Colloidal gold kit, preparation method and application of colloidal gold kit in rapid sperm motility detection

Abstract

The invention belongs to the field of biological detection, and particularly relates to a colloidal gold kit, a preparation method and application of the colloidal gold kit in rapid sperm motility detection. The preparation method of the colloidal gold kit comprises the following steps: step 1, firing colloidal gold particles by using a trisodium citrate reduction method; step 2, protein labeling of colloidal gold and coating of an NC membrane antibody; and 3, assembling the colloidal gold test strip to obtain the colloidal gold kit. The colloidal gold kit fills the blank of rapid detection of sperm motility, sperm motility can be detected at home without a professional microscope and complex sperm automatic detection and analysis system software, the cost can be saved, and detection can be conveniently and rapidly carried out.

Description

Technical Field

[0001] The invention belongs to the field of biological detection, and particularly relates to a colloidal gold kit, a preparation method and an application in rapid detection of sperm motility. Background Art

[0002] Sperm motility is a prerequisite for sperm and egg to meet and fertilize. However, the sperm motility of patients with asthenozoospermia is poor, especially those with severe and critical asthenozoospermia. At present, the detection of sperm motility requires the use of a microscope and is carried out under the software of the sperm automatic detection and analysis system. Due to the expensive equipment, the test can only be performed in the hospital. Summary of the Invention

[0003] In view of the shortcomings of the existing technology, the present invention provides a colloidal gold kit to solve the problem in the existing technology that patients can operate and test at home by themselves without the need for expensive professional instruments and professionals.

[0004] In order to achieve the above object, the technical solution adopted by the present invention is as follows: a method for preparing a colloidal gold kit, comprising the following steps: Step 1: sintering of colloidal gold; Colloidal gold particles were sintered using trisodium citrate reduction method; Step 2: Protein labeling with colloidal gold and antibody coating of NC membrane (nitrocellulose membrane); 2.1. Protein labeling with colloidal gold; 2.2、NC membrane antibody coating; Step 3: Assemble the colloidal gold test strips to obtain a colloidal gold test kit.

[0005] Preferably, in the step 1, the colloidal gold particles are sintered using a trisodium citrate reduction method, which specifically includes: Heat the chloroauric acid (HAuCl4) aqueous solution to boiling, add trisodium citrate aqueous solution, continue heating to keep boiling, and after heating is completed, cool and adjust the volume to obtain a colloidal gold solution containing colloidal gold particles.

[0006] Preferably, the volume ratio of the aqueous solution of chloroauric acid to the aqueous solution of trisodium citrate is 100:1.5; The chloroauric acid aqueous solution includes a 0.01wt% chloroauric acid aqueous solution; The trisodium citrate aqueous solution includes a 1 wt % trisodium citrate aqueous solution.

[0007] Preferably, the particle size of the colloidal gold particles in the colloidal gold solution is 30 nm.

[0008] Preferably, in step 2, the protein labeling with colloidal gold specifically includes: 2.1.1. Protein processing; 2.1.2, marking; 2.1.3. Centrifugal purification.

[0009] Preferably, the above 2.1.1 specifically includes: The protein is dialyzed using a buffer with low ionic strength to obtain the treated protein, i.e., the protein to be labeled; The protein is a SKAP2 polyclonal antibody; The buffer with low ionic strength includes any one of a 10 mM phosphate buffer with a pH value of 7.4 and a 2 mM borate buffer with a pH value of 9.

[0010] In this process, since high concentrations of salt components will affect the adsorption of colloidal gold particles to proteins and can cause the colloidal gold solution containing colloidal gold particles to precipitate, the protein solution should be dialyzed with a low ionic strength buffer before labeling the colloidal gold with protein. The protein solution should be absolutely clear and free of fine particles, otherwise they should be removed first using a microporous filter membrane or ultracentrifugation.

[0011] Preferably, the above 2.1.2 specifically includes: 2.1.2.1. pH adjustment of colloidal gold; adjusting the pH value of the colloidal gold solution to a target value to obtain a colloidal gold solution after adjusting the pH value; 2.1.2.2. Add the protein to be labeled to the pH-adjusted colloidal gold solution, and then add NaCl aqueous solution to label. 2.1.3, centrifugal purification; After labeling, the solution was centrifuged and resuspended to obtain a colloidal gold solution labeled with SKAP2 protein.

[0012] In this process, proteins are adsorbed on the surface of colloidal gold particles at the isoelectric point or slightly alkaline. According to the principle of positive and negative charges attract each other, they are firmly bound. Generally, the surface of colloidal gold particles is negatively charged, and they are combined with the positively charged groups carried by proteins by electrostatic attraction. Since protein molecules are firmly bound to the surface of gold particles, a protein layer is formed, which prevents the colloidal gold particles from contacting each other, making the gold probe labeled with protein more stable.

[0013] Preferably, in step 2, the NC membrane is coated with antibodies, specifically comprising: Draw C lines and T lines on the NC membrane and let it dry to obtain a coated NC membrane; Among them, line C is goat anti-mouse secondary antibody, and line T is SKAP2 Polyclonal Antibody.

[0014] Preferably, in step 3, the assembly of the colloidal gold test strips specifically includes: S1. Select glass cellulose membrane as the gold label pad material, cut it, immerse it in colloidal gold solution labeled with SKAP2 protein, soak it, and dry it to obtain a gold-plated gold label pad; S2. Take the PVC base plate, stick the coated NC film on the PVC base plate, scratch the film, and dry it; S3. Stick absorbent paper on the PVC base plate, covering part of the area on one side of the NC membrane; S4. Stick the gold label pad after gold coating on the PVC base plate, covering part of the area on the other side of the NC film; S5. Stick the sample pad on the PVC base plate, covering the gold label pad area after gold paving; S6. After assembly is completed, cut the test strips into pieces, place the test strips in the plastic card, cover the plastic card cover, and complete the assembly.

[0015] The invention also discloses a colloidal gold kit prepared by adopting the preparation method of the colloidal gold kit.

[0016] The present invention also discloses an application of the colloidal gold kit in rapid detection of sperm motility.

[0017] Compared with the prior art, the present invention has the following beneficial effects: Since the seminal plasma protein SKAP2 plays an important role in sperm motility, it can promote sperm motility by initiating the polymerization of microfilaments at the sperm tail. The concentration of the seminal plasma protein SKAP2 has a certain correlation with sperm motility. Therefore, the purpose of detecting sperm motility can be achieved by detecting the concentration of seminal plasma SKAP2 through colloidal gold technology. The present invention fills the gap in the rapid detection of sperm motility. It does not require the aid of professional microscopes and complex sperm automatic detection and analysis system software. Sperm motility can be detected at home, which can save costs and can be detected conveniently and quickly. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The following are photos of the results of sperm motility testing using a colloidal gold kit in Experimental Group 1 and Experimental Group 2 of Example 2; Figure 2 This is a photograph of the results of sperm motility testing using a colloidal gold kit in Experimental Group 3 of Example 2; Figure 3 This is a photograph of the results of sperm motility testing using a colloidal gold kit in Experimental Group 4 of Example 2; Figure 4 This is a photograph of the results of sperm motility testing using a colloidal gold kit in Experimental Group 5 of Example 2; Figure 5This is a photograph of the results of sperm motility detection using a colloidal gold kit in Experimental Group 1 of Example 3; Figure 6 This is a photo of the sperm motility test results of Experimental Group 2 in Example 3 using a colloidal gold kit; Figure 7 This is a photograph of the results of sperm motility detection using a colloidal gold kit in Experimental Group 1 of Example 4; Figure 8 This is a photograph of the results of sperm motility testing using a colloidal gold kit in Experimental Group 2 of Example 4; Figure 9 This is a photo of the results of sperm motility detection using a colloidal gold kit in the experimental group (1) of Example 5; Figure 10 This is a photo of the results of sperm motility detection using a colloidal gold kit in experimental group (2) of Example 5; Figure 11 This is a photo of the results of detecting sperm motility using a colloidal gold kit in the experimental group (3) of Example 5. DETAILED DESCRIPTION

[0019] Example 1

[0020] This embodiment discloses a method for preparing a colloidal gold kit, comprising the following steps: Step 1: sintering of colloidal gold; Colloidal gold particles were prepared by the trisodium citrate reduction method: 100 mL of a 0.01 wt% aqueous solution of chloroauric acid was heated to boiling, and 1.5 mL of a 1 wt% aqueous solution of trisodium citrate was added. The solution was heated and kept boiling until the color of the solution changed from black to blue and finally to dark red. After the color of the solution turned to dark red, the solution was boiled for 2 minutes, the heating was stopped, and the solution was naturally cooled to room temperature. The volume was then adjusted to 100 mL to obtain a colloidal gold solution containing colloidal gold particles. According to the experimental requirements, a colloidal gold solution containing colloidal gold particles with a particle size of 30 nm was selected. The absorption wavelength λmax (nm) of the colloidal gold particles with a particle size of 30 nm is 525 nm. The colloidal gold solution containing colloidal gold particles with a particle size of 30 nm was recorded as 525 colloidal gold solution and stored in the dark at 4°C for future use. Step 2: Protein labeling with colloidal gold and antibody coating of NC membrane; 2.1. Protein labeling with colloidal gold; 2.1.1. Protein processing; Since commercial or pre-made antibody storage solutions contain preservatives and glycerol, high concentrations of salt components can affect the adsorption of 525 colloidal gold to SKAP2 polyclonal antibodies. Dialysis treatment is required before labeling. 0.6 mg / mL SKAP2 polyclonal antibody was dialyzed against 1x PBS buffer (0.01 M, pH 7.4) at 4°C for 24 h to remove salt ions and other components. The dialysate was changed twice during the dialysis. After dialysis, the protein was filtered through a 0.22 μm microporous membrane to remove fine particles. 2.1.2, marking; 2.1.2.1. pH adjustment of colloidal gold; Take 525 colloidal gold solution, add 0.1M hydrochloric acid solution to adjust the pH of the 525 colloidal gold solution to the target value of 5.8, and obtain a colloidal gold solution after adjusting the pH value; 2.1.2.2. Take 0.5 mL of the colloidal gold solution after adjusting the pH in 2.1.2.1, add 0.5 mL of the protein to be labeled (i.e., the treated protein obtained in 2.1.1), and then add 0.1 mL of 10 wt% NaCl aqueous solution. Let it stand for 2 h for labeling at room temperature. During this process, pay attention to the color changes. Stable red indicates effective, and blue indicates aggregation. 2.1.3, centrifugal purification; After labeling, the aggregates were removed by centrifugation at 4°C, 1500 g, and 20 min. The red supernatant was aspirated and subjected to high-speed centrifugation at 4°C, 14,000 g, and 1 h. After centrifugation, the colorless supernatant was discarded, and the loose red precipitate was collected and resuspended in PBS containing 1 wt% PEG to obtain a colloidal gold solution labeled with SKAP2 protein. Among them, PBS containing 1 wt% PEG is obtained by dissolving PEG (polyethylene glycol) with a Mw of 20,000 in 1xPBS buffer, and the mass percentage of PEG is 1%; 2.2、NC membrane antibody coating; Take a 200mm*25mm NC film, starting from the wide end, and draw a C line and a T line at 10mm and 16mm from top to bottom to the other end respectively, so that the NC film is moist. After the marking is completed, let it dry naturally to obtain a well-coated NC film; Among them, line C is goat anti-mouse secondary antibody, the concentration is 1.0 mg / mL; line T is SKAP2 Polyclonal Antibody, the concentration is 0.3 mg / mL; Step 3: assembling colloidal gold test strips to obtain a colloidal gold test kit; The assembly of the colloidal gold test strip specifically includes: S1. Select GL-b03 glass cellulose membrane as the gold label pad material, cut the gold label pad into 6mm*200mm, immerse it in the colloidal gold solution labeled with SKAP2 protein, and soak it for 5 minutes to allow the gold label pad to evenly absorb the colloidal gold solution labeled with SKAP2 protein. After soaking, take it out and place it in a vacuum at 37°C and a vacuum degree of 0.08 MPa for vacuum drying to obtain the gold-plated gold label pad; S2. Take a 200mm*60mm (20mm+25mm+15mm) PVC baseboard (model DB-6), peel off the 25mm wide release paper on the PVC baseboard, stick the coated NC film on the PVC baseboard, scratch the film, and dry it; The smooth side of the NC film is glued to the adhesive of the PVC base plate, and the matte side is used for scratching. S3. Remove the 15mm wide release paper on the PVC base plate and stick a 200mm*16mm absorbent paper on the PVC base plate, covering one side of the NC membrane by 2mm. S4. Remove the 20mm wide release paper on the PVC base plate and stick the gold label pad after gold coating on the PVC base plate, covering the other side of the NC film by 2mm. S5. Stick the sample pad cut into 200mm*16mm on the PVC base plate, covering the gold label pad by 2mm. S6. After assembly is completed, cut the test paper into 60mm*4mm strips, place them in the plastic card, cover the plastic card cover, and complete the assembly.

[0021] By verifying the sensitivity, specificity and repeatability of the colloidal gold kit prepared in Example 1 in detecting SKAP2 protein in seminal plasma (asthenospermia and normal sperm), the optimal detection conditions of the colloidal gold kit were determined.

[0022] Example 2

[0023] This embodiment discloses a method for detecting sperm motility using a colloidal gold kit, comprising the following steps: 1. Prepare experimental materials; Semen sample, SKAP2 protein detection kit (including: colloidal gold kit prepared in Example 1, isotonic PBS buffer (0.01 mol / L, pH = 7.4), pipette), constant temperature water bath, centrifuge tubes of various specifications, pipette, pipette tips, etc.; The extraction of semen samples includes the following steps: Two subjects were selected and all semen was ejaculated into a 50 mL sterile centrifuge tube by masturbation. The tube was placed in a 37°C constant temperature water bath for 20 minutes to allow the semen to liquefy naturally. After liquefaction is completed, the liquefied semen is transferred into EP tubes (Eppendorf tubes, microcentrifuge tubes) and the semen samples are numbered (No. 1, No. 2); Among them, sample 1 was obtained from a middle-aged person (43 years old, 21% anterior motility sperm, 5% non-anterior motility sperm) with poor semen quality; Sample 2 was obtained from a young population (28 years old, 41% anterior motility sperm, 6% non-anterior motility sperm) with excellent semen quality; 2. Detection experiment; 2.1 Experimental Group 1: Open the test kit and use the pipette included in the kit to aspirate liquefied semen from the EP tube. Place one drop of semen into the designated well of the test kit. Add two drops of isotonic PBS buffer. Count the time until a positive result appears (both the C and T lines are colored). Wait 20 minutes and observe the results. 2.2 Experimental Group 2: Repeat Experimental Group 1; 2.3 Experimental Group 3: 2.3.1 Pipette the liquefied semen and mix it with isotonic PBS buffer at a volume ratio of 1:5 in a new EP tube to obtain a mixed solution (test immediately); 2.3.2 Open the test kit and use the pipette provided in the kit to draw up the mixture prepared in 2.3.1. Drop it into the designated well of the test kit. Count the time until a positive result appears, wait 20 minutes, and then observe the result. 2.4 Experimental Group 4: 2.4.1 Prepare hypotonic buffer: Dilute isotonic PBS buffer 10-fold with distilled water at a volume ratio of 1:9 to obtain hypotonic buffer, and place it in an EP tube for later use; 2.4.2 Pipette the liquefied semen and mix it with hypotonic buffer in a 1:2 volume ratio in a new EP tube to obtain a mixed solution (test immediately). 2.4.3 Open the test kit and use the pipette provided in the kit to draw up the mixture prepared in 2.3.2. Drop it into the designated well of the test kit. Count the time until a positive result appears, wait 20 minutes, and then observe the result. 2.5 Experimental Group 5: 2.5.1 Prepare hypotonic buffer: Dilute isotonic PBS buffer 10-fold with distilled water at a volume ratio of 1:9 to obtain hypotonic buffer, and place it in an EP tube for later use; 2.5.2 Pipette the liquefied semen and mix it with hypotonic buffer in a 1:2 volume ratio in a new EP tube. Mix for 5 minutes to obtain a mixed solution. 2.5.3 Open the test kit and use the pipette provided in the kit to draw up the mixture prepared in 2.3.2. Drop it into the designated well of the test kit. Count the time until a positive result appears, wait 20 minutes, and then observe the result. In both experimental groups 1 and 2, the results appeared after 5 minutes. Both the C line and the T line were colored, and the color of the T line was weaker. Figure 1 As shown, 1-1 is the measurement result of sample No. 1 in experimental group 1, 1-2 is the measurement result of sample No. 1 in experimental group 2, 2-1 is the measurement result of sample No. 2 in experimental group 1, and 2-2 is the measurement result of sample No. 2 in experimental group 2. The color of the T line of sample No. 1 is stronger than that of sample No. 2. In experimental group 3, the results appeared after 5 minutes, the C line showed color, and the T line showed very weak color, such as Figure 2 As shown, 1 is the measurement result of sample No. 1 in experimental group 3, and 2 is the measurement result of sample No. 2 in experimental group 3. The color of the T line of sample No. 1 is stronger than that of sample No. 2, and the color development is very weak; In experimental group 4, the results appeared after 5 minutes. Both C line and T line showed color, and the color of T line was very weak. Figure 3 As shown, 1-4 are the test results of sample No. 1 in the experimental group 4, and 2-4 are the test results of sample No. 2 in the experimental group 4. The color of the T line of sample No. 1 is stronger than that of sample No. 2. In experimental group 5, the results appeared after 5 min, and both the C line and the T line showed color, e.g. Figure 4 As shown, 1-5 is the measurement result of sample No. 1 in experimental group 5, and 2-5 is the measurement result of sample No. 2 in experimental group 5. The color of the T line of sample No. 2 is stronger than that of sample No. 1.

[0024] This example illustrates that when detecting SKAP2 protein in seminal plasma, compared to experimental group three in which the liquefied semen was diluted with isotonic PBS buffer at a volume ratio of 1:5 and then immediately tested, and experimental group four in which the liquefied semen was diluted with hypotonic buffer at a volume ratio of 1:2 and then immediately tested, in experimental group five, the liquefied semen was diluted with hypotonic buffer at a volume ratio of 1:2 and then waited for several minutes before testing, and the detection effect was better; after waiting for several minutes after dilution with hypotonic buffer and then testing, the sample with good sperm motility had brighter bands.

[0025] Example 3

[0026] This embodiment discloses a method for detecting sperm motility using a colloidal gold kit, comprising the following steps: 1. Prepare experimental materials; Semen sample, SKAP2 protein detection kit (including: colloidal gold kit prepared in Example 1, isotonic PBS buffer, pipette), constant temperature water bath, centrifuge tubes of various specifications, pipette, pipette tips, etc.; The extraction of semen samples includes the following steps: Two subjects were selected and all semen was ejaculated into a 50 mL sterile centrifuge tube by masturbation. The tube was placed in a 37°C constant temperature water bath for 20 minutes to allow the semen to liquefy naturally. After liquefaction is completed, the liquefied semen is transferred to EP tubes and the semen samples are numbered (No. 1, No. 2); Among them, sample 1 was obtained from a middle-aged person (45 years old, 11% anterior motility sperm, 7% non-anterior motility sperm) with poor semen quality; Sample 2 was obtained from a young population (25 years old, 43% anterior motility sperm, 7% non-anterior motility sperm) with excellent semen quality; 2. Detection experiment; 2.1 Experimental Group 1: 2.1.1 Take liquefied semen and drip one drop into a new EP tube. Add 9 drops of isotonic PBS buffer and mix thoroughly to obtain a mixed solution (test immediately). 2.1.2 Open the test kit and use the pipette provided in the kit to draw up the mixture prepared in 2.1.1. Drop it into the designated well of the test kit. Count the time until a positive result appears, wait 20 minutes, and then observe the result. 2.2 Experimental Group 2: 2.3.1 Prepare hypotonic buffer: Dilute isotonic PBS buffer 10-fold with distilled water at a volume ratio of 1:9 to obtain hypotonic buffer, and place it in an EP tube for later use; 2.3.2 Pipette the liquefied semen and mix it with hypotonic buffer in a volume ratio of 1:9 in a new EP tube to obtain a mixed solution (test immediately); 2.3.3 Open the test kit and use the pipette provided in the kit to draw up the mixture prepared in 2.3.2. Drop it into the designated well of the test kit. Count the time until a positive result appears, wait 20 minutes, and then observe the result. In experimental group 1, the results appeared after 5 minutes. Both C line and T line showed color, but T line was weaker. Figure 5 As shown, 1 is the measurement result of sample No. 1 in experimental group 1, and 2 is the measurement result of sample No. 2 in experimental group 1. The color of the T line of sample No. 2 is stronger than that of sample No. 1; In experimental group 2, the C line of sample 1 showed, but the T line did not show; the result of sample 2 appeared after 5 minutes, and both the C line and the T line showed color, but the T line was weaker. Figure 6 As shown, 1 is the measurement result of sample No. 1 in experimental group 2, and 2 is the measurement result of sample No. 2 in experimental group 2; Based on Example 2, this example found that SKAP2 protein in seminal plasma can be detected when the hypotonicity of the seminal plasma (i.e., the volume ratio of liquefied semen to hypotonic buffer) is 1:4. At this ratio, the sample band indicating poor sperm motility after hypotonicity disappears, indicating that increasing the hypotonicity of the seminal plasma can shorten the waiting time.

[0027] Example 4

[0028] This embodiment discloses a method for detecting sperm motility using a colloidal gold kit, comprising the following steps: 1. Prepare experimental materials; Semen sample, SKAP2 protein detection kit (including: colloidal gold kit prepared in Example 1, isotonic PBS buffer, 0.1 M acetic acid solution, 0.01 M hydrochloric acid solution, NaOH, pipette), constant temperature water bath, centrifuge tubes of various specifications, pipette, pipette tips, etc.; The extraction of semen samples includes the following steps: Two subjects were selected and all semen was ejaculated into a 50 mL sterile centrifuge tube by masturbation. The tube was placed in a 37°C constant temperature water bath for 20 minutes to allow the semen to liquefy naturally. After liquefaction is completed, the liquefied semen is transferred to EP tubes and the semen samples are numbered (No. 1, No. 2); Among them, sample 1 was obtained from a middle-aged person (45 years old, 11% anterior motility sperm, 7% non-anterior motility sperm) with poor semen quality; Sample 2 was obtained from a young population (25 years old, 43% anterior motility sperm, 7% non-anterior motility sperm) with excellent semen quality; 2. Detection experiment; 2.1 Experimental Group 1: 2.1.1 Prepare hypotonic buffer: Dilute isotonic PBS buffer 10-fold with distilled water at a volume ratio of 1:9, then adjust the pH to 8.7 with NaOH and place in an EP tube for later use; 2.1.2 Aspirate the liquefied semen and adjust the pH to 2 by adding 0.01 M hydrochloric acid solution. After waiting for 5 minutes, mix the mixture with hypotonic buffer at a volume ratio of 1:4 in a new EP tube and mix for 3 minutes to obtain a mixed solution. 2.1.3 Open the test kit and use the pipette provided in the kit to draw up the mixture prepared in 2.1.2. Drop it into the designated well of the test kit. Count the time until a positive result appears, wait 20 minutes, and then observe the result. 2.2 Experimental Group 2: 2.2.1 Prepare hypotonic buffer: Dilute isotonic PBS buffer 10-fold with distilled water at a volume ratio of 1:9, then adjust the pH to 8.7 with NaOH and place in an EP tube for later use; 2.2.2 Aspirate the liquefied semen and adjust the pH to 3 by adding 0.1 M acetic acid solution. After waiting for 30 minutes, mix the mixture with hypotonic buffer at a volume ratio of 1:4 in a new EP tube and mix for 3 minutes to obtain a mixed solution. 2.2.3 Open the test kit and use the pipette provided in the kit to draw up the mixture prepared in 2.2.2. Drop it into the designated well of the test kit. Count the time until a positive result appears, wait 20 minutes, and observe the result. In experimental group 1, the results appeared after 5 minutes, e.g. Figure 7 As shown, 1 is the measurement result of sample No. 1 in experimental group 1, and 2 is the measurement result of sample No. 2 in experimental group 1. The C line of sample No. 1 is colored, but the T line is not colored; both the C line and T line of sample No. 2 are colored, but the color of the T line is weaker, which is comparable. The sperm has poor motility and is not colored. This method takes a short time, but the bands are weak.

[0029] In experimental group 2, the results appeared after 5 minutes, e.g. Figure 8 As shown, 1 is the measurement result of sample No. 1 in experimental group 2, and 2 is the measurement result of sample No. 2 in experimental group 2. The C line of sample No. 1 is colored, but the T line is not colored; both the C line and T line of sample No. 2 are colored, but the color of the T line is weaker; they are comparable, the sperm has poor motility and is not colored. This method takes a long time, but the bands are bright.

[0030] Based on Examples 2 and 3, this Example found that when detecting SKAP2 protein in seminal plasma, a rapid decrease in the pH of semen can cause denaturation of the SKAP2 protein in seminal plasma. However, SKAP2 in the extracellular vesicles of seminal plasma is not affected because it is in the vesicles. Therefore, the semen is first acidified and then mixed with a hypotonic buffer at a volume ratio of 1:4. The pH value is restored to about 7.4, which can effectively detect the SKAP2 protein in the extracellular vesicles of seminal plasma.

[0031] Example 5

[0032] This embodiment discloses a method for detecting sperm motility using a colloidal gold kit, including three groups of detection experiments: Experimental group (1): 1. Prepare experimental materials; Semen sample, SKAP2 protein detection kit (including: colloidal gold kit prepared in Example 1, isotonic PBS buffer, 0.01 M hydrochloric acid solution, NaOH, pipette), constant temperature water bath, centrifuge tubes of various specifications, pipette, pipette tips, etc.; The extraction of semen samples includes the following steps: Twenty subjects were selected and all semen was ejaculated into a 50 mL sterile centrifuge tube by masturbation. The tube was placed in a 37°C constant temperature water bath for 20 minutes to allow the semen to liquefy naturally. After liquefaction is completed, the liquefied semen is transferred into EP tubes and the semen samples are numbered (1-20); Among them, semen samples 1-10 were collected from 10 men with normal sperm motility, and semen samples 11-20 were collected from 10 men with weak sperm motility; Subject 1: 25 years old, sperm motility rate 60.8%, non-motility 22%; Subject 2: 27 years old, sperm motility rate 60.8%, non-motility 22%; Subject 3: age 30 years, sperm motility rate 48.8%, non-motility 22%; Subject 4: age 31 years, sperm motility rate 47.8%, non-motility 22%; Subject 5: age 24 years, sperm motility rate 39.5%, non-motility 22%; Subject 6: age 33 years, sperm motility rate 24.9%, non-motility 21%; Subject 7: age 28 years, sperm motility rate 21.9%, non-motility 21%; Subject 8: age 25 years, sperm motility rate 21.1%, non-motility 22%; Subject 9: 29 years old, sperm motility rate 59.5%, non-motility 22%; Subject 10: age 28 years, sperm motility rate 53%, non-motility 22%; Subject 11: age 27 years, sperm motility rate 23.6%, non-motility 1.4%; Subject 12: age 30 years, sperm motility rate 22.5%, non-motility 1.4%; Subject 13: age 30 years, sperm motility rate 0.4%, non-motility 2.1%; Subject 14: age 38 years, sperm motility rate 6.1%, non-motility 3.7%; Subject 15: age 37 years, sperm motility rate 9.9%, non-motility rate 4%; Subject 16: age 31 years, sperm motility rate 20.6%, non-motility 4.4%; Subject 17: age 28 years, sperm motility rate 3.3%, non-motility 4.4%; Subject 18: Age 27, sperm motility rate 9.9%, non-motility 4.7%; Subject 19: age 33 years, sperm motility rate 6.6%, non-motility 5.1%; Subject 20: age 25 years, sperm motility rate 1.7%, non-motility 5.1%; 2. Detection experiment; 2.1.1 Prepare hypotonic buffer: Dilute isotonic PBS buffer 10-fold with distilled water at a volume ratio of 1:9, then adjust the pH to 8.7 with NaOH and place in an EP tube for later use; 2.1.2 Aspirate the liquefied semen and adjust the pH to 2 by adding 0.01 M hydrochloric acid solution. After waiting for 5 minutes, mix the mixture with hypotonic buffer at a volume ratio of 1:4 in a new EP tube and mix for 3 minutes to obtain a mixed solution. 2.1.3 Open the test kit and use the pipette provided in the kit to draw up the mixture prepared in 2.1.2. Drop it into the designated well of the test kit. Count the time until a positive result appears, wait 20 minutes, and then observe the result. according to Figure 9 It can be seen that the results appeared after 5 minutes. Among them, groups 1-10 with good sperm motility basically showed color, with C line showing color and T line showing color; groups 11-20 with poor sperm motility basically did not show color, with C line showing color and T line not showing color; No. 2 had both C line and T line showing color, with the T line color being weaker; they were comparable, and the sperm with poor motility did not show color. Among them, No. 11, No. 16, No. 17, and No. 20 showed faint color, and the bands were negligible. This method can be used to detect sperm motility simply and quickly. Sperm with good motility will show color in T, while sperm with poor motility will not show color in T. Experimental group (2): The experiment was commissioned to Henan Provincial Reproductive Center (Henan Provincial People's Hospital) for testing and verification; 1. Prepare experimental materials; Semen sample, SKAP2 protein detection kit (including: colloidal gold kit prepared in Example 1, isotonic PBS buffer, 0.01 M hydrochloric acid solution, NaOH, pipette), constant temperature water bath, centrifuge tubes of various specifications, pipette, pipette tips, etc.; The extraction of semen samples includes the following steps: Twenty subjects were selected and all semen was ejaculated into a 50 mL sterile centrifuge tube by masturbation. The tube was placed in a 37°C constant temperature water bath for 20 minutes to allow the semen to liquefy naturally. After liquefaction is completed, the liquefied semen is transferred into EP tubes and the semen samples are numbered (21-40); Among them, semen samples 21-30 were collected from 10 men with normal sperm motility, and semen samples 31-40 were collected from 10 men with weak sperm motility; Subject 21: age 22 years, sperm motility rate 51%, non-motility 12%; Subject 22: age 21 years, sperm motility rate 62%, non-motility 23%; Subject 23: age 32 years, sperm motility rate 44%, non-motility 22%; Subject 24: age 31 years, sperm motility rate 45%, non-motility 22%; Subject 25: age 25 years, sperm motility rate 34%, non-motility 22%; Subject 26: age 31 years, sperm motility rate 37%, non-motility 11%; Subject 27: age 27, sperm motility rate 39%, non-motility 21%; Subject 28: age 25 years, sperm motility rate 31%, non-motility 12%; Subject 29: age 30 years, sperm motility rate 49%, non-motility 12%; Subject 30: age 28 years, sperm motility rate 43%, non-motility 12%; Subject 31: age 32 years, sperm motility rate 14%, non-motility 11%; Subject 32: age 30 years, sperm motility rate 13%, non-motility 11%; Subject 33: age 40 years, sperm motility rate 2%, non-motility rate 12%; Subject 34: age 28 years, sperm motility rate 6%, non-motility rate 13%; Subject 35: age 37 years, sperm motility rate 11%, non-motility 14%; Subject 36: age 31 years, sperm motility rate 20%, non-motility 10%; Subject 37: age 28 years, sperm motility rate 11%, non-motility 8%; Subject 38: age 27 years, sperm motility rate 11%, non-motility 14%; Subject 39: age 33 years, sperm motility rate 17%, non-motility 15%; Subject 40: age 27 years, sperm motility rate 12%, non-motility 15%; The specific operation process of the detection experiment is the same as that of the experimental group (1); Experimental group (3): The experiment was commissioned to the Shandong Provincial Reproductive Center (Shandong Provincial Maternal and Child Health Hospital) for testing and verification; 1. Prepare experimental materials; Semen sample, SKAP2 protein detection kit (including: colloidal gold kit prepared in Example 1, isotonic PBS buffer, 0.01 M hydrochloric acid solution, NaOH, pipette), constant temperature water bath, centrifuge tubes of various specifications, pipette, pipette tips, etc.; The extraction of semen samples includes the following steps: Twenty subjects were selected and all semen was ejaculated into a 50 mL sterile centrifuge tube by masturbation. The tube was placed in a 37°C constant temperature water bath for 20 minutes to allow the semen to liquefy naturally. After liquefaction is completed, the liquefied semen is transferred into EP tubes and the semen samples are numbered (41-60); Among them, semen samples 41-50 were collected from 10 men with normal sperm motility, and semen samples 51-60 were collected from 10 men with weak sperm motility; Subject 41: age 21 years, sperm motility rate 43%, non-motility 12%; Subject 42: age 28 years, sperm motility rate 36%, non-motility 12%; Subject 43: age 31 years, sperm motility rate 43%, non-motility 12%; Subject 44: age 21 years, sperm motility rate 41%, non-motility 20%; Subject 45: age 34 years, sperm motility rate 34%, non-motility 10%; Subject 46: age 23 years, sperm motility rate 35%, non-motility 21%; Subject 47: age 38 years, sperm motility rate 32%, non-motility 11%; Subject 48: age 22 years, sperm motility rate 41%, non-motility 12%; Subject 49: age 39 years, sperm motility rate 49%, non-motility 12%; Subject 50: age 22 years, sperm motility rate 43%, non-motility 21%; Subject 51: age 27 years, sperm motility rate 12%, non-motility 8%; Subject 52: age 20 years, sperm motility rate 12%, non-motility 1%; Subject 53: age 20 years, sperm motility rate 4%, non-motility rate 12%; Subject 54: age 38 years, sperm motility rate 16%, non-motility 3%; Subject 55: age 33 years, sperm motility rate 10%, non-motility 14%; Subject 56: age 33 years, sperm motility rate 21%, non-motility 4%; Subject 57: age 28 years, sperm motility rate 13%, non-motility 14%; Subject 58: age 23 years, sperm motility rate 20%, non-motility 4%; Subject 59: age 34 years, sperm motility rate 17%, non-motility 8%; Subject 60: age 26 years, sperm motility rate 12%, non-motility 5%; The specific operation process of the detection experiment is the same as that of the experimental group (1); Depend on Figure 10 and Figure 11 It can be seen that the group with good sperm motility basically showed color, with both the C line and the T line showing color; the group with poor sperm motility basically did not show color, with both the C line and the T line showing color.

[0033] contrast Figure 9 、 Figure 10 and Figure 11 , indicating that for semen samples from different regions, the colloidal gold kit prepared in Example 1 of the present invention has good repeatability when detecting sperm motility using the detection method in Example 5, which proves the detection accuracy of the colloidal gold kit and the representativeness of the detection method.

[0034] Although the present invention has been disclosed above with reference to preferred embodiments, this is not intended to limit the present invention. Anyone familiar with this technology may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for preparing a colloidal gold kit, characterized in that: The following steps are involved: Step 1: sintering of colloidal gold; Colloidal gold particles were sintered using trisodium citrate reduction method; Step 2: Protein labeling with colloidal gold and antibody coating of NC membrane; 2.

1. Protein labeling with colloidal gold; The protein labeling of colloidal gold is specifically include: 2.1.

1. Protein treatment: The protein is dialyzed using a low ionic strength buffer to obtain the treated protein; The protein is a SKAP2 polyclonal antibody; 2.1.2, Marking: 2.1.2.

1. pH adjustment of colloidal gold; adjusting the pH value of the colloidal gold solution to a target value to obtain a colloidal gold solution after adjusting the pH value; 2.1.2.

2. Add the protein to be labeled to the pH-adjusted colloidal gold solution, and then add NaCl aqueous solution to label. 2.1.

3. Centrifugal purification: After labeling, centrifuge and resuspend to obtain a colloidal gold solution labeled with SKAP2 protein; 2.2、NC membrane antibody coating; The NC membrane antibody coating specifically includes: Draw C lines and T lines on the NC membrane and let it dry to obtain a coated NC membrane; Among them, line C is goat anti-mouse secondary antibody, and line T is SKAP2 polyclonal antibody; Step 3: Assemble the colloidal gold test strips to obtain a colloidal gold test kit.

2. The method for preparing a colloidal gold kit according to claim 1, wherein In the step 1, colloidal gold particles are sintered using a trisodium citrate reduction method, which specifically includes: The chloroauric acid aqueous solution is heated to boiling, and a trisodium citrate aqueous solution is added, and the heating is continued to maintain boiling. After the heating is completed, the solution is cooled and the volume is constant to obtain a colloidal gold solution containing colloidal gold particles.

3. The method for preparing a colloidal gold kit according to claim 2, wherein: The volume ratio of the chloroauric acid aqueous solution to the trisodium citrate aqueous solution is 100:1.5; The chloroauric acid aqueous solution includes a 0.01wt% chloroauric acid aqueous solution; The trisodium citrate aqueous solution includes a 1 wt % trisodium citrate aqueous solution.

4. The method for preparing a colloidal gold kit according to claim 1, wherein In the above 2.1.1, the low ionic strength buffer includes any one of a 10 mM phosphate buffer with a pH value of 7.4 and a 2 mM borate buffer with a pH value of 9.

5. The method for preparing a colloidal gold kit according to claim 1, wherein In step 3, the assembly of the colloidal gold test strips specifically includes: S1. Select glass cellulose membrane as the gold label pad material, cut it, immerse it in colloidal gold solution labeled with SKAP2 protein, soak it, and dry it to obtain a gold-plated gold label pad; S2. Take the PVC base plate, stick the coated NC film on the PVC base plate, scratch the film, and dry it; S3. Stick absorbent paper on the PVC base plate, covering part of the area on one side of the NC membrane; S4. Stick the gold label pad after gold coating on the PVC base plate, covering part of the area on the other side of the NC film; S5. Stick the sample pad on the PVC base plate, covering the gold label pad area after gold paving; S6. After assembly is completed, cut the test strips into pieces, place the test strips in the plastic card, cover the plastic card cover, and complete the assembly.

6. A colloidal gold kit prepared by the method for preparing a colloidal gold kit according to any one of claims 1 to 5.

7. Use of the colloidal gold kit as claimed in claim 6 in rapid detection of sperm motility.

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