A toxin four-item microarray chip ELISA plate and its preparation method and application

Abstract

The present invention provides a toxin four-item microarray chip ELISA plate and its preparation method and application, belonging to the technical field of toxin detection. Each sample well of the toxin four-item microarray chip ELISA plate provided by the present invention contains a biomolecule recognition site, and the biomolecule recognition site is coated with a quality control protein, aflatoxin antigen, zeaxanthin antigen, deoxynivalenol antigen and fumonisin antigen, respectively, and the coating liquid used is composed of 0.01M PB pH7.5 and 1% concentration of glycerol. The toxin four-item microarray chip ELISA plate provided by the present invention can simultaneously perform quantitative detection of four toxins in a single sample, and can minimize the workload compared with traditional ELISA, and has the advantages of high throughput, multiple indicators, microquantification, intelligence, visualization and low cost.

Description

Technical Field

[0001] The invention belongs to the technical field of toxin detection, and in particular relates to a toxin four-item microarray chip ELISA plate and a preparation method and application thereof. Background Art

[0002] Aflatoxin (AFB1) is a type of fungal toxin that is highly carcinogenic and widely present in food. Zearalenone (ZEN) mainly contaminates grains such as corn, wheat, rice, barley, millet and oats. It has estrogen-like effects and can cause acute and chronic poisoning in animals, causing abnormal reproductive function or even death in animals, which can cause huge economic losses to livestock farms. Deoxynivalenol (DON) is a secondary metabolite of the genus Fusarium. It inhibits protein synthesis and causes toxic reactions such as vomiting, diarrhea, anorexia, nausea, and nervous disorders. The nature of this toxin is relatively stable, and food processing methods such as baking at high temperature and high pressure have little effect on it. Fumonisin (FB) is a fungal toxin. Fumonisin contamination is universal and poses great harm to humans and animals. It mainly contaminates corn and its products. Therefore, it is crucial to detect and effectively separate the above four toxins in a timely, rapid and efficient manner and control the content within the permitted range.

[0003] When multiple toxins in the same sample are tested simultaneously, how to ensure that different toxins do not affect each other and how to ensure that multiple toxins can be successfully tested simultaneously under the same conditions have always been the difficulties in this field. For example, putting antibodies to multiple toxins in the same antibody diluent will lead to problems such as too high or too low recovery of individual items, background or super-negative of individual items. Therefore, the methods for detecting the above four toxins disclosed in the prior art are all single toxin detection methods. While meeting the requirements of testing a large number of samples, they cannot meet the requirements of simultaneous detection of multiple toxins in the same sample. There are disadvantages such as single target, complex sample pretreatment, cumbersome operation, large amount of sample required, and long time consumption, which in turn affects the efficiency of detection. In addition, the results of existing toxin detection kits need to be read as soon as possible and cannot be stored for a long time. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide a toxin four-item microarray chip ELISA plate, which can simultaneously perform quantitative detection of multiple toxins in a single sample. Compared with traditional ELISA, it has the advantages of high throughput, multiple indicators, micro-quantification, intelligence, visualization, low cost, etc., and can minimize the workload.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The invention provides a toxin four-item microarray chip ELISA plate, wherein each sample well of the ELISA plate contains a biomolecule recognition site, wherein the biomolecule recognition sites are respectively coated with a quality control protein, an aflatoxin antigen, a zeaxanthin antigen, a vomitoxin antigen and a fumonisin antigen, and the coating solution used is composed of 0.01M PB pH7.5 and 1% glycerol.

[0007] Preferably, the coating titers of the quality control protein, aflatoxin antigen, zeaxanthin antigen, deoxynivalenol antigen and fumonisin antigen in each sample well of the ELISA plate are 1:2000-4000, 1:1000-3000, 1:3000-7000, 1:5000-9000 and 1:60000-10000, respectively.

[0008] The present invention also provides a method for preparing the above-mentioned ELISA plate, comprising the following steps: mixing the coating solution with quality control protein, aflatoxin antigen, zeaxanthin antigen, deoxynivalenol antigen and fumonisin antigen respectively to obtain five sample solutions, spotting the plate using a biochip spotter, coating at 37°C for 2h, washing the plate and sealing.

[0009] Preferably, the conditions for spotting by the biochip spotter are as follows: the spotting volume of each recognition site is 4 nL, the spot spacing is 600 μm, and the spotting needle moving speed is 4000 μm / s.

[0010] The present invention also provides a method for detecting four toxin items, comprising the following steps: adding four toxin standard items or samples to be tested to the above-mentioned ELISA plate, adding four toxin enzyme labels and four toxin anti-reagents to react, washing the plate, adding color developing solution to react, washing the plate, and obtaining the results.

[0011] Preferably, the volume ratio of the four toxin standards or test samples, the four toxin enzyme labels, and the four toxin anti-reagents is 1:1:1.

[0012] The present invention also provides an application of the above-mentioned ELISA plate or the above-mentioned preparation method in preparing a toxin four-item detection product.

[0013] The present invention also provides a four-item visualization microarray chip kit for toxins, comprising the above-mentioned ELISA plate, four-item toxin standard substances, antibody diluent, ELISA diluent and sample diluent.

[0014] Preferably, the antibody diluent consists of 0.1M PB pH 7.5, 1% sucrose, 0.025% sodium thimerosal and 1% bovine serum.

[0015] Preferably, the preparation method of the four toxin standards comprises the following steps: mixing the standard dilution solution with the four toxins, setting five concentration gradient solutions, wherein the concentrations of the four toxins in the first concentration gradient solution are all 0 ppb, the concentrations of aflatoxin, zearalenone, vomitoxin, and fumonisin in the second concentration gradient solution are 1.5, 20, 200, and 200 ppb, respectively, the concentrations of aflatoxin, zearalenone, vomitoxin, and fumonisin in the third concentration gradient solution are 5, 70, 700, and 600 ppb, respectively, the concentrations of aflatoxin, zearalenone, vomitoxin, and fumonisin in the fourth concentration gradient solution are 15, 250, 2100, and 1800 ppb, respectively, and the concentrations of aflatoxin, zearalenone, vomitoxin, and fumonisin in the fifth concentration gradient solution are 50, 750, 7000, and 5400 ppb, respectively.

[0016] Beneficial effects of the present invention:

[0017] The present invention is the first to apply ELISA microarray chip technology to develop a product that can detect four toxins simultaneously. The toxin four-item microarray chip ELISA plate provided by the present invention can simultaneously perform quantitative detection of four toxins in a single sample. Compared with traditional ELISA, it can minimize the workload and has the following advantages:

[0018] High throughput: one multi-well biochip ELISA plate can detect up to 90 samples simultaneously;

[0019] Multi-indicator: Sample analysis can simultaneously detect four toxins: aflatoxin, zearalenone, vomitoxin and fumonisin;

[0020] Micro-quantification: Only 50μL of sample is needed to meet the detection needs;

[0021] Intelligent: experimental data can be obtained within 90 seconds and analysis reports can be provided;

[0022] Visualization: The positive and negative of the test items can be distinguished by naked eyes, and the results can be retained for a long time;

[0023] Low cost: Only a few nL are required for each biomolecule recognition site. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a schematic diagram of the four-item microarray chip ELISA plate of the toxin of the present invention;

[0025] Figure 2 is the result of spotting of biomolecule recognition sites, where af represents different sizes of biomolecule recognition sites, tailing of spots, few spots, hollow spots, scattered spots, and uneven spots, respectively;

[0026] Figure 3are the shapes of different coating solution recognition sites, wherein ac represent the shapes of the coating solution recognition sites of Example 1, Comparative Example 1 and Comparative Example 2, respectively. DETAILED DESCRIPTION

[0027] The invention provides a toxin four-item microarray chip ELISA plate, wherein each sample well of the ELISA plate contains a biomolecule recognition site, wherein the biomolecule recognition sites are respectively coated with a quality control protein, an aflatoxin antigen, a zeaxanthin antigen, a vomitoxin antigen and a fumonisin antigen, and the coating solution used is composed of 0.01M PB pH7.5 and 1% glycerol.

[0028] In the present invention, each sample well is coated with five biomolecule recognition sites, namely, quality control protein, aflatoxin antigen, zeaxanthin antigen, vomitoxin antigen and fumonisin antigen, in the form of a microarray. The present invention does not specifically limit the specific positions of the above five biomolecule recognition sites in the sample well. In a specific embodiment, 96 sample wells are arranged on each plate of the toxin four-item ELISA plate, and the sample wells are neatly arranged in an array with twelve horizontally and eight vertically. Five biomolecule recognition sites, namely, quality control point, aflatoxin antigen, zeaxanthin antigen, vomitoxin antigen and fumonisin antigen, are fixed in each sample well, and each item has three recognition sites, such as Figure 1 As shown. The present invention achieves the purpose of throughput, multi-index and visual detection of biological samples through a combination of multiple sample wells and multiple biomolecule recognition points in the sample wells, and can be widely used in fields such as food detection. In the present invention, the purpose of setting quality control proteins and quality control points is to serve as a standard for judging the recognition sites of the remaining items. After the scanner scans the image, the number of items needs to be selected, and the software will use the location of the quality control points as a standard to judge the location of the three recognition sites of each item. The present invention does not specifically limit the specific type of quality control protein. In a specific embodiment, the quality control protein is mouse IgG. The present invention does not specifically limit the specific sources of mouse IgG, aflatoxin antigen, zeaxanthin antigen, vomitoxin antigen and fumonisin antigen. In the present invention, the coating titers of quality control protein, aflatoxin antigen, zeaxanthin antigen, deoxynivalenol antigen and fumonisin antigen in each sample well of the ELISA plate are preferably 1:2000-4000, 1:1000-3000, 1:3000-7000, 1:5000-9000 and 1:60000-100000, respectively, and more preferably 1:3000, 1:1500, 1:5000, 1:7000 and 1:80000, respectively.

[0029] The present invention also provides a method for preparing the above-mentioned ELISA plate, comprising the following steps: mixing the coating solution with quality control protein, aflatoxin antigen, zeaxanthin antigen, deoxynivalenol antigen and fumonisin antigen respectively to obtain five sample solutions, spotting the plate using a biochip spotter, coating at 37°C for 2h, washing the plate and sealing.

[0030] In the present invention, the conditions for spotting by the biochip spotter are preferably as follows: the spotting volume of each recognition site is 4 nL, the spot spacing is 600 μm, and the spotting needle moving speed is 4000 μm / s.

[0031] In the present invention, a biochip spotter is used to perform automated micro-spotting, but the spotting cannot be guaranteed to be completely neat, and the following may occur: Figure 2 The biological molecule recognition sites shown are of different sizes, tailing, few points, hollow points, scattered points, and uneven points. The present invention solves the above technical problems through the following technical means: ① Change the viscosity of the coating solution, and select a coating solution suitable for biochip spotting instrument spotting, specifically 0.01M PB pH7.5 + 1% glycerol as the coating solution. ② Control the speed of the spotting instrument, change the up and down movement speed from the initial 2000μm / s to 4000μm / s, and change the front and back and left and right movement speeds from 6000μm / s to 4000μm / s. ③ Change the volume of the biochip spotting instrument spotting, and change the volume of each point from 12nL to 4nL to reduce the hollow phenomenon caused by uneven drying speed. ④ The traditional method of coating at 4℃ for 16-20h and then sealing the plate easily leads to the problem of shallow edges and deep in the middle. The present invention changes the coating mode to a method of washing the plate after coating for 2h in a 37℃ constant temperature incubator and then sealing. ⑤ The spotting range of the micro-hole is very small. The spotting needle of the biochip spotter is made of fragile glass. If the spot spacing is too large, the spotting needle will hit the plate hole and break. If the spot spacing is too small, the spots of different projects will overlap and affect each other, thus affecting the experimental results. The present invention gradually changes the spot spacing and the arrangement of the spots, and finally selects the parameters of 600μm spot spacing and 6×8 spot arrangement for spotting.

[0032] The present invention also provides a method for detecting four toxin items, comprising the following steps: adding four toxin standard items or samples to be tested to the above-mentioned ELISA plate, adding four toxin enzyme labels and four toxin anti-reagents to react, washing the plate, adding color developing solution to react, washing the plate, and obtaining the results.

[0033] In the present invention, the volume ratio of the four toxin standards or samples to be tested, the four toxin enzyme labels, and the four toxin anti-reagents is preferably 1:1:1. The present invention does not specifically limit the specific sources of aflatoxin antibodies, zearalenone antibodies, vomitoxin antibodies, and fumonisin antibodies in the four toxin anti-reagents. In the four toxin anti-reagents of the present invention, the titers of aflatoxin antibodies, zearalenone antibodies, vomitoxin antibodies, and fumonisin antibodies are preferably 1:60,000, 900,000, 250,000, and 50,000, respectively, and the antibody diluent in the four toxin anti-reagents is preferably composed of 0.1MPB pH7.5+1% sucrose+0.025% sodium thimerosal+1% bovine serum. The present invention has no special limitation on the specific types of the four enzyme markers for toxins. The enzyme marker is an enzyme-labeled secondary antibody. The corresponding enzyme-labeled secondary antibody can be selected according to the type of quality control protein. In a specific embodiment of the present invention, the quality control protein is mouse IgG, so the enzyme marker selected is enzyme-labeled sheep anti-mouse. In the present invention, the enzyme marker diluent is preferably composed of 1×PBS+1% bovine serum+0.025% sodium thimerosal, and the four enzyme markers for toxins are preferably prepared according to a titer of 1:1000.

[0034] In the present invention, after adding four toxin standard substances or samples to the toxin four-item enzyme label plate, four toxin enzyme labels and four toxin anti-reagents are added to react. The temperature of the reaction is preferably 37°C, and the time of the reaction is preferably 30min. Then wash the plate, and the present invention does not specifically limit the specific method of washing the plate. After the plate is washed, a color developing solution is added for reaction, and the temperature of the reaction is preferably 37°C, and the time of the reaction is preferably 15min. The present invention does not specifically limit the specific source of the color developing solution. The color-developed chip is placed in a scanner to obtain an image, and a dedicated analysis software is enabled for automated analysis, and the result report will be automatically generated. Specifically: after the reaction is completed, only the recognition site in the plate well is colored, and the rest is not colored. Therefore, there is no need for a specific wavelength, but it is scanned by a scanner to determine the grayscale of the color-developed recognition site, select the number of items on the analysis software, and according to the location of the quality control point, the software can locate the position of each recognition site of the remaining items, and interpret the grayscale of the 96 areas of the position, and take the average grayscale value of the 96 areas, which is the signal value of the site. First, after scanning the image, the software will output the project and automatically locate the positions of 12 recognition sites in 4 projects. Then, enter the curve concentration, dilution factor and detection limit of each project according to the instructions. For example, if Project 1 is AFB1, fill in the corresponding project, curve concentration, dilution factor and detection limit in the Project 1 position. Once filled in once, the data can be automatically generated.

[0035] The present invention also provides an application of the above ELISA plate or the above preparation method in preparing a toxin four-item detection product. The present invention has no special limitation on the type of the product, including a test kit.

[0036] The present invention also provides a four-item visualization microarray chip kit for toxins, comprising the above-mentioned ELISA plate, four-item toxin standard substances, antibody diluent, ELISA diluent and sample diluent.

[0037] In the present invention, since the four toxins in the sample to be tested need to be detected at the same time, it means that the standard diluent, antibody diluent, enzyme label diluent and sample diluent used for the four toxins must be kept uniform. However, after all, the biological characteristics of the four toxins are different, so how to ensure that each toxin is properly recovered and there are no problems such as background or super negative is actually very difficult. The present invention preferably uses an antibody diluent composed of 0.1M PB pH7.5, 1% sucrose, 0.025% sodium thimerosal and 1% bovine serum, which can overcome the above technical obstacles. If the antibody diluent defined in the present invention is replaced with other antibody diluents, it will cause problems such as too high or too low recovery of individual items, high background or super negative of individual items. In addition, the four standard diluents of toxins provided by the present invention are preferably 1×PBS, the sample diluent is preferably 2×PBS, and the enzyme label diluent is preferably 1×PBS+1% bovine serum+0.025% sodium thimerosal.

[0038] In the present invention, the preparation method of the four toxin standards preferably includes the following steps: mixing the standard dilution solution with the four toxins, setting five concentration gradient solutions, wherein the concentrations of the four toxins in the first concentration gradient solution are all 0 ppb, the concentrations of aflatoxin, zearalenone, vomitoxin, and fumonisin in the second concentration gradient solution are 1.5, 20, 200, and 200 ppb, respectively, the concentrations of aflatoxin, zearalenone, vomitoxin, and fumonisin in the third concentration gradient solution are 5, 70, 700, and 600 ppb, respectively, the concentrations of aflatoxin, zearalenone, vomitoxin, and fumonisin in the fourth concentration gradient solution are 15, 250, 2100, and 1800 ppb, respectively, and the concentrations of aflatoxin, zearalenone, vomitoxin, and fumonisin in the fifth concentration gradient solution are 50, 750, 7000, and 5400 ppb, respectively. It shows that the four-item visualization microarray chip kit for toxins of the present invention includes 5 kinds of standard substances, each of which is placed in a bottle. When the 5 bottles of standard substances are used to prepare the standard curve, the 5 bottles of standard substance liquid are respectively taken and dripped into 5 different wells, for example, the first concentration gradient liquid is dripped into the first well, the second concentration gradient liquid is dripped into the second well, and so on. After scanning, the standard curves of aflatoxin, zearalenone, vomitoxin and fumonisin can be obtained respectively.

[0039] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0040] In the following embodiments, unless otherwise specified, all of them are conventional methods.

[0041] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0042] Example 1

[0043] Preparation of four-item microarray ELISA plate for toxins

[0044] According to the raw materials and titers in Table 1, prepare the coating solutions separately, shake and mix them, and then add them separately to the liquid adding area of ​​the biochip spotter. The biochip spotter is equipped with a liquid adding area and a spotting area. The liquid adding area refers to the plate wells in which the prepared coating solution needs to be added. The spotter can automatically take liquid from this area for spotting. There are many blank and clean plate wells in the liquid adding area. The above-prepared 5 kinds of coating solutions need to be added to different plate wells respectively.

[0045] The spotting needle first aspirates liquid from the first plate well (mouse IgG coating solution), and then spots samples in each blank plate well in the spotting area in sequence. After spotting is completed, the spotting needle is automatically cleaned. After cleaning, the spotting needle will move to the second plate well (B1 raw material coating solution) to aspirate liquid, and then spot samples in each plate well in the spotting area. After spotting is completed, it will be rinsed, and then different types of coating solutions will be aspirated and spotted in turn.

[0046] Table 1 Preparation method of four toxin microarray chip ELISA plate

[0047]

[0048]

[0049] The schematic diagram of the toxin four-item microarray chip ELISA plate prepared according to the above preparation method is shown in Figure 1 .

[0050] Example 2

[0051] The difference from Example 1 is that the coating titers of mouse IgG, aflatoxin antigen, zeaxanthin antigen, vomitoxin antigen and fumonisin antigen in each sample well are 1:2000, 1:1000, 1:3000, 1:5000 and 1:60000 respectively, and the rest are the same as Example 1.

[0052] Example 3

[0053] The difference from Example 1 is that the coating titers of mouse IgG, aflatoxin antigen, zeaxanthin antigen, vomitoxin antigen and fumonisin antigen in each sample well are 1:4000, 1:3000, 1:7000, 1:9000 and 1:100000 respectively, and the rest are the same as Example 1.

[0054] Comparative Example 1

[0055] The difference from Example 1 is that the coating solution is 0.01M PB+glycine, and the rest is the same as Example 1.

[0056] Comparative Example 2

[0057] The difference from Example 1 is that the coating solution is CB solution, and the rest is the same as Example 1.

[0058] Comparative Example 3

[0059] The difference from Example 1 is that the coating solution is 0.01M PB+casein, and the rest is the same as Example 1.

[0060] The shapes of the recognition sites obtained in Example 1 and Comparative Examples 1-3 are as follows Figure 3 As shown, after using the coating solution of Comparative Example 3, needle blocking occurred and no recognition site was formed. Figure 3 It can be seen that the sites formed by the coating solution of Example 1 are relatively neat, without scattered points, etc., while the comparative example 1 has scattered points, and the comparative example 2 has scattered points and point tailing phenomenon, and in actual operation, the comparative example 2 is prone to needle blocking. In addition, the present invention conducts a single factor comparison experiment on the concentration of glycerol in the coating solution of Example 1, and the results show that the recognition sites corresponding to 1% glycerol are relatively neat, and the lower the glycerol content, the more obvious the hollow effect.

[0061] Example 4

[0062] The toxin four-item microarray chip ELISA plate obtained in Example 1 and Comparative Examples 1-2 was used for toxin four-item detection. The specific detection method comprises the following steps: adding 50 μL of toxin four-item standard / sample (the sample was diluted with 2×PBS sample diluent) to the toxin four-item ELISA plate, adding 50 μL of toxin four-item enzyme label, and then adding 50 μL of toxin four-item anti-reagent, reacting at 37°C for 30 minutes, washing the plate 4 times with 250 μL / well washing solution, patting dry with absorbent paper, adding 100 μL / well color development solution, reacting at 37°C for 15 minutes, carefully uncovering the cover film, drying the liquid in the well, washing twice with 250 μL / well washing solution, patting dry with absorbent paper, placing the color-developed chip in a scanner to obtain an image, activating a dedicated analysis software for automated analysis, and the result report will be automatically generated. The biochip scanner can scan the grayscale of the corresponding recognition sites on the plate after the reaction is completed. The chip analysis software we developed can be divided according to the rule of 3 recognition sites as one project, and 3 recognition sites can be manually selected as one project to obtain the average grayscale value of the corresponding project, and finally analyze and generate an Excel table, which contains 4 sheets, each of which is the data of a project and the linearity obtained by analysis.

[0063] The preparation method of the above four toxin standards is as follows: the standard diluent is DX standard dilution (1×PBS), the concentration of aflatoxin curve is 0 / 1.5 / 5 / 15 / 50ppb, the concentration of zearalenone curve is 0 / 20 / 70 / 250 / 750ppb, the concentration of vomitoxin curve is 0 / 200 / 700 / 2100 / 7000ppb, the concentration of fumonisin curve is 0 / 200 / 600 / 1800 / 5400ppb, and the curve can be prepared according to the above concentrations. Note: The same standard point is prepared in the same solution. For example: the concentrations of the second standard of the four items are 1.5 / 20 / 200 / 200ppb, then the 1.5ppb point of AFB1, the 20ppb point of ZEN, the 200ppb point of DON, and the 200ppb point of FB are prepared in the same solution.

[0064] The preparation method of the four anti-reagents for the above toxins is as follows: the antibody diluent is D1 anti-dilute (0.1M PB pH7.5 + 1% sucrose + 0.025% sodium thimerosal + 1% bovine serum), the aflatoxin antibody titer is 6w, the zearalenone antibody titer is 90w, the vomitoxin antibody titer is 25w, and the fumonisin antibody titer is 5w. The anti-reagents are prepared according to the above titers (all antibodies are prepared in the same solution).

[0065] The preparation methods of the four enzyme labels of the above-mentioned toxins are as follows: the enzyme label diluent is H1 (1×PBS+1% bovine serum+0.025% sodium thimerosal), and the enzyme label is an enzyme-labeled secondary antibody, which is prepared according to a titer of 1k.

[0066] The signal values ​​and sensitivities of the recognition sites of Example 1 and Comparative Examples 1-2 were compared. The results showed that Example 1 had a normal signal value and a high sensitivity, while Comparative Example 1 had a too light color and no obvious sensitivity, and Comparative Example 2 had a normal signal value and a low sensitivity.

[0067] Comparative Example 4

[0068] The difference from Example 1 is that the coating temperatures are 25°C and 45°C respectively, and the rest are the same as Example 1. Two parallel experiments are performed at each coating temperature. The toxin four-item microarray chip ELISA plate obtained in Example 1 and Comparative Example 4 is used for toxin four-item detection. The specific detection method is the same as Example 4 (the 0 mark point and the inhibition point (i.e., the first point and the second point of the standard curve) as well as the enzyme label and the anti-reagent are added to the ELISA plate wells, and the reaction is completed, and the color-developed ELISA plate is scanned). The detection results of Example 1 and Comparative Example 4 are shown in Table 2.

[0069] Table 2 Signal values ​​and inhibition rates of ELISA plates under different coating temperature conditions

[0070]

[0071] Among them, inhibition rate = inhibition point signal value / zero mark signal value × 100%. At the same inhibition point, the lower the inhibition rate, the higher the sensitivity; the higher the signal value, the higher the protein content adsorbed on the ELISA plate. As can be seen from Table 2, when the coating temperature is 37°C, the signal values ​​of the four items are higher than those at 25°C and 45°C. When the coating temperature is 25°C and 37°C, the inhibition rate is lower than that at 45°C, and the sensitivity is high, indicating that the coating temperature of the present invention has the best results.

[0072] Comparative Example 5

[0073] The difference from Example 1 is that the coating time is 0.5h, 1h, 3h, and 4h respectively, and the rest is the same as Example 1. Two parallel experiments are performed for each coating time. The toxin four-item microarray chip ELISA plate obtained in Example 1 and Comparative Example 5 is used for toxin four-item detection. The specific detection method is the same as Example 4 (the 0 mark point of the standard curve (i.e., the first point of the standard curve) and the enzyme label and the anti-reagent are added to the ELISA plate hole, and the reaction is completed, and the ELISA plate after color development is scanned). The results obtained in Example 1 and Comparative Example 5 are shown in Table 3.

[0074] Table 3 Signal values ​​of ELISA plates at different coating times

[0075]

[0076] It can be seen from Table 3 that the signal values ​​of the four items all show a trend of first increasing and then decreasing with the increase of the coating time. The signal value is the highest when the coating is 2 hours, indicating that the coating time result of the present invention is the best.

[0077] Comparative Example 6

[0078] The ELISA plate was prepared by the coating method commonly used in the art: the raw materials were added to the coating solution, mixed, and added directly to the ELISA plate, 100 μL / well, covered with a cover film, coated in a 37°C constant temperature incubator for 2 hours, washed twice with the washing solution, spun dry, blocked with 200 μL / well of the blocking solution, 37°C for 3 hours, spun dry, dried, and then vacuum packed. This is recorded as coating method ①.

[0079] Comparative Example 6 and Example 1 (referred to as coating method ②) were tested using the same four standard products of antigen, antibody, enzyme label, and toxin and the same spot plate method, and the detection method was the same as Example 4. Comparative Example 6 used a 450nm / 630nm dual-wavelength microplate reader for reading, so the signal value was 0-3.0; Example 1 used a biochip dedicated reader for scanning, and the gray value of the recognition site was used as the judgment standard, so the signal value was different from that of Comparative Example 6. The results are shown in Table 4.

[0080] Table 4 Test results of two different ELISA plate preparation methods

[0081]

[0082]

[0083] According to Table 4, for the three items AFB1, DON and FB, the inhibition rate of each curve concentration point of coating method ② is lower than that of coating method ①, and the inhibition rate of the ZEN item except the second point of the curve concentration points is lower than that of coating method ①. It can be seen that the coating method of the biochip spotter of the present invention improves the sensitivity of the raw material compared with the existing ELISA kit coating method.

[0084] Example 5

[0085] Comparison of test results from different antibody dilutions

[0086] The ELISA plate obtained in Example 1 was used to detect the same sample according to the detection method of Example 4. The difference from Example 4 was that different antibody diluents were used, specifically:

[0087] D1 antibody dilute: 0.1M PB pH7.5 + 1% sucrose + 0.025% sodium thimerosal + 1% bovine serum (Example 4); DON-K antibody dilute: 0.1M PB pH7.5 + 0.1% sodium chloride + 0.025% sodium thimerosal + 1% bovine serum; H1 antibody dilute: 0.1M PB pH7.5 + 0.1% calcium chloride + 0.025% sodium thimerosal + 1% bovine serum + 1% trehalose. The results are shown in Table 5.

[0088] Table 5 Detection results of different antibody dilutions

[0089]

[0090]

[0091] For the microarray chip test kit, the detection recovery rate is between 70% and 130%, which is in compliance with the requirements. As can be seen from Table 5, when the antibody diluent is D1 anti-dilution, 95% of the test results of all items meet the requirements; when the antibody diluent is DON-K anti-dilution, except for DON, the remaining items all have background (background refers to the sample liquid phase fixed value is negative, while the biochip test result is positive), and only 80% of the test results of all items meet the requirements, and the recovery of most items is high; when the antibody diluent is H1 anti-dilution, only 80% of the test results of all items meet the requirements, and the recovery of 4 items is low, indicating that the D1 anti-dilution of the present invention has significant advantages as an antibody diluent for the four anti-toxin reagents.

[0092] Example 6

[0093] 100 96T ELISA plates were coated with the ELISA plate preparation method of Example 1, and the 1st, 50th, and 100th ELISA plates were extracted. The first and last strips of each plate were extracted, and the plates were spotted for color development reaction. 50 μL of the third point solution of the four-item toxin standard curve was added to each well (using an 8-channel pipette for liquid addition), and then 50 μL of the four-item toxin enzyme label was added (using an 8-channel pipette for liquid addition), and then 50 μL of the four-item toxin anti-reagent was added (using an 8-channel pipette for liquid addition), wherein the four-item toxin standard The preparation method of the sample curve, four toxin enzyme labels and four toxin anti-reagents is the same as that in Example 4. The reaction was carried out at 37°C and 300 r / min for 30 min, the liquid in the plate was discarded, the plate was washed 4 times with 250 μL / well washing solution, and patted dry; then 100 μL / well color development solution was added, the reaction was carried out at 37°C and 300 r / min for 15 min, the plate was washed twice with 250 μL / well washing solution, and patted dry. The color-developed chip was placed in a scanner to obtain an image, and the dedicated analysis software was enabled for automated analysis. The results are shown in Tables 6-9.

[0094] Table 6 Aflatoxin B1 inter-plate and intra-plate variation test results

[0095]

[0096]

[0097] Table 7 Results of inter- and intra-plate variation test of zearalenone

[0098]

[0099] Table 8 Deoxynivalenol inter- and intra-plate variation test results

[0100]

[0101]

[0102] Table 9 Results of inter- and intra-plate variation tests on fumonisin

[0103]

[0104] The coefficient of variation = standard deviation / mean value × 100% (if the coefficient of variation <10%, the batch of ELISA plates is judged to meet the requirements); intra-plate variation: the coefficient of variation of the two strips extracted from an ELISA plate is the intra-plate variation of the plate; inter-plate variation: the coefficient of variation of all ELISA strips extracted from the batch of ELISA plates is the inter-plate variation of the batch of ELISA plates.

[0105] It can be seen from Tables 6-9 that the intra-plate variation of the three ELISA plates for the four toxins of the present invention is 2%-6%, and the inter-plate variation is 3%-8%, both of which are less than 10%, indicating that the ELISA plates coated by the method of the present invention meet the requirements and can be used for batch product debugging and production.

[0106] Example 7

[0107] Add 1 part of the four-item toxin concentrated sample dilution solution (2×PBS) to 9 parts of water and mix well to obtain the four-item toxin sample dilution solution.

[0108] Weigh 2g of sample that is negative for all four toxin items, add spikes according to the concentrations of the four toxin standard points (the same standard point concentrations for different items are added to the same sample), then add 10mL of 60% methanol, oscillate for 5min, centrifuge at 4000r / min at room temperature for 5min, take 200μL of supernatant, add it to 800μL of the four toxin sample dilution solution, mix thoroughly, and take 50μL for spot plate analysis.

[0109] The toxins in the samples were detected using the detection method obtained in Example 4, and the results are shown in Tables 10-13.

[0110] Note: Negative sample spike recovery rate = (sample concentration - sample concentration when spike value is 0) / spike value × 100%

[0111] Table 10 Aflatoxin B1 original data and recovery rate

[0112]

[0113] Table 11 Original data and recovery rate of zearalenone

[0114]

[0115] Table 12 Original data and recovery rate of vomitoxin

[0116]

[0117] Table 13 Fumonisin raw data and recovery rate

[0118]

[0119] It can be seen from Tables 10-13 that the linearity of the present invention in detecting spiked samples is >0.995, and the contents of four items in a single sample can be detected simultaneously, greatly improving the detection efficiency.

[0120] Example 8

[0121] The difference from Example 7 is that the sample is a sample with one of the four toxins being positive, and the rest is the same as Example 7. The results are shown in Tables 14-17. In the table, inf indicates that the detection value is 0, and the recovery rate of positive samples = detection sample concentration / liquid phase fixed value × 100%.

[0122] Table 14 Aflatoxin B1 original data and recovery rate

[0123]

[0124]

[0125] Table 15 Original data and recovery rate of zearalenone

[0126]

[0127]

[0128]

[0129] Table 16 DON raw data and recovery rate

[0130]

[0131]

[0132] Table 17 Fumonisin raw data and recovery rate

[0133]

[0134]

[0135]

[0136] As can be seen from Tables 14-17, the present invention simultaneously analyzed and detected 42 samples, and the linearity of the spiked samples detected by the method of the present invention was >0.995, and the recovery of positive samples with different toxins was 80%±120%, indicating that the method of the present invention can simultaneously detect the contents of multiple toxins in one sample, and can also simultaneously detect the contents of multiple toxins in multiple samples, thus achieving high throughput.

[0137] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A toxin four-item visualization microarray chip kit, characterized in that: Includes ELISA plate, four toxin standards, antibody diluent, ELISA diluent and sample diluent; Each sample well of the ELISA plate contains a biomolecule recognition site, and the biomolecule recognition sites are respectively coated with a quality control protein, aflatoxin antigen, zeaxanthin antigen, deoxynivalenol antigen and fumonisin antigen, and the coating solution used is composed of 0.01M PB pH7.5 and 1% glycerol; The preparation method of the ELISA plate comprises the following steps: mixing the coating solution with the quality control protein, aflatoxin antigen, zeaxanthin antigen, deoxynivalenol antigen and fumonisin antigen respectively to obtain five sample solutions, spotting the plate using a biochip spotter, coating at 37°C for 2h, washing the plate and sealing; The conditions for spotting the biochip spotter are as follows: the spotting volume of each recognition site is 4 nL, the spot spacing is 600 μm, and the spotting needle movement speed is 4000 μm / s; The antibody diluent consisted of 0.1 M PB pH 7.5, 1% sucrose, 0.025% sodium thimerosal and 1% bovine serum.

2. The kit according to claim 1, characterized in that The coating titers of the quality control protein, aflatoxin antigen, zeaxanthin antigen, deoxynivalenol antigen and fumonisin antigen in each sample well of the ELISA plate are 1:2000-4000, 1:1000-3000, 1:3000-7000, 1:5000-9000 and 1:60000-100000 respectively.

3. The kit according to claim 1, characterized in that The preparation method of the four toxin standards comprises the following steps: mixing the standard dilution solution with the four toxins, setting five concentration gradient solutions, wherein the concentrations of the four toxins in the first concentration gradient solution are all 0 ppb, the concentrations of aflatoxin, zearalenone, vomitoxin, and fumonisin in the second concentration gradient solution are 1.5, 20, 200, and 200 ppb, respectively, the concentrations of aflatoxin, zearalenone, vomitoxin, and fumonisin in the third concentration gradient solution are 5, 70, 700, and 600 ppb, respectively, the concentrations of aflatoxin, zearalenone, vomitoxin, and fumonisin in the fourth concentration gradient solution are 15, 250, 2100, and 1800 ppb, respectively, and the concentrations of aflatoxin, zearalenone, vomitoxin, and fumonisin in the fifth concentration gradient solution are 50, 750, 7000, and 5400 ppb, respectively.

4. A method for detecting four toxins using the kit according to any one of claims 1 to 3, characterized in that: The method comprises the following steps: adding four toxin standard substances or samples to be tested to the ELISA plate, adding four toxin enzyme markers and four toxin anti-reagents to react, washing the plate, adding color developing solution to react, washing the plate, and obtaining the result.

5. The method according to claim 4, characterized in that The volume ratio of the four toxin standards or test samples, the four toxin enzyme labels, and the four toxin anti-reagents is 1:1:1.

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