A lysozyme activity detection method and kit

Abstract

The present invention belongs to the field of biomedicine technology, and in particular, relates to a lysozyme activity detection method and a kit thereof, wherein the lysozyme activity detection method mainly comprises the following steps: first, a quantitative ATP bioluminescent liquid in the kit is mixed with a sample to be tested, and incubated at room temperature for a period of time; then, an active substrate is added to the mixed solution and the mixture is continued to be incubated at room temperature for a certain period of time; finally, the bioluminescent intensity value of the mixed solution is determined. The kit comprises a box body, wherein a culture chamber and an injection chamber 1 and an injection chamber 2 connected to the culture chamber are provided in the box body, and the ATP bioluminescent liquid and the active substrate are respectively located in the injection chamber 1 and the injection chamber 2. The kit provided by the present invention can quickly and quantitatively add the ATP bioluminescent liquid and the active substrate to the test object, making the operation more convenient and standardized, and making the test results more accurate.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and particularly relates to a lysozyme activity detection method and a kit thereof. Background Art

[0002] Lysozyme is a low molecular weight alkaline protein with antibacterial effects. It is widely present in the tissue fluids of animals and plants and in certain microorganisms. Lysozyme can hydrolyze the β-1,4-glycosidic bond between N-acetylglucosamine and N-acetylmuramic acid in the bacterial cell wall, destroying the structure of the cell wall, allowing water to enter, and ultimately leading to bacterial disintegration. In the human body, lysozyme is present in neutrophils, monocytes, and macrophages. It is also common in various secretions of the human body and is one of the body's surface defense factors. In clinical practice, lysozyme can be used as a diagnostic marker for a variety of diseases. For example, the determination of serum lysozyme activity can be used to identify the type of acute leukemia, the determination of urine lysozyme can be used to diagnose urinary tract infection or injury, and the determination of cerebrospinal fluid lysozyme content can be used to determine whether the central nervous system is infected or has a tumor. The determination of lysozyme activity is increasingly valued in clinical practice.

[0003] At present, the activity of lysozyme is mostly detected by agar plate method and turbidimetry. The agar plate method is to make a small hole on an agar plate mixed with micrococci, and quantitatively drop the sample enzyme to be tested into the small hole. The lysozyme in the sample will diffuse on the agar plate and react with the lysococci to produce an inhibition zone. The diameter of the inhibition zone is linearly related to the activity concentration of lysozyme in the sample to be tested, thereby obtaining the activity concentration of lysozyme in the unknown sample. The agar plate method is not only cumbersome to operate, but also has a long detection cycle, and has low sensitivity, and requires sample enrichment. Turbidimetry is a standard method for determining the activity of lysozyme. Its principle is that lysozyme lyses the lysococci, resulting in a decrease in the turbidity of the bacterial solution. A standard curve is made according to the change in turbidity or absorbance, and then the turbidity of the solution after the reaction of the sample to be tested with the micrococci is substituted into the standard curve to obtain the enzyme activity of the sample to be tested. Turbidimetry is easily affected by various factors such as natural sedimentation of the bacteria, growth state of the bacteria, reaction time, etc., so the test results are often not accurate and have poor repeatability. In order to solve the defects of the above methods, some researchers use dyes or enzyme molecules to mark lysodeiktic micrococci. When lysozyme lyses lysodeiktic micrococci, the dyes or enzyme molecules are released. The amount of dyes or enzyme molecules released can be measured to quantitatively analyze the activity of lysodeiktic micrococci. These methods require the labeling of lysodeiktic micrococci, which increases the cost of detection.

[0004] Adenosine triphosphate is the most direct source of energy in organisms and is widely present in all kinds of life forms. Bacteria also contain ATP, and the ATP in bacterial cells will only be released after the bacteria are lysed. Currently, the most commonly used method for ATP determination is bioluminescence. Its basic principle is that D-luciferin undergoes a series of biochemical reactions with ATP under the catalysis of luciferase, converting the chemical energy in ATP into light energy. By measuring the luminescence intensity of the solution, the ATP content in the sample can be quantified. Summary of the invention

[0005] In view of the above problems, the present invention proposes a lysozyme activity detection method and a kit thereof, which effectively solves the technical problems of low accuracy and high detection cost in the prior art.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is as follows: a method for detecting lysozyme activity, the method for detecting lysozyme activity comprises the following steps:

[0007] Step 1: Open the cover of the test kit and add the sample to be tested into the culture chamber of the test kit;

[0008] Step 2: Mix the quantitative ATP bioluminescent solution in the kit with the sample to be tested, and then incubate the mixture at room temperature for a period of time;

[0009] Step 3: Mix the quantitative active substrate in the kit with the mixed solution in step 2, and continue to incubate at room temperature for a period of time;

[0010] Step 4: Use a chemiluminescence meter to measure the luminescence intensity of the solution;

[0011] Step 5: Calculate the lysozyme activity in the sample based on the standard curve of the luminescence intensity value and the lysozyme activity;

[0012] The test kit comprises a box body, a culture chamber is arranged in the box body, an openable cover is arranged in the culture chamber, an injection chamber is also arranged in the box body, the injection chamber comprises an injection chamber 1 and an injection chamber 2, a pushing piston is arranged in the injection chamber 1 and the injection chamber 2, a sliding chamber is arranged on the upper part of the injection chamber 1 and the injection chamber 2, a piston rod is arranged on the pushing piston, an injection pipe is arranged in the piston rod, the lower end of the injection pipe is connected to the injection chamber, the upper end of the piston rod is slidably connected in the sliding chamber, the upper and lower ends of the sliding chamber are respectively provided with fitting parts that can fit with the upper end of the injection pipe, a connecting groove is arranged on the side of the sliding chamber, the sides of the connecting groove are connected with drainage channels, the other ends of the drainage channels are connected to the culture chamber, and the ATP bioluminescent liquid and the active substrate are respectively located in the injection chamber 1 and the injection chamber 2.

[0013] Furthermore, in the step 2, the volume ratio of the added amount of the sample to be tested to the ATP bioluminescent solution is 1:2, and in the step 3, the volume ratio of the active substrate to the sample to be tested is 1:1.

[0014] Furthermore, the ATP bioluminescent solution in step 2 includes luciferase, D-luciferin and a buffer solution containing Mg2+.

[0015] Furthermore, the ATP bioluminescent solution includes luciferase, D-luciferin and Mg at corresponding concentrations of 1-100 μg / ml, 0.05-10 mM and 5-25 mM, respectively. 2+ .

[0016] Furthermore, the Mg 2+ The buffer is one or more combinations of PBS, Tris-HCl or Hepes, with a pH of 6.5-8.5.

[0017] Furthermore, the incubation time in step 2 is 0.5-2.5 minutes.

[0018] Furthermore, the active substrate in step three is a bacterial solution of Micrococcus lysodeikticus.

[0019] Further, the OD of the lysodeiktic micrococcus liquid is 600 Between 0.05-1.5.

[0020] Furthermore, the incubation time in step three is 1-25 minutes.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The detection sensitivity of the present invention is high: the detection method provided by the present invention can detect lysozyme as low as 1.03U / mL, which can meet the sensitivity requirements of clinical tests;

[0023] The present invention is simple and quick to operate, and is suitable for high-throughput detection of large quantities of samples: the detection method provided by the present invention only requires simple operations to complete the detection, and the detection cycle of a single sample is about 6 minutes;

[0024] The detection cost of the present invention is low: all reagents used in the detection method provided by the present invention can be purchased through commercial channels, and the bacterial substrate used does not need to be additionally labeled, which effectively reduces the cost of lysozyme activity detection;

[0025] The present invention facilitates standardized operation. The kit provided by the present invention can quickly and quantitatively add ATP bioluminescent liquid and active substrate to the detection object, making the operation more convenient and standardized and the detection result more accurate. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 : Schematic diagram of the principle of the method for detecting lysozyme activity of the present invention;

[0027] Figure 2 :Step 2: Incubation time optimization results;

[0028] Figure 3 :Step 3: Incubation time optimization results;

[0029] Figure 4 : Active substrate concentration optimization results;

[0030] Figure 5 :Standard curve for lysozyme activity detection;

[0031] Figure 6 : Schematic diagram of the kit structure;

[0032] Figure 7 : Figure 6 Enlarged view of the structure of part A in the middle.

[0033] In the figure: 1. box body; 2. culture chamber; 3. cover; 4. injection chamber; 41. injection chamber one; 42. injection chamber two; 5. push piston; 51. piston head; 52. piston push rod; 6. piston rod; 7. injection pipeline; 8. fitting part; 81. fitting part one; 82. fitting part two; 9. connecting groove; 10. drainage channel. DETAILED DESCRIPTION

[0034] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0035] A method for detecting lysozyme activity, comprising a kit for detecting lysozyme activity, the kit comprising a box body 1, a culture chamber 2 is arranged in the box body 1, two injection ports are arranged at the upper part of the culture chamber 2, a sealing cap 3 is arranged at the injection port, the sealing cap 3 is made of rubber material, the lower part of the bottle cap is two rubber soft columns clamped in the injection port, an injection chamber 4 is also arranged in the box body 1, the injection chamber 4 comprises an injection chamber 1 41 and an injection chamber 2 42, a push piston 5 is arranged in the injection chamber 1 41 and the injection chamber 2 42, the push piston 5 comprises a piston arranged in the injection chamber 1 41 and the injection chamber 2 42, and the piston 5 comprises a piston arranged in the injection chamber 1 41 and the injection chamber 2 42. The piston head 51 in the cavity 4 and the piston push rod 52 connected to the piston head 51 are connected to the piston head 51. The piston head 51 is made of rubber, and a card interface is provided under the piston head 51. The upper end of the piston push rod 52 is inserted into the card interface of the piston head 51 and connected to the card interface. The piston head 51 is slidably connected to the inner wall of the injection cavity 4. The lower parts of the injection cavity 1 41 and the injection cavity 2 42 are both provided with through holes connected to the outside of the cavity. The through holes are also provided with a slideway located on the upper side of the box body 1. The piston push rod 52 passes through the through holes, and the lower end of the piston push rod 52 is upward to the box body 1. The side is integrally bent and leaks out of the upper part of the slideway. The upper parts of the injection chamber 1 41 and the injection chamber 2 42 are both provided with sliding chambers. The piston is provided with a piston rod 6 integrally connected to the piston push rod 52. An injection pipe 7 is provided in the piston rod 6. The lower end of the injection pipe 7 is bent toward the side of the piston rod 6 and connected to the injection chamber 4. The upper end of the piston rod 6 is slidably connected in the sliding chamber. The upper part of the injection pipe 7 is also connected to the side of the piston rod 6. The upper and lower ends of the sliding chamber are respectively provided with a fitting portion 8 that can fit with the upper end of the injection pipe 7. The fitting portion 8 includes It includes a fitting portion 81 located at the upper part of the side of the sliding cavity and a fitting portion 82 located at the lower part of the side of the sliding cavity. A connecting groove 9 is opened on the side of the sliding cavity. The sides of the connecting groove 9 are connected to a drainage channel 10 opened inside the box body 1. The other end of the drainage channel 10 is connected to the culture cavity 2. The ATP bioluminescent liquid and the active substrate are respectively located in the injection cavity 41 and the injection cavity 42. A ball head is provided at the upper end of the piston rod 6. A rubber sleeve is adhered to the upper end of the sliding cavity. The ball head is connected to the rubber sleeve and the piston rod 6 is fixed.

[0036] Example 1

[0037] A method for detecting lysozyme activity is implemented as follows:

[0038] Step 1: Open the cover 3 of the reagent kit and add the sample to be tested into the culture chamber 2 of the reagent kit from the two injection ports provided at the upper part of the culture chamber 2;

[0039] Step 2: Push the push piston 5 in the injection chamber 41 to mix the quantitative ATP bioluminescent liquid in the injection chamber 41 in the reagent kit with the sample to be tested, and then incubate the mixed liquid at room temperature for a period of time;

[0040] Step 3: Push the push piston 5 in the second injection chamber 42 to mix the quantitative active substrate in the kit with the mixed solution in step 2, and continue to incubate at room temperature for a period of time;

[0041] Step 4: Use a chemiluminescence meter to measure the luminescence intensity of the solution;

[0042] Step 5: Calculate the lysozyme activity in the sample based on the luminescence intensity value and the standard curve of lysozyme activity.

[0043] Example 2

[0044] Optimize the incubation time of steps 2 and 3

[0045] There may be a certain concentration of exogenous ATP in the sample. If these exogenous ATP remain in the sample, it will affect the accuracy of lysozyme activity detection. Therefore, before adding the active substrate (Micrococcus lysodeikticus), the sample needs to be incubated with ATP bioluminescent solution for a sufficient time to remove the exogenous ATP. The experiment first mixed one ATP solution or a solution without ATP (blank control) with 2 parts of ATP bioluminescent solution, and then incubated the mixture at room temperature, and the luminescence intensity of the system was detected every 30 seconds. Figure 2 It can be seen that after the incubation time reaches 120s, the luminescence intensity value of the ATP solution group is close to the blank value, indicating that the exogenous ATP can be completely consumed after incubation for 120s.

[0046] After adding the optimized active substrate in step three, the lysozyme in the sample to be tested will decompose the cell wall of the lysodeiktic micrococcus, which will burst after excessive water absorption, thereby releasing intracellular ATP. In order to obtain the best detection sensitivity, it is necessary to determine the reasonable incubation time of step three. The experiment first mixed one portion of the sample to be tested with 2 portions of ATP bioluminescent solution, added one portion of lysodeiktic micrococcus solution to the mixture at room temperature for 120 seconds, and then measured the luminescence intensity of the detection system at regular intervals. The results are as follows Figure 3 As shown in the figure, within 0-30 minutes, the luminescence intensity value increases rapidly, reaches the maximum value at 4 minutes and remains basically stable, while after 12 minutes, the luminescence intensity value shows a trend of slow decrease, probably due to the consumption of the reaction substrate. Therefore, the luminescence intensity value of the detection system should be detected within 4-12 minutes after the addition of the active substrate.

[0047] Example 3 Optimizing the concentration of active substrate

[0048] Lysodeiktic micrococci are the active substrate for detecting lysozyme, so it is necessary to ensure that there are enough lysodeiktic micrococci in the reaction system. Figure 4 It can be seen that in the substrate OD 600When the OD value was less than 0.509, the luminescence signal intensity increased with the increase of the concentration of Micrococcus lysodeikticus. 600 When it is greater than 0.509, the luminescence signal intensity shows a decreasing trend. This may be due to the high concentration of Micrococcus lysodeikticus, which leads to a large turbidity in the reaction system and interferes with the detection of luminescence intensity. 600 =0.509 was taken as the optimal concentration for lysozyme activity detection.

[0049] Example 4 Linear range and detection limit

[0050] First, the lysozyme standard was prepared into a standard series with concentrations of 0, 1, 5, 50, 250, and 500 U / mL, and then the luminescence intensity of the solution was determined according to the process described in Example 1. Three parallel experiments were performed for each concentration standard solution, and the average value was calculated to draw a standard curve of enzyme activity lg value (lg C)-luminescence intensity (BL intensity). The results are shown in Figure 1. Figure 5 As shown in Figure 2, within the range of 1-500 U / mL, lg C and luminescence intensity showed a good linear relationship, with a correlation coefficient of R 2 =0.9948.

[0051] The limit of detection (LOD) of the method of the present invention is calculated according to Formula 1:

[0052] LOD=3.3δ / S Formula 1

[0053] Wherein, δ is the standard deviation of the blank value; S is the slope of the standard curve. According to calculation, the detection limit (LOD) of the method of the present invention is 1.03U / mL.

[0054] Example 5 Accuracy and stability evaluation of the detection method

[0055] In order to evaluate the stability of the test results of the method described in the present invention, the inventors conducted three concentration gradient spike experiments, and each concentration was measured 6 times in parallel. In the intra-day stability experiment, 6 parallel determinations were completed in one batch experiment; in the inter-day stability experiment, 6 parallel determinations were completed in 3 days. The recovery rate and relative standard deviation (RSD) of the test results were calculated, and the results are shown in Table 1.

[0056] Table 1 Recovery and relative standard deviation of intra-day and inter-day spike experiments

[0057]

[0058] Note: ND=No detected, RSD=Relative standard deviation

[0059] As shown in Table 1, the intra-day and inter-day spiked test recovery rates of the lysozyme spiked solution were between 93% and 105.60%, indicating that the method of the present invention has good detection accuracy. At the same time, the intra-day standard deviation is less than 4.8%, and the inter-day standard deviation is less than 8.4%, indicating that the detection method of the present invention has good detection stability.

[0060] Example 6 Detection of actual samples

[0061] In order to further verify the detection performance of the method of the present invention for lysozyme activity in actual samples, we took urine samples from three healthy adults and conducted three concentration spike experiments, and each sample was measured in parallel three times. The recovery rate and relative standard deviation of the test results are shown in Table 2. As can be seen from Table 2, for three adult urines, the spike recovery rates of different concentrations were between 87% and 112.20%, and the relative standard deviation was less than 10%, indicating that the method of the present invention can be well used for the detection of lysozyme activity in urine.

[0062] Table 2 Results of spike recovery experiments on human biological samples

[0063]

[0064] Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for detecting lysozyme activity, characterized in that: The lysozyme activity detection method comprises the following steps: Step 1: Open the cover of the test kit and add the sample to be tested into the culture chamber of the test kit; Step 2: Mix the quantitative ATP bioluminescent solution in the kit with the sample to be tested, and then incubate the mixture at room temperature for a period of time. The ATP bioluminescent solution includes luciferase, D-luciferin and Mg 2+ Buffer solution; Step 3: Mixing the active substrate quantified in the kit with the mixed solution in step 2, and continuing to incubate for a period of time at room temperature, wherein the active substrate is a bacterial solution of Micrococcus lysodeikticus; Step 4: Use a chemiluminescence meter to measure the luminescence intensity of the solution; Step 5: Calculate the lysozyme activity in the sample based on the standard curve of the luminescence intensity value and the lysozyme activity; The test kit comprises a box body, a culture chamber is arranged in the box body, an openable cover is arranged in the culture chamber, an injection chamber is also arranged in the box body, the injection chamber comprises an injection chamber 1 and an injection chamber 2, a pushing piston is arranged in the injection chamber 1 and the injection chamber 2, a sliding chamber is arranged on the upper part of the injection chamber 1 and the injection chamber 2, a piston rod is arranged on the pushing piston, an injection pipe is arranged in the piston rod, the lower end of the injection pipe is connected to the injection chamber, the upper end of the piston rod is slidably connected in the sliding chamber, the upper and lower ends of the sliding chamber are respectively provided with fitting parts that can fit with the upper end of the injection pipe, a connecting groove is arranged on the side of the sliding chamber, the sides of the connecting groove are connected with drainage channels, the other ends of the drainage channels are connected to the culture chamber, and the ATP bioluminescent liquid and the active substrate are respectively located in the injection chamber 1 and the injection chamber 2.

2. The method for detecting lysozyme activity according to claim 1, characterized in that: The volume ratio of the added amount of the sample to be tested in step 2 to the ATP bioluminescent solution is 1:2, and the volume ratio of the active substrate to the sample to be tested in step 3 is 1:

1.

3. The method for detecting lysozyme activity according to claim 1, characterized in that: The ATP bioluminescent solution includes luciferase, D-luciferin and Mg at corresponding concentrations of 1-100 μg / ml, 0.05-10 mM and 5-25 mM, respectively. 2+ .

4. The method for detecting lysozyme activity according to claim 1, characterized in that: The Mg 2+ The buffer is one or more combinations of PBS, Tris-HCl or Hepes, with a pH of 6.5-8.

5.

5. The method for detecting lysozyme activity according to claim 1, characterized in that: The incubation time in step 2 is 0.5-2.5 minutes.

6. The method for detecting lysozyme activity according to claim 1, characterized in that: The OD of the lysodeiktic micrococcus bacterial solution 600 Between 0.05-1.

5.

7. The method for detecting lysozyme activity according to claim 1, characterized in that: The incubation time in step 3 is 1-25 minutes.

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