Leukocyte esterase detection test paper, preparation method thereof, vaginitis detection kit and urinary system infection detection kit

Abstract

The invention provides leukocyte esterase detection test paper, a preparation method thereof, a vaginitis detection kit and a urinary system infection detection kit. The leukocyte esterase detection test paper comprises a liquid A and a liquid B. The liquid A is a water phase, and the liquid B is a solvent or a non-polar organic phase of which the polarity is less than or equal to that of ethanol; the solution A is a buffer solution; and the solution B comprises a polyvinylpyrrolidone solution which is dissolved in a solvent or a non-polar organic solvent with polarity less than or equal to that of ethanol. The invention has the advantages of wide application range, long period of validity, stable performance and high sensitivity.

Description

Technical Field

[0001] The invention relates to the technical field of in vitro diagnostic reagents, and in particular to a leukocyte esterase detection test paper and a preparation method thereof, a vaginitis detection kit and a urinary system infection detection kit. Background Art

[0002] Leukocyte esterase is a specific enzyme present in neutrophils, mainly in the cytoplasm of neutrophils, and is present in very little or no content in other cells such as monocytes and eosinophils. When an inflammatory response occurs in the human body, neutrophils will gather in large numbers at the site of inflammation and release leukocyte esterase. It is often used clinically to assist in the diagnosis of some diseases, especially those related to the urinary and reproductive systems. In urine, urine leukocyte esterase detection is mainly used to diagnose urinary tract infections. Leukocyte esterase detection in the vagina can reflect the degree of vaginal inflammation, and can also be used to evaluate the cleanliness of vaginal secretions and vaginal mucosal damage. However, in the current detection of leukocyte esterase, different samples use different test strips, different test items must use different detection methods, the substrate is sensitive to light and temperature, the sensitivity is low, and the validity period is short. Therefore, it is particularly important to develop a leukocyte esterase test strip with a wide range of applications, a long validity period, stable performance, and high sensitivity. Summary of the invention

[0003] The technical problem to be solved by the present invention is to overcome the above-mentioned defects of the prior art and provide a leukocyte esterase detection test strip and its preparation method, a vaginitis detection kit and a urinary system infection detection kit, which have the advantages of wide application range, long validity period, stable performance and high sensitivity.

[0004] The present invention is achieved through the following technical solutions: The first aspect of the present invention provides a leukocyte esterase detection test strip, comprising: liquid A and liquid B, The liquid A is an aqueous phase, and the liquid B is a solvent with a polarity ≤ ethanol or a non-polar organic phase; The A solution is a buffer solution; the B solution includes a polyvinyl pyrrolidone solution, which is dissolved in a solvent with a polarity ≤ ethanol or a non-polar organic solvent.

[0005] As an optimization, the K value of polyvinyl pyrrolidone in the B solution is not limited to a specific numerical range.

[0006] As an optimization, lithium bromide is also added to the B solution.

[0007] As an optimization, the B solution is a 0.5%-5% polyvinyl pyrrolidone solution, added with 0.1%-1% lithium bromide, dissolved in a solvent with polarity ≤ ethanol or a non-polar organic solvent, and 0.5-5g / L of substrate is added.

[0008] As an optimization, the substrate is 5-bromo-4-chloro-3-indoleacetic acid ester.

[0009] As an optimization, the A solution is a buffer solution with a pH of 6-9 and a concentration of 50 mM to 400 mM.

[0010] As an optimization, sodium sulfate and ProClin300 are also added to the A solution.

[0011] The second aspect of the present invention provides a method for preparing a leukocyte esterase test strip, comprising the following steps: S1. First, use the liquid A to spray / soak the filter paper so that the liquid A evenly covers the filter paper or the carrier, and then thoroughly dry it for later use; S2, then use the B solution to spray / soak the filter paper obtained in S1 so that the B solution evenly covers it, dry it with hot air, cut it, and seal it for storage to obtain a leukocyte esterase detection test paper.

[0012] The K value of polyvinyl pyrrolidone in the B solution is not limited to a specific numerical range.

[0013] As an optimization, the solution A described in S1 is a 200 mM, pH = 8.0 phosphate buffer containing 5% sodium sulfate and 0.1% ProClin300; The solution B in S2 is a 2.5% anhydrous ethanol solution of polyvinylpyrrolidone-K30, containing 1% lithium bromide, and 2 g / L of 5-bromo-4-chloro-3-indoleacetate; The drying temperature of S2 is not higher than 47°C.

[0014] As an optimization, the A solution described in S1 is a 50 mM, pH = 8.0 phosphate buffer containing 2% sodium sulfate and 0.03% ProClin300; The solution B in S2 is a 0.5% solution of polyvinylpyrrolidone-K18 in anhydrous ethanol, containing 0.1% lithium bromide and 1 g / L of 5-bromo-4-chloro-3-indoleacetate; The drying temperature of S2 is not higher than 47°C.

[0015] The third aspect of the present invention provides a vaginitis detection kit, comprising the leukocyte esterase detection test paper provided in the first aspect.

[0016] The fourth aspect of the present invention provides a urinary system infection detection kit, comprising the leukocyte esterase detection test strip provided by the first aspect above.

[0017] The beneficial effects of the present invention are: The present invention provides a leukocyte esterase detection test strip and a preparation method thereof, a vaginitis detection kit and a urinary system infection detection kit. A leukocyte esterase detection test strip comprises: liquid A and liquid B, wherein the liquid A is an aqueous phase, and the liquid B is a solvent with a polarity ≤ ethanol or a non-polar organic phase; the liquid A is a buffer solution; the liquid B comprises a polyvinyl pyrrolidone solution, which is dissolved in a solvent with a polarity ≤ ethanol or a non-polar organic solvent. The present invention provides an optimal pH environment for leukocyte esterase reaction through the liquid A buffer solution, the solvent with a smaller polarity of the liquid B can maintain the X-based chromogen structure in the substrate, and the polyvinyl pyrrolidone can significantly improve the thermal stability and reaction sensitivity of the X-based chromogen in the liquid B, so that the product performance is more stable, the validity period is longer, and the application range is wide. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The following is a further description of a leukocyte esterase test strip and a preparation method thereof, a vaginitis detection kit and a urinary system infection detection kit in conjunction with the accompanying drawings: Figure 1 This is the situation after Example 1 of the present invention and Comparative Examples 1, 2 and 3 were thermally accelerated at 47° C. for 5 weeks.

[0019] Wherein: A: Example 1 of the present invention after 5 weeks of heat acceleration at 47°C. Upper: Positive quality control Lower: Negative quality control B: Comparative Example 1 after 5 weeks of heat acceleration at 47°C. Top: Positive quality control. Bottom: Negative quality control. C: Comparative Example 2 after 5 weeks of heat acceleration at 47°C. Top: Positive quality control. Bottom: Negative quality control. D: Comparative Example 3 after 9 weeks of heat acceleration at 47°C. Top: Positive quality control. Bottom: Negative quality control. Figure 2 This is the color development of 0.1U leukocyte esterase after thermal acceleration at 47°C in Example 2 of the present invention.

[0020] Wherein: A: Color development of 0.1U leukocyte esterase after heat acceleration at 47°C for 42 days in Example 2 of the present invention B: Color development of 0.1U leukocyte esterase after heat acceleration at 47°C for 91 days in Example 2 of the present invention Figure 3 The color development is compared between Example 1 of the present invention and a commercial product using 0.1U and 0.5U leukocyte esterase, respectively.

[0021] Wherein: A: Color development of leukocyte esterase in Example 1 of the present invention Left: 0.1U Right: 0.5U B: Color development of commercially available product No. 1 leukocyte esterase Left: 0.1U Right: 0.5U C: Color development of commercially available product No. 2 leukocyte esterase Left: 0.1U Right: 0.5U D: Color development of commercially available product No. 3 leukocyte esterase Left: 0.1U Right: 0.5U E: Color development of commercially available product No. 4 leukocyte esterase Left: 0.1U Right: 0.5U F: Color development of commercially available product No. 5 leukocyte esterase Left: 0.1U Right: 0.5U G: Color development of commercially available product No. 6 leukocyte esterase Left: 0.1U Right: 0.5U H: Color development of commercially available product No. 7 leukocyte esterase Left: 0.1U Right: 0.5U DETAILED DESCRIPTION

[0022] The present application is further described in detail below in conjunction with the accompanying drawings and examples. It is particularly noted that the following examples are only used to illustrate the present application, but are not intended to limit the scope of the present application. Similarly, the following examples are only some embodiments of the present application rather than all embodiments, and all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present application.

[0023] In the following examples, various processes and methods not described in detail are conventional methods known in the art. All required components are commercially available products and can be purchased through commercial channels.

[0024] Example 1

[0025] Prepare solution A, which is 200 mM phosphate buffer, pH = 8.0, containing 5% sodium sulfate and 0.1% ProClin300; Prepare solution B, which is a 2.5% solution of polyvinylpyrrolidone-K30 in anhydrous ethanol, containing 1% lithium bromide, and then add 2g / L of 5-bromo-4-chloro-3-indoleacetate; The method for preparing a leukocyte esterase test strip comprises the following steps: S1. Spray / soak the prepared liquid A on the filter paper so that the liquid A evenly covers the filter paper or carrier, and then dry it thoroughly for later use; S2. Spray / soak the prepared liquid B on the filter paper obtained in S1 so that the liquid B evenly covers the filter paper or carrier. Dry the filter paper or carrier with hot air not higher than 47° C., cut and seal the filter paper or carrier to obtain a leukocyte esterase test paper.

[0026] A vaginitis detection kit comprises the above-mentioned leukocyte esterase detection test strip.

[0027] Example 2

[0028] Prepare solution A, which is 50 mM phosphate buffer, pH = 8.0, containing 2% sodium sulfate and 0.03% ProClin300; Prepare solution B, which is a 0.5% solution of polyvinylpyrrolidone-K18 in anhydrous ethanol, containing 0.1% lithium bromide, and then add 1g / L 5-bromo-4-chloro-3-indoleacetate; The method for preparing a leukocyte esterase test strip comprises the following steps: S1. Spray / soak the prepared liquid A on the filter paper so that the liquid A evenly covers the filter paper or carrier, and then dry it thoroughly for later use; S2. Spray / soak the prepared liquid B on the filter paper obtained in S1 so that the liquid B evenly covers the filter paper or carrier. Dry the filter paper or carrier with hot air not higher than 47° C., cut and seal the filter paper or carrier to obtain a leukocyte esterase test paper.

[0029] A urinary tract infection detection kit comprises the above-mentioned leukocyte esterase detection test strip.

[0030] Example 3

[0031] The leukocyte esterase test paper prepared in Example 2 was subjected to a thermal accelerated stability test at a temperature of 47°C for 91 days, with weekly sampling, and was tested with purified water (negative quality control) and leukocyte esterase (purchased from Merck brand) 0.1U / ml (positive quality control). The purified water should be colorless (-), and the leukocyte esterase should be blue (+). The experimental results are shown in Table 1 below: Table 1 Results of thermal accelerated stability test of the leukocyte esterase test paper prepared in Example 2

[0032] From Table 1 and Figure 2 It can be seen that the leukocyte esterase detection test paper provided by the present invention can still be used normally after being heat accelerated at 47° C. for 91 days, which shows that it has high thermal stability, long validity period and stable performance.

[0033] Comparative Example 1 The 2.5% anhydrous ethanol solution of polyvinyl pyrrolidone-K30 in Example 1 was removed, and the other components remained unchanged. The operation steps were also the same as in Example 1.

[0034] The results of thermal acceleration comparison with the leukocyte esterase test strip prepared in Example 1 are shown in Table 2.

[0035] Comparative Example 2 The anhydrous ethanol solution in Example 1 was replaced with an aqueous solution, and the other components remained unchanged. The operation steps were also the same as in Example 1.

[0036] The results of thermal acceleration comparison with the leukocyte esterase test strip prepared in Example 1 are shown in Table 2.

[0037] Comparative Example 3 1% of lithium bromide in Example 1 was removed, and the other components remained unchanged. The operation steps were also the same as in Example 1.

[0038] The results of thermal acceleration comparison with the leukocyte esterase test strip prepared in Example 1 are shown in Table 2.

[0039] Table 2 Comparison of thermal acceleration of leukocyte esterase test strips prepared in Comparative Example 1, Comparative Example 2, Comparative Example 3 and Example 1

[0040] From Table 2 and Figure 1 It can be seen that the X-based chromogen of the substrate in Comparative Example 1 after 47°C heat acceleration for 4 weeks, Comparative Example 2 after 47°C heat acceleration for 5 weeks, and Comparative Example 3 after 47°C heat acceleration for 9 weeks was destroyed, and the negative quality control spontaneously developed color and could no longer be used normally. Comparative Example 1 after 47°C heat acceleration for 5 weeks, the positive quality control color began to fade. It can be seen that the leukocyte esterase detection test paper provided by the present invention has high thermal stability, long validity period, and stable performance. Polyvinyl pyrrolidone can significantly improve the stability of the X-based chromogen in liquid B in an organic phase with polarity ≤ ethanol or a non-polar organic phase. In addition, lithium bromide plays the role of drying and fluffy filter paper / carrier after fixation, which can extend the shelf life on the one hand, and increase the contact area between the enzyme and the substrate during the reaction on the other hand, further improving the sensitivity.

[0041] Comparative Example 4 There are 7 commercial products, namely, commercial product No. 1, commercial product No. 2, commercial product No. 3, commercial product No. 4, commercial product No. 5, commercial product No. 6, and commercial product No. 7, and 0.1 U and 0.5 U of leukocyte esterase (purchased from Merck brand) are used for comparison with Example 1. The operations are carried out according to the product instructions, and the interpretation is based on the colorimetric card attached to the product.

[0042] Table 3 Comparison of the sensitivity of Example 1 of the present invention and commercially available products using 0.1 U and 0.5 U leukocyte esterase respectively

[0043] From Table 3 and Figure 3 It can be seen that the leukocyte esterase detection test paper provided by the present invention has high sensitivity, which is 5 times more sensitive than commercially available product No. 2, commercially available product No. 4, commercially available product No. 5, commercially available product No. 6, and commercially available product No. 7.

[0044] Comparative Example 5 A total of 7 commercially available products, namely Commercially Available Product No. 1, Commercially Available Product No. 2, Commercially Available Product No. 3, Commercially Available Product No. 4, Commercially Available Product No. 5, Commercially Available Product No. 6, and Commercially Available Product No. 7, were subjected to heat accelerated stability tests at 47°C and tested with purified water and 0.1 U / ml of leukocyte esterase (purchased from Merck), respectively.

[0045] Table 4 Comparison of thermal accelerated stability test at 47°C for Example 3 of the present invention and commercially available products

[0046] As shown in Table 4, after being heat accelerated at 47°C for 4 weeks, all the commercially available products showed a decrease in sensitivity and a lighter color, and some even changed color and became unusable. By comparing with Example 3 of the present invention, it can be seen that the leukocyte esterase detection test paper provided by the present invention has high thermal stability, a long effective period, and more stable performance.

[0047] Different from the prior art, the present application provides a leukocyte esterase test strip and a preparation method thereof, a vaginitis detection kit and a urinary tract infection detection kit. A leukocyte esterase test strip comprises: A liquid and B liquid, wherein the A liquid is an aqueous phase, and the B liquid is a solvent with a polarity ≤ ethanol or a non-polar organic phase; the A liquid is a buffer solution; the B liquid comprises a polyvinyl pyrrolidone solution, which is dissolved in a solvent with a polarity ≤ ethanol or a non-polar organic solvent; and lithium bromide is also added to the B liquid. The present application provides the optimum pH environment for the leukocyte esterase reaction through the A liquid buffer solution, and the solvent with a smaller polarity of the B liquid can maintain the X-based chromogen structure in the substrate, while polyvinyl pyrrolidone can significantly improve the thermal stability and reaction sensitivity of the X-based chromogen in the B liquid, so that the sensitivity is increased by 5 times compared with the existing products, the product performance is stable, and it can still be used normally after being accelerated at 47°C for 91 days. In addition, by adding lithium bromide to the B liquid, it plays the role of drying and fluffy filter paper / carrier after fixation, which can extend the shelf life on the one hand, and increase the contact area between the enzyme and the substrate during the reaction on the other hand, thereby improving the sensitivity. Therefore, the above-mentioned implementation mode of the present application has the advantages of wide application range, long validity period, stable performance and high sensitivity.

[0048] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with the technology to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Any changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. A leukocyte esterase test strip, characterized in that: include: Liquid A and Liquid B, The liquid A is an aqueous phase, and the liquid B is a solvent with a polarity ≤ ethanol or a non-polar organic phase; The A solution is a buffer solution; the B solution includes a polyvinyl pyrrolidone solution, which is dissolved in a solvent with a polarity ≤ ethanol or a non-polar organic solvent.

2. A leukocyte esterase test strip as claimed in claim 1, characterized in that: The K value of polyvinyl pyrrolidone in the B solution is not limited to a specific numerical range.

3. A leukocyte esterase test strip as claimed in claim 2, characterized in that: Lithium bromide is also added to the B solution.

4. A leukocyte esterase test strip as claimed in claim 3, characterized in that: The B solution is a 0.5%-5% polyvinyl pyrrolidone solution, added with 0.1%-1% lithium bromide, dissolved in a solvent with polarity ≤ ethanol or a non-polar organic solvent, and 0.5-5g / L of substrate is added.

5. A leukocyte esterase test strip as claimed in claim 4, characterized in that: The substrate is 5-bromo-4-chloro-3-indoleacetic acid ester.

6. A leukocyte esterase test strip according to any one of claims 1 to 5, characterized in that: The A solution is a buffer solution with a pH of 6-9 and a concentration of 50 mM to 400 mM.

7. A leukocyte esterase test strip as claimed in claim 6, characterized in that: Sodium sulfate and ProClin300 are also added to the A solution.

8. The method for preparing a leukocyte esterase test paper according to any one of claims 1 to 7, characterized in that: The following steps are involved: S1. First, use the liquid A to spray / soak the filter paper so that the liquid A evenly covers the filter paper or the carrier, and then thoroughly dry it for later use; S2, then use the B solution to spray / soak the filter paper obtained in S1 so that the B solution evenly covers it, dry it with hot air, cut it, and seal it for storage to obtain a leukocyte esterase test paper; The K value of polyvinyl pyrrolidone in the B solution is not limited to a specific numerical range.

9. A method for preparing a leukocyte esterase test strip as claimed in claim 8, characterized in that: Solution A described in S1 is a 200 mM, pH = 8.0 phosphate buffer containing 5% sodium sulfate and 0.1% ProClin300; The solution B in S2 is a 2.5% anhydrous ethanol solution of polyvinylpyrrolidone-K30, containing 1% lithium bromide, and 2 g / L of 5-bromo-4-chloro-3-indoleacetate; The drying temperature of S2 is not higher than 47°C.

10. The method for preparing a leukocyte esterase test strip according to claim 8, characterized in that: Solution A described in S1 is a 50 mM, pH = 8.0 phosphate buffer containing 2% sodium sulfate and 0.03% ProClin300; The solution B in S2 is a 0.5% solution of polyvinylpyrrolidone-K18 in anhydrous ethanol, containing 0.1% lithium bromide and 1 g / L of 5-bromo-4-chloro-3-indoleacetate; The drying temperature of S2 is not higher than 47°C.

11. A vaginitis detection kit, characterized in that: The vaginitis detection kit comprises the leukocyte esterase detection test paper according to any one of claims 1-7.

12. A urinary system infection detection kit, characterized in that: The urinary system infection detection kit comprises the leukocyte esterase detection test paper according to any one of claims 1-7.

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