An immune detection solution, kit for preventing fluorescence leakage and its application

Abstract

The present invention provides an immune detection solution, kit and application thereof for preventing fluorescence leakage. The immune detection solution is characterized by comprising: a buffer solution with a concentration of 5 mM to 100 mM; cyclodextrin or its derivative with a concentration of 0.1% to 10% (W / V); and a surfactant with a concentration of 0.05% to 0.2% (W / V). The present invention also provides an application of the immune detection solution for preventing fluorescence leakage in droplet-based immunoassay. The beneficial effects of the present invention are as follows: cyclodextrin or its derivative is added to the immune detection solution, effectively controlling the leakage of resorufin and ensuring the accuracy of the single-molecule droplet imaging immunoassay results; by controlling the magnesium ion concentration in the immune detection solution, the positive rate is significantly increased, thereby improving the sensitivity of the droplet system; the immune detection solution is stable and can be stored at room temperature, avoiding the use of related temperature control components on the instrument; the preparation process of the immune detection solution is simple and the cost is controllable.

Description

Technical Field

[0001] The present invention belongs to the technical field of immunoassay, and particularly relates to an immunoassay liquid, a kit and an application thereof for preventing fluorescence leakage. Background Art

[0002] Droplet-based immunoassay is an immunoassay method based on microdroplet technology. This method uses microfluidics technology to generate droplets with a volume ranging from nanoliters to picoliters, and independent immunoassays are carried out in each droplet. Droplet-based immunoassay has broad application prospects in the fields of clinical diagnosis, food safety, environmental monitoring, etc. In droplet-based immunoassay using galactosidase conjugates as reagent components, the most commonly used fluorescent substrates are fluorescein di-β-D-galactoside (FDG) and resorufin-β-D-galactoside (RGP). Under the catalysis of galactosidase, the fluorescent product of FDG is fluorescein, which is highly hydrophilic and stable in the "oil-in-water" structure formed by the microfluidic chip, and there will be no obvious fluorescence leakage. However, FDG shows the characteristic of slow reaction in the catalytic reaction of galactosidase, and it shows obvious disadvantages in the development of instrument reagents pursuing detection speed. Under the catalysis of galactosidase, the fluorescent product of RGP is resorufin, which has both certain hydrophilicity and certain hydrophobicity, and it is easy to have fluorescence product leakage in the "oil-in-water" structure formed by the microfluidic chip, thus affecting the accuracy of immunoassay results. However, RGP shows the characteristic of rapid reaction in the catalytic reaction of galactosidase, which meets the requirement of instrument reagents for speed measurement.

[0003] Regarding the problem of fluorescence product leakage, some research results are given in the literature. The research by Philipp Gurner et al. shows that different fluorescent substances show different leakage rates in the same droplet oil, and the droplet oil containing different concentrations of surfactants also affects the leakage rate. The research in this direction is time-consuming and costly, and in the view of the test results of the author, its effect in preventing resorufin leakage is very limited. Yin Kun et al. used Pickering emulsification to solve the problem of resorufin leakage. This method has a certain effect, but it has the disadvantages of complex preparation process, time-consuming and unstable finished products of Pickering emulsion, and it is difficult to be used in commercialization. The research by Yousr Skhiri et al. shows that adding bovine serum albumin (BSA) to the buffer solution can control the leakage of resorufin to a certain extent. In the author's experiment, it was found that this method has a certain effect, but the droplets generated after using the buffer solution containing BSA showed the phenomenon of fusion and enlargement, which had an adverse impact on the experimental data analysis.

[0004] Therefore, there is an urgent need for a reagent that can effectively control fluorescence leakage and is suitable for commercialization to solve the fluorescence leakage problem in droplet immunoassay. Summary of the Invention

[0005] In view of the above-mentioned disadvantages of the prior art, the purpose of the present invention is to provide an immune detection solution, a kit and its application for preventing fluorescence leakage, so as to solve the problems of fluorescence leakage, poor stability of droplets, and inaccurate test results in current droplet immune detection.

[0006] To achieve the above object and other related objects, the present invention provides an immune detection solution for preventing fluorescence leakage, which is characterized by comprising:

[0007] A buffer solution of 5 mM to 100 mM;

[0008] Cyclodextrin or its derivative with a mass-volume fraction of 0.1% to 10%;

[0009] A surfactant with a mass-volume fraction of 0.05% to 0.2%

[0010] A preservative with a mass-volume fraction of 0.05% to 0.2%.

[0011] As a preference of the present application, the immune detection solution comprises:

[0012] A buffer solution of 10 mM to 100 mM;

[0013] Cyclodextrin or its derivative with a mass-volume fraction of 0.1% to 10%;

[0014] A soluble magnesium salt of 1 mM to 20 mM;

[0015] A surfactant with a mass-volume fraction of 0.05% to 0.2%;

[0016] A preservative with a mass-volume fraction of 0.05% to 0.2%.

[0017] As a preference of the present application, the cyclodextrin or its derivative is one or more of α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, hydroxypropyl-β-cyclodextrin, carboxymethyl-β-cyclodextrin, sodium sulfobutyl-β-cyclodextrin. More preferably, it is hydroxypropyl-β-cyclodextrin.

[0018] As a preference of the present application, the buffer solution is one or more of PBS buffer solution, Tri-HCl buffer solution, HEPES buffer solution. More preferably, it is PBS buffer solution.

[0019] As a preference of the present application, the pH value of the buffer solution is 7.0 to 8.0. More preferably, it is 7.4.

[0020] As a preference of the present application, the surfactant is one or more of anionic surfactant, cationic surfactant, amphoteric surfactant or nonionic surfactant.

[0021] Preferably in the present application, the surfactant is one or more of Triton X-100, Tween 20, Tween 80, Pluronic F-127, and sodium dodecyl sulfate. More preferably, the surfactant is Tween 20.

[0022] Preferably in the present application, the soluble magnesium salt is one or more of magnesium chloride, magnesium nitrate, and magnesium sulfate. More preferably, it is magnesium chloride.

[0023] Preferably in the present application, the preservative is Proclin 950.

[0024] Preferably in the present application, the immunoassay solution includes:

[0025] 10 mM PBS buffer;

[0026] 1% (W / V) hydroxypropyl-β-cyclodextrin;

[0027] 5 mM magnesium chloride;

[0028] 0.05% (W / V) Tween 20;

[0029] 0.1% (W / V) Proclin 950.

[0030] The present invention also provides a fluorescence immunoassay kit, characterized by including the immunoassay solution for preventing fluorescence leakage.

[0031] The present invention also provides an application of the fluorescence immunoassay kit in droplet-based immunoassay.

[0032] The present invention also provides an application of the immunoassay solution for preventing fluorescence leakage in droplet-based immunoassay.

[0033] Preferably in the present application, the application method includes the following partial or all steps:

[0034] Step 1, preparing a test sample dilution: diluting the test sample by a known multiple with a buffer to obtain a test sample dilution;

[0035] Step 2, preparing a magnetic bead immune complex: capturing the antigen in the test sample dilution with immune magnetic beads coated with a specific antibody, then mixing and incubating with a biotin-conjugated specific antibody, washing, and then mixing and incubating with a streptavidin-galactosidase complex, and washing again to obtain a magnetic bead immune complex;

[0036] Step 3, resuspending the magnetic bead immune complex with the immunoassay solution described above, oscillating and mixing evenly to obtain a resuspended magnetic bead solution;

[0037] Step 4: Use the resuspended magnetic bead solution as the magnetic bead phase, the substrate solution as the substrate phase, and the droplet generation oil as the oil phase. After passing through the microfluidic chip, water-in-oil droplets are generated; the substrate solution is a resorufin-β-D-galactoside solution.

[0038] Step 5: Leave the water-in-oil droplets in the droplet storage channel of the microfluidic chip to stand and incubate at 37°C - 60°C for 2 - 5 minutes.

[0039] Step 6: After the incubation is completed, place the microfluidic chip in an inverted microscope to take bright-field and dark-field photos, obtain the bright-field photos and dark-field fluorescence photos of the sample dilutions with different concentrations, and based on the results of the bright-field photos and dark-field photos of the sample dilutions, determine the total number of magnetic beads, the number of positive magnetic beads, and the number of magnetic beads with fluorescence leakage in each group. Then calculate the positive rate and the fluorescence leakage rate according to formula (1) and formula (2).

[0040] Positive rate = number of positive magnetic beads / total number of magnetic beads (1);

[0041] Fluorescence leakage rate = number of magnetic beads with fluorescence leakage / number of positive magnetic beads (2);

[0042] Step 7: Look up the corresponding antigen concentration value according to the antigen concentration - positive rate standard curve and then multiply it by the dilution factor of the test sample to obtain the concentration and content of the antigen in the test sample.

[0043] As a preference of this application, the volume ratio of the magnetic bead phase to the substrate phase is 1:1.

[0044] As a preference of this application, in Step 3, the droplet generation oil is HFE7500.

[0045] As a preference of this application, the substrate solution is the RGP reagent (product number: 103159) of the company quanterix.

[0046] As a preference of this application, the material of the microfluidic chip is PDMS.

[0047] As a preference of this application, the height of the droplet storage channel of the microfluidic chip is 10 μm - 25 μm. More preferably, it is 15 μm.

[0048] As a preference of this application, the connection medium between the microfluidic chip and the sample is an elastic quartz capillary with an inner diameter of 100 μm.

[0049] As a preference of this application, in Step 3, the diameter of the water-in-oil droplets is 7 μm - 15 μm. More preferably, it is 10 μm.

[0050] As a preference of this application, in Step 4, the incubation temperature of the water-in-oil droplets is 50°C and the incubation time is 3 minutes.

[0051] Preferably in the present application, the antigen concentration - positive rate standard curve is established according to the following method:

[0052] The antigen with known purity and concentration is serially diluted with a buffer to obtain an antigen dilution solution;

[0053] Prepare a magnetic bead immune complex: capture the antigen in the antigen dilution solution with immunomagnetic beads coated with a specific antibody, then mix and incubate with a biotin - conjugated specific antibody, wash, and then mix and incubate with a streptavidin - galactosidase complex, and wash again to obtain a magnetic bead immune complex;

[0054] Resuspend the magnetic bead immune complex with the aforementioned immunodetection solution, shake and mix well to obtain a resuspended magnetic bead solution;

[0055] Use the resuspended magnetic bead solution as the magnetic bead phase, the substrate solution as the substrate phase, and the droplet - generating oil as the oil phase, and generate water - in - oil droplets through a microfluidic chip; the substrate solution is a resorufin - β - D - galactoside solution;

[0056] Leave the water - in - oil droplets collected in the droplet storage channel of the microfluidic chip to stand and incubate at 37 °C - 60 °C for 2 - 5 min;

[0057] After the incubation is completed, place the microfluidic chip in an inverted microscope to take bright - field and dark - field photos, obtain bright - field photos and dark - field fluorescence photos of sample dilution solutions with different concentrations, automatically fuse and analyze the bright - field pictures and the corresponding dark - field pictures. According to the results of the bright - field photos and dark - field photos of the sample dilution solutions, determine the total number of magnetic beads, the number of positive magnetic beads, and the number of magnetic beads with fluorescence leakage in each group, and then calculate the positive rate and the fluorescence leakage rate according to formula (1) and formula (2) to establish the antigen concentration - positive rate standard curve;

[0058] Positive rate = number of positive magnetic beads / total number of magnetic beads (1);

[0059] Fluorescence leakage rate = number of magnetic beads with fluorescence leakage / number of positive magnetic beads (2).

[0060] Preferably in the present application, in the step of establishing the antigen concentration - positive rate standard curve, the antigen is serially diluted with a PBST solution containing 1% BSA to obtain an antigen dilution solution.

[0061] The present invention also provides a microfluidic chip, including a chip body, characterized in that: the chip body is provided with a liquid inlet part and a droplet storage channel communicating with the liquid inlet part. The liquid inlet part includes an oil - phase inlet, a magnetic - bead - phase inlet, a substrate - phase inlet, and a droplet - generating port that are connected to each other. The droplet - generating port is connected to the liquid inlet of the droplet storage channel, and the liquid outlet of the droplet storage channel is located at one end far from the liquid inlet part.

[0062] As a preference of this application, the microfluidic chip further includes a temperature control module, which includes a microscope stage, a heating module, a temperature sensing probe and a heat conducting block arranged on the microscope stage. There is a chip droplet generation observation window on the microscope stage for observing individual droplets flowing through the chip droplet generation observation window; the temperature sensing probe is arranged on the heat conducting block for detecting the temperature of the heat conducting block; the heat conducting block is in contact with the heating module for realizing heat conduction.

[0063] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0064] 1. Cyclodextrin or its derivative is added to the immunoassay solution, effectively controlling the leakage of resorufin.

[0065] 2. Overcoming the droplet fusion caused by the presence of bovine serum albumin in the solution, ensuring the accuracy of the single-molecule droplet photography immunoassay results.

[0066] 3. By controlling the magnesium ion concentration in the immunoassay solution, the positive rate is significantly increased, and thus the sensitivity of the droplet system is improved.

[0067] 4. The immunoassay solution is stable and can be stored at room temperature, avoiding the use of related temperature control components on the instrument.

[0068] 5. The preparation process of the immunoassay solution is simple and the cost is controllable. Description of the Drawings

[0069] Figure 1 and Figure 2 are respectively schematic diagrams of fluorescence leakage of the immunoassay solution in Example 1 ( Figure 1 is the bright-field image, Figure 2 is the dark-field fluorescence image).

[0070] Figure 3 and Figure 4 are respectively schematic diagrams of fluorescence leakage of Detection Liquid 1 in Comparative Example 1 ( Figure 3 is the bright-field image, Figure 4 is the dark-field fluorescence image).

[0071] Figure 5 and Figure 6 are respectively schematic diagrams of fluorescence leakage of Detection Liquid 2 in Comparative Example 2 ( Figure 5 is the bright-field image, Figure 6 is the dark-field fluorescence image).

[0072] Figure 7 is the antigen concentration-positive rate standard curve of the immunoassay solution in Example 5 in the detection of human IL-6 antigen.

[0073] Figure 8 is the structural schematic diagram of the microfluidic chip of this application.

[0074] Figure 9 This is a schematic diagram of the back side of the temperature control module of the microfluidic chip of the present application. Specific embodiments

[0075] The following uses specific specific examples to illustrate the implementation manners of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific implementation manners. Various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0076] It should be noted that the process equipment or devices not specifically noted in the following embodiments all adopt conventional equipment or devices in the art.

[0077] In addition, it should be understood that one or more method steps mentioned in the present invention do not exclude the existence of other method steps before and after the combined steps or the insertion of other method steps between these clearly mentioned steps, unless otherwise stated; it should also be understood that the combined connection relationship between one or more devices / devices mentioned in the present invention does not exclude the existence of other devices / devices before and after the combined devices / devices or the insertion of other devices / devices between these two clearly mentioned devices / devices, unless otherwise stated. Moreover, unless otherwise stated, the numbers of the method steps are only convenient tools for identifying the method steps, rather than limiting the arrangement order of the method steps or the scope in which the present invention can be implemented. The change or adjustment of their relative relationship, without substantial change in technical content, should also be regarded as the scope in which the present invention can be implemented.

[0078] Example 1

[0079] Step 1: Prepare the antigen dilution solution: Use a PBST solution of 1% (W / V) BSA (MERCK, product number: B2064-100g) as the antigen dilution solution;

[0080] Step 2: Preparation of magnetic bead immune complexes: The immunomagnetic beads coated with human IL-6 antibody (GenScript Biotech, product number: V06401) at 500,000 per test are used as reagent 1, the biotin-conjugated human IL-6 antibody (GenScript Biotech, product number: V06403) at 50 ng / mL is used as reagent 2, the streptavidin galactosidase complex (Thermo Fisher Scientific, product number: S931) at 50 ng / mL is used as reagent 3, and the solution after dissolving NIBSC / WHO(89 / 548) INTERLEUKIN-6 is used as the antigen, which is diluted to 30 pg / mL using the antigen diluent prepared in Step 1. A three-step immunological reaction is carried out with an incubation program of 30 minutes + 15 minutes + 15 minutes. Then, the obtained product is placed on a magnetic stand for magnetic absorption, and the supernatant solution is discarded to obtain immune complex magnetic beads;

[0081] Step 3: Resuspend the magnetic bead immune complexes with the immunodetection solution, shake and mix well to obtain a resuspended magnetic bead solution for subsequent droplet fluorescence leakage testing; The immunodetection solution includes: 10 mM PBS solution (pH 7.4), 1% (W / V) hydroxypropyl-β-cyclodextrin, 0.05% (W / V) Tween 20, 0.1% Proclin950;

[0082] Step 4: Take the resuspended magnetic bead solution as the magnetic bead phase, the RGP reagent from Quanterix Corporation (product number: 103159) as the substrate phase, and HFE7500 as the oil phase. The microfluidic chip is connected to the sample tube through a quartz capillary with an inner diameter of 100 μm, and the sample tube is then connected to the air pump through a hose. The volume ratio of the magnetic bead phase to the substrate phase is controlled to be 1:1 by air pressure. The air pump applies an air pressure of 115 kPa to the substrate phase and the magnetic bead phase in the microfluidic chip respectively, and an air pressure of 140 kPa to the oil phase. Under these conditions, water-in-oil droplets are generated, and the size of the droplets is about 10 μm;

[0083] Step 5: When the generated droplets completely fill the droplet storage channel of the microfluidic chip for storing droplets, turn off the air pump and incubate at 50 °C for 3 min in the temperature control module of the microfluidic chip;

[0084] Step 6: After the incubation is completed, place the microfluidic chip in an inverted microscope to take bright-field and dark-field photos; Obtain bright-field photos and dark-field fluorescence photos of sample diluents with different concentrations;

[0085] Specifically, adjust the inverted fluorescence microscope to the 10x objective lens. First, find the position with dense droplets in the chip through the bright field and take a bright-field photo; Then turn off the bright-field light source, switch to the green laser channel, set the exposure time to 1000 milliseconds, and take a dark-field fluorescence photo; Note that multiple groups of photos are taken for each sample to obtain sufficient analysis data;

[0086] Process the photos: Automatically fuse and analyze a set of pictures (including bright field and dark field), identify the droplets containing only a single magnetic bead in the pictures by setting the magnetic bead particle size parameter, identify the fluorescent positive droplets in the pictures by setting the fluorescence intensity parameter, analyze the average fluorescence intensity of the surrounding droplets within the diameter range of the center of the magnetic bead-containing droplets to determine the droplet leakage situation, and finally output the total number of magnetic beads, the number of positive magnetic beads, and the number of magnetic beads with fluorescence leakage for each group of pictures, and then calculate the positive rate and the fluorescence leakage rate according to formula (1) and formula (2);

[0087] Positive rate = number of positive magnetic beads / total number of magnetic beads (1);

[0088] Fluorescence leakage rate = number of magnetic beads with fluorescence leakage / number of positive magnetic beads (2).

[0089] The test results are as Figure 1 and Figure 2 .

[0090] Examples 2 to 14

[0091] The differences between the examples and Example 1 are as follows: The immunoassay solutions are different (the immunoassay solution formulations are shown in Table 1), and two groups of test antigen concentrations are 30 pg / mL and 0.37 pg / mL, and the other process conditions are exactly the same. The test results are shown in Table 2.

[0092] Table 1 Formulation table of the detection solution

[0093] Buffer Soluble salt Cyclodextrin or its derivative ((W / V)) Surfactant (W / V) Preservative (W / V) Example 1 10 mM PBS pH 7.4 - 1% Hydroxypropyl-β-cyclodextrin 0.05% Tween 20 0.1% Proclin 950 Example 2 10 mM PBS pH 7.4 - 1% α-Cyclodextrin 0.05% Tween 20 0.1% Proclin 950 Example 3 10 mM PBS pH 7.4 - 0.1% Hydroxypropyl-β-cyclodextrin 0.05% Tween 20 0.1% Proclin 950 Example 4 10 mM PBS pH 7.4 - 10% Hydroxypropyl-β-cyclodextrin 0.05% Tween 20 0.1% Proclin 950 Example 5 10 mM PBS pH 7.4 Magnesium chloride, 5 mM 1% Hydroxypropyl-β-cyclodextrin 0.05% Tween 20 0.1% Proclin 950 Example 6 10 mM PBS pH 7.4 Magnesium chloride, 20 mM 1% Hydroxypropyl-β-cyclodextrin 0.05% Tween 20 0.1% Proclin 950 Example 7 10 mM PBS pH 7.4 Magnesium chloride, 5 mM 1% Hydroxypropyl-β-cyclodextrin 0.1% Pluronic F-127 0.1% Proclin 950 Example 8 20 mM HEPES, pH 7.4 Magnesium chloride, 5 mM 1% Hydroxypropyl-β-cyclodextrin 0.05% Tween 20 0.1% Proclin 950 Example 9 20 mM Tris, pH 8.0 Magnesium chloride, 5 mM 1% Hydroxypropyl-β-cyclodextrin 0.05% Tween 20 0.1% Proclin 950 Example 10 5 mM PBS pH 7.4 Magnesium chloride, 5 mM 1% Hydroxypropyl-β-cyclodextrin 0.05% Tween 20 0.1% Proclin 950 Example 11 100 mM PBS pH 7.4 Magnesium chloride, 5 mM 1% Hydroxypropyl-β-cyclodextrin 0.05% Tween 20 0.1% Proclin 950 Example 12 10 mM PBS pH 7.4 Magnesium sulfate, 5 mM 1% Hydroxypropyl-β-cyclodextrin 0.05% Tween 20 0.1% Proclin 950 Example 13 10 mM PBS pH 7.4 Magnesium chloride, 1 mM 1% Hydroxypropyl-β-cyclodextrin 0.05% Tween 20 0.1% Proclin 950 Example 14 10 mM PBS pH 7.4 - 1% β-Cyclodextrin 0.05% Tween 20 0.1% Proclin 950

[0094] Comparative Example 1

[0095] The difference between this comparative example and Example 1 is that: Detection solution 1 is used to replace the immunoassay solution. Detection solution 1 includes: 10 mM PBS solution, 0.05% Tween 20, 0.1% (W / V) Proclin 950, and the pH of the PBS solution is 7.4. The other processes are exactly the same. The test results are as Figure 3 and Figure 4 .

[0096] Comparative Example 2

[0097] The difference between this comparative example and Example 1 is that: Detection solution 2 is used to replace the immunoassay solution. Detection solution 2 includes: 10 mM PBS solution, 5% (W / V) BSA, 0.05% Tween 20, 0.1% (W / V) Proclin 950, and the pH of the PBS solution is 7.4. The other processes are exactly the same. The test results are as Figure 5 and Figure 6 .

[0098] Comparative Example 3

[0099] The difference between this comparative example and Example 1 is that 1% (W / V) PVP K30 is used instead of 1% (W / V) cyclodextrin. The remaining processes are exactly the same.

[0100] Comparative Example 4

[0101] The difference between this comparative example and Example 1 is that 1% (W / V) PEG6000 is used instead of 1% (W / V) cyclodextrin. The remaining processes are exactly the same.

[0102] Comparative Example 5

[0103] The difference between this comparative example and Example 1 is that 1% (W / V) urea is used instead of 1% (W / V) cyclodextrin. The remaining processes are exactly the same.

[0104] Comparative Example 6

[0105] The difference between this comparative example and Example 1 is that 1% (W / V) DMSO is used instead of 1% (W / V) cyclodextrin. The remaining processes are exactly the same.

[0106] Result analysis:

[0107] As shown in Figures 1 - 6 in the first detection solution of Comparative Example 1 ( Figure 3 and Figure 4 ), the leakage of the fluorescent product resorufin is obvious (the positive droplets and adjacent droplets form a "flower" shape); in the second detection solution of Comparative Example 2 ( Figure 5 and Figure 6 ), there is no obvious leakage of the fluorescent product, but the droplet sizes are uneven (further research found that it is caused by the fusion of droplets during the incubation process); in the immunoassay solution of Example 1, there is no obvious leakage of the fluorescent substance, and the droplet sizes are uniform (as shown in Figure 1 and Figure 2 ). The above experimental results show that the immunoassay solution of the present application has the superiority of solving the fluorescence leakage in droplet immunoassay, and at the same time can overcome the droplet fusion caused by the presence of bovine serum albumin in the solution.

[0108] Table 2 Leakage rates of detection solutions with different ratios at antigen dilution concentrations of 30 pg / mL and 0.37 pg / mL

[0109]

[0110] From the positive rate and / or leakage rate data of Examples 1 to 14 and Comparative Examples 1 to 6 tabulated in Table 2, it can be seen that the addition of cyclodextrin or its derivatives significantly controls the fluorescence leakage rate of the 30 pg / mL antigen system, which can be reduced from the original 89.42% to below 40.78%. The anti-fluorescence leakage rates of Examples 1, 4 to 13 are controlled below 1%, and almost no fluorescence leakage problem occurs. Thus, it can be judged that cyclodextrin or its derivatives play a key role in the detection solution, and the detection solution added with cyclodextrin or its derivatives can effectively control the leakage of the fluorescent product (resorufin). Further, from the positive rate and leakage rate data of Example 2 and Example 1, even when the concentration of hydroxypropyl-β-cyclodextrin is 0.1%, the leakage rate is better than that of Example 2. Thus, it can be seen that hydroxypropyl-β-cyclodextrin has a better effect than cyclodextrin. From Examples 1, 3 to 4, it can be seen that as the concentration of hydroxypropyl-β-cyclodextrin increases, the 30 pg / mL leakage rate decreases significantly. However, when the concentration of hydroxypropyl-β-cyclodextrin reaches 10%, the positive rate of 0.37 pg / mL is significantly inhibited. Considering comprehensively, the concentration of hydroxypropyl-β-cyclodextrin at 1% is the optimal concentration. According to the changes in the types of buffer solutions, pH variations, and surfactant replacements of Examples 5, 7 to 9 tabulated in Table 2, the effects on the leakage rate and positive rate changes are not significant. From Examples 1, 5, 6, and 12 tabulated in Table 2, it can be seen that the addition of magnesium chloride or magnesium sulfate improves the positive rate of the 0.37 pg / mL antigen system to a certain extent and the effects are comparable, but there is no obvious effect on the leakage rate. It is analyzed that this may be caused by the promotion of magnesium ions in the activity of galactosidase. Considering the results of Examples 2 to 13 comprehensively, it is determined that Example 5 is the best choice for the immunodetection solution to prevent fluorescence leakage.

[0111] Example 15

[0112] Prepare a detection solution according to Example 5 again for use in a human IL-6 antigen detection kit to determine the effect of this formulation in actual application. The preparation of the sample refers to the steps for obtaining the immunocomplex magnetic beads in Example 1. The specific antigen concentrations are set as 30 pg / mL, 10 pg / mL, 3.333 pg / mL, 1.111 pg / mL, 0.37 pg / mL, 0.123 pg / mL, 0.041 pg / mL, and 0 pg / mL. The subsequent steps refer to the other contents of Example 1.

[0113] Table 3 Effect verification of the detection solution of Formulation 6 in the detection of human IL-6 antigen

[0114] Antigen concentration (pg / mL) Total number of magnetic beads Number of positive magnetic beads Number of magnetic beads with fluorescence leakage Positive rate Fluorescence leakage rate 0.000 6482 90 0 1.39% 0.00% 0.041 6031 122 0 2.02% 0.00% 0.123 5975 245 0 4.10% 0.00% 0.370 6981 641 0 9.18% 0.00% 1.111 6589 1439 0 21.84% 0.00% 3.333 7210 3824 2 53.04% 0.03% 10.000 5844 5256 6 89.94% 0.10% 30.000 6571 6525 11 99.30% 0.17%

[0115] From the statistical data in Table 3, it can be seen that under various antigen concentration conditions of the human IL-6 standard curve, the leakage rate of the droplets is extremely low, indicating that this solution can effectively control the leakage of droplet fluorescence and ensure the accuracy of the positive rate statistics of the experimental results. In addition, from Figure 4 the drawn standard curve, the discrimination of each concentration point is good, and the sensitivity can reach the fg / mL level, indicating that this group of immunoassay solutions can meet the product requirements.

[0116] The above embodiments are intended to illustrate the embodiments disclosed in the present invention and should not be construed as limitations on the present invention. In addition, various modifications listed herein and changes in the methods and compositions of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been specifically described in connection with various specific preferred embodiments of the present invention, it should be understood that the present invention should not be limited to these specific embodiments. In fact, all such modifications that are obvious to those skilled in the art as described above to obtain the invention should be included within the scope of the present invention.

Claims

1. An immune detection solution for preventing fluorescence leakage, characterized in that, Comprising: A buffer solution of 5 mM to 100 mM; Hydroxypropyl-β-cyclodextrin with a mass-volume fraction of 0.1% to 10%; A soluble magnesium salt of 1 mM to 20 mM; A surfactant with a mass-volume fraction of 0.05% to 0.2%; A preservative with a mass-volume fraction of 0.05% to 0.2%.

2. The immunodetection solution for preventing fluorescence leakage according to claim 1, wherein: The buffer solution is one or more of PBS buffer solution, Tri-HCl buffer solution, and HEPES buffer solution.

3. The immunodetection solution for preventing fluorescence leakage according to claim 2, wherein: The pH value of the buffer solution is 7.0 to 8.

0.

4. The immunodetection solution for preventing fluorescence leakage according to claim 2, wherein: The soluble magnesium salt is one or more of magnesium chloride, magnesium sulfate, and magnesium nitrate.

5. The immunodetection solution for preventing fluorescence leakage according to claim 1, wherein Comprising: 10 mM of PBS buffer solution; Hydroxypropyl-β-cyclodextrin with a mass-volume fraction of 1%; 5 mM of magnesium chloride; Tween 20 with a mass-volume fraction of 0.05%; Proclin 950 with a mass-volume fraction of 0.1%.

6. A fluorescence immunoassay quantitative determination kit, characterized in that, Comprising the immunodetection solution for preventing fluorescence leakage according to any one of claims 1 to 5.

7. Use of the immunodetection solution for preventing fluorescence leakage according to any one of claims 1 to 5 in the preparation of the fluorescence immunoassay kit.

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