Collection and preservation of specimens
Usually, there are many types of samples that we can use for ELISA detection, such as serum, plasma, urine, cell culture supernatant or tissue homogenate. Different types of test samples have different pretreatment methods. Correctly handling samples is the first step to ensure the correctness and accuracy of ELISA detection. The following briefly introduces the treatment methods of different types of samples.
serum
Serum is the most commonly used sample for ELISA detection, and its treatment is relatively simple.
Collect blood samples with a test tube or centrifuge tube without pyrogen and endotoxin, and leave the test tube or centrifuge tube at room temperature for 2 hours or 4℃ overnight to precipitate the serum. (It is best to put the test tube or centrifuge tube obliquely to increase the cross section of the liquid surface, which can precipitate the serum to a greater extent. ) centrifuge at 4℃ 1000×g for 20min, and carefully collect the supernatant. It is suggested that the serum be divided into multiple parts and stored at -20℃ or -80℃ to avoid repeated freezing and thawing.
Hemolysis should be avoided in the process of blood collection, because substances with peroxidase activity will be released when red blood cells are dissolved, and HRP-labeled ELISA will show non-specific color development, which will lead to inaccurate detection. At the same time, bacterial contamination should be avoided, because bacteria may contain endogenous HRP, which may lead to false positive detection.
blood plasma
Blood samples were collected by blood collection tube or centrifugal tube containing anticoagulant, and centrifuged at 4℃ 1000×g 15 minutes within 30 minutes after the sample was collected, and the supernatant was taken as plasma. Divide the supernatant into multiple parts and store it at -20℃ or -80℃ to avoid repeated freezing and thawing. Avoid using hemolytic or hyperlipidemia samples.
Commonly used anticoagulants are EDTA, heparin sodium and sodium citrate. When testing, you should also read the instructions of the kit carefully to check whether the kit has special requirements for anticoagulants.
Cell culture supernatant
Take cell culture supernatant to centrifuge tube, centrifuge at 1000×g for 20min, remove cell debris and impurities, take supernatant, and store it at -20℃ or -80℃ to avoid repeated freezing and thawing.
Cell lysate
1) Suck the culture medium from the culture plate, digest the cells with trypsin, and add an appropriate amount of culture medium to blow the cells off the culture plate. Suspended cells can be omitted.
2) Collect cell suspension, centrifuge for 1000×g 10 minute, discard the culture medium, and moisten it with precooled PBS for three times.
3) Add a proper amount of precooled PBS or cell lysate (add protease inhibitor before use) to resuspend the cells. Generally, the amount of cells in one well of a 6-well plate needs 150~250μL PBS to resuspend.
4) Freeze the sample at -20℃ or -80℃, then thaw it at room temperature, and freeze it several times repeatedly to fully lyse the cells. The sample can also be broken by ultrasonic wave to achieve the purpose of cracking.
5) Centrifuge at 4℃ 10000×g 10 minute, remove cell debris, take supernatant, and store at -20℃ or -80℃ to avoid repeated freezing and thawing.
tissue homogenate
1) Rinse the tissue sample with PBS (0.0 1M, PH 7.4) to wash off the residual blood or impurities on the tissue surface.
2) Weigh the tissue block, record it and cut it into pieces. The smaller the fragments, the better, so as to be even and full.
3) Add pre-cooled PBS (add protease inhibitor before use) into the tissue according to a certain proportion for homogenization, and put it on ice or ice bath during homogenization. (Generally, the tissue weight: PBS volume is equal to 1: 9, for example, 1g tissue sample corresponds to 9mL PBS, and the specific volume can be appropriately adjusted according to the experimental needs, and the sample concentration after detection should be multiplied by the corresponding dilution multiple).
4) Suck the homogenate into a centrifuge tube, centrifuge at 4℃ for 5~ 10min at 5000× g, and take the supernatant and store it at -20℃ or -80℃ to avoid repeated freezing and thawing.
Urine, saliva and other liquid biological samples
Centrifuge with 1000×g for 20min, and take the supernatant for detection.
Generally speaking, because ELISA can only detect the content of soluble protein, it is necessary to ensure that all samples are clear liquids and remove sediments or suspended substances by centrifugation.
To ensure the accuracy of detection, samples stored at -20℃ or -80℃ should be detected within 1~6 months. Samples stored at 4℃ shall be inspected within 1 week.
In addition, it is also necessary to ensure that the sample does not contain NaN3, because NaN3 will inhibit the activity of HRP, which will lead to false negative results.
Preparation of reagent
Prepare the reagents needed for the experiment according to the requirements of the kit instructions. Distilled water or deionized water used in ELISA, including washing, should be fresh and of high quality. Self-made buffer and washing liquid were measured with an acid meter. The test reagent taken out of the refrigerator should be used after the temperature and room temperature are balanced, and the room temperature balance is generally not less than 30min. The unnecessary parts in the kit should be put back into the refrigerator in time for preservation.
application of sample
In ELISA, there are generally three steps to add samples, namely, adding samples, adding enzyme conjugates and adding substrates. When adding samples, the added substances should be added to the bottom of Reza plate hole to avoid being added to the upper part of hole wall, and be careful not to overflow or generate bubbles.
Specimens are generally added with a micro sampler, and the plate holes are added according to the specified amount. The suction nozzle should be replaced every time the sample is added to avoid cross contamination, and the sample can also be added with disposable quantitative plastic tubes (generally not recommended). Some indicators (such as indirect ELISA) need to dilute the serum, which can be diluted in the test tube according to the prescribed dilution before adding samples. You can also add diluent to the plate hole, then add the serum sample, and then shake it evenly on a micro-shaker 1min. Multi-channel pipettes can be used when adding enzyme conjugate working solution and substrate working solution, so that the liquid adding process can be completed in the shortest time.
thermal insulation
There are generally two antigen-antibody reactions in ELISA, that is, after adding the sample and the enzyme conjugate. It takes a certain temperature and time to complete the antigen-antibody reaction. This heat preservation process is called incubation, and some people call it incubation.
ELISA is a solid-phase immunoassay, and the binding of antigen and antibody only occurs on the surface of solid phase. Take the sandwich method of antibody coating as an example. Not all antigens in the sample added to the plate hole have equal opportunities for solid-phase anti-binding, and only the antigen in the solution closest to the hole wall is in direct contact with the antibody. This is a gradual equilibrium process, so diffusion is needed to reach the end of the reaction. The same is true for the binding of enzyme-labeled antibodies to solid-phase antigens. This is why ELISA always needs a certain period of incubation.
The common incubation temperatures are 43℃, 37℃, room temperature and 4℃ (refrigerator temperature). 37℃ is a commonly used temperature in the laboratory, and it is also a suitable temperature for the combination of most antigens and antibodies. When establishing ELISA method to study the reaction kinetics, the reaction of two antigens and antibodies usually takes 1-2 hours at 37℃, and the product can reach the peak. In order to accelerate the reaction, the reaction temperature can be increased. Some experiments are carried out at 43℃, but higher temperature is not suitable. The reaction between antigen and antibody is more thorough at 4℃, and the reaction in radioimmunoassay often stays overnight in the refrigerator, forming the most precipitate. But because it takes too long, it is generally not used for ELISA.
Except for some ELISA instruments with special electric heating blocks, water bath insulation is generally used. The enzyme-labeled plate can be placed in a water bath box, and the bottom of the enzyme-labeled plate should be attached to the water surface, so that the temperature can be quickly balanced. In order to avoid evaporation, the plate should be covered, and the holes of the plate can also be covered with plastic sealing paper or plastic wrap to make the reaction plate float on the water. If an incubator is used, the ELISA plate should be placed in a wet box. The wet box should be made of metal and other materials with good thermal conductivity, and the bottom of the box should be padded with wet gauze. Finally, the enzyme-labeled plate should be placed on wet gauze. The wet box should be preheated to the specified temperature in the incubator, especially when the temperature is low. In order to avoid the reading error caused by water vapor or wear at the bottom of the enzyme label plate, a blower is generally used to keep the temperature constant. In this process, paper must be pasted above the enzyme-labeled plate to avoid evaporation. Whether in 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 incubated at room temperature, the room temperature during operation should be strictly limited within the specified range. The standard room temperature refers to 20-25℃, but the specific operation can be controlled according to the requirements of the manual. When incubating at room temperature, the enzyme-labeled plate can only be placed flat on the operating table. It should be noted that the incubation temperature and time should be as accurate as possible according to the regulations. In order to ensure accurate time, one should not operate and measure more than two boards at the same time.
wash
Although washing is not a reaction step in ELISA, it also determines the success or failure of the experiment. ELSIA can separate free and bound enzyme markers by washing. By washing, the substances remaining in the plate holes that can not be combined with solid-phase antigen or antibody and the interfering substances that are nonspecific adsorbed on the solid-phase carrier during the reaction are removed. The adsorption of protein by plastics such as polystyrene is universal, and this nonspecific adsorption interfering substance should be washed away when washing. It can be said that washing is the most important key technology in ELISA operation, which should be paid great attention by operators, and washing should be carried out in strict accordance with the requirements, and it should not be sloppy.
In addition to some ELISA instruments equipped with special automatic washing instruments, there are two washing methods: soaking and running water washing. The process is as follows:
(1) Immersion type: a. Absorb or spin dry the reaction solution in the hole; B. Overwash with washing liquid (after the washing liquid fills the plate hole, throw it away); C) Soaking, that is, filling the plate hole with washing liquid, standing for 1-2min and shaking intermittently, and the soaking time cannot be shortened at will; Suck up the liquid in the hole. Suction should be thorough, you can use a water pump or vacuum pump, or pat dry with a clean towel or absorbent paper after the liquid is poured out; E repeat operations c and d and wash for 3-4 times (as specified in the instructions). In the indirect method, if the background is high, the washing times or soaking time can be increased.
Immersion washing method is often used for microtiter plates. The washing liquid is mainly neutral buffer containing nonionic detergent. The combination of polystyrene carrier and protein is hydrophobic. The nonionic detergent contains both hydrophobic groups and hydrophilic groups, and its hydrophobic groups are combined with those of protein through hydrophobic bonds, thus weakening the combination of protein and solid support. With the combination of hydrophilic groups and water molecules, protein returned to the state of aqueous solution, thus being separated from the solid carrier. The nonionic detergent in the washing liquid is generally Tween 20, and its concentration can be between 0.05% and 0.2%. When it is higher than 0.2%, the antigen or antibody coated on the solid phase will be desorbed, thus reducing the sensitivity of the test.
(2) Running water washing method: The running water washing method was originally used to clean the bead carrier, and the washing liquid was only distilled water or even tap water. When washing, attach a special device to let the beads roll under the impact of running water. After continuous washing for 2 minutes, drain the liquid, then soak it in distilled water for 2 minutes, and then drain it. Soaking is like taking a bath, and running water is like taking a shower. Its washing effect is more thorough, simple and fast. The existing experiments show that running water washing is also suitable for washing microtiter plates. When cleaning, increase the water flow or water pressure as much as possible to make the water flow impact the surface of the plate hole, and the cleaning effect is better (this method is rarely used in the kit).
Color development and colorimetry
put colour on
Color development is the last incubation reaction in ELISA, when the enzyme catalyzes the colorless substrate to produce colored products. Reaction temperature and time are still the factors affecting color development. In a certain period of time, the negative pores can remain colorless, while the positive pores will become more colorful over time. Increasing the temperature properly is helpful to accelerate the color development. In quantitative determination, the reaction temperature and time after adding substrate should be as accurate as possible. The color development of qualitative determination can be carried out at room temperature, and the time generally does not need strict control. Sometimes, the reaction time can be shortened or extended according to the color development of the positive control hole and the negative control hole, so as to judge in time.
Generally, the color of OPD substrate will not deepen after 20-30 minutes of reaction at outdoor temperature or 37℃, and the background value will increase when the reaction time is prolonged. The substrate solution of OPD will automatically change color when exposed to light, and the color reaction should be carried out in the dark. At the end of the color reaction, the reaction was terminated by adding a termination solution. After the OPD product was terminated with sulfuric acid, the color changed from orange to brown.
TMB is not affected by light, so it can be placed on the operating table at room temperature, and the results can be observed during the reaction. However, in order to ensure the stability of the experimental results, it is advisable to read the results at an appropriate time. After TMB was treated with HRP, the color development reached its peak in about 40 minutes, then gradually weakened, and disappeared completely after 2 hours. There are many kinds of TMB termination solutions, and enzyme inhibitors such as sodium azide and sodium dodecyl sulfate (SDS) can terminate the reaction. This terminator can stay blue for a long time (12-24 hours) and is a good terminator for visual judgment. In addition, all kinds of acidic termination solutions will change from blue to yellow, and the absorbance can be measured at a specific wavelength (450nm).
[Chemistry] Chromatic ...
Before colorimetry, use clean absorbent paper to absorb the liquid attached to the bottom of the plate, but try to avoid the surface, and then put the plate on the colorimetric rack of the enzyme-labeled colorimeter correctly. In the experiment with soft board as the carrier, the color comparison can only be carried out if the board is placed on the standard 96-hole pedestal. It is best to cut off the edge of the soft board before adding the substrate solution to develop color, so that the board can be completely seated in the seat frame.
When comparing colors, first calibrate the zero point with distilled water, measure and read the substrate hole (the hole where only the substrate solution is added without any reaction) and the blank hole (the hole of the sample is replaced by normal saline or diluent in the whole process), and record the reagent state of this test. After that, the zero point can be calibrated by distilled water with a blank hole, and the absorbance of the above hole can be subtracted from the absorbance of the blank hole, and then calculated.
The expression of colorimetric results used to be optical density (OD), but now absorbance (A) is used according to regulations, which means the same thing. 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".
Enzyme-labeled colorimeter
Enzyme-labeled colorimetric instrument, referred to as enzyme-labeled instrument for short, usually refers to a photometer dedicated to measuring the absorbance of ELISA results. According to the different forms of solid carriers, there are special designs suitable for flat plates, beads and small test tubes. Many reagent companies supply microplate readers. The main performance indexes of enzyme-labeled instrument 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 0.0 0 1, with accuracy 1% and 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 .0830.01(1.073 ~1.093), and its A value should be/after repeated measurement. The measurable range of enzyme-labeled instrument varies with the performance of each enzyme-labeled instrument. The general microplate reader is 0.000~2.000, and the upper limit of the new microplate reader is widened to 2.900 or even higher. Values exceeding the measurable upper limit are usually indicated by "*" or "exceeding" or other symbols. We should pay attention to the difference between measurable range and linear range, which is often smaller than measurable range. For example, the measurable range of an enzyme-labeled instrument is 0.000~2.900, while its linear range is only 0.000~2.000, which should be paid attention to when quantitative ELISA makes standard curves.
The microplate reader should not be placed in sunlight or strong light, and the room temperature should be 15~30℃ when operating. Preheat the reader for 15-30 minutes before use, so that the reading result will be more stable (some microplate reader instructions clearly indicate that preheating is not needed).
When reading the A value, we should choose the sensitive absorption peak of the product, such as OPD with a wavelength of 492nm. Some microplate readers can use dual-wavelength reading, that is, each hole is read twice, the first time at the optimal wavelength (W 1) and the second time at the insensitive wavelength (W2), and the position of the microplate does not move between the two measurements. For example, OPD uses 492nm as W 1 and 630nm as W 2, and the final measured value of a is the difference between them (W 1-W2). Dual-wavelength reading can reduce optical interference caused by scratches or fingerprints on containers.
The performance of various enzyme-labeled instruments is different, so the reporter's instructions should be detailed when using them.
Result judgment
qualitative determination
The result of qualitative judgment is to give a simple answer of "yes" or "no" to whether the specimen contains the antigen or antibody to be detected, which is represented by "positive" and "negative" respectively. "Positive" means that the sample reacts in the assay system. "No" means no response. Qualitative judgment can also get semi-quantitative results, that is, the titer is used to express the strength of the reaction, which is essentially a qualitative test. In this semi-quantitative determination, samples are diluted in series and tested, and the highest dilution of positive reaction is titer. According to the titer, the reactivity of the sample can be judged, which is more quantitative than observing the color depth of the undiluted sample to judge whether it is strong positive or weak positive.
In indirect method and sandwich method ELSIA, the color of positive hole is darker than that of negative hole. On the contrary, in competitive ELISA, the color of negative wells is darker than that of positive wells. The results of the two reactions are judged in different ways, as follows.
(1) indirect method and sandwich method
The qualitative results of such reactions can be judged by naked eyes. If the visual specimen is colorless or nearly colorless, it is judged as negative, and if the color is clear, it is positive. However, in ELSIA, a colored background often appears after normal human serum reaction, and the depth of this background varies with reagent composition and experimental conditions, so negative control must be added in the experiment. The components of negative control should be normal serum or analogues without test substance. When judging the results with naked eyes, it is more appropriate to use the color darker than the negative control as the positive index of the specimen.
The visual method is simple and clear, but it is quite subjective. When conditions permit, the absorbance value can be measured by colorimeter, so that objective data can be obtained. First, read the absorbance values of the sample (S), positive control (P) and negative control (N), and then calculate. There are many calculation methods, which can be roughly divided into two categories: positive judgment value method and ratio method of specimen and negative control.
A. Positive judgment value
Generally, the positive critical value is the negative control A value plus a specific constant, which is used as the standard to judge whether the result is positive or negative.
Judging the results by this method requires that the experimental conditions are very constant, the preparation of reagents must be standardized, the positive and negative reference materials must meet certain specifications, and precise instruments must be used and the operation must be strictly in accordance with the regulations. The constants in the formula of positive judgment value are obtained through the experimental detection of a large number of samples in this specific system. Let's take the kit for detecting HBsAg as an example. The negative reference substance in the kit is recalcified human plasma without HBsAg, and the content of HBsAg in the positive reference substance is marked as P = 9±2 ng/ml. There are 2 positive controls and 3 negative controls in each experiment. After measuring the A value, the average value of negative control A value (NC X) and the average value of positive control A value (PCX) are calculated first, and the difference (P-N) between the two averages must be greater than a specific value (for example, 0.400) before the test is effective. The a values of the three negative controls should be ≥0.5×NCX and ≤ 1.5×NCX. If one of them is out of this range, it should be discarded and the ncx should be recalculated with the other two negative controls. If one of the two negative controls exceeds the above range, the experiment is invalid. Calculate the positive judgment value according to the following formula:
Positive judgment value =NCX+0.05
Specimens with a value greater than the positive judgment value are positive, while those with a value less than the positive judgment value are negative. It should be noted that 0.05 in the formula is the constant of the kit, which is only applicable to this specific condition and is not applicable to all kinds of reagents.
According to the above description, it can be seen that negative control and positive control also play a role in the inspection quality control of this method, and both reagent deterioration and improper operation will lead to the result of "invalid inspection".
B. Sample/negative quality control ratio
This judgment method is more applicable when the experimental conditions (including reagents) are difficult to ensure constant. After obtaining the A value of the sample and the negative control, calculate the S/N value. Others write P/N, where P is not for masculine gender, but for patient. Don't get me wrong. In order to avoid confusion, s/n is more suitable. In the early indirect ELISA, some authors defined S/N as a positive standard, and now it is commonly used in various tests. In fact, each measurement system should find its own SNR threshold through experiments. It should be noted that the negative control denoted by n is human serum containing no test substance. The negative control in some kits is a buffer containing no protein or protein, so the background after the reaction may be much lower than that of normal human serum. Therefore, such kit specifications, such as N
(2) Competition Law
In the competition law ELISA, the color of the negative hole is darker than that of the positive hole. The intensity of negative coloration depends on the concentration of enzyme conjugate and the amount of competition inhibitor added in the reaction. Generally, the absorbance of negative control is adjusted between 1.0- 1.5, and the reaction is the most sensitive at this time.
Competitive ELISA is not easy to judge the results by its own observation, because it is difficult for naked eyes to distinguish the color difference between weak positive reaction and negative control. Generally, it is determined by reading the absorption values of S, P and N by colorimeter. There are two main calculation methods, namely, positive judgment value method and inhibition rate method.
A. positive judgment method
It is basically the same as the positive judgment value method in indirect method and sandwich method, except that the positive control A value is introduced into the calculation formula. Now take the kit for detecting anti-HBc as an example. The negative control in the kit is recalcified human plasma without anti -HBc, and the anti -HBc content in the positive control is 125 100u/ml. There are 2 positive controls and 3 negative controls in each experiment. After measuring the A value, first calculate the average value of the negative control A value (NC X) and the average value of the positive control A value (PCX), and the difference (N-P) between the two averages must be greater than a specific value (for example, 0.300) before the test is effective. The a values 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 is out of this range, it should be discarded and × ncx should be recalculated with the other two negative controls. If there are two negative controls A beyond the above range, the experiment is invalid. Calculate the positive judgment value according to the following formula:
Negative judgment value =0.4×NCX+0.6×PCX
The reaction of a value ≤ positive judgment value is positive, and the reaction of a > positive judgment value is negative.
B. inhibition rate method
The inhibition rate indicates the degree of inhibition of negative reaction color development in competitive binding, which is calculated as follows:
Inhibition rate (%) = (negative control A value-specimen A value) × 100%/ negative control A value.
Generally speaking, inhibition rate ≥50% is positive and < 50% is negative.
quantitative determination
The operation steps of ELSIA are complicated, and there are many factors that affect the reaction, especially the coating of solid carrier is difficult to achieve the consistency among individuals. Therefore, in quantitative determination, a series of reference standards with different concentrations must be used in each batch of experiments to make standard curves under the same conditions. Sandwich ELISA for the determination of high molecular weight substances generally has a wide standard curve range, and the absorbance at the highest point of the curve can be close to 2.0. Semi-logarithmic paper is often used in drawing, and the concentration of the measured substance is taken as the abscissa and the absorbance as the ordinate to connect the concentration values point by point. The obtained curve is generally S-shaped, and its head and tail curves tend to be flat, and the straight line in the middle is the most ideal detection area.
Competition method is often used to determine small molecular weight substances, and the absorbance in its standard curve is negatively correlated with the concentration of the substances to be measured. Due to the different models used in the kit, the shape of the standard curve is slightly different.