absorbent paper after removing the liquid; e. Repeat operations c and d, wash 3-4 times (or as specified). In the indirect method, if the background is higher, the number of washings or the soaking time can be increased.

Microtiter plates are mostly immersed. The washing solution is mostly a neutral buffer containing a nonionic detergent. The binding of the polystyrene carrier to the protein is hydrophobic. The nonionic detergent contains both a hydrophobic group and a hydrophilic group, and the hydrophobic group binds to the hydrophobic group of the protein through a hydrophobic bond, thereby weakening the protein and The solid phase carrier is combined, and by the combination of the hydrophilic group and the water molecule, the protein is returned to the aqueous solution state, thereby being separated from the solid phase carrier. The non-ionic detergent in the washing liquid is generally Tween 20, and its concentration may be between 0.05% and 0.2%. When the concentration is higher than 0.2%, the antigen or antibody coated on the solid phase may be desorbed to reduce the test. Sensitivity.

(2) Flow-through flushing The flow-through flushing method is initially used for the washing of the bead carrier, and the washing liquid is only distilled water or even tap water. A special device is attached during washing to continuously roll and wash the beads under the impact of running water. After rinsing for 2 minutes, the liquid is drained, soaked in distilled water for 2 minutes, and blotted dry. The immersion is like a bath, and the running water is like a shower. The washing effect is more thorough and simple and fast. It has been shown in experiments that the flow rinse type is also suitable for the washing of microtiter plates. Try to increase the water flow or increase the water pressure during washing, so that the water flow impacts the surface of the plate hole, and the washing effect is better.

6 color and colorimetric

(1) Color development

Color development is the last incubation reaction in the ELISA where the enzyme catalyzes the colorless substrate to produce a colored product. The temperature and time of the reaction are still factors that affect color development. The negative holes remain colorless for a certain period of time, while the positive holes are enhanced in color over time. Properly increasing the temperature helps accelerate color development. In the quantitative determination, the reaction temperature and time after the addition of the substrate should be determined according to the regulations. The color of the qualitative measurement can be carried out at room temperature, and the time generally does not need to be strictly controlled. Sometimes, the reaction time can be appropriately shortened or extended according to the coloration of the positive control hole and the negative control hole, and judged in time.

The color development of the OPD substrate is generally no longer deepened after 20-30 minutes of outdoor temperature or 37 ° C reaction, and the reaction time is prolonged to increase the background value. The OPD substrate liquid will change color by itself, and the color reaction should be carried out in the dark. At the end of the color reaction, the stop solution is added to terminate the reaction. After the OPD product was terminated with sulfuric acid, the color developed from orange to brown.

TMB is not affected by light and can be placed on the console at room temperature to reflect the observations. However, in order to ensure the stability of the experimental results, it is advisable to read the results at the appropriate time. After TMB was subjected to HRP, the color developed to a peak in about 40 minutes, and then gradually weakened, and after 2 hours, it completely disappeared to colorless. There are a variety of stop solutions for TMB, and enzyme inhibitors such as sodium azide and sodium dodecyl sulfate (SDS) can terminate the reaction. This type of terminator still keeps the blue color for a long time (12-24 hours) and is a good terminator for visual judgment. In addition, various types of acidic stop solutions turn the blue color into yellow, at which point the absorbance can be read at a specific wavelength (450 nm).

(2) colorimetric

Before colorimetry, dry the liquid attached to the bottom of the plate with a clean absorbent paper, and then place the plate correctly in the colorimetric frame of the enzyme-based colorimeter. For the test with soft board as the carrier, the board needs to be placed in a standard 96-hole frame before colorimetry can be performed. It is best to cut the edge of the soft board before adding the liquid to the substrate, so that the board can be seated completely in the seat.

When colorimetric, first check the zero point with distilled water, and read the substrate hole (the hole without adding any substrate liquid without any reaction) and the blank hole (the hole for the whole process by replacing the sample with physiological saline or diluent) to record the book. The reagent status of the subtest. Thereafter, a blank hole can be used to calibrate the zero point of the distilled water, and the absorbance of each of the above holes is subtracted from the absorbance of the blank hole, and then calculation is performed.

The colorimetric result is expressed by the conventional general optical density (OD). The absorbance (A) is used as specified, and the meanings of the two are the same. The usual representation is to write the absorption wavelength in the lower right corner of the A letter, such as the absorption wavelength of OPD is 492 nm, which means the method is "A492nm" or "OD492nm".

(3) Enzyme standard colorimeter

The enzyme standard colorimeter is referred to as a microplate reader, and generally refers to a photometer dedicated to reading the absorbance of an ELISA result. For the different forms of solid support, each has a special design for plates, beads and small tubes. Many reagent companies supply microplate readers. The main performance indicators of the microplate reader are: reading speed, accuracy of reading, repeatability, accuracy and measurable range, linearity and so on. Excellent microplate readers typically have a reading accuracy of 0.001, an accuracy of ±1%, and a repeatability of 0.5%. For example, if the measured A value of a hole is 1.083, the true A value of the hole relative to air should be 1.083±0.01 (1.073~1.093), repeated several times, and the A value should be 1.083±0.05 (1.078). ~1.088) between. The measurable range of the microplate reader varies depending on the performance of each microplate reader. The common microplate reader is 0.000~2.000, and the new model of the microplate reader is extended to 2.900 or even higher. A values that exceed the measurable upper limit are often indicated by "*" or "over" or other symbols. It should be noted that the measurable range is different from the linear range. The linear range is often smaller than the measurable range. For example, the measurable range of a microplate reader is 0.000~2.900, and the linear range is only 0.000~2.000, which is the standard in quantitative ELISA. Pay attention to the curve.

The microplate reader should not be placed under sunlight or strong light. The room temperature should be 15~30°C during operation. Preheat the instrument for 15-30 minutes before use. The test results are more stable.

When reading the A value, the sensitive absorption peak of the product should be selected, such as OPD with a wavelength of 492 nm. Some microplate readers can be used for dual-wavelength reading, that is, two readings per well, the first time at the optimum wavelength (W1), the second at the insensitive wavelength (W2), and the ELISA is not moved between the two measurements. The position of the board. For example, OPD uses W1 at 492 nm and W2 at 630 nm, and the final measured A value is the difference between the two (W1-W2). Dual-wavelength reading reduces light interference caused by scratches or fingerprints on the container.

The performance of various microplate readers is different, and the journalist's instructions should be detailed in use.

7 result judgment

The result of the qualitative determination is a simple answer to whether there is "yes" or "none" in the test specimen containing the antigen or antibody to be tested, and is indicated by "positive" or "negative", respectively. "Positive" indicates that the specimen is responsive in the assay system. "negative" is no response. A semi-quantitative result can also be obtained by qualitative judgment, that is, the titer is used to indicate the intensity of the reaction, and the essence is still a qualitative test. In this semi-quantitative assay, the specimen is tested as a series of dilutions, and the highest dilution that is positive is the titer. According to the level of the titer, the reactivity of the specimen can be judged, which is more quantitative than the observation of the depth of the undiluted specimen is strongly positive and weakly positive.

In the indirect method and the sandwich ELSIA, the positive holes are darker than the negative holes. In the competition ELISA, the negative hole was darker than the positive well. The results of the two types of reactions are judged differently and are described below.

(1) Indirect method and sandwich method

The qualitative results of such reactions can be judged by the naked eye. Visual specimens were also colorless or nearly colorless and negative, and those with clear color were positive. However, in ELSIA, the background of the coloration often appears after normal human serum reaction. The depth of the background varies depending on the composition of the reagent and the experimental conditions. Therefore, a negative control must be added in the experiment. The composition of the negative control should be normal serum or analog free of the test substance (see 3.6). When judging the results with the naked eye, it is better to use a color deeper than the negative control as an indicator of positive specimens.

The visual method is simple and clear, but quite subjective. When conditions permit, the absorbance should be measured with a colorimeter so that objective data can be obtained. The absorbance values of the sample (S, S), the positive control (P), and the negative control (N) are read first, and then calculated. There are many calculation methods, which can be roughly divided into positive judgment value method and specimen and negative control ratio method.

a. Positive judgment value

The cut-off value is generally the negative control A value plus a specific constant as a criterion for positive or negative judgment.

Judging the results by this method requires that the experimental conditions are very constant, the preparation of the reagents must be standardized, and the positive and negative control materials should meet certain specifications, and must be equipped with precision instruments and strictly in accordance with the regulations. The constants in the positive decision value formula are obtained by experimental testing of a large number of specimens in this particular system. Here is an example of a kit for detecting HBsAg. The negative control in the kit was recalcified human plasma without HBsAg, and the content of the positive control HBsAg was indicated as P=9±2 ng/ml. Two positive controls and three negative controls were set up for each test. After measuring the A value, first calculate the mean of the negative control A value (NCX) and the positive control A value (PCX), the difference between the two averages (PN) must be greater than a specific value (example 0.400), The test is effective. The values of the three negative control A should be ≥0.5×NCX and ≤1.5×NCX. If one of them exceeds this range, discard it, and the other two negative controls recalculate NCX; if there are two negative controls, the A value exceeds The above range is invalid for this experiment. The positive judgment value is calculated as follows:

Positive judgment value = NCX + 0.05

The specimen A value > positive judgment value is positive, and less than the positive judgment value is negative. It should be noted that 0.05 in the formula is a constant of the kit and is only suitable for the specific conditions, and is not versatile for various reagents.

As can be seen from the above description, in this method, the negative control and the positive control also play a quality control role in the test, and the deterioration of the reagent and the improper operation may result in "invalidation of the test".

b. Specimen/negative control ratio

This method of judgment is more suitable when the experimental conditions (including reagents) are difficult to ensure. After the A values of the specimen (S) and the negative control (N) are obtained, the S/N value is calculated. There are also writing P/N, where P does not represent positive, but a patient abbreviation, which should not be misunderstood. To avoid confusion, it is better to use S/N. In the early indirect ELISA, some authors determined that S/N was a positive standard and are now used in various assays. In fact, each measurement system should experimentally determine the respective S/N threshold. It should be noted that the negative control represented by N is human serum containing no test substance. In some kits, the negative control is a buffer containing no protein or protein content, so that the background generated after the reaction may be much lower than the background of normal human serum. Therefore, such kit rules, such as N < 0.05 (or other values), are calculated as 0.05, otherwise false positive results will occur.

(2) Competition law

In the competition ELISA, the negative wells were darker than the positive wells. The intensity of the negative coloration depends on the concentration of the enzyme conjugate in the reaction and the amount of the competitive inhibitor added. The absorbance of the negative control is generally adjusted between 1.0 and 1.5, at which time the reaction is most sensitive.

The competitive ELISA is not easy to judge by self-viewing. It is difficult for the naked eye to distinguish the difference between the weak positive reaction and the negative control. Generally, the colorimetric values of S, P and N are read by a colorimeter. There are two main calculation methods, namely the positive judgment value method and the inhibition rate method.

a. Positive judgment method

It is basically the same as the positive judgment value method in the indirect method and the sandwich method, but the positive control A value is introduced in the calculation formula, and a kit for detecting anti-HBc is taken as an example. The negative control in the kit was recalcified human plasma without anti-HBc, and the anti-HBc content in the positive control was 125 ± 100 u/ml. Two positive controls and three negative controls were set up for each test. After the A value is measured, the average of the negative control A value (NCX) and the positive control A value (PCX) are calculated first, and the difference between the two averages (NP) must be greater than a specific value (for example, 0.300). The test is effective. The values of the three negative control A should be less than 2.000, and should be ≥0.5×NCX and ≤1.5×NCX. If one of them exceeds this range, discard it and recalculate ×NCX with the other 2 negative controls; if there are 2 If the negative control A exceeds the above range, the experiment is invalid. The positive judgment value is calculated as follows:

Negative judgment value = 0.4 × NCX + 0.6 × PCX

The reaction of specimen A value ≤ positive judgment value was positive, and the reaction of A> positive judgment value was negative.

b. inhibition rate method

The inhibition rate indicates the degree of inhibition of the coloration of the negative reaction by the specimen in the competitive binding, and is calculated by the following formula:

Inhibition rate (%) = (negative control A value - specimen A value) × 100% / negative control A value

Generally, the inhibition rate is ≥50% positive, and <50% is negative.

Quantitative determination

ELSIA has complicated operation steps and affects many reaction factors. In particular, the coating of solid phase carriers is difficult to achieve uniformity among the individual bodies. Therefore, in the quantitative measurement, each batch of tests must be in the same series with different concentrations of reference standards. A standard curve is produced under the conditions. Sandwich ELISA for the determination of large molecular weight substances, the range of the standard curve is generally wide, the absorbance at the highest point of the curve can be close to 2.0, the semi-logarithmic paper is usually used for drawing, the concentration of the detected substance is the abscissa, and the absorbance is the ordinate. The values of the respective concentrations are connected point by point, and the obtained curve is generally S-shaped, and the head and tail curves tend to be flat, and the portion with a straight line at the center is the most ideal detection area.