Detection method of trace protein

Abstract

The invention provides a trace protein detection method. According to the embodiment of the invention, the method comprises the following steps: diluting a fluorescent dye stock solution in the NanoOrange protein quantitative detection kit to obtain a fluorescent dye diluent; respectively mixing a standard substance and a to-be-detected sample with the fluorescent dye diluent so as to obtain a standard substance mixed solution and a to-be-detected sample mixed solution; carrying out fluorescence intensity detection on the standard substance mixed solution and the to-be-detected sample mixed solution so as to obtain a fluorescence value; and determining the protein content in the to-be-detected sample based on the fluorescence value. By adopting the method disclosed by the invention, the sample loading concentration of the sample can be effectively improved, so that the detection sensitivity of residual protein in the mRNA sample is greatly improved, and the method is used for detecting trace protein.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a method for detecting trace amounts of protein in mRNA samples. Background Art

[0002] At present, the detection limits of protein detection methods such as BCA, Brandford, and Lorry are relatively high, and cannot meet the requirements for trace detection of protein residues in mRNA samples. Although the BCA method developed by Thermo has a detection limit of 0.5μg / ml, experimental tests have found that the detection limit can only be 1μg / ml, and there is a relatively serious matrix interference phenomenon for mRNA samples, and the tolerable mRNA concentration is lower than 0.25mg / ml; the detection limit of the CBQCA method can be as low as 0.1μg / ml, but because its main component contains cyanide, it is a highly toxic chemical with high toxicity, so it is not suitable for protein detection.

[0003] Currently, NanoOrange is a new type of fluorescent dye for protein quantification. According to the operating method provided in the existing test kit, when detecting mRNA samples, the samples need to be diluted at least 25 times (that is, the maximum sample loading concentration is 0.08 mg / ml) to ensure that the fluorescent detection reagent will not be affected. Such an operation will lead to limited sample injection volume, thereby limiting the sensitivity of residual protein detection in mRNA samples. Therefore, it is not conducive to the detection of trace proteins in mRNA samples.

[0004] Therefore, there is an urgent need to provide a detection method that can be used to detect trace amounts of protein in mRNA samples. Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art to at least a certain extent.

[0006] Based on the above problems, the inventors optimized the operation process in the kit. Specifically, the method of the present invention optimizes the preparation concentration of the fluorescent dye diluent, increases the concentration of the fluorescent dye diluent from 2× to 5×, and increases the sample loading volume by reducing the volume ratio of the fluorescent dye diluent mixed with the sample. After experimental verification, the optimized operation can increase the sample loading concentration by more than 10 times without affecting the detection limit, thereby greatly improving the sensitivity of residual protein detection in mRNA samples, which can be used for trace detection.

[0007] In one aspect of the present invention, a method for detecting the protein content of a sample to be tested is proposed. According to an embodiment of the present invention, the method includes: diluting the fluorescent dye stock solution in the NanoOrange protein quantitative detection kit to obtain a fluorescent dye dilution, wherein the dilution is performed using protein diluent 1; mixing the standard and the sample to be tested with the fluorescent dye dilution respectively to obtain a standard mixed solution and a sample mixed solution to be tested; performing fluorescence intensity detection on the standard mixed solution and the sample mixed solution to be tested to obtain a fluorescence value; and determining the protein content in the sample to be tested based on the fluorescence value. The method of the present invention can effectively increase the sample loading concentration, thereby greatly improving the sensitivity of residual protein detection in the mRNA sample, and can be used for the detection of trace proteins.

[0008] According to an embodiment of the present invention, the method for detecting the protein content of a sample to be tested further includes at least one of the following additional technical features:

[0009] According to an embodiment of the present invention, the dilution multiple is 100 to 250 times. The inventors have continuously optimized the operation process in the kit and increased the concentration of the fluorescent dye diluent to increase the sample loading concentration, thereby effectively increasing the sample loading concentration and further improving the sensitivity of residual protein detection.

[0010] It should be noted that the "dilution multiple of 100 to 250 times" mentioned in the present invention means diluting the fluorescent dye stock solution in the NanoOrange protein quantitative detection kit, that is, using protein diluent 1 to dilute the 500× fluorescent dye stock solution 100 to 250 times to obtain a (2× to 5×) fluorescent dye dilution solution.

[0011] According to an embodiment of the present invention, the standard substance and the sample to be tested are mixed with the fluorescent dye diluent in a volume ratio of (1-4):1 respectively.

[0012] It should be noted that the volume ratio of the standard and the sample to be tested to the fluorescent dye diluent depends on the concentration of the prepared fluorescent dye diluent. If it is 2×, the standard and the sample to be tested are mixed with the fluorescent dye diluent in a ratio of 1:1. If it is 4×, the standard and the sample to be tested are mixed with the fluorescent dye diluent in a ratio of 1:3. It is necessary to ensure that the fluorescent dye diluent in the final reaction solution is 1×.

[0013] According to an embodiment of the present invention, the volume ratios of the standard and the sample to be tested to the fluorescent dye diluent include but are not limited to 1:1, 2:1, 3:1, and 4:1.

[0014] According to an embodiment of the present invention, before the standard and the sample to be tested are mixed with the fluorescent dye diluent respectively, the process further includes: first mixing the standard with protein diluent 1; second mixing the sample to be tested with protein diluent 2 to obtain a second mixed solution; and third mixing the second mixed solution with the protein diluent 1 to obtain a third mixed solution.

[0015] According to an embodiment of the present invention, the volume ratio of the standard substance to the protein diluent 1 is 1:(200-5100).

[0016] According to an embodiment of the present invention, the volume ratio of the standard to the protein diluent 1 is 1: (240-260), 1: (300-320), 1: (400-420), 1: (610-630), 1: (1200-1300), 1: (2400-2600), 1: (4900-5100).

[0017] It should be noted that the volume ratio of the standard product to the protein diluent 1 is set to obtain a standard product with a concentration of (0.39-10) μg / ml. The specific concentration of the standard product can be set according to the instructions in the kit.

[0018] According to an embodiment of the present invention, the volume ratio of the sample to be tested to the protein diluent 2 is (8-10):1.

[0019] According to an embodiment of the present invention, the volume ratio of the sample to be tested to the protein diluent 2 includes but is not limited to 8:1, 9:1, and 10:1.

[0020] According to an embodiment of the present invention, the volume ratio of the sample to be tested to the protein diluent 2 is 9: 1. The inventors have found through experiments that diluting the sample to be tested by 0.9 times with the protein diluent 2 can ensure that the matrix of the sample to be tested is consistent with the matrix of the standard solution, both of which are protein diluent 1.

[0021] According to an embodiment of the present invention, the concentration of the sample to be tested in the third mixed solution is 1.5-1.8 μg / ml.

[0022] According to an embodiment of the present invention, the concentration of the sample to be tested in the third mixed solution is 1.6 μg / ml.

[0023] It should be noted that there is no fixed volume ratio when the second mixed solution and the protein diluent 1 are mixed. It is only necessary to make the concentration of the sample to be tested in the third mixed solution after mixing finally reach 1.5-1.8 μg / ml.

[0024] According to an embodiment of the present invention, the concentration of the standard is 2 mg / mL.

[0025] According to an embodiment of the present invention, the concentration of the sample to be tested is 2 mg / mL.

[0026] According to an embodiment of the present invention, the concentration of the sample to be tested includes but is not limited to 1.3 mg / mL, 1.4 mg / mL, 1.5 mg / mL, 1.6 mg / mL, 1.7 mg / mL, 1.8 mg / mL, 1.9 mg / mL, and 2 mg / mL.

[0027] According to an embodiment of the present invention, the protein diluent 1 is a 1× protein diluent.

[0028] According to an embodiment of the present invention, the protein diluent 2 is a 10× protein diluent.

[0029] According to an embodiment of the present invention, before the fluorescence intensity detection is performed, the standard mixed solution and the sample mixed solution are further incubated and cooled.

[0030] According to an embodiment of the present invention, the incubation process is carried out under light-proof conditions.

[0031] According to an embodiment of the present invention, the incubation temperature is 90-96°C.

[0032] According to an embodiment of the present invention, the incubation temperature includes but is not limited to 90°C, 91°C, 92°C, 93°C, 94°C, 95°C, and 96°C.

[0033] According to an embodiment of the present invention, the incubation time is 8 to 12 minutes.

[0034] According to an embodiment of the present invention, the incubation time includes but is not limited to 8 min, 9 min, 10 min, 11 min, and 12 min.

[0035] According to an embodiment of the present invention, the cooling process is performed under light-proof conditions.

[0036] According to an embodiment of the present invention, the cooling temperature is 20-25°C.

[0037] According to an embodiment of the present invention, the cooling temperature includes but is not limited to 20°C, 21°C, 22°C, 23°C, 24°C, and 25°C.

[0038] According to an embodiment of the present invention, the cooling time is 15 to 25 minutes.

[0039] According to an embodiment of the present invention, the cooling time includes but is not limited to 15 min, 16 min, 17 min, 18 min, 19 min, 20 min, 21 min, 22 min, 23 min, 24 min, and 25 min.

[0040] According to an embodiment of the present invention, the fluorescence intensity detection is performed by an ELISA instrument.

[0041] According to an embodiment of the present invention, the excitation wavelength of the microplate reader is 460-480 nm.

[0042] According to an embodiment of the present invention, the excitation wavelength of the microplate reader is 470 nm.

[0043] According to an embodiment of the present invention, the emission wavelength is 580-600 nm.

[0044] According to an embodiment of the present invention, the emission wavelength is 590 nm.

[0045] According to an embodiment of the present invention, based on the fluorescence value, determining the protein content in the sample to be tested is carried out in the following manner: establishing a standard curve according to the concentration of the standard and the fluorescence value of the standard to obtain a linear equation; and obtaining the protein content of the sample to be tested according to the fluorescence value of the sample to be tested and the linear equation.

[0046] Beneficial effects:

[0047] By optimizing the entire operation process, the mRNA sample is treated with 10× protein diluent, so that its buffer matrix becomes 1× protein diluent, and the standard is diluted with 1× protein diluent. The loading concentration is increased by 10 times by reducing the dilution multiple of the mRNA sample. In addition, the loading volume of the sample is increased by further increasing the concentration of the fluorescent dye diluent, that is, the loading concentration can be further increased. Therefore, the entire optimization process increases the loading concentration of the sample by 16 times, thereby greatly improving the sensitivity of protein content detection in mRNA samples, and can be used for trace detection of protein residues in mRNA samples (the matrix is ​​water).

[0048] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0050] Figure 1is a diagram of the operation process of a conventional NanoOrange experimental method according to an embodiment of the present invention;

[0051] Figure 2 is an operation process diagram of the optimized NanoOrange experimental method according to an embodiment of the present invention;

[0052] Figure 3 is an operation process diagram of the NanoOrange experimental method after optimization of Example 2 according to an embodiment of the present invention;

[0053] Figure 4 is a diagram of the operation process of the NanoOrange experimental method after optimization of Example 3 according to an embodiment of the present invention;

[0054] Figure 5 is a standard curve diagram obtained by using a conventional NanoOrange experimental method according to an embodiment of the present invention;

[0055] Figure 6 is a standard curve obtained by using the NanoOrange experimental method optimized in Example 2 according to an embodiment of the present invention;

[0056] Figure 7 is a standard curve obtained by using the NanoOrange experimental method optimized in Example 3 according to an embodiment of the present invention;

[0057] Figure 8 This is a standard curve obtained by using the NanoOrange experimental method optimized in Example 5 according to an embodiment of the present invention. DETAILED DESCRIPTION

[0058] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. If no specific techniques or conditions are specified in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used that do not specify the manufacturer are all conventional products that can be obtained commercially.

[0059] The reagents used in the following examples are all from Thermo Protein Quantitation Kit (Cat. No.: N6666). Water is nuclease-free water or water for injection

[0060] Without changing the matrix component (1× protein diluent + 1× dye) in the final reaction solution, the effects of the three different operation processes were compared. See Examples 1, 2 and 3 for details.

[0061] Example 1 Conventional NanoOrange Experimental Method

[0062] The experimental operation process is as follows Figure 1 As shown, the specific steps are as follows:

[0063] (1) Prepare 1× protein diluent: dilute 10× protein diluent with water to 1× protein diluent and mix by inverting.

[0064] (2) Prepare fluorescent dye diluent 1: dilute 500× fluorescent dye stock solution to 1× using 1× protein diluent, mix by inverting the solution to obtain fluorescent dye diluent 1, and place away from light.

[0065] (3) Dilution of BSA standard: 2 mg / ml BSA standard was prepared into a series of standard solutions using fluorescent dye diluent 1, with concentrations of 10 μg / ml, 6 μg / ml, 3 μg / ml, 1 μg / ml, 0.6 μg / ml, and 0.3 μg / ml, respectively. Fluorescent dye diluent 1 was used as a blank control.

[0066] (4) Sample processing: Use fluorescent dye diluent 1 to dilute the mRNA sample (concentration: 2 mg / ml) to 0.08 mg / ml (since the dilution factor has reached the maximum of 25 times), mix well, and centrifuge briefly.

[0067] (5) The standard solution and sample solution were reacted in a constant temperature mixer at 92°C for 10 min. After cooling at room temperature in the dark for 20 min, they were mixed by inverting.

[0068] (6) The reaction solution was transferred to a 96-well black microplate, with 120 μl in each well. Three replicate wells were set for each sample and detected on an ELISA reader with the excitation wavelength set to 470 nm and the emission wavelength set to 590 nm.

[0069] (7) Deduct the fluorescence value of the blank sample, and establish a standard curve with the BSA concentration in the standard solution as the horizontal axis and the calibration fluorescence value as the vertical axis. The standard curve is fitted using a binomial equation, and the concentration in the mRNA sample is calculated based on the calibration fluorescence value of the sample to be tested and the linear equation.

[0070] Example 2 Optimized NanoOrange Experimental Method

[0071] The optimized experimental operation process is as follows Figure 3 As shown, the specific steps are as follows:

[0072] (1) Prepare 1× protein diluent: dilute 10× protein diluent with water to 1× protein diluent and mix by inverting.

[0073] (2) Prepare fluorescent dye diluent 2: dilute 500× fluorescent dye stock solution to 2× using 1× protein diluent to obtain fluorescent dye diluent 2, invert the solution to mix well, and place away from light.

[0074] (3) Dilution of BSA standard: 2 mg / ml BSA standard was prepared into a series of standard solutions using 1× protein diluent, with concentrations of 10 μg / ml, 8 μg / ml, 6.4 μg / ml, 4.8 μg / ml, 3.2 μg / ml, 1.6 μg / ml, and 0.8 μg / ml, respectively. Among them, 1× protein diluent was used as a blank control. After mixing with fluorescent dye diluent 2, the BSA concentrations in the final reaction solution were 5 μg / ml, 4 μg / ml, 3.2 μg / ml, 2.4 μg / ml, 1.6 μg / ml, 0.8 μg / ml, and 0.4 μg / ml, respectively.

[0075] (4) Sample processing: The mRNA sample (concentration: 2 mg / ml) was diluted 0.9 times using 10× protein diluent, and then diluted to 1.6 mg / ml using 1× protein diluent for subsequent testing.

[0076] (5) The prepared standard solutions and the sample solutions were mixed with the fluorescent dye diluent 2 at a ratio of 1:1 (the concentration of the sample to be tested becomes 0.8 mg / ml after mixing), mixed by inversion, centrifuged briefly, reacted in a 92°C constant temperature mixer for 10 min, cooled in the dark at room temperature for 20 min, and mixed by inversion.

[0077] (6) Detection and data processing are the same as in Example 1.

[0078] Example 3 Optimized NanoOrange Experimental Method

[0079] The optimized experimental operation process is as follows Figure 4 As shown, the specific steps are as follows:

[0080] (1) Prepare 2× protein diluent: dilute 10× protein diluent with water to 2× protein diluent and gently invert to mix.

[0081] (2) Prepare fluorescent dye diluent 3: dilute the 500× fluorescent dye stock solution to 2× using 2× protein diluent to obtain fluorescent dye diluent 3, and gently invert the solution to mix.

[0082] (3) Dilution of BSA standard: 2 mg / ml BSA standard was prepared into a series of standard solutions with water, and the concentrations were 8 μg / ml, 6.4 μg / ml, 4.8 μg / ml, 3.2 μg / ml, 1.6 μg / ml, and 0.8 μg / ml, respectively. Water was used as a blank control. After mixing with the dye, the BSA concentrations in the final reaction solution were 4 μg / ml, 3.2 μg / ml, 2.4 μg / ml, 1.6 μg / ml, 0.8 μg / ml, and 0.4 μg / ml, respectively.

[0083] (4) Sample treatment: mRNA sample (concentration: 2 mg / ml) was diluted to 1.6 μg / ml using water.

[0084] (5) The prepared standard solutions and the sample solutions were mixed with the fluorescent dye diluent 3 at a ratio of 1:1 (the concentration of the sample to be tested becomes 0.8 mg / ml after mixing), mixed by inversion, centrifuged briefly, reacted in a 92°C constant temperature mixer for 10 min, cooled at room temperature in the dark for 20 min, and mixed by inversion.

[0085] (6) Detection and data processing are the same as in Example 1.

[0086] Example 4 Comparison of the results of conventional NanoOrange experimental method and optimized NanoOrange experimental method

[0087] (1) The standard curves of Examples 1, 2 and 3 are as follows Figure 5 , Figure 6 and Figure 7 The fluorescence value and detection limit results are shown in Table 1. It can be seen that the correlation coefficients R of the standard curves of the three different operation modes are 2 All reached ≥0.99, but the trend and shape of the standard curve of Example 2 were basically consistent with those of Example 1 (kit instructions), and there were differences between Example 3 and Example 1; and when the fluorescence values ​​were detected using the same concentration of standard products, there was basically no difference between Example 2 and Example 1, but the fluorescence value of Example 3 was halved, and there was basically no difference in the fluorescence value at low concentrations (0.4-1.6 μg / ml), and there was no distinction. There was no difference in the detection limit between Example 2 and Example 1, and the detection limit of Example 3 deteriorated.

[0088] (2) Improved detection sensitivity: Compared with Example 1, the detection limit of Example 2 was basically unchanged. Without changing the detection limit of the method, the sample loading concentration was increased from 0.08 mg / mL to 0.8 mg / ml, which increased the sensitivity of the method by 10 times. Although the loading concentration of Example 3 was also increased to 0.8 mg / ml, its detection limit deteriorated, affecting its detection sensitivity. In general, the optimized operation of Example 2 not only had no effect on the detection limit, but also increased the loading concentration.

[0089] Table 1

[0090]

[0091] Example 5 Further optimization of NanoOrange experimental method

[0092] Based on the optimized operation example 2, it is further explored whether it is feasible to increase the sample volume by increasing the concentration of the fluorescent dye diluent (from 2× to 5×) and reducing the volume ratio of the fluorescent dye diluent to the sample (from 1:1 to 1:4) to further increase the sample concentration and thus increase the detection sensitivity. Figure 2 The specific operation process is as follows:

[0093] (1) Prepare 1× protein diluent: dilute 10× protein diluent with water to 1× protein diluent and mix by inverting.

[0094] (2) Prepare fluorescent dye diluent 4: dilute 500× fluorescent dye stock solution to 5× using 1× protein diluent to obtain fluorescent dye diluent 4, invert the solution to mix well, and place away from light.

[0095] (3) Dilution of BSA standard: 2 mg / ml BSA standard was prepared into a series of standard solutions using 1× protein diluent, with concentrations of 8 μg / ml, 6.4 μg / ml, 4.8 μg / ml, 3.2 μg / ml, 1.6 μg / ml, 0.8 μg / ml, and 0.4 μg / ml, respectively. Among them, 1× protein diluent was used as a blank control. After mixing with fluorescent dye diluent 4, the BSA concentrations in the final reaction solution were 4 μg / ml, 3.2 μg / ml, 2.4 μg / ml, 1.6 μg / ml, 0.8 μg / ml, 0.4 μg / ml, and 0.2 μg / ml, respectively.

[0096] (4) Sample processing: The mRNA sample (concentration: 2 mg / ml) was diluted 0.9 times using 10× protein diluent, and then diluted to 1.6 mg / ml using 1× protein diluent for subsequent testing.

[0097] (5) The prepared standard solutions and the sample solutions were mixed with the fluorescent dye diluent 4 at a ratio of 4:1 (the concentration of the sample to be tested becomes 1.28 mg / ml after mixing), mixed by inversion, centrifuged briefly, reacted in a 92°C constant temperature mixer for 10 min, cooled in the dark at room temperature for 20 min, and mixed by inversion.

[0098] (6) Detection and data processing are the same as in Example 1.

[0099] The results are as follows Figure 8 As shown in Table 2, it can be seen that by increasing the sample loading volume by increasing the concentration of the fluorescent dye preparation, the standard curve, fluorescence signal value and detection limit are similar to the results of Example 2, and the standard curve, fluorescence signal value and detection limit are not affected. The mRNA sample loading concentration is increased from 0.8 mg / ml to 1.28 mg / ml, and the detection sensitivity is further increased.

[0100] Table 2

[0101]

[0102] Example 6 Other protein detection methods

[0103] The BCA method was compared with the optimized NanoOrange experimental method (Example 5). The experimental steps of the BCA method are as follows:

[0104] (1) Reagents: BCA Protein Assay Kit (Cat. No. 23235) produced by Thermo Fisher Scientific. Water used was nuclease-free water or water for injection.

[0105] (2) Standard dilution: BSA standard (concentration: 2 mg / ml) was prepared into a series of standard solutions with concentrations of 40 μg / ml, 20 μg / ml, 10 μg / ml, 5 μg / ml, 2.5 μg / ml, 1 μg / ml, and 0.5 μg / ml, respectively. The aqueous solution was used as a blank control.

[0106] (3) Sample treatment: mRNA samples were diluted with water to 1 mg / ml, 0.5 mg / ml, and 0.25 mg / ml, respectively.

[0107] (4) Prepare working solution: Mix BCA reagent A:BCA reagent B:BCA reagent C in a volume ratio of 25:24:1.

[0108] (5) The standard solution and sample solution were mixed with the working solution in a 1:1 volume ratio and reacted at 37°C for 2 h.

[0109] (6) After cooling, the reaction solution was transferred to a microplate, and three replicate wells were set for each sample. The absorbance value was measured at 562 nm using an ELISA reader.

[0110] (7) Subtract the fluorescence value of the blank sample, and establish a standard curve with the BSA concentration in the standard solution as the horizontal axis and the absorbance value as the vertical axis. The standard curve is fitted linearly, and the concentration in the mRNA sample is calculated based on the absorbance value of the sample to be tested and the linear equation.

[0111] The optimized NanoOrange and BCA methods for mRNA sample detection are shown in Table 3. Compared with the BCA method, the optimized NanoOrange method has a low detection limit, can tolerate a high concentration of mRNA samples, and has greatly improved detection sensitivity.

[0112] Table 3

[0113]

[0114] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0115] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A method for detecting trace protein, characterized in that: include: The fluorescent dye stock solution in the NanoOrange protein quantitative detection kit is diluted to obtain a fluorescent dye dilution solution, wherein the dilution is performed using protein dilution solution 1; The standard substance and the sample to be tested are mixed with the fluorescent dye diluent respectively to obtain a standard substance mixed solution and a sample mixed solution to be tested; Performing fluorescence intensity detection on the standard mixed solution and the sample mixed solution to obtain a fluorescence value; Based on the fluorescence value, the protein content in the sample to be tested is determined.

2. The method according to claim 1, characterized in that The dilution multiple is 100 to 250 times.

3. The method according to claim 1, characterized in that The standard substance and the sample to be tested are mixed with the fluorescent dye diluent in a volume ratio of (1-4):1 respectively.

4. The method according to claim 1, characterized in that: Before the standard and the sample to be tested are mixed with the fluorescent dye diluent respectively, the method further comprises: The standard substance is first mixed with protein diluent 1; The sample to be tested is mixed with the protein diluent 2 for a second time to obtain a second mixed solution; The second mixed solution is mixed with the protein diluent 1 for a third time to obtain a third mixed solution.

5. The method according to claim 4, characterized in that The volume ratio of the standard to the protein diluent 1 is 1:(200-5100); Optionally, the volume ratio of the standard to the protein diluent 1 is 1:(240-260), 1:(300-320), 1:(400-420), 1:(610-630), 1:(1200-1300), 1:(2400-2600), 1:(4900-5100); Optionally, the volume ratio of the sample to be tested to the protein diluent 2 is (8-10):1; Optionally, the concentration of the sample to be tested in the third mixed solution is 1.5-1.8 μg / ml; Optionally, the concentration of the standard is 2 mg / mL; Optionally, the concentration of the sample to be tested is 2 mg / mL.

6. The method according to any one of claims 1 to 5, characterized in that: The protein diluent 1 is a 1× protein diluent; Optionally, the protein diluent 2 is a 10× protein diluent.

7. The method according to claim 1, characterized in that Before the fluorescence intensity detection is performed, the standard mixed solution and the sample mixed solution are further incubated and cooled.

8. The method according to claim 7, characterized in that The incubation treatment is carried out under light-proof conditions; Optionally, the incubation temperature is 90-96°C; Optionally, the incubation time is 8 to 12 minutes; Optionally, the cooling treatment is carried out under light-proof conditions; Optionally, the cooling temperature is 20-25°C; Optionally, the cooling time is 15 to 25 minutes.

9. The method according to claim 1, characterized in that: The fluorescence intensity detection is performed by an enzyme-labeled instrument; Optionally, the excitation wavelength of the microplate reader is 460 to 480 nm; Optionally, the emission wavelength of the microplate reader is 580-600 nm.

10. The method according to claim 1, characterized in that Based on the fluorescence value, the protein content in the sample to be tested is determined in the following manner: A standard curve is established according to the concentration of the standard and the fluorescence value of the standard to obtain a linear equation; The protein content of the sample to be tested is obtained according to the fluorescence value of the sample to be tested and the linear equation.

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