A leukocyte esterase detection test strip, its preparation method, a vaginitis detection kit and a urinary tract infection detection kit

Abstract

The present invention provides a leukocyte esterase test strip, a preparation method thereof, a vaginitis detection kit and a urinary tract infection detection kit. A leukocyte esterase test strip includes: liquid A and liquid B, wherein liquid A is an aqueous phase, and liquid B is a solvent with a polarity ≤ ethanol or a non-polar organic phase; liquid A is a buffer solution; liquid B includes a polyvinylpyrrolidone solution, which is dissolved with a solvent with a polarity ≤ ethanol or a non-polar organic solvent. The present invention has the advantages of wide application range, long shelf life, stable performance and high sensitivity.

Description

Technical Field

[0001] The present invention relates to the technical field of in vitro diagnostic reagents, and particularly relates to a leukocyte esterase test strip and its preparation method, a vaginitis detection kit, and a urinary tract 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 small amounts or not at all in other cells such as monocytes and eosinophils. When an inflammatory reaction occurs in the human body, neutrophils will accumulate in large numbers at the inflammatory site and release leukocyte esterase. Clinically, it is often used to assist in the diagnosis of some diseases, especially those related to the urinary and reproductive systems. In urine, the detection of urinary leukocyte esterase is mainly used for the diagnosis of urinary tract infections. The detection of leukocyte esterase 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 test strips are used for different samples, and different detection methods are required for different detection items. The substrate is sensitive to light and temperature, with low sensitivity and a short shelf life. Therefore, it is particularly important to develop a leukocyte esterase test strip with a wide application range, a long shelf life, 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 test strip and its preparation method, a vaginitis detection kit, and a urinary tract infection detection kit, which have the advantages of a wide application range, a long shelf life, stable performance, and high sensitivity.

[0004] The present invention is achieved by the following technical solutions:

[0005] In the first aspect of the present invention, a leukocyte esterase test strip is provided, comprising: liquid A and liquid B,

[0006] Liquid A is an aqueous phase, and liquid B is a solvent with a polarity ≤ ethanol or a non-polar organic phase;

[0007] Liquid A is a buffer solution; liquid B includes a polyvinylpyrrolidone solution and is dissolved with a solvent with a polarity ≤ ethanol or a non-polar organic solvent.

[0008] As an optimization, the K value of polyvinylpyrrolidone in liquid B is not limited to a specific numerical range.

[0009] As an optimization, lithium bromide is also added to liquid B.

[0010] As an optimization, the liquid B is a 0.5%-5% polyvinylpyrrolidone solution, added with 0.1%-1% lithium bromide, dissolved in a solvent with a polarity ≤ ethanol or a non-polar organic solvent, and 0.5-5 g / L of a substrate is added.

[0011] As an optimization, the substrate is 5-bromo-4-chloro-3-indolyl acetate.

[0012] As an optimization, the liquid A is a buffer solution with a pH of 6-9 and a concentration of 50 mM to 400 mM.

[0013] As an optimization, sodium sulfate and ProClin300 are also added to the liquid A.

[0014] The second aspect of the present invention provides a method for preparing a leukocyte esterase detection test strip, comprising the following steps:

[0015] S1. First, spray / soak the filter paper with the liquid A so that the liquid A uniformly covers the filter paper or the carrier, and then thoroughly dry it for standby;

[0016] S2. Then, spray / soak the filter paper obtained in S1 with the liquid B so that the liquid B uniformly covers it, dry it with hot air, cut it, and seal it for storage to obtain a leukocyte esterase detection test strip.

[0017] Among them, the K value of polyvinylpyrrolidone in the liquid B is not limited to a specific numerical range.

[0018] As an optimization, in S1, the liquid A is a phosphate buffer solution with a concentration of 200 mM and a pH of 8.0, containing 5% sodium sulfate and 0.1% ProClin300;

[0019] In S2, the liquid B is an absolute ethanol solution of 2.5% polyvinylpyrrolidone-K30, containing 1% lithium bromide, and 2 g / L of 5-bromo-4-chloro-3-indolyl acetate is added;

[0020] The drying temperature in S2 is not higher than 47 °C.

[0021] As an optimization, in S1, the liquid A is a phosphate buffer solution with a concentration of 50 mM and a pH of 8.0, containing 2% sodium sulfate and 0.03% ProClin300;

[0022] In S2, the liquid B is an absolute ethanol solution of 0.5% polyvinylpyrrolidone-K18, containing 0.1% lithium bromide, and 1 g / L of 5-bromo-4-chloro-3-indolyl acetate is added;

[0023] The drying temperature in S2 is not higher than 47 °C.

[0024] The third aspect of the present invention provides a vaginitis detection kit, which includes the leukocyte esterase test strip provided in the first aspect above.

[0025] The fourth aspect of the present invention provides a urinary tract infection detection kit, which includes the leukocyte esterase test strip provided in the first aspect above.

[0026] The beneficial effects of the present invention are as follows:

[0027] The present invention provides a leukocyte esterase test strip, its preparation method, a vaginitis detection kit and a urinary tract infection detection kit. A leukocyte esterase test strip includes: liquid A and liquid B. Liquid A is an aqueous phase, and liquid B is a solvent with a polarity ≤ ethanol or a non-polar organic phase; liquid A is a buffer solution; liquid B includes a polyvinylpyrrolidone solution, which is dissolved in a solvent with a polarity ≤ ethanol or a non-polar organic solvent. The present invention provides the optimal pH environment for the leukocyte esterase reaction through the buffer solution of liquid A. The solvent with a smaller polarity in liquid B can maintain the X chromogen structure in the substrate, and polyvinylpyrrolidone can significantly improve the thermal stability and reaction sensitivity of the X chromogen in liquid B. The product performance is more stable, the validity period is longer, and the application range is wide. Description of the Drawings

[0028] The following further describes a leukocyte esterase test strip, its preparation method, a vaginitis detection kit and a urinary tract infection detection kit in conjunction with the drawings:

[0029] Figure 1 This shows the situation after 5 weeks of thermal acceleration at 47°C for Example 1 of the present invention, Comparative Example 1, Comparative Example 2, and Comparative Example 3.

[0030] Among them: A: The situation after 5 weeks of thermal acceleration at 47°C for Example 1 of the present invention. Above: positive control. Below: negative control

[0031] B: The situation after 5 weeks of thermal acceleration at 47°C for Comparative Example 1. Above: positive control. Below: negative control

[0032] C: The situation after 5 weeks of thermal acceleration at 47°C for Comparative Example 2. Above: positive control. Below: negative control

[0033] D: The situation after 9 weeks of thermal acceleration at 47°C for Comparative Example 3. Above: positive control. Below: negative control

[0034] Figure 2 This shows the color development of 0.1U leukocyte esterase after thermal acceleration at 47°C for Example 2 of the present invention.

[0035] Among them: A: The color development of 0.1U leukocyte esterase after 42 days of thermal acceleration at 47°C for Example 2 of the present invention

[0036] B: Color development of 0.1U leukocyte esterase after 91 days of thermal acceleration at 47°C in Example 2 of the present invention

[0037] Figure 3 This is the comparative color development of 0.1U and 0.5U leukocyte esterase used in Example 1 of the present invention and commercially available products respectively.

[0038] Among them: A: Color development of leukocyte esterase in Example 1 of the present invention Left: 0.1U Right: 0.5U

[0039] B: Color development of leukocyte esterase of commercially available product No. 1 Left: 0.1U Right: 0.5U

[0040] C: Color development of leukocyte esterase of commercially available product No. 2 Left: 0.1U Right: 0.5U

[0041] D: Color development of leukocyte esterase of commercially available product No. 3 Left: 0.1U Right: 0.5U

[0042] E: Color development of leukocyte esterase of commercially available product No. 4 Left: 0.1U Right: 0.5U

[0043] F: Color development of leukocyte esterase of commercially available product No. 5 Left: 0.1U Right: 0.5U

[0044] G: Color development of leukocyte esterase of commercially available product No. 6 Left: 0.1U Right: 0.5U

[0045] H: Color development of leukocyte esterase of commercially available product No. 7 Left: 0.1U Right: 0.5U Detailed implementation manners

[0046] The following further describes the present application in detail in conjunction with the drawings and examples. It should be specifically pointed out that the following examples are only used to illustrate the present application, but do not limit the scope of the present application. Similarly, the following examples are only partial examples of the present application rather than all examples. All other examples obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present application.

[0047] In the following examples, various processes and methods not described in detail are conventional methods well known in the art. All required components are commercially available products and can be obtained through commercial channels.

[0048] Example 1

[0049] Prepare Solution A. Solution A is a phosphate buffer solution with 200 mM and pH = 8.0, containing 5% sodium sulfate and 0.1% ProClin300;

[0050] Prepare Solution B. Solution B is an absolute ethanol solution of 2.5% polyvinylpyrrolidone-K30, containing 1% lithium bromide, and then add 5-bromo-4-chloro-3-indolyl acetate at 2 g / L;

[0051] A method for preparing a leukocyte esterase test strip, comprising the following steps:

[0052] S1. Spray / soak the prepared Solution A on the filter paper so that Solution A uniformly covers the filter paper or the carrier, and then thoroughly dry it for standby;

[0053] S2. Then spray / soak the filter paper obtained in S1 with the prepared Solution B so that Solution B uniformly covers the filter paper or the carrier, dry the above filter paper or carrier with hot air not higher than 47°C, and then cut and seal it for storage to obtain a leukocyte esterase test strip.

[0054] A vaginitis detection kit, comprising the above-mentioned leukocyte esterase test strip.

[0055] Example 2

[0056] Prepare Solution A. Solution A is a phosphate buffer solution of 50 mM and pH = 8.0, containing 2% sodium sulfate and 0.03% ProClin300;

[0057] Prepare Solution B. Solution B is an absolute ethanol solution of 0.5% polyvinylpyrrolidone-K18, containing 0.1% lithium bromide, and then add 5-bromo-4-chloro-3-indolyl acetate at 1 g / L;

[0058] A method for preparing a leukocyte esterase test strip, comprising the following steps:

[0059] S1. Spray / soak the prepared Solution A on the filter paper so that Solution A uniformly covers the filter paper or the carrier, and then thoroughly dry it for standby;

[0060] S2. Then spray / soak the filter paper obtained in S1 with the prepared Solution B so that Solution B uniformly covers the filter paper or the carrier, dry the above filter paper or carrier with hot air not higher than 47°C, and then cut and seal it for storage to obtain a leukocyte esterase test strip.

[0061] A urinary tract infection detection kit, comprising the above-mentioned leukocyte esterase test strip.

[0062] Example 3

[0063] Perform a thermal accelerated stability experiment on the leukocyte esterase test strip prepared in Example 2. The experimental temperature is 47°C and the duration is 91 days. Sampling is carried out weekly. Test with purified water (negative quality control) and leukocyte esterase (purchased from Merck brand) at 0.1 U / ml (positive quality control) respectively. Adding purified water should be colorless (-), and adding leukocyte esterase should be blue (+). The experimental results are shown in Table 1 below:

[0064] Table 1 Results table of the thermal acceleration stability test of the leukocyte esterase test strip prepared in Example 2

[0065]

[0066] From Table 1 and Figure 2 it can be seen that the leukocyte esterase test strip provided by the present invention can still be used normally after 91 days of thermal acceleration at 47°C, indicating that it has high thermal stability, a long shelf life, and stable performance.

[0067] Comparative Example 1

[0068] Remove the absolute ethanol solution of 2.5% polyvinylpyrrolidone-K30 in Example 1, and keep other components unchanged. The operation steps are the same as those in Example 1.

[0069] Compare with the leukocyte esterase test strip prepared in Example 1 for thermal acceleration, and the results are shown in Table 2.

[0070] Comparative Example 2

[0071] Replace the absolute ethanol solution in Example 1 with an aqueous solution, and keep other components unchanged. The operation steps are the same as those in Example 1.

[0072] Compare with the leukocyte esterase test strip prepared in Example 1 for thermal acceleration, and the results are shown in Table 2.

[0073] Comparative Example 3

[0074] Remove 1% lithium bromide in Example 1, and keep other components unchanged. The operation steps are the same as those in Example 1.

[0075] Compare with the leukocyte esterase test strip prepared in Example 1 for thermal acceleration, and the results are shown in Table 2.

[0076] Table 2 Comparative table of thermal acceleration of the leukocyte esterase test strips prepared in Comparative Example 1, Comparative Example 2, Comparative Example 3 and Example 1

[0077]

[0078] From Table 2 and Figure 1It can be seen that in Comparative Example 1, after heat acceleration at 47°C for 4 weeks, in Comparative Example 2, after heat acceleration at 47°C for 5 weeks, and in Comparative Example 3, after heat acceleration at 47°C for 9 weeks, the X chromogen of the substrate was destroyed and the negative control showed spontaneous color development, making it unable to be used normally. In Comparative Example 1, after heat acceleration at 47°C for 5 weeks, the color development of the positive control began to fade. Thus, it can be seen that the leukocyte esterase detection test strip provided by the present invention has high thermal stability, a long shelf life, and stable performance. Polyvinylpyrrolidone can significantly improve the stability of the X chromogen in Solution B in an organic phase with polarity ≤ ethanol or a nonpolar organic phase. In addition, lithium bromide, after being fixed, plays the role of drying and fluffing the filter paper / carrier. On the one hand, it can extend the storage period, and on the other hand, it increases the contact area between the enzyme and the substrate during the reaction process, further improving the sensitivity.

[0079] Comparative Example 4

[0080] There are 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. They were compared with Example 1 using 0.1 U and 0.5 U of leukocyte esterase (purchased from the Merck brand). The operations were carried out according to the product instructions respectively, and the judgment was based on the color comparison card attached to the product.

[0081] Table 3 Sensitivity comparison table of Example 1 of the present invention and commercially available products using 0.1 U and 0.5 U of leukocyte esterase

[0082]

[0083] From Table 3 and Figure 3 It can be seen that the leukocyte esterase detection test strip provided by the present invention has high sensitivity and 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.

[0084] Comparative Example 5

[0085] There are 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. They were subjected to a thermal acceleration stability experiment at 47°C and tested with purified water and 0.1 U / ml of leukocyte esterase (purchased from the Merck brand) respectively.

[0086] Table 4 Comparative table of the thermal acceleration stability experiment of Example 3 of the present invention and commercially available products at 47°C

[0087]

[0088] As can be seen from Table 4, after the commercially available products were thermally accelerated at 47°C for 4 weeks, all of them showed a decrease in sensitivity and a lighter color development. Some products even changed color directly and became unusable. By comparing Example 3 of the present invention, it can be seen that the leukocyte esterase detection test strip provided by the present invention has higher thermal stability, a longer shelf life, and more stable performance.

[0089] Different from the prior art, the present application provides a leukocyte esterase detection test strip, its preparation method, a vaginitis detection kit, and a urinary tract infection detection kit. A leukocyte esterase detection test strip includes: Solution A and Solution B. Solution A is an aqueous phase, and Solution B is a solvent with a polarity ≤ ethanol or a non-polar organic phase; Solution A is a buffer solution; Solution B includes a polyvinylpyrrolidone solution, which is dissolved in a solvent with a polarity ≤ ethanol or a non-polar organic solvent; Lithium bromide is also added to Solution B. The present application provides the optimal pH environment for the leukocyte esterase reaction through the buffer solution of Solution A. The solvent with a relatively small polarity in Solution B can maintain the X chromogen structure in the substrate, while polyvinylpyrrolidone can significantly improve the thermal stability and reaction sensitivity of the X chromogen in Solution B, resulting in a 5-fold increase in sensitivity compared to existing products. The product has stable performance and can still be used normally after being accelerated at 47°C for 91 days. In addition, by adding lithium bromide to Solution B, it plays a role in drying and fluffing the filter paper / carrier after fixation. On the one hand, it can extend the storage period, and on the other hand, it increases the contact area between the enzyme and the substrate during the reaction process, improving the sensitivity. Therefore, the above embodiments of the present application have the advantages of a wide application range, a long shelf life, stable performance, and high sensitivity.

[0090] The above embodiments are only used to illustrate the technical concept and characteristics of the present invention, and their purpose is to enable those familiar with this technology to understand the content of the present invention and implement it accordingly. It should not be used to limit the protection scope of the present invention. Any transformation or modification made in accordance with the spirit and essence of the present invention should be covered within the protection scope of the present invention.

Claims

1. A leukocyte esterase detection test strip, characterized in that, It includes Liquid A and Liquid B. Liquid A is an aqueous phase, and Liquid B is a solvent with a polarity ≤ ethanol or a non-polar organic phase. Liquid A is a buffer solution; Liquid B includes a polyvinylpyrrolidone solution, which is dissolved in a solvent with a polarity ≤ ethanol or a non-polar organic solvent, and 5-bromo-4-chloro-3-indolyl acetate is added. The preparation method of the leukocyte esterase test strip is as follows: S1. First, use Liquid A to spray / soak the filter paper so that Liquid A evenly covers the filter paper, and then dry it thoroughly for standby. S2. Then use Liquid B to spray / soak the filter paper obtained in S1 so that Liquid B evenly covers the filter paper. After drying it with hot air, cut and seal it for storage to obtain the leukocyte esterase test strip. Among them, the K value of polyvinylpyrrolidone in Liquid B is not limited to a specific numerical range.

2. The leukocyte esterase detection test strip according to claim 1, wherein: Lithium bromide is also added to Liquid B.

3. The leukocyte esterase test strip according to claim 2, wherein: Liquid B is a 0.5%-5% polyvinylpyrrolidone solution, added with 0.1%-1% lithium bromide, dissolved in a solvent with a polarity ≤ ethanol or a non-polar organic solvent, and 0.5-5 g / L of 5-bromo-4-chloro-3-indolyl acetate is added.

4. The leukocyte esterase test strip according to claim 3, characterized in that: Liquid A is a buffer solution with a pH of 6-9 and a concentration of 50 mM to 400 mM.

5. The leukocyte esterase test strip according to claim 4, wherein: Sodium sulfate and ProClin300 are also added to Liquid A.

6. The leukocyte esterase test strip according to claim 5, wherein, In S1 of the preparation method of the leukocyte esterase test strip, Liquid A is a phosphate buffer solution with a concentration of 200 mM and a pH of 8.0, containing 5% sodium sulfate and 0.1% ProClin300; in S2, Liquid B is an absolute ethanol solution of 2.5% polyvinylpyrrolidone-K30, containing 1% lithium bromide, and 2 g / L of 5-bromo-4-chloro-3-indolyl acetate is added; the drying temperature in S2 is not higher than 47°C.

7. The leukocyte esterase test strip according to claim 5, characterized in that, In S1 of the preparation method of the leukocyte esterase test strip, Liquid A is a phosphate buffer solution with a concentration of 50 mM and a pH of 8.0, containing 2% sodium sulfate and 0.03% ProClin300; in S2, Liquid B is an absolute ethanol solution of 0.5% polyvinylpyrrolidone-K18, containing 0.1% lithium bromide, and 1 g / L of 5-bromo-4-chloro-3-indolyl acetate is added; the drying temperature in S2 is not higher than 47°C.

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

9. A urinary tract infection detection kit, characterized in that: The urinary tract infection detection kit includes the leukocyte esterase test strip according to any one of claims 1-7.

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