A novel lactose intolerance assay kit and its application

Abstract

The present invention discloses a novel lactose intolerance determination kit and application thereof. The kit uses urine as a sample for detection, has simple steps, high detection efficiency, and can be used for large-scale screening of a population. The present invention proves through comparative experiments that the self-developed kit provided by the present invention has excellent detection performance and has broad application prospects in screening of lactose intolerance populations.

Description

Technical Field

[0001] The invention belongs to the technical field of lactose intolerance detection, and in particular relates to a novel lactose intolerance detection kit and application thereof. Background Art

[0002] Lactose intolerance (LI) is a common clinical sugar-derived diarrheal disease. It mainly refers to one or more digestive tract discomfort symptoms caused by taking lactose or foods containing lactose due to insufficient lactase secretion in the patient's small intestine, such as abdominal distension, diarrhea, abdominal pain, nausea, vomiting, etc. The incidence of this disease in the population is related to age, etc. Premature infants are live births born before 37 weeks of gestational age. Their gastrointestinal tract is immature and their metabolic function is weak. They are susceptible to various diseases, so they are more likely to show feeding intolerance during feeding, among which lactose intolerance is a disease with a higher incidence. Lactose intolerance will affect the absorption and utilization of dairy products by infants and young children. If it is not corrected for a long time, it will delay the growth and development of the children.

[0003] At present, there are four common methods for diagnosing lactose intolerance. The first is the breath test, but this method requires 4-8 samplings, and the sampling lasts for 2-4 hours. The sampling process is cumbersome and time-consuming; it requires high cooperation from the subjects, and it is impossible to test young children; diabetic patients affect the test results; it is greatly affected by diet and is prone to false positives. The second is the determination of stool reducing sugar and pH value, but because the sugars ingested by older children and adults, in addition to lactose, also include sucrose, maltose, galactose, fructose, L-(-)-glucose, etc. are all reducing sugars. At this time, a positive lead acetate method can only indicate poor sugar absorption. This method must be combined with a dietary history to confirm the diagnosis of lactase deficiency. The third is the lactose tolerance test, but this method requires multiple blood draws, which is difficult for children to accept, has a high false positive rate, and poor specificity. The fourth is jejunal biopsy to determine the activity of lactase and pathological histological observation, but this method is an invasive examination and is difficult to use as a routine diagnostic method. The fifth is the urine galactose detection method, but this method can only perform qualitative detection but not quantitative detection, and the operation process is cumbersome and time-consuming, and has not been applied in clinical practice.

[0004] In addition, a survey of commercialized urine galactose detection kits at home and abroad found that there is currently no lactose intolerance diagnostic reagent using the urine galactose detection method in domestic clinical practice. The only urine galactose detection kit used in the country to detect lactose intolerance can only perform qualitative detection of galactose in urine, but cannot perform quantitative detection of the galactose content in urine, and is not used clinically. Other urine galactose detection kits that can be quantitatively used in the country are only used for scientific research and cannot be used for clinical testing. It can be seen that how to develop a lactose intolerance assay kit that can be used for clinical detection of lactose intolerance patients based on the urine galactose detection kit for scientific research disclosed in the prior art is still one of the important unresolved technical problems facing this field.

[0005] Therefore, it is of great significance to find and develop a new, faster and more effective lactose intolerance detection method and its related detection kit, and apply it to the clinical detection of lactose intolerance patients. Summary of the invention

[0006] In view of this, in order to fill the technical gap in the art that there is no lactose intolerance determination kit that can be used for clinical testing, the purpose of the present invention is to provide a new lactose intolerance determination kit for the art to overcome the many technical problems existing in the existing common methods for diagnosing lactose intolerance.

[0007] The present invention adopts the following technical solutions to achieve the above-mentioned invention objectives:

[0008] The first aspect of the present invention provides a novel lactose intolerance assay kit.

[0009] Further, the kit comprises working solution 1, working solution 2, working solution 3, working solution 4, and working solution 5, and the components and corresponding contents of the reagents are as follows;

[0010] Working solution 1: galactose oxidase, 1.2 mg / mL; NAD coenzyme, 2 mg / mL; H2O2 protective agent, 0.36 mg / mL; diluent is phosphate buffered saline (PBS);

[0011] Working solution 2: 3-methyl-2-benzothiazolone hydrazone hydrochloride, 60 mg / mL; N,N-dimethylaniline, 20 mg / mL; Photofrin ® , 0.36 mg / mL; galactose protective agent, 0.36 mg / mL; diluent is PBS;

[0012] Working solution 3: peroxidase, 2.6 mg / mL; 2-chlorophenol, 100 mg / mL; phenol, 25 mmol / L; diluent is PBS;

[0013] Working solution 4: galactose, 1 mg / mL; solution is PBS;

[0014] Working solution 5: phosphate buffered saline (PBS);

[0015] The components and contents of the H2O2 protective agent are as follows: oxidized glutathione, 0.36 mg / mL;

[0016] The components contained in the galactose protective agent and the content of each component are as follows: L-(-)-glucose, 0.36 mg / mL; L-(-)-galactose, 0.36 mg / mL.

[0017] In some embodiments, the H2O2 protective agent is composed of oxidizing and reducing glutathione, silver ions, sodium persulfate, and potassium persulfate in specific proportions, which can protect the generated H2O2 from being decomposed by the reducing substances in the system, and can eliminate the interference of reducing substances such as Vc in the sample to a certain extent, thereby making the reaction more sensitive and accurate.

[0018] In a specific embodiment of the present invention, the components and contents of the H2O2 protective agent are as follows: oxidized glutathione, 0.36 mg / mL.

[0019] In some embodiments, the galactose protective agent includes, but is not limited to, one or more of the following sugars: L-(-)-glucose, fructose, trehalose, L-galactose, glycosaminoglycans, etc., which are substances similar in structure or properties to D-galactose and can reduce the degree of degradation of galactose by interfering substances.

[0020] In a specific embodiment of the present invention, the components contained in the galactose protective agent and the content of each component are as follows: L-(-)-glucose, 0.36 mg / mL; L-(-)-galactose, 0.36 mg / mL.

[0021] Furthermore, the sample detected by the kit is a urine sample.

[0022] Furthermore, the kit also comprises a detachable nested chamber device with a filter membrane.

[0023] Furthermore, the filter membrane in the detachable nested chamber device with filter membrane is a 0.4-1 μM filter membrane.

[0024] Furthermore, the kit comprises a reaction device, a urine purification device and a standard solution reagent bottle;

[0025] The reaction device is provided with a reaction hole and a calibration hole, and the reaction hole and the calibration hole are loaded with reaction reagents, and the reaction reagents are the working solution 1, working solution 2, and working solution 3 as described above;

[0026] The urine purification device is a detachable nested chamber device with filter membrane as described above;

[0027] The content of the standard solution reagent bottle is the standard solution, which is obtained by diluting the working solution 4 described above with the working solution 5 described above. The corresponding working solution 4 (1 mg / mL standard) is added to each reaction well to obtain standards of different concentrations.

[0028] In some embodiments, the standard solutions of different concentrations are 0.08, 0.12, 0.16, 0.18, 0.20, and 0.24 mg / mL galactose standard solutions.

[0029] In some embodiments, the reaction well is a microwell for performing the actual detection reaction. During the detection process of the kit, samples (such as blood, urine, tissue extracts, etc.) and detection reagents are added to the reaction wells, where specific biochemical reactions or immune reactions occur to detect the presence and content of the target substance. For example, in an enzyme-linked immunosorbent assay (ELISA) kit, the binding reaction between antigen and antibody, the reaction between enzyme and substrate, etc. are all carried out in the reaction wells, and the signals generated by these reactions (such as color changes, fluorescence or chemiluminescence, etc.) are used to judge the situation of the target molecule in the sample.

[0030] In some embodiments, the calibration wells are mainly used to establish a standard curve to ensure the accuracy and quantification of the test results. Standard substances (also called calibrators) of known concentrations are usually added to these wells, and the concentrations of these standard substances are accurately measured. During the detection process, the calibration wells and the reaction wells will undergo the same experimental conditions and operating steps. By comparing the signal generated by the sample in the reaction well with the signal generated by the standard substance in the calibration well, the concentration of the target substance in the sample is calculated according to the standard curve.

[0031] In some embodiments, the signal intensity (such as absorbance, fluorescence intensity, chemiluminescence intensity, etc.) in the reaction well is unknown and needs to be interpreted by comparing with the standard curve established in the calibration well. For example, in a kit for detecting protein content, the absorbance value generated by the sample in the reaction well needs to find the corresponding protein concentration point on the standard curve to determine the protein content in the sample.

[0032] In some embodiments, the signal generated by the calibration well is the reaction result of the known concentration standard, and these results are used to draw a standard curve. Generally, the standard concentration is used as the horizontal axis and the corresponding signal intensity is used as the vertical axis, and the standard curve equation is established by linear regression or other methods. The signal of the calibration well is directly related to the accuracy of the standard curve, which in turn affects the accuracy of the sample detection results in the reaction well.

[0033] In some embodiments, the reaction wells and calibration wells are covered with aluminum foil.

[0034] In some embodiments, the urine sample is derived from a subject in need. The subject of the present invention refers to any animal, and also refers to humans and non-human animals. Non-human animals include all vertebrates, for example, mammals, such as non-human primates (especially higher primates), sheep, dogs, rodents (such as mice or rats), guinea pigs, goats, pigs, cats, rabbits, cows, and any livestock or pets; and non-mammals, such as chickens, amphibians, reptiles, etc. In a preferred embodiment, the subject of the present invention refers in particular to humans.

[0035] In some embodiments, the detachable nested chamber device with filter membrane is an experimental device commonly used in biological experiments and research, and is also commonly referred to as a nested dish. The detachable nested chamber device with filter membrane comprises a nested chamber and a filter membrane. Among them, the nested chamber is generally composed of two parts, the upper part is a structure similar to a small cup or a small cylinder, and the lower part is a porous plate. The chamber can be inserted into the hole of the porous plate to form a nested structure. The filter membrane is located at the bottom of the chamber and is a key component of this device. The filter membrane is usually made of materials such as polyethylene terephthalate membrane (PET) or polycarbonate membrane (PC), and has a certain pore size, ranging from 0.1 μm to 8.0 μm, which can be selected according to experimental requirements. In a specific embodiment of the present invention, the pore size of the filter membrane in the detachable nested chamber device with filter membrane is 0.4-1 μM.

[0036] The detachable nested chamber device with filter membrane can be easily removed or inserted from the porous plate by the experimenter according to the experimental needs due to its detachability, which is convenient for observation, detection and other operations. It is also convenient for the chamber and the porous plate to be cleaned and reused separately, thereby reducing the experimental cost.

[0037] The presence of the filter membrane in the detachable nested chamber device with filter membrane can limit the movement of cells or macromolecular substances, that is, it can filter impurities in the urine sample, further improving the accuracy of the test result.

[0038] In the present invention, the Photofrin ® Refers to Porfimer sodium, trade name Photofrin ® , whose generic name is porphyrin sodium, is a mixture of porphyrin oligomers extracted and chemically modified from bovine blood. It is also the world's first photodynamic therapy (PDT) photosensitizer approved for clinical treatment.

[0039] In some embodiments, Photofrin is administered intravenously® After that, it will selectively accumulate in tumor tissue. This is because tumor tissue has some special physiological characteristics, such as rich blood vessels and high permeability, which makes Photofrin ® can more easily penetrate and remain around tumor cells.

[0040] In some embodiments, under irradiation with laser light of a specific wavelength, Photofrin ® Absorbing light energy, it produces singlet oxygen and other reactive oxygen species. These reactive oxygen species have strong oxidizing properties and can destroy the cell membrane, mitochondria, nucleic acid and other structures of tumor cells, leading to necrosis or apoptosis of tumor cells, thereby achieving the purpose of treating tumors.

[0041] Currently, the Photofrin ® It has been successfully used to treat various tumors on the body surface, oral and maxillofacial area, and in the cavity. It can also be used to treat liver cancer, pancreatic cancer, kidney cancer, peripheral lung cancer, etc. under B-ultrasound or CT-guided intervention or direct surgery. In addition, it is also used for precancerous lesions and some benign diseases, such as Barrett's esophagus, port wine stains, psoriasis, lupus erythematosus, age-related macular degeneration, etc. However, Photofrin has not yet been seen ® Photofrin is not used in the determination of lactose intolerance. ® The application of the lactose intolerance test kit is reported for the first time.

[0042] Furthermore, when loading the sample to be tested, the detachable nested chamber device with filter membrane is clamped above the reaction hole, the sample is loaded through the detachable nested chamber device with filter membrane, and the detachable nested chamber device with filter membrane is removed after loading.

[0043] In some embodiments, when the kit provided by the present invention is used, when the urine sample to be tested is loaded, the detachable nested chamber device with filter membrane is stuck above the reaction well, and the sample is loaded through the detachable nested chamber device with filter membrane. After loading, the detachable nested chamber device with filter membrane is removed, that is, the urine sample to be tested is added to the reaction well, and the standard liquid is dropped into the calibration well. After the calibration well and the reaction well are colored, the color of the reaction well is compared with the color of the calibration well with the color of the calibration well as the standard.

[0044] In some embodiments, the kit for determining lactose intolerance includes the following steps and operation sequences, as well as random adjustment combinations of the operation sequences of the steps:

[0045] (1) diluting working solution 4 with working solution 5 to obtain galactose of different concentrations;

[0046] (2) Take 50 μL of each concentration of galactose and working solution 5 and add them to the multi-well plate;

[0047] (3) Add 20 μL of working solution 1 to each well and incubate for 10 min.

[0048] (4) Add 100 μL of working solution 2 and 20 μL of working solution 3 to each well and incubate for 10 min.

[0049] (5) Determine the absorbance value at a wavelength of 450-600 nm and fit the standard curve formula;

[0050] (6) Determine the absorbance value corresponding to the sample to be tested and calculate the galactose concentration in the sample to be tested.

[0051] In some embodiments, the present invention has no particular restrictions on the operation sequence of the above steps, and those skilled in the art can make adjustments according to actual needs. Therefore, methods related to random adjustment combinations of the operation sequence of the above steps all fall within the protection scope of the present invention.

[0052] In some embodiments, the corresponding working solution 4 is added to each reaction well to obtain the different concentrations of 0.08, 0.12, 0.16, 0.18, 0.20, and 0.24 mg / mL galactose standard solutions.

[0053] In some embodiments, the multi-well plate is not particularly limited, including but not limited to: a 24-well plate or a 96-well plate.

[0054] In some embodiments, the addition of reagents as described above can protect galactose and eliminate the interference of reducing substances such as Vc, so that the test samples can be exempted from all sample pre-treatment steps, such as filtration, centrifugation and other operations, thereby reducing the overall steps of the detection process, shortening the time of the entire detection process, and improving the accuracy of the detection.

[0055] In some embodiments, the above-mentioned method can be used for large-scale testing in medical facilities. The 96-well plate loading test meets the requirements of automated loading and incubation, and the microplate reader is used for batch testing, which greatly saves manpower, material resources and costs; and the microplate reader is a common instrument in medical facilities, and does not increase the testing cost, personnel, venue, etc.

[0056] In some embodiments, the experimental time of the method described above is significantly shortened and the influencing factors are reduced: the experiment does not include sample preparation and operation time, the experimental reaction time is within 0.5-1 hour, the test sample is urine, and is not affected by factors such as diabetes and smoking.

[0057] In the present invention, after adding the substrate of the enzyme reaction, the substrate is catalyzed by the enzyme to form a colored product, and the amount of the product is proportional to the amount of the object under test, so qualitative or quantitative analysis can be performed based on the color depth. The method provided by the present invention has the characteristics of high sensitivity, high specificity, good repeatability, and fast detection speed, and is particularly suitable for large-volume sample detection, and is an internationally recognized standardized diagnostic method.

[0058] In some embodiments, the source of the reagents described above in the present invention is not particularly limited, and the reagents can be obtained through conventional commercial purchase channels or by self-preparation.

[0059] The second aspect of the present invention provides a reaction reagent for preparing a lactose intolerance assay kit.

[0060] Furthermore, the reaction reagent is the working solution 1, working solution 2, or working solution 3 described in the first aspect of the present invention.

[0061] The third aspect of the present invention provides a method for preparing the kit described in the first aspect of the present invention.

[0062] Furthermore, the method comprises the following steps: dropping the reaction reagent described in the second aspect of the present invention into the reaction well and the calibration well respectively, and after drying, successfully loading the reaction reagent into the reaction well and the calibration well.

[0063] In some embodiments, the drying condition is drying at 37-40° C. in a N 2 environment for 20-60 min.

[0064] The fourth aspect of the present invention provides use of the reaction reagent described in the second aspect of the present invention in preparing a lactose intolerance assay kit.

[0065] In addition, the present invention also provides the use of the reaction reagent described in the second aspect of the present invention in detecting lactose intolerance or screening people with lactose intolerance.

[0066] In addition, the present invention also provides a method for detecting lactose intolerance or screening a lactose intolerant population.

[0067] Furthermore, the method comprises the following steps: using the kit described in the first aspect of the present invention or the reaction reagent described in the second aspect of the present invention to detect a urine sample to be tested from a subject in need.

[0068] Compared with the prior art, the present invention has the following beneficial effects:

[0069] The present invention provides a novel lactose intolerance determination kit, which is the first lactose intolerance quantitative detection kit applicable to clinical diagnosis in China, and fills the technical gap of the lack of lactose intolerance quantitative detection kit applicable to clinical diagnosis in China. The kit uses urine as sample detection, the steps are simple, and it can be used for large-scale screening of the population; the filter membrane used to filter urine impurities can improve the accuracy of the results; the detachable nested chamber with filter membrane is used, and the chamber is directly removed after filtering urine, and the filtrate is continuously tested, which can simplify the experimental steps and is easy to operate; the final test result is calculated by machine reading, and the result judgment is simple and reliable, reducing manual errors, improving detection efficiency, and is conducive to large-scale population screening. In addition, the present invention proves through comparative experiments that the self-developed kit provided by the present invention has excellent detection performance and broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 : Comparison of results with the addition of galactose protective agent;

[0071] Figure 2 : Comparison of results with adding H2O2 protective agent;

[0072] Figure 3 :Galactose oxidase concentration optimization experimental results;

[0073] Figure 4 :The result diagram of the relationship between absorbance and concentration of quality standard detected by photometric microplate reader;

[0074] Figure 5 : Comparison of standard curves between the self-developed kit and other similar kits. DETAILED DESCRIPTION

[0075] The present invention is further described below in conjunction with specific embodiments, which are only used to explain the present invention and cannot be understood as limiting the present invention. It can be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and purpose of the present invention, and the scope of the present invention is defined by the claims and their equivalents.

[0076] The reagents and raw materials used in the present invention are easily obtained by those of ordinary skill in the art, and can be obtained from commercial sources unless otherwise specified. The experimental methods for which specific conditions are not specified in the present invention are usually tested under conventional conditions or under conditions recommended by the manufacturer. In particular, the following examples are only used to illustrate the present invention and should not limit the scope of the present invention in any way. It should be noted that the experimental conditions and results described in the following examples are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0077] Example 1 Optimization effect of adding galactose protective agent

[0078] 1. Experimental Materials

[0079] 24 / 96-well plate, filter membrane (0.4-1 μM), reagent 1, reagent 2A, reagent 2B, reagent 2C, reagent 3, reagent 4, reagent 5. All of the above are domestically available consumables and reagents.

[0080] Reagent 1: galactose oxidase (purchased from Sigma; CAS No. 9028-79-9), NAD coenzyme (purchased from Sigma; CAS No. 53-84-9);

[0081] Reagent 2A: 3-methyl-2-benzothiazolone hydrazone hydrochloride (purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; CAS No. 4338-98-1); N,N-dimethylaniline (purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; CAS No. 121-69-7); Photofrin ® (gift; CAS No. 87806-31-3);

[0082] Reagent 2B: galactose protective agent (D-(+)-glucose (purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; CAS No. 50-99-7));

[0083] Reagent 2C: galactose protective agent (L-(-)-glucose (purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; CAS No. 921-60-8); L-(-)-galactose (purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; CAS No. 15572-79-9));

[0084] Reagent 3: Peroxidase (purchased from Sigma; CAS No. 9003-99-0); 2-chlorophenol (purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; CAS No. 120-83-2); phenol (purchased from MacLean; CAS No. 108-95-2);

[0085] Reagent 4: galactose (purchased from Sigma; CAS No. 59-23-4);

[0086] Reagent 5: PBS buffer (purchased from Beijing Solebow Technology Co., Ltd.).

[0087] The novel lactose intolerance assay kit comprises reagent 1, reagent 2A, reagent 2B / reagent 2C, reagent 3, and reagent 4, which are as follows:

[0088] Reagent 1: galactose oxidase, 0.8 mg / mL; NAD coenzyme, 2 mg / mL; diluent is phosphate buffered saline (PBS).

[0089] Reagent 2A: 3-Methyl-2-benzothiazolone hydrazone hydrochloride, 60 mg / mL; N,N-dimethylaniline, 20 mg / mL; Photofrin ® , 0.36 mg / mL; the diluent was PBS.

[0090] Reagent 2B: the above reagent 2B, i.e., galactose protective agent 1, 0.36 mg / mL; the diluent is PBS.

[0091] Reagent 2C: the above reagent 2C, i.e. galactose protective agent 2, 0.36 mg / mL; the diluent is PBS.

[0092] Reagent 3: Peroxidase, 2.6 mg / mL; 2-chlorophenol, 100 mg / mL; Phenol, 25 mmol / L. Diluent: PBS.

[0093] Reagent 4: Galactose, 1 mg / mL.

[0094] Reagent 5: Phosphate buffered saline (PBS).

[0095] The components and contents of the galactose protective agent 1 are as follows: D-(+)-glucose, 0.36 mg / mL.

[0096] The components contained in the galactose protective agent 2 and the contents of each component are as follows: L-(-)-glucose, 0.36 mg / mL; L-(-)-galactose, 0.36 mg / mL.

[0097] 2. Experimental methods

[0098] Dilute reagent 4 with reagent 5 to obtain a 0.18 mg / mL standard solution;

[0099] Add 50 μL of 0.18 mg / mL standard solution to a 96-well plate;

[0100] Take 20 μL of reagent 1 and add it to each well and incubate for 10 minutes;

[0101] Add 100 μL of Reagent 2A to the first column of wells;

[0102] Add 70 μL of Reagent 2A to the wells in the second column, followed by 30 μL of Reagent 2B;

[0103] Add 70 μL of Reagent 2A to the third column of wells, and then add 30 μL of Reagent 2C;

[0104] Take 20 μL of reagent 3 and add it to each well and incubate for 10 minutes;

[0105] After incubation for 10 minutes, the absorbance was measured at a wavelength of 450-600 nm.

[0106] 3. Experimental results

[0107] After the reaction, the reaction wells without galactose protective agent and with D-(+)-glucose as galactose protective agent showed no obvious color change, while the reaction wells with L-(-)-glucose and L-(-)-galactose as galactose protective agents showed obvious color change. Figure 1 as shown).

[0108] Example 2 Optimization effect of adding H2O2 protective agent

[0109] 1. Experimental Materials

[0110] 24 / 96-well plate, filter membrane (0.4-1 μM), reagent 1A, reagent 1B, reagent 1C, reagent 2, reagent 3, reagent 4, reagent 5, all of the above are domestically available consumables and reagents.

[0111] Reagent 1A: galactose oxidase (purchased from Sigma; CAS No. 9028-79-9), NAD coenzyme (purchased from Sigma; CAS No. 53-84-9);

[0112] Reagent 1B: H2O2 protective agent (reduced glutathione (purchased from Sigma, USA; CAS No. 70-18-8));

[0113] Reagent 1C: H2O2 protective agent (oxidized glutathione (purchased from Shanghai MacLean Biochemical Technology Co., Ltd., CAS No. 121-24-4));

[0114] Reagent 2: 3-methyl-2-benzothiazolone hydrazone hydrochloride (purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; CAS No. 4338-98-1); N,N-dimethylaniline (purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; CAS No. 121-69-7); Photofrin ® (gift; CAS No. 87806-31-3); galactose protective agent (L-(-)-glucose (purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; CAS No. 921-60-8); L-(-)-galactose (purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; CAS No. 15572-79-9));

[0115] Reagent 3: Peroxidase (purchased from Sigma; CAS No. 9003-99-0); 2-chlorophenol (purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; CAS No. 120-83-2); phenol (purchased from MacLean; CAS No. 108-95-2);

[0116] Reagent 4: galactose (purchased from Sigma; CAS No. 59-23-4);

[0117] Reagent 5: PBS buffer (purchased from Beijing Solebow Technology Co., Ltd.).

[0118] The novel lactose intolerance assay kit comprises reagent 1A, reagent 1B / reagent 1C, reagent 2, reagent 3, and reagent 4, which are as follows:

[0119] Reagent 1A: galactose oxidase, 0.8 mg / mL; NAD coenzyme, 2 mg / mL; diluent is phosphate buffered saline (PBS).

[0120] Reagent 1B: H2O2 protective agent 1, 0.36 mg / mL.

[0121] Reagent 1C: H2O2 protective agent 2, 0.36 mg / mL.

[0122] Reagent 2: 3-Methyl-2-benzothiazolone hydrazone hydrochloride, 60 mg / mL; N,N-dimethylaniline, 20 mg / mL; Photofrin ® , 0.36 mg / mL; galactose protective agent, 0.36 mg / mL; diluent is PBS.

[0123] Reagent 3: Peroxidase, 2.6 mg / mL; 2-chlorophenol, 100 mg / mL; Phenol, 25 mmol / L. Diluent: PBS.

[0124] Reagent 4: Galactose, 1 mg / mL.

[0125] Reagent 5: Phosphate buffered saline (PBS).

[0126] The components and contents of the H2O2 protective agent 1 are as follows: reduced glutathione, 0.36 mg / mL.

[0127] The components and contents of the H2O2 protective agent 2 are as follows: oxidized glutathione, 0.36 mg / mL.

[0128] The components contained in the galactose protective agent and the content of each component are as follows: L-(-)-glucose, 0.36 mg / mL; L-(-)-galactose, 0.36 mg / mL.

[0129] 2. Experimental methods

[0130] Dilute reagent 4 with reagent 5 to obtain a 0.18 mg / mL standard solution;

[0131] Add 50 μL of 0.18 mg / mL standard solution to a 96-well plate;

[0132] Add 20 μL of reagent 1A to the first column of wells and incubate for 10 minutes;

[0133] Add 15 μL of Reagent 1A to the second column of wells, then add 5 μL of Reagent 1B and incubate for 10 minutes;

[0134] Add 15 μL of Reagent 1A to the third column of wells, then add 5 μL of Reagent 1C and incubate for 10 minutes;

[0135] Take 100 μL of reagent 2 and 20 μL of reagent 3 and add them to each well and incubate for 10 minutes;

[0136] After incubation for 10 minutes, the absorbance was measured at a wavelength of 450-600 nm.

[0137] 3. Experimental results

[0138] After the reaction, it was observed with the naked eye that the color change of the reaction wells adding oxidized glutathione as a H2O2 protective agent was the most obvious, and its effect was significantly better than that of the reaction wells without adding H2O2 protective agent and adding reduced glutathione. Oxidized glutathione and reduced glutathione are both H2O2 protective agents. However, the present invention has verified through comparative experiments that the detection effect of oxidized glutathione added to the kit provided by the present invention is significantly better than that of reduced glutathione. This result is a technical effect that is unexpected by those skilled in the art based on the prior art. It can be seen that oxidized glutathione is effective as a H2O2 protective agent (such as Figure 2 as shown).

[0139] Example 3 Effect of Optimizing Galactose Oxidase Concentration

[0140] 1. Experimental Materials

[0141] 24 / 96-well plate, filter membrane (0.4-1 μM), reagent 1C, reagent 1D, reagent 2, reagent 3, reagent 4, reagent 5, all of the above are domestically available consumables and reagents.

[0142] Reagent 1C: NAD coenzyme (purchased from Sigma; CAS No. 53-84-9); H2O2 protective agent (oxidized glutathione (purchased from Shanghai MacLean Biochemical Technology Co., Ltd., CAS No. 121-24-4));

[0143] Reagent 1D: galactose oxidase (purchased from Sigma; CAS number 9028-79-9);

[0144] Reagent 2: 3-methyl-2-benzothiazolone hydrazone hydrochloride (purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; CAS No. 4338-98-1); N,N-dimethylaniline (purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; CAS No. 121-69-7); Photofrin ® (gift; CAS No. 87806-31-3); galactose protective agent (L-(-)-glucose (purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; CAS No. 921-60-8); L-(-)-galactose (purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; CAS No. 15572-79-9));

[0145] Reagent 3: Peroxidase (purchased from Sigma; CAS No. 9003-99-0); 2-chlorophenol (purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; CAS No. 120-83-2); phenol (purchased from MacLean; CAS No. 108-95-2);

[0146] Reagent 4: galactose (purchased from Sigma; CAS No. 59-23-4);

[0147] Reagent 5: PBS buffer (purchased from Beijing Solebow Technology Co., Ltd.).

[0148] The novel lactose intolerance assay kit comprises reagent 1C, reagent 1D, reagent 2, reagent 3, and reagent 4, and the reagent 1C, reagent 1D, reagent 2, reagent 3, reagent 4, and reagent 5 are as follows:

[0149] Reagent 1C: NAD coenzyme, 2 mg / mL; H2O2 protective agent, 0.36 mg / mL; diluent is phosphate buffered saline (PBS).

[0150] Reagent 1D: Galactose oxidase, diluted in phosphate buffered saline (PBS).

[0151] Reagent 2: 3-Methyl-2-benzothiazolone hydrazone hydrochloride, 60 mg / mL; N,N-dimethylaniline, 20 mg / mL; Photofrin ® , 0.36 mg / mL; galactose protective agent, 0.36 mg / mL; diluent is PBS.

[0152] Reagent 3: Peroxidase, 2.6 mg / mL; 2-chlorophenol, 100 mg / mL; Phenol, 25 mmol / L. Diluent: PBS.

[0153] Reagent 4: Galactose, 1 mg / mL.

[0154] Reagent 5: Phosphate buffered saline (PBS).

[0155] The components and contents of the H2O2 protective agent are as follows: oxidized glutathione, 0.36 mg / mL.

[0156] The components contained in the galactose protective agent and the content of each component are as follows: L-(-)-glucose, 0.36 mg / mL; L-(-)-galactose, 0.36 mg / mL.

[0157] 2. Experimental methods

[0158] Dilute reagent 4 with reagent 5 to obtain a 0.18 mg / mL standard solution;

[0159] Add 50 μL of 0.18 mg / mL standard solution to a 96-well plate;

[0160] Reagent 1D was diluted with reagent 5 to obtain 0.8, 1.0, and 1.2 mg / mL of galactose oxidase;

[0161] Add 15 μL of reagent 1C to each well above;

[0162] Add 5 μL of 0.8, 1.0, or 1.2 mg / mL galactose oxidase to each well and incubate for 10 minutes;

[0163] Take 100 μL of reagent 2 and 20 μL of reagent 3 and add them to each well and incubate for 10 minutes;

[0164] After incubation for 10 minutes, the absorbance was measured at a wavelength of 450-600 nm.

[0165] 3. Experimental results

[0166] After the reaction, the color of the reaction wells with 1.2 mg / mL galactose oxidase was observed with the naked eye and found to change most significantly. This shows that the optimal concentration of galactose oxidase is 1.2 mg / mL (e.g. Figure 3 as shown).

[0167] Example 4 A novel lactose intolerance assay kit and the effect of the assay quality standard thereof

[0168] 1. Experimental Materials

[0169] 24 / 96-well plate, filter membrane (0.4-1 μM), working solution 1, working solution 2, working solution 3, working solution 4, working solution 5. All of the above are domestically available consumables and reagents.

[0170] Working solution 1: galactose oxidase (purchased from Sigma; CAS No. 9028-79-9), NAD coenzyme (purchased from Sigma; CAS No. 53-84-9); H2O2 protective agent (oxidized glutathione (purchased from Shanghai MacLean Biochemical Technology Co., Ltd., CAS No. 121-24-4));

[0171] Working solution 2: 3-methyl-2-benzothiazolone hydrazone hydrochloride (purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; CAS No. 4338-98-1); N,N-dimethylaniline (purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; CAS No. 121-69-7); Photofrin ® (gift; CAS No. 87806-31-3); galactose protective agent (L-(-)-glucose (purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; CAS No. 921-60-8); L-(-)-galactose (purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; CAS No. 15572-79-9));

[0172] Working solution 3: peroxidase (purchased from Sigma; CAS No. 9003-99-0); 2-chlorophenol (purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; CAS No. 120-83-2); phenol (purchased from MacLean; CAS No. 108-95-2);

[0173] Working solution 4: galactose (purchased from Sigma; CAS number 59-23-4);

[0174] Working solution 5: PBS buffer (purchased from Beijing Solebow Technology Co., Ltd.).

[0175] The novel lactose intolerance assay working solution box comprises working solution 1, working solution 2, working solution 3, and working solution 4, and the working solution 1, working solution 2, working solution 3, working solution 4, and working solution 5 are as follows:

[0176] Working solution 1: galactose oxidase, 1.2 mg / mL; NAD coenzyme, 2 mg / mL; H2O2 protective agent, 0.36 mg / mL; diluent is phosphate buffered saline (PBS).

[0177] Working solution 2: 3-methyl-2-benzothiazolone hydrazone hydrochloride, 60 mg / mL; N,N-dimethylaniline, 20 mg / mL; Photofrin ®, 0.36 mg / mL; galactose protective agent, 0.36 mg / mL; diluent is PBS.

[0178] Working solution 3: peroxidase, 2.6 mg / mL; 2-chlorophenol, 100 mg / mL; phenol, 25 mmol / L. Diluent is PBS.

[0179] Working Solution 4: Galactose, 1 mg / mL.

[0180] Working solution 5: phosphate buffered saline (PBS).

[0181] The components and contents of the H2O2 protective agent are as follows: oxidized glutathione, 0.36 mg / mL.

[0182] The components contained in the galactose protective agent and the content of each component are as follows: L-(-)-glucose, 0.36 mg / mL; L-(-)-galactose, 0.36 mg / mL.

[0183] 2. Experimental methods

[0184] Working solution 4 was diluted with working solution 5 to obtain standard solutions of 0.08, 0.12, 0.16, 0.18, 0.20, and 0.24 mg / mL;

[0185] 50 μL of each concentration standard solution and working solution 5 (as a blank control group) were added to a 96-well plate;

[0186] Take 20 μL of working solution 1 and add it to each well and incubate for 10 minutes;

[0187] Take 100 μL of working solution 2 and 20 μL of working solution 3 and add them to each well and incubate for 10 minutes;

[0188] After incubation for 10 minutes, the absorbance was measured at a wavelength of 450-600 nm.

[0189] 3. Experimental results

[0190] The results were read out by a photometric microplate reader. After deducting the blank control value from each well, the concentration of galactose standard was used as the X-axis, and the value corresponding to each concentration after deducting the blank control value was used as the Y-axis to construct an XY coordinate system, and the standard curve formula was fitted. The results are shown in the figure. Figure 4 As shown, the fitted formula is: y = 7.3971x + 0.1069, R² = 0.999, indicating that there is an excellent linear covariance relationship between galactose concentration and OD value. Figure 4 as shown).

[0191] Example 5 Effect of a novel lactose intolerance assay kit on the determination of a quality standard

[0192] 1. Experimental Materials

[0193] Self-developed kit and purchased kit 1 (purchased from Company A).

[0194] Wherein, the self-developed kit includes:

[0195] Working solution 1: galactose oxidase, 1.2 mg / mL; NAD coenzyme, 2 mg / mL; H2O2 protective agent, 0.36 mg / mL; diluent is phosphate buffered saline (PBS).

[0196] Working solution 2: 3-methyl-2-benzothiazolone hydrazone hydrochloride, 60 mg / mL; N,N-dimethylaniline, 20 mg / mL; Photofrin ® , 0.36 mg / mL; galactose protective agent, 0.36 mg / mL; diluent is PBS.

[0197] Working solution 3: peroxidase, 2.6 mg / mL; 2-chlorophenol, 100 mg / mL; phenol, 25 mmol / L. Diluent is PBS.

[0198] Working solution 4: Galactose, 1 mg / mL; solution is PBS.

[0199] Working solution 5: phosphate buffered saline (PBS).

[0200] The components contained in the H2O2 protective agent and the content of each component are as follows: oxidized glutathione, 0.36 mg / mL.

[0201] The components contained in the galactose protective agent and the content of each component are as follows: L-(-)-glucose, 0.36 mg / mL; L-(-)-galactose, 0.36 mg / mL.

[0202] 2. Experimental methods

[0203] The standard curve was constructed and the detection experiment was performed as described in the previous examples, and the standard purchased from kit 1 was used as a sample for the experiment.

[0204] 3. Experimental results

[0205] The self-developed kit provided by the present invention can accurately calculate the concentration of each standard point of the purchased kit, indicating that the results of the two kits have a good match (such as Figure 5 as shown).

[0206] Application example: The application effect of a new lactose intolerance test kit in the determination of actual samples

[0207] 1. Experimental Materials

[0208] Self-developed test kit, purchased test kit 1 (purchased from Company A), volunteer urine, and pure milk.

[0209] Wherein, the self-developed kit includes:

[0210] Working solution 1: galactose oxidase, 1.2 mg / mL; NAD coenzyme, 2 mg / mL; H2O2 protective agent, 0.36 mg / mL; diluent is phosphate buffered saline (PBS).

[0211] Working solution 2: 3-methyl-2-benzothiazolone hydrazone hydrochloride, 60 mg / mL; N,N-dimethylaniline, 20 mg / mL; Photofrin ® , 0.36 mg / mL; galactose protective agent, 0.36 mg / mL; diluent is PBS.

[0212] Working solution 3: peroxidase, 2.6 mg / mL; 2-chlorophenol, 100 mg / mL; phenol, 25 mmol / L. Diluent is PBS.

[0213] Working solution 4: Galactose, 1 mg / mL; solution is PBS.

[0214] Working solution 5: phosphate buffered saline (PBS).

[0215] The components contained in the H2O2 protective agent and the content of each component are as follows: oxidized glutathione, 0.36 mg / mL.

[0216] The components contained in the galactose protective agent and the content of each component are as follows: L-(-)-glucose, 0.36 mg / mL; L-(-)-galactose, 0.36 mg / mL.

[0217] 2. Experimental methods

[0218] The self-developed test kit uses the method described in the previous examples. When loading the sample, the guide nested chamber device with the filter membrane (i.e., a detachable nested chamber with a filter membrane) is stuck above the urine sample loading hole, and the sample is loaded through the guide nested chamber device. After loading, the guide nested chamber device is removed; the purchased test kit uses the operating method recommended by the kit to detect human urine respectively.

[0219] 3. Experimental results

[0220] The experimental results are shown in Table 1. The results show that in the population samples, the detection tolerance rate of Kit 1 (self-developed kit) was 19.3%, and the detection tolerance rate of Kit 2 (purchased kit) was 20.7%, Kappa=0.958>0.75, indicating that the consistency of the two kits is very good.

[0221] Table 1. The consistency of the two kits

[0222]

[0223] Comparative Example Comparison of the measurement results of the novel lactose intolerance measurement kit provided by the present invention and the commercialized kit

[0224] 1. Experimental Materials

[0225] Self-developed test kit, other test kit 2 (purchased from Company B in the United States), volunteer urine, pure milk.

[0226] Wherein, the self-developed kit includes:

[0227] Working solution 1: galactose oxidase, 1.2 mg / mL; NAD coenzyme, 2 mg / mL; H2O2 protective agent, 0.36 mg / mL; diluent is phosphate buffered saline (PBS).

[0228] Working solution 2: 3-methyl-2-benzothiazolone hydrazone hydrochloride, 60 mg / mL; N,N-dimethylaniline, 20 mg / mL; Photofrin®, 0.36 mg / mL; galactose protective agent, 0.36 mg / mL; diluent is PBS.

[0229] Working solution 3: peroxidase, 2.6 mg / mL; 2-chlorophenol, 100 mg / mL; phenol, 25 mmol / L. Diluent is PBS.

[0230] Working solution 4: Galactose, 1 mg / mL; solution is PBS.

[0231] Working solution 5: phosphate buffered saline (PBS).

[0232] The components contained in the H2O2 protective agent and the content of each component are as follows: oxidized glutathione, 0.36 mg / mL.

[0233] The components contained in the galactose protective agent and the content of each component are as follows: L-(-)-glucose, 0.36 mg / mL; L-(-)-galactose, 0.36 mg / mL.

[0234] 2. Experimental methods

[0235] The self-developed test kit uses the method described in the previous examples to detect human urine. When loading the sample, the guide nested chamber device with the filter membrane is stuck above the urine sample loading hole, and the sample is loaded through the guide nested chamber device. After loading, the guide nested chamber device is removed; the imported test kit uses the operating method recommended by the kit to detect human urine.

[0236] 3. Experimental results

[0237] The results are shown in Table 2-3. When the urinary galactose concentration of the volunteers reached 0.18 mg / mL, they were considered lactose tolerant. The self-developed kit provided by the present invention and other kits 2 were used to simultaneously detect the above-mentioned human urine. The results showed that the self-developed kit described in the present invention can detect that the urine galactose concentration of the volunteers is greater than the threshold value of 0.18 mg / mL, and the detection performance is excellent. In summary, the detection performance of the self-developed kit provided by the present invention is excellent, and this result belongs to the technical effect that the technicians in this field could not expect based on the content disclosed in the prior art.

[0238] Table 2 Galactose concentration in urine of volunteers detected by self-developed kit (n=30)

[0239]

[0240] Table 3 Galactose concentration in urine of volunteers detected by other kit 2 (n=30)

[0241]

Claims

1. A novel lactose intolerance assay kit, characterized in that: The kit comprises working solution 1, working solution 2, working solution 3, working solution 4 and working solution 5, and the components and corresponding contents of the reagents are as follows; Working solution 1: galactose oxidase, 1.2 mg / mL; NAD coenzyme, 2 mg / mL; H2O2 protective agent, 0.36 mg / mL; diluent is phosphate buffered saline (PBS); Working solution 2: 3-methyl-2-benzothiazolone hydrazone hydrochloride, 60 mg / mL; N,N-dimethylaniline, 20 mg / mL; Photofrin ® , 0.36 mg / mL; galactose protective agent, 0.36 mg / mL; diluent is PBS; Working solution 3: peroxidase, 2.6 mg / mL; 2-chlorophenol, 100 mg / mL; phenol, 25 mmol / L; diluent is PBS; Working solution 4: galactose, 1 mg / mL; solution is PBS; Working solution 5: phosphate buffer; The components and contents of the H2O2 protective agent are as follows: oxidized glutathione, 0.36 mg / mL; The components contained in the galactose protective agent and the content of each component are as follows: L-(-)-glucose, 0.36 mg / mL; L-(-)-galactose, 0.36 mg / mL.

2. The kit according to claim 1, characterized in that The sample detected by the kit is a urine sample.

3. The kit according to claim 1, characterized in that The kit also comprises a detachable nested chamber device with a filter membrane.

4. The kit according to claim 3, characterized in that The filter membrane in the detachable filter membrane nested chamber device is a 0.4-1 μM filter membrane.

5. The kit according to claim 4, characterized in that The kit comprises a reaction device, a urine purification device and a standard solution reagent bottle; The reaction device is provided with a reaction hole and a calibration hole, and the reaction hole and the calibration hole are loaded with reaction reagents, and the reaction reagents are the working solution 1, the working solution 2, and the working solution 3; The urine purification device is the detachable nested chamber device with filter membrane; The content of the standard solution reagent bottle is a standard solution, and the standard solution is galactose of different concentrations obtained by diluting the working solution 4 with the working solution 5.

6. The kit according to claim 4, characterized in that When loading the sample to be tested, the detachable nested chamber device with filter membrane is clamped above the reaction hole, the sample is loaded through the detachable nested chamber device with filter membrane, and the detachable nested chamber device with filter membrane is removed after loading.

7. The kit according to claim 1, characterized in that The test kit for lactose intolerance includes the following steps and operation sequence and random adjustment combinations of the operation sequence of each step: (1) Diluting working solution 4 with working solution 5 to obtain galactose of different concentrations; (2) Take 50 μL of each concentration of galactose and working solution 5 and add them to the multi-well plate; (3) Add 20 μL of working solution 1 to each well and incubate for 10 min. (4) Add 100 μL of working solution 2 and 20 μL of working solution 3 to each well and incubate for 10 min. (5) Determine the absorbance value at a wavelength of 450-600 nm and fit the standard curve formula; (6) Determine the absorbance value corresponding to the sample to be tested and calculate the galactose concentration in the sample to be tested.

8. A reaction reagent for preparing a lactose intolerance assay kit, characterized in that: The reaction reagents are the working solution 1, working solution 2, and working solution 3 described in claim 1.

9. A method for preparing the kit according to any one of claims 1 to 7, characterized in that: The method comprises the following steps: dripping the reaction reagent described in claim 8 into the reaction well and the calibration well respectively, and after drying, successfully loading the reaction reagent into the reaction well and the calibration well.

10. Use of the reaction reagent according to claim 8 in preparing a lactose intolerance assay kit.

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