Key points of enzyme-linked immunosorbent assay (ELISA) operation

1 Collection and preservation of specimens
Specimens that can be used for ELISA measurement are very wide. Body fluids (such as serum), secretions (saliva), and excreta (such as urine and feces) can be used as specimens to determine certain antibody or antigen components. Some specimens can be directly measured (such as serum, urine), and some require pretreatment (such as feces and certain secretions). Most ELISA tests use serum as the specimen. Except for fibrinogen and anticoagulants, other components in plasma are equivalent to serum. The preparation of plasma specimens requires the use of anticoagulants, while serum specimens can be obtained only after the serum naturally coagulates and the blood clots contract. Except in special circumstances, serum is used as the test specimen in medical tests. Plasma and serum can be used equally in ELISA. Serum samples can be collected according to conventional methods, and care should be taken to avoid hemolysis. When erythrocytes are lysed, substances with peroxidase activity will be released. In ELISA assays labeled with HRP, hemolysis samples may increase non-specific coloration.

Serum specimens should be tested when fresh. If there is bacterial contamination, the bacteria may contain endogenous HRP, which will also produce false positive reactions. If stored in the refrigerator for too long, polymerization can occur, and the background can be deepened in the indirect ELISA. Generally speaking, serum specimens measured within 5 days can be placed at 4 ° C, and cryopreservation should be kept at low temperature for more than one week. After the frozen serum is thawed, the protein is locally concentrated and unevenly distributed. It should be mixed thoroughly and gently, to avoid air bubbles, and the mixture can be inverted upside down. Serum specimens that are cloudy or precipitated should be centrifuged or filtered, and then clarified before testing. Repeated freezing and thawing will make the antibody titer drop, so if the serum samples for antibody detection need to be stored for multiple tests, it should be stored in small quantities in ice. Attention should be paid to aseptic operation when storing serum since collection, and appropriate preservatives can also be added.

2 Preparation of reagents
Prepare the reagents needed in the experiment according to the requirements of the kit instructions. Distilled or deionized water used in ELISA, including those used for washing, should be fresh and of high quality. The self-made buffer solution should be measured and corrected with a pH meter. The test reagents removed from 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.

3 Add sample
In ELISA, there are generally 3 sample addition steps, namely adding specimen, adding enzyme conjugate, and adding substrate. When adding the sample, add the added substance to the bottom of the hole of the LEISA plate, avoid adding it to the upper part of the hole wall, and pay attention not to splash or generate bubbles.
For adding specimens, generally use a micro sampler, and add it to the well of the plate in the required amount. The nozzle should be replaced each time a specimen is added to avoid cross-contamination, and a disposable quantitative plastic tube can also be used to add samples. For this determination (such as indirect ELISA), diluted serum is required, and the sample can be added after dilution in a test tube at the prescribed dilution. You can also add the diluent to the wells, and then add the serum sample to it, and then shake it on the micro-shaker for 1 minute to ensure mixing. When adding enzyme conjugate application liquid and substrate application liquid, a quantitative multi-channel liquid addition device can be used to quickly complete the liquid addition process.

4 Insulation
There are generally two antigen-antibody reactions in ELISA, that is, after adding specimen and adding enzyme conjugate. The completion of the antigen-antibody reaction requires a certain temperature and time. This incubation process is called incubation, and some people call it incubation, which seems inappropriate in ELISA.
ELISA is a solid-phase immunoassay, and the binding of antigen and antibody only occurs on the surface of the solid phase. Taking the antibody-coated sandwich method as an example, the specimens added to the wells of the plates do not all have equal opportunities to bind to the solid phase. Only the antigen in the layer of solution closest to the well wall directly contacts the antibody . This is a gradual equilibrium process, so it takes diffusion to reach the end of the reaction. The same is true for the binding of the enzyme-labeled antibody and the solid-phase antigen added thereafter. This is why ELISA reactions always require a certain period of incubation.

The temperatures commonly used for incubation are 43 ° C, 37 ° C, room temperature, and 4 ° C (refrigerator temperature). 37 ℃ is a commonly used holding temperature in the laboratory, and it is also a suitable temperature for most antigen-antibody binding. When establishing an ELISA method for the reaction kinetics study, the experiment showed that the two antigen-antibody reactions were generally at 37 ℃ for 1-2 hours, and the production of the product reached the peak. In order to accelerate the reaction, the temperature of the reaction can be increased. Some tests are conducted at 43 ° C, but higher temperatures should not be used. The antigen-antibody reaction is more thorough at 4 ° C. In radioimmunoassay, the reaction is usually left in the refrigerator overnight to form the most precipitate. However, due to the long time required, it is generally not used in ELISA.

In addition to the special electric heating block attached to some ELISA instruments, the method of heat preservation is generally a water bath. The ELISA plate can be placed in a water bath box. The bottom of the ELISA plate should be attached to the water surface to quickly balance the temperature. In order to avoid evaporation, the plate should be covered, and the hole of the plate can also be covered with plastic sealing paper or plastic wrap. At this time, the reaction plate can be floated on the water. If an incubator is used, the ELISA plate should be placed in a wet box. Materials with good heat transfer properties such as metal should be used in the wet box. Wet gauze should be placed on the bottom of the box. Finally, the ELISA plate should be placed on the wet gauze. The wet box should be placed in an incubator to pre-heat to the specified temperature, especially when the temperature is low. 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 room temperature incubation reactions, the room temperature during operation should be strictly limited to the specified range. The standard room temperature refers to 20-25 ° C, but the incubation can be controlled according to the instructions in the specific operation. When incubating at room temperature, the ELISA plate only needs to be placed flat on the operating table. It should be noted that the temperature and time of incubation should be as accurate as possible. To ensure this, it is not advisable to measure more than two boards at the same time when operating by one person.

5 Washing
Although washing is not a reaction step in the ELISA process, it also determines the success or failure of the experiment. ELSIA is to separate free and bound enzyme markers by washing. Washing to remove substances remaining in the wells of the plate that could not bind to the solid-phase antigen or antibody, as well as interfering substances that were non-specifically adsorbed to the solid-phase carrier during the reaction. The adsorption of polystyrene and other plastics to proteins is universal, and this non-specifically adsorbed interfering substance should be washed down 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, and the operator should strictly follow the requirements for washing, not sloppy.

Washing methods Except that some ELISA instruments are equipped with special automatic washing instruments, there are two types of manual operation: immersion and running water rinse. The process is as follows:
(1) Immersion type a. Absorb or spin-dry the reaction solution in the hole; b. Over-wash with the washing solution (after the washing solution is filled into the plate hole, it will be thrown away); c. Soak, the washing solution will be filled into the plate Place the hole for 1-2 minutes with intermittent shaking. The soaking time cannot be shortened at will; d. Absorb the liquid in the hole. Drying should be thorough, you can use a water pump or vacuum pump to suck, you can also throw off the liquid on a clean towel or absorbent paper; e. Repeat operations c and d, wash 3-4 times (or as specified). In the indirect method, if the background is high, the washing times can be increased or the soaking time can be extended.

Most microtiter plates use the immersion washing method. The washing solution is mostly a neutral buffer solution containing a non-ionic detergent. The combination of the polystyrene carrier and the protein is hydrophobic. The non-ionic detergent contains both hydrophobic and hydrophilic groups. The hydrophobic group and the hydrophobic group of the protein are combined by hydrophobic bonds, thereby weakening the protein and the protein. The combination of the solid-phase carrier and the combination of the hydrophilic group and the water molecule restores the protein to the state of the aqueous solution, thereby leaving the solid-phase carrier. The non-ionic detergent in the washing liquid is generally Tween 20, and its concentration can be between 0.05% -0.2%. When it is higher than 0.2%, it can desorb the antigen or antibody coated on the solid phase and reduce the test. Sensitivity.

(2) Flowing water rinsing method Flowing water rinsing method is initially used for washing the bead carrier. The washing solution is only distilled water or even tap water. A special device is attached to the washing, so that the beads are continuously rolled and washed under the impact of running water. After continuous rinsing for 2 minutes, the liquid is absorbed, and then soaked in distilled water for 2 minutes, and then dried. The immersion type is like a bathtub, and the running water rinse type is like a shower. Its washing effect is more thorough, and it is also simple and fast. Existing experiments show that the running water flushing method is also suitable for washing microtiter plates. When washing, try to increase the water flow or increase the water pressure, so that the water flow hits the surface of the plate hole, and the washing effect is better.
6 Color rendering and color comparison
(1) Color rendering
Color development is the last incubation reaction in ELISA, at which time the enzyme catalyzes the colorless substrate to produce a colored product. The temperature and time of the reaction are still factors affecting the color development. For a certain period of time, the female wells can remain colorless, while the male wells become more colored with time. Appropriately increasing the temperature will help accelerate the color development. In quantitative determination, the reaction temperature and time after adding the substrate should be as accurate as possible. The color development of the qualitative determination can be carried out at room temperature, and the time generally does not need to be strictly controlled. Sometimes, the reaction time can be shortened or extended appropriately according to the color development of the positive control well and the negative control well, and judged in time.

The color development of OPD substrates generally does not deepen after 20-30 minutes of reaction at outdoor temperature or 37 ° C. Prolonging the reaction time can increase the background value. The OPD substrate solution will change color by light. The color reaction should be carried out in the dark. When the color reaction is over, a termination solution is added to stop the reaction. After the OPD product was terminated with sulfuric acid, the color developed from orange to brown.

TMB is not greatly affected by light, and can be placed on the operating table at room temperature to observe the results while reacting. However, in order to ensure the stability of the experimental results, the results should be read at the appropriate time specified. After TRP is treated by HRP, the color development reaches its peak in about 40 minutes, and then gradually weakens. After 2 hours, it completely disappears to colorless. There are many termination solutions for TMB, and enzyme inhibitors such as sodium azide and sodium dodecyl sulfate (SDS) can terminate the reaction. This type of terminator can still maintain the blue color for a long time (12-24 hours) and it is a good terminator for visual judgment. In addition, all kinds of acidic stop solution will change the blue into yellow, at this time, the absorbance value can be measured with a specific wavelength (450nm).

(2) Color comparison
Before the color comparison, the liquid attached to the bottom of the plate should be dried with clean absorbent paper, and then the plate should be correctly placed in the colorimetric rack of the enzyme label colorimeter. For the test with soft board as the carrier, the board must be placed in a standard 96-well holder before color comparison. It is best to cut off the edge of the soft board before adding the substrate liquid to develop the color, so that the board can be completely seated in the seat frame.

For color comparison, the zero point should be calibrated with distilled water, and the substrate hole (the hole to which only the substrate liquid is added without any reaction) and the blank hole (the saline or diluent instead of the sample for the whole process of the hole) should be read to record Reagent status for the second test. After that, the blank hole can be used to calibrate the zero point with distilled water. The absorbance of the above holes must be subtracted from the blank hole, and then calculated.

The expression of colorimetric results used to be the universal optical density (OD), and now the absorbance (A) is used according to the regulations. Both have the same meaning. The usual representation method is to write the absorption wavelength in the lower right corner of the letter A. For example, the absorption wavelength of OPD is 492nm, and the representation method is "A492nm" or "OD492nm".

(3) Enzyme label colorimeter
The microplate reader is abbreviated as microplate reader, usually referring to a photometer dedicated to reading the absorbance of ELISA results. According to the different forms of solid support, each has its own special design for plates, beads and small test tubes. Many reagent companies supply microplate readers. The main performance indicators of the microplate reader are: reading speed, reading accuracy, repeatability, accuracy and measurable range, linearity and so on. The reading of an excellent microplate reader can generally be accurate to 0.001, with 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). Repeat the measurement several times, and the A value should be 1.083 ± 0.05 (1.078 ~ 1.088) in between. The measurable range of the microplate reader depends on the performance of each microplate reader. The common microplate reader is between 0.000 and 2.000, and the upper limit of the new type microplate reader has been widened to 2.900, or even higher. A value exceeding the measurable upper limit is 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 less 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. Attention should be paid to the curve.

The microplate reader should not be placed under sunlight or strong light. The room temperature should be 15 ~ 30 ℃ during operation. The instrument should be preheated for 15-30 minutes before use. The reading result is more stable.

When measuring the value of A, the sensitive absorption peak of the product should be selected. For example, OPD uses 492nm wavelength. Some microplate readers can use dual-wavelength reading, that is, each well is read twice in succession, the first time at the optimal wavelength (W1), the second time at the insensitive wavelength (W2), and the ELISA does not move between the two measurements The location of the board. For example, OPD uses 492nm as W1 and 630nm as W2. The final measured A value is the difference between the two (W1-W2). Dual-wavelength reading can reduce light interference caused by scratches or fingerprints on the container.
The performance of various microplate readers is different, and the reporter's instructions should be detailed during use.

7 Result judgment
Qualitative determination
The result of qualitative determination is to give a simple answer of "yes" or "no" to whether the tested specimen contains the antigen or antibody to be tested, and respectively indicate "positive" and "negative". "Positive" means that the specimen reacted in the measurement system. "Negative" means no response. The qualitative judgment method can also obtain semi-quantitative results, that is, the titer is used to indicate the strength of the reaction, and its essence is still a qualitative test. In this semi-quantitative determination, the specimen is tested after a series of dilutions, and the highest dilution that is positive is the titer. According to the level of titer, the strength of the reactivity of the specimen can be judged, which is more quantitative than judging the strong and weak positive by observing the shade of the undiluted specimen.

In the indirect method and the sandwich method ELSIA, the positive holes appear darker than the negative holes. In the competition ELISA, on the contrary, the negative wells are darker than the positive wells. The methods for judging the results of the two types of reactions are different and are described below.

(1) Indirect method and sandwich method
The qualitative results of such reactions can be judged with the naked eye. The visual specimen is also colorless or nearly colorless, and it is negative, and the color is clear and positive. However, in ELSIA, a colored background often appears after a normal human serum reaction. The depth of this background varies depending on the composition of the reagent and the conditions of the experiment. Therefore, a negative control must be added to the experiment. The composition of the negative control should be normal serum or similar without test substance (see 3.6). When judging the results with the naked eye, it is more appropriate to use the color development darker than the negative control as the positive index of the specimen.

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. First read the absorbance of the specimen (sample, S), positive control (P), and negative control (N), and then calculate. There are many calculation methods, which can be roughly divided into two types: positive judgment value method and specimen and negative control ratio method.
a. Positive judgment value
The positive judgment value (cut-off value) is generally the negative control A value plus a specific constant, which is used as the criterion for judging the positive or negative result.

Judging the results by this method requires that the experimental conditions are very constant, the preparation of the reagents must be standardized, the positive and negative controls should meet certain specifications, must be equipped with sophisticated instruments, and operate in strict accordance with regulations. The constants in the positive judgment 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 substance in the kit is calcium human plasma without HBsAg, and the content of the positive control substance HBsAg is indicated as P = 9 ± 2ng / ml. Each test has 2 positive controls and 3 negative controls. After measuring the A value, first calculate the average 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 (eg 0.400), The test is effective. The three negative control A values ​​should be ≥0.5 × NCX, and ≤1.5 × NCX, if one of them exceeds this range, it is discarded, and the other two negative controls have been recalculated NCX; if two negative controls A value exceeds Above the range, the experiment is invalid. The positive judgment value is calculated as follows: positive judgment value = NCX + 0.05 A value of specimen> positive judgment value is positive, and less than positive judgment value is negative. It should be noted that 0.05 in the formula is a constant of the kit, which is only suitable for the specific conditions, and is not universal for various reagents.

According to the above description, it can be seen that in this method, the negative control and the positive control also play a role in the quality control of the test. Both the deterioration of the reagent and the improper operation will result in "test invalid".
b. Specimen / negative control ratio
In the case where the experimental conditions (including reagents) are difficult to ensure constant, this judgment method is more appropriate. After obtaining the A value of the specimen (S) and the negative control (N), calculate the S / N value. There are also writing P / N, where P does not mean positive, but the abbreviation of patient, which should not be misunderstood. To avoid confusion, it is better to use S / N. In the early indirect ELISA, some authors set the S / N as a positive standard, which is now mostly used in various determinations. In fact, each measurement system should find their own S / N threshold by experiment. It should be more noted that the negative control represented by N is human serum without test substance. The negative control set in some kits contains no protein or a buffer with a low protein content, so that the background produced after the reaction may be much lower than that of normal human serum. Therefore, such a kit rule, if N <0.05 (or other value), it is calculated as 0.05, otherwise false positive results will appear.

(2) Competition Law
In the competitive ELISA, the negative wells are darker than the positive wells. The intensity of negative coloration depends on the concentration of the enzyme conjugate in the reaction and the amount of competition inhibitor added. Generally, the absorbance of the negative control is adjusted between 1.0-1.5, at which time the reaction is most sensitive.

Competitive ELISA is not easy to judge the results by self-view. Because it is difficult to distinguish the color difference between the weak positive reaction and the negative control with the naked eye, it is generally measured with a colorimeter and the absorbance values ​​of S, P and N are read. There are also two main calculation methods, namely the positive judgment value method and the inhibition rate method.
a. Positive judgment value 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 into the calculation formula, and now a certain kit for detecting anti-HBc is taken as an example. The negative control in the kit is reconstituted human plasma without anti-HBc, and the anti-HBc content in the positive control is 125 ± 100u / ml. Each test has 2 positive controls and 3 negative controls. After measuring the A value, first calculate the average value of the negative control A value (NCX) and the average value of the positive control A value (PCX). The difference between the two averages (NP) must be greater than a specific value (for example, 0.300), The test is effective. The A value of the three negative controls should be less than 2.000, and should be ≥0.5 × NCX and ≤1.5 × NCX, if one of them exceeds this range, it is discarded, and the other 2 negative controls are recalculated × NCX; A negative control A exceeds the above range, the experiment is invalid. The positive judgment value is calculated according to the following formula: negative judgment value = 0.4 × NCX + 0.6 × PCX The reaction with A value ≤ positive judgment value is positive, and the reaction with A> positive judgment value is negative.
b. Inhibition rate method
The inhibition rate indicates the degree of inhibition of the negative reaction coloration of the specimen in the competitive combination, calculated by the following formula: Inhibition rate (%) = (negative control A value-specimen A value) × 100% / negative control A value generally specifies inhibition Rate ≥50% is positive, <50% is negative.

Quantitative determination
The ELSIA procedure is complicated, and there are many factors that affect the reaction. Especially the coating of the solid phase carrier is difficult to achieve consistency between the individual bodies. Therefore, in the quantitative determination, each batch of tests must use a series of reference standards with different concentrations in the same Standard curve under the conditions of For the sandwich ELISA for the determination of large molecular weight substances, the range of the standard curve is generally wider, and the absorbance at the highest point of the curve can be close to 2.0. Semi-logarithmic paper is often used when drawing. The values ​​of each concentration are connected point by point. The resulting curve is generally S-shaped, and the curve of the head and tail tends to be flat. The more linear part in the center is the most ideal detection area.

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