A diluent for a fecal occult blood test kit and a preparation method and application thereof

Abstract

The application relates to the field of diluents, in particular to a diluent for a fecal occult blood detection kit and a preparation method and application thereof. The diluent for the fecal occult blood detection kit comprises the following components: 0.05-0.3 g / L of a chelating agent, 0.05-5 g / L of serum albumin, 0-0.5 mL / L of a preservative, 0.01-0.03 g / L of sodium cholate, 0.02-0.05 g / L of 3-sulfopropyl tetradecyl dimethyl betaine and 0.005-0.02 g / L of sodium lauryl polyoxyethylene ether sulfate; the balance is a buffer solution, and the pH is maintained at 7.0-7.8. The application can improve the fecal occult blood detection accuracy and expand the application range of the diluent.

Description

Technical Field

[0001] This application relates to the field of diluents, and more specifically, to a diluent for a fecal occult blood test kit, its preparation method, and its application. Background Technology

[0002] In normal individuals, approximately 0.5-1.5 mL of blood is lost daily during the renewal of the gastrointestinal mucosa, which is considered normal. However, when the daily bleeding is as small as about 5 mL, and the blood is not visible to the naked eye in the stool but can only be detected through chemical testing, it is called fecal occult bleeding (FOB). Usually, this small, inconspicuous amount of gastrointestinal bleeding is accompanied by normal stool color and no symptoms, but it is indeed an important indication of organic lesions in the digestive tract.

[0003] Many rapid testing kits are available on the market, with colloidal gold assay kits being among the most commonly used. Immunogold assays use a double-antibody sandwich method to detect hemoglobin in human feces. During testing, when the sample contains human hemoglobin, it reacts with a labeled Hb2 monoclonal antibody to form a complex. Under chromatography, the reaction complex moves along a nitrocellulose membrane to the detection zone, where it is captured by Hb1 monoclonal monomers on the nitrocellulose membrane. A red reaction line may or may not appear in the detection zone, yielding the test result.

[0004] After sampling, the sample needs to be diluted with a diluent to release the hemoglobin from the red blood cells. In actual research and development, it was found that some positive results could not be accurately detected due to incomplete hemoglobin release or low initial hemoglobin levels, thus affecting the accuracy of the test. Therefore, further improvement is needed. Summary of the Invention

[0005] To improve the accuracy of fecal occult blood detection, this application provides a diluent for a fecal occult blood detection kit, its preparation method, and its application.

[0006] In a first aspect, this application provides a diluent for a fecal occult blood test kit, employing the following technical solution: A diluent for a fecal occult blood test kit comprises the following components: 0.05-0.3 g / L chelating agent, 0.05-5 g / L serum albumin, 0-0.5 mL / L preservative, 0.01-0.03 g / L sodium cholate, 0.02-0.05 g / L 3-sulfopropyltetradecyl dimethyl betaine, and 0.005-0.02 g / L sodium lauryl polyoxyethylene ether sulfate; the buffer is replenished to the remaining amount, and the pH is maintained at 7.0-7.8.

[0007] When using the colloidal gold method to test samples, surfactants are needed to break down the red blood cells in the sample, releasing hemoglobin. However, when the hemoglobin content is too low, a positive result may not be detected, and there are limitations in sensitivity and specificity. In such cases, microscopic examination (using a stool analyzer) combined with the number and morphology of red blood cells can improve the accuracy of the analysis.

[0008] However, if machine testing is required, the red blood cells cannot be damaged too severely; their shape needs to be preserved to a certain extent in order to achieve accurate results.

[0009] Therefore, the idea of ​​developing a diluent that can be used for both reagent kit detection and instrumental testing was conceived, thus improving the compatibility of the diluent.

[0010] By adopting the above technical solution, with the combined use of sodium cholate, 3-sulfopropyltetradecyl dimethyl betaine, and sodium lauryl polyoxyethylene ether sulfate, the treatment is gentler and does not completely destroy red blood cells, resulting in a lower degree of damage, but it can still release hemoglobin so that it can be detected by the kit.

[0011] Furthermore, by further limiting the amount of sodium cholate, 3-sulfopropyltetradecyl dimethyl betaine, and sodium lauryl polyoxyethylene ether sulfate used, the kit can more sensitively capture small amounts of hemoglobin, which can effectively improve the detection accuracy of the colloidal gold method kit.

[0012] Preferably, the diluent comprises the following components: 0.1-0.2 g / L chelating agent, 0.05-3 g / L serum albumin, 0-0.5 mL / L preservative, 0.01-0.02 g / L sodium cholate, 0.025-0.035 g / L 3-sulfopropyltetradecyl dimethyl betaine, and 0.005-0.01 g / L sodium lauryl polyoxyethylene ether sulfate.

[0013] Preferably, the mass ratio of sodium cholate, 3-sulfopropyltetradecyl dimethyl betaine, and sodium lauryl polyoxyethylene ether sulfate is 1:(2-3):(0.5-1.0), based on the mass of sodium cholate.

[0014] By adopting the above technical solution, the content of each component in the diluent is further limited, especially the amount of sodium cholate, 3-sulfopropyltetradecyl dimethyl betaine, and sodium lauryl polyoxyethylene ether sulfate. This provides a more stable and suitable treatment effect on red blood cells with different contents in the sample, which is beneficial to maintaining the general shape of red blood cells and releasing the internal hemoglobin. Thus, it can be used in both kit detection and instrument detection.

[0015] Preferably, the buffer solution is at least one of phosphate buffer, borate buffer, and citrate buffer.

[0016] Preferably, the chelating agent is at least one or a mixture of EDTA-dipowdrine or EDTA-disodium.

[0017] Preferably, the serum albumin is at least one or a mixture of bovine serum albumin, horse serum albumin, and human serum albumin.

[0018] Preferably, the serum albumin is bovine serum albumin.

[0019] By adopting the above technical solution and adding an appropriate amount of bovine serum albumin to the system, antibodies can bind to non-specific proteins, thereby effectively improving the accuracy of detection.

[0020] Secondly, this application provides a method for preparing a diluent for a fecal occult blood test kit, employing the following technical solution: A method for preparing a diluent for a fecal occult blood test kit includes the following steps: mixing and stirring various components until homogeneous to obtain the finished product.

[0021] The diluent provided in this application is very simple to prepare. It only requires mixing all the components evenly and does not require too many harsh conditions. It is suitable for widespread use in the industry.

[0022] Thirdly, this application provides a diluent for a fecal occult blood test kit for use in fecal analysis instruments.

[0023] As described above, the diluent provided in this application can be used not only in reagent kit detection but also in instrumental analysis, which significantly improves the versatility and application range of the diluent and effectively simplifies the detection steps.

[0024] In summary, this application has the following beneficial effects: 1. The diluent provided in this application can be used as a diluent for reagent kits using the colloidal gold method, and can also be used as a diluent in subsequent fecal analysis instruments. This diluent can better maintain the morphology of red blood cells for on-machine observation, and can also release hemoglobin for rapid detection by the reagent kit, making the diluent more widely applicable.

[0025] 2. The diluent of this application, by further selecting specific sodium cholate, 3-sulfopropyltetradecyl dimethyl betaine, and sodium lauryl polyoxyethylene ether sulfate, and limiting the content of the three in the system, together enables the kit to more sensitively capture small amounts of hemoglobin, which can effectively improve the detection accuracy of the colloidal gold method kit. Attached Figure Description

[0026] Figure 1This is a graph showing the test results of the diluent of Example 1 of this application in Experiment 1.

[0027] Figure 2 This is a graph showing the test results of the diluent of Example 4 of this application in Experiment 1.

[0028] Figure 3 This is a graph showing the test results of the diluted solution of Comparative Example 4 in Experiment 1.

[0029] Figure 4 This is a graph showing the test results of the diluted solution of Comparative Example 5 in Experiment 1.

[0030] Figure 5 This is an observation graph of the diluted solution of Example 1 of this application after 60 minutes in Experiment 2.

[0031] Figure 6 This is an observation graph of the diluted solution of Example 4 of this application after 60 minutes in Experiment 2.

[0032] Figure 7 This is an observation graph of the diluted solution of Comparative Example 1 in this application after 60 minutes in Experiment 2.

[0033] Figure 8 This is an observation graph of the diluted solution of Comparative Example 5 of this application after 60 minutes in Experiment 2.

[0034] Figure 9 This is an observation graph of the diluted solution of Comparative Example 6 of this application after 60 minutes in Experiment 2. Detailed Implementation

[0035] The present application will be further described in detail below with reference to the accompanying drawings and embodiments.

[0036] The raw materials used in the following examples and comparative examples are all commercially available products.

[0037] A diluent for a fecal occult blood test kit comprises the following components: (1) Chelating agents: The chelating agent can be one or more of EDTA-dipotassium, EDTA-disodium, etc.

[0038] The amount of chelating agent used in the diluent can be any value among 0.05 g / L, 0.08 g / L, 0.10 g / L, 0.12 g / L, 0.15 g / L, 0.18 g / L, 0.20 g / L, 0.22 g / L, 0.25 g / L, 0.28 g / L, and 0.3 g / L.

[0039] (2) Serum albumin: Serum albumin can be one of bovine serum albumin, horse serum albumin, or human serum albumin.

[0040] The amount of serum albumin used in the diluent can be any value from 0.05 g / L, 0.08 g / L, 0.12 g / L, 0.35 g / L, 0.68 g / L, 0.92 g / L, 1.25 g / L, 2.35 g / L, 3.00 g / L, 4.32 g / L, 5.0 g / L, etc.

[0041] (3) Preservatives: The preservative is mainly used for corrosion prevention and can be Proclin 300.

[0042] The amount of preservative used in the diluent can be any value among 0, 0.01 mL / L, 0.05 mL / L, 0.1 mL / L, 0.2 mL / L, 0.3 mL / L, 0.4 mL / L, 0.5 mL / L, etc.

[0043] (4) Sodium cholate: The amount of sodium cholate used in the diluent can be any value among 0.01 g / L, 0.015 g / L, 0.02 g / L, 0.025 g / L, 0.03 g / L, etc.

[0044] (5) 3-Sulfopropyltetradecyl dimethyl betaine: The amount of 3-sulfopropyltetradecyl dimethyl betaine used can be any value among 0.02 g / L, 0.025 g / L, 0.03 g / L, 0.035 g / L, 0.04 g / L, 0.045 g / L, 0.05 g / L, etc.

[0045] (6) Sodium lauryl polyoxyethylene ether sulfate: The amount of sodium lauryl polyoxyethylene ether sulfate used in the diluent can be any value among 0.005 g / L, 0.008 g / L, 0.01 g / L, 0.015 g / L, 0.02 g / L, etc.

[0046] (7) Buffer solution: The buffer solution can be one or more of phosphate buffer, borate buffer, and citrate buffer.

[0047] (8) The mass ratio of sodium cholate, 3-sulfopropyltetradecyl dimethyl betaine, and sodium lauryl polyoxyethylene ether sulfate can be any of the following: 1:2:0.5, 1:2.5:0.8, 1:3:1, or 1:2:1. Example

[0048] Example 1 A fecal occult blood test kit diluent, per 1L, comprises the following components: 0.15g chelating agent, 1g serum albumin, 0.1mL preservative, 0.015g sodium cholate, 0.032g 3-sulfopropyltetradecyl dimethyl betaine, 0.009g sodium lauryl polyoxyethylene ether sulfate, with the buffer added to a final volume of 1L.

[0049] The pH of the diluent is 7.4.

[0050] The chelating agent was EDTA-disodium, the serum albumin was bovine serum albumin, the preservative was Proclin 300, and the buffer was phosphate buffer (0.1M, 0.85% NaCl).

[0051] The specific dosage of each component is detailed in Table 1.

[0052] This application also provides a method for preparing a diluent for a fecal occult blood test kit, comprising the following steps: adding a chelating agent, serum albumin, preservative, sodium cholate, 3-sulfopropyltetradecyl dimethyl betaine, and sodium lauryl polyoxyethylene ether sulfate into a buffer solution and stirring until homogeneous to obtain a diluent.

[0053] Example 2 A diluent for a fecal occult blood test kit differs from that in Example 1 in that the mass ratio of sodium cholate, 3-sulfopropyltetradecyl dimethyl betaine, and sodium lauryl polyoxyethylene ether sulfate is 1:2:0.5. Specifically, sodium cholate is 0.015 g, 3-sulfopropyltetradecyl dimethyl betaine is 0.030 g, and sodium lauryl polyoxyethylene ether sulfate is 0.0075 g.

[0054] The specific dosage of each component is detailed in Table 1.

[0055] Example 3 A diluent for a fecal occult blood test kit differs from that in Example 1 in that the mass ratio of sodium cholate, 3-sulfopropyltetradecyl dimethyl betaine, and sodium lauryl polyoxyethylene ether sulfate is 1:3:1. Specifically, sodium cholate is 0.015 g, 3-sulfopropyltetradecyl dimethyl betaine is 0.045 g, and sodium lauryl polyoxyethylene ether sulfate is 0.015 g.

[0056] The specific dosage of each component is detailed in Table 1.

[0057] Examples 4-5 A diluent for a fecal occult blood test kit differs from that in Example 1 in that the amounts of each component are different, as detailed in Table 1.

[0058] Table 1 Comparative Example Comparative Example 1 A diluent for a fecal occult blood test kit differs from that in Example 1 in that sodium cholate is replaced with Tween 80, i.e., sodium cholate is 0g and Tween 80 is 0.015g.

[0059] Comparative Example 2 A diluent for a fecal occult blood test kit differs from that in Example 1 in that 3-sulfopropyltetradecyl dimethyl betaine is replaced with cocamidopropyl betaine, i.e., 0g of 3-sulfopropyltetradecyl dimethyl betaine and 0.032g of cocamidopropyl betaine.

[0060] Comparative Example 3 A diluent for a fecal occult blood test kit differs from that in Example 1 in that sodium lauryl polyoxyethylene ether sulfate is replaced with sodium dodecyl sulfate, i.e., sodium lauryl polyoxyethylene ether sulfate is 0g and sodium dodecyl sulfate is 0.009g.

[0061] Comparative Example 4 A fecal occult blood test kit diluent differs from Example 1 in that it omits sodium cholate, 3-sulfopropyltetradecyl dimethyl betaine, and sodium lauryl polyoxyethylene ether sulfate, and uses a buffer solution to replenish the amount.

[0062] Comparative Example 5 A diluent for a fecal occult blood test kit, differing from that in Example 1, contains 0.005 g of sodium cholate, 0.01 g of 3-sulfopropyltetradecyl dimethyl betaine, and 0.001 g of sodium lauryl polyoxyethylene ether sulfate.

[0063] Comparative Example 6 A diluent for a fecal occult blood test kit, differing from that in Example 1, contains 0.15 g of sodium cholate, 0.32 g of 3-sulfopropyltetradecyl dimethyl betaine, and 0.09 g of sodium lauryl polyoxyethylene ether sulfate.

[0064] Application Example 1 Application of a fecal occult blood test kit diluent in fecal analysis instruments.

[0065] Use a sampling spoon to take a level spoonful (about 0.3-0.5g) of sample from 6 different locations in the fecal sample. Insert the sampling spoon with the fecal sample into the specimen collection cup. Place the specimen collection cup in the instrument's sample rack and then place it on the sample inlet tray of the fecal analyzer. Set the fecal analyzer parameters and start the detection operation.

[0066] The fecal analyzer automatically adds 5-10 ml of diluent and mixes the sample.

[0067] In this application example, the diluent used is the diluent from Example 1.

[0068] Performance testing 1. Rapid detection kit: Prepare human hemoglobin at 1 mg / ml, and then dilute it with the diluents of Examples 1-5 and Comparative Examples 1-6 to obtain a test sample of 6 μg / ml.

[0069] Take 3 drops (approximately 120 μL) of the sample to be tested and place them into the sample well of the FOB test kit (colloidal gold method).

[0070] Wait 3-5 minutes, interpret the results, check if a red reaction line appears in the detection area, and record the results in Table 2.

[0071] 2. Instrumental Testing: Take 10 mL of the diluted solutions from Examples 1-5 and Comparative Examples 1-6 and place them in a conical-bottom test tube. Add 10 μL of fresh blood using a pipette and mix well. Immediately observe the sample using a fecal analyzer (built-in microscope) and record the image. After 30 minutes, repeat the test using the same method and record the image. After 60 minutes, repeat the test using the same method and record the image. Compare the results at 30 minutes and 60 minutes with the image taken immediately after the test. Determine the differences in erythrocyte morphology between the initial and final images and record the results in Table 2.

[0072] If the cell morphology is not significantly different, and there is no swelling or shrinkage, it indicates a good effect; if the cell morphology is significantly different, it indicates a poor effect.

[0073] 3. Reagent kit accuracy testing: Human hemoglobin was prepared at a concentration of 1 mg / ml and then diluted with the diluents of Examples 1-5 and Comparative Examples 1-6 to obtain test samples of 200 ng / ml, 400 ng / ml, 600 ng / ml, 800 ng / ml, 1000 ng / ml, 1200 ng / ml, 1500 ng / ml, 2 μg / ml, 4 μg / ml, 6 μg / ml, and 8 μg / ml.

[0074] Then, take 3 drops (approximately 120 μL) of the test sample and place them into the sample well of the FOB test kit (colloidal gold method). Wait 3-5 minutes, interpret the results, and check if a red reaction line appears in the detection area. The lowest concentration of the test sample that produces a positive result is the sensitivity of the kit. Record the results in Table 2.

[0075] The lower the concentration of human hemoglobin corresponding to the sensitivity of the kit, the better the effect of the diluent.

[0076] Table 2 According to the detection results of Examples 1-5 in Table 2, the diluents of Examples 1-5 can produce positive results when used in the rapid detection kit; they also maintain the morphology of red blood cells well during instrumental detection, allowing for observation. Comparative Example 4 is based on Example 1, but omits sodium cholate, 3-sulfopropyltetradecyl dimethyl betaine, and sodium lauryl polyoxyethylene ether sulfate. The detection results show that although the diluent of Comparative Example 4 can maintain the state of red blood cells well during instrumental detection, it is not compatible with the rapid detection kit. This indicates that the diluent provided in this application, by selecting a specific combination of sodium cholate, 3-sulfopropyltetradecyl dimethyl betaine, and sodium lauryl polyoxyethylene ether sulfate, and further limiting the amount of each in the system, can be used simultaneously for both kit detection (colloidal gold method) and instrumental microscopy.

[0077] Comparative Examples 1-3 are diluents prepared by arbitrarily replacing one of sodium cholate, 3-sulfopropyltetradecyl dimethyl betaine, or sodium lauryl polyoxyethylene ether sulfate, based on Example 1. Although the diluents of Comparative Examples 1-3 can all be used in rapid detection kits, they severely damage the morphology of red blood cells during instrumental testing, making microscopic examination and disease diagnosis impossible.

[0078] Comparative Examples 5 and 6 are based on Example 1, but with changes to the specific amounts of sodium cholate, 3-sulfopropyltetradecyl dimethyl betaine, and sodium lauryl polyoxyethylene ether sulfate in the system. The resulting diluents also disrupted the morphology of red blood cells.

[0079] The inventors speculate that the issue may be related to the permeability of the red blood cell membrane. It's possible that the specially prepared diluent in this application increases the permeability of the red blood cell membrane, allowing hemoglobin to permeate out without causing excessive swelling and rupture of the red blood cells. The red blood cells retain their good shape, making them suitable for instrumental observation and enabling rapid detection with the kit. Disrupting the formulation of this application would disrupt this special balance, making it impossible to release hemoglobin for detection while maintaining the red blood cell morphology.

[0080] Regarding the accuracy of the reagent kit, the diluents of Examples 1-5 showed better accuracy than the diluents of Comparative Examples 1-3 and 5-6, and were able to detect lower concentrations of hemoglobin.

[0081] Specifically, a comparison of the detection data from Examples 1 and 2 with Examples 3-5 in Table 2 shows that the diluted solution (Examples 1-2) prepared by limiting the dosage and ratio of sodium cholate, 3-sulfopropyltetradecyl dimethyl betaine, and sodium lauryl polyoxyethylene ether sulfate has higher sensitivity. Although the diluted solution of Example 3 limits the specific ratio of the three components, it is not within the optimal dosage range, so its sensitivity is not as good as that of Examples 1 and 2; however, Example 3 still has good sensitivity, which is undeniable.

[0082] according to Figure 1-4 It can be seen that Examples 1, 4, and 5 all yielded positive results, with Example 1 showing the best effect and the most obvious red line, followed by Example 4, while the red line in Comparative Example 5 was the faintest. Comparative Example 4, however, failed to yield a positive result.

[0083] according to Figure 5-9 It can be seen that the red blood cells using the diluents of Examples 1 and 4 have good and normal morphology and can be observed under a microscope. The number and morphology of the red blood cells can be used for comprehensive judgment. The red blood cells using the diluents of Comparative Examples 1 and 5 are all ruptured and cannot be observed under a microscope. The red blood cells using the diluent of Comparative Example 6 are all ruptured and the degree of rupture is large, making it impossible to make a judgment.

[0084] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A diluent for a fecal occult blood test kit, characterized in that, Includes the following components: 0.05-0.3 g / L chelating agent, 0.05-5 g / L serum albumin, 0-0.5 mL / L preservative, 0.01-0.03 g / L sodium cholate, 0.02-0.05 g / L 3-sulfopropyltetradecyl dimethyl betaine, 0.005-0.02 g / L sodium lauryl polyoxyethylene ether sulfate; Replenish the buffer solution to the remaining amount and maintain the pH at 7.0-7.

8.

2. The diluent for the fecal occult blood test kit according to claim 1, characterized in that: The diluent comprises the following components: 0.1-0.2 g / L chelating agent, 0.05-3 g / L serum albumin, 0-0.5 mL / L preservative, 0.01-0.02 g / L sodium cholate, 0.025-0.035 g / L 3-sulfopropyltetradecyl dimethyl betaine, and 0.005-0.01 g / L sodium lauryl polyoxyethylene ether sulfate.

3. The diluent for the fecal occult blood test kit according to claim 1, characterized in that: The mass ratio of sodium cholate, 3-sulfopropyltetradecyl dimethyl betaine, and sodium lauryl polyoxyethylene ether sulfate is 1:(2-3):(0.5-1.0), with the mass of sodium cholate as the reference.

4. The diluent for the fecal occult blood test kit according to claim 1, characterized in that: The buffer solution is at least one of phosphate buffer, borate buffer, and citrate buffer.

5. The diluent for the fecal occult blood test kit according to claim 1, characterized in that: The chelating agent is at least one or a mixture of EDTA-dipowdrine or EDTA-disodium.

6. The diluent for the fecal occult blood test kit according to claim 1, characterized in that: The serum albumin is at least one or a mixture of bovine serum albumin, horse serum albumin, and human serum albumin.

7. A method for preparing a diluent for a fecal occult blood test kit according to any one of claims 1-6, characterized in that, Includes the following steps: Mix all the components until homogeneous to obtain the finished product.

8. The application of a diluent for a fecal occult blood test kit according to any one of claims 1-6 in fecal analysis instrument detection.

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