WO2023246254A1 - 精子唾液酸酶1/3检测试剂盒及其制备方法、检测精子唾液酸酶1/3表达水平的方法 - Google Patents

精子唾液酸酶1/3检测试剂盒及其制备方法、检测精子唾液酸酶1/3表达水平的方法 Download PDF

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WO2023246254A1
WO2023246254A1 PCT/CN2023/088755 CN2023088755W WO2023246254A1 WO 2023246254 A1 WO2023246254 A1 WO 2023246254A1 CN 2023088755 W CN2023088755 W CN 2023088755W WO 2023246254 A1 WO2023246254 A1 WO 2023246254A1
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solution
sperm
sialidase
concentration
antibody
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PCT/CN2023/088755
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English (en)
French (fr)
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马芳
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成都思瑞多医疗科技有限公司
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label

Definitions

  • the present disclosure relates to the field of detection technology, specifically, to a sperm sialidase 1/3 detection kit and its preparation method, and a method for detecting the expression level of sperm sialidase 1/3.
  • the present disclosure provides a sperm sialidase 1/3 detection kit, which is characterized in that the sperm sialidase 1/3 detection kit includes: fixative solution, permeabilization solution, buffer solution, blocking solution, cleaning solution, Antibody solution and fluorescent probe solution;
  • the antibody solution includes biotinylated antibody NEU1 or biotinylated antibody NEU3.
  • the concentration of biotinylated antibody NEU1 or biotinylated antibody NEU3 in the antibody solution is 5 to 10 mg/L.
  • the fluorescent probe solution includes R-phycoerythrin labeled streptavidin.
  • the concentration of R-phycoerythrin-labeled streptavidin in the fluorescent probe solution is 4 to 8 mg/L.
  • the fixative includes sodium dihydrogen phosphate, sodium hydrogen phosphate, paraformaldehyde and water.
  • the buffer includes sodium dihydrogen phosphate, sodium hydrogen phosphate, sodium chloride and water.
  • the permeating liquid includes polyethylene glycol octylphenyl ether, sodium dihydrogen phosphate, sodium hydrogen phosphate, sodium chloride and water.
  • the blocking solution includes bovine serum albumin, sodium dihydrogen phosphate, sodium hydrogen phosphate, sodium chloride and water.
  • the cleaning solution includes Tween 20, sodium dihydrogen phosphate, sodium hydrogen phosphate, sodium chloride and water.
  • the antibody solution includes bovine serum albumin, water and biotinylated antibody NEU1 or biotinylated antibody NEU3.
  • the fluorescent probe solution includes bovine serum albumin, R-phycoerythrin labeled streptavidin and water.
  • the concentration of paraformaldehyde in the fixative is 35 to 45 g/L
  • the concentration of sodium dihydrogen phosphate is 0.015 to 0.02 mol/L
  • the concentration of sodium hydrogen phosphate is 0.13 to 0.14 mol/L.
  • the concentration of sodium hydrogen phosphate in the buffer is 0.15-0.17 mol/L
  • the concentration of sodium dihydrogen phosphate is 0.035-0.04 mol/L
  • the concentration of sodium chloride is 0.15-0.16 mol/L.
  • the concentration of polyethylene glycol octyl phenyl ether in the permeabilization solution is 0.8-1.2 mL/L
  • the concentration of sodium dihydrogen phosphate is 0.005-0.0056 mol/L
  • the concentration of sodium hydrogen phosphate is 0.00117-0.00133 mol/L
  • the concentration of sodium chloride is 0.005 ⁇ 0.0053mol/L.
  • the concentration of bovine serum albumin in the blocking solution is 10-14g/L
  • the concentration of sodium dihydrogen phosphate is 0.005-0.0056mol/L
  • the concentration of sodium hydrogenphosphate is 0.00117-0.00133mol/L
  • the concentration of sodium chloride The concentration is 0.005 ⁇ 0.0053mol/L.
  • the concentration of Tween 20 in the cleaning solution is 0.8 ⁇ 1.2mL/L
  • the concentration of sodium dihydrogen phosphate is 0.005 ⁇ 0.0056mol/L
  • the concentration of sodium hydrogenphosphate is 0.00117 ⁇ 0.00133mol/L
  • the concentration of sodium chloride The concentration is 0.005 ⁇ 0.0053mol/L.
  • the present disclosure also provides a sperm sialidase 1/3 detection reagent.
  • the sperm sialidase 1/3 detection kit includes: fixative solution, permeabilization solution, buffer solution, blocking solution, cleaning solution, antibody solution and fluorescence Probe fluid;
  • the antibody solution includes biotinylated antibody NEU1 or biotinylated antibody NEU3.
  • the concentration of biotinylated antibody NEU1 or biotinylated antibody NEU3 in the antibody solution is 5 to 10 mg/L.
  • the fluorescent probe solution includes R-phycoerythrin labeled streptavidin.
  • the concentration of R-phycoerythrin-labeled streptavidin in the fluorescent probe solution is 4 to 8 mg/L.
  • the fixative includes sodium dihydrogen phosphate, sodium hydrogen phosphate, paraformaldehyde and water.
  • the buffer includes sodium dihydrogen phosphate, sodium hydrogen phosphate, sodium chloride and water.
  • the permeating liquid includes polyethylene glycol octylphenyl ether, sodium dihydrogen phosphate, sodium hydrogen phosphate, sodium chloride and water.
  • the blocking solution includes bovine serum albumin, sodium dihydrogen phosphate, sodium hydrogen phosphate, sodium chloride and water.
  • the cleaning solution includes Tween 20, sodium dihydrogen phosphate, sodium hydrogen phosphate, sodium chloride and water.
  • the antibody solution includes bovine serum albumin, water and biotinylated antibody NEU1 or biotinylated antibody NEU3.
  • the fluorescent probe solution includes bovine serum albumin, R-phycoerythrin labeled streptavidin and water.
  • the present disclosure also provides a method for preparing the sperm sialidase 1/3 detection kit described in any one of the above.
  • the method for preparing the sperm sialidase 1/3 detection kit includes: respectively preparing the fixative, The permeabilization solution, the buffer solution, the blocking solution, the cleaning solution, the antibody solution and the fluorescent probe solution, and then the fixative solution, the permeability solution, and the buffer solution , the blocking solution, the cleaning solution, the antibody solution and the fluorescent probe The liquid is divided and assembled into the sperm sialidase 1/3 detection kit.
  • the present disclosure also provides a method for detecting the expression level of sperm sialidase 1/3 using any one of the above sperm sialidase 1/3 detection kits, which detects the expression level of sperm sialidase 1/3
  • the method includes: taking a semen sample, using the cleaning solution to perform a first washing process on the semen sample to prepare a first sample liquid, and using the fixative solution to perform a fixation process on the first sample liquid.
  • the first sample is permeabilized using the permeabilizing liquid to obtain a third sample liquid, and the third sample liquid is washed for the second time using the cleaning liquid to obtain the third sample liquid.
  • sample liquids the fourth sample liquid is sealed with the blocking liquid to obtain a fifth sample liquid, the antibody liquid is added to the fifth sample liquid and the sixth sample liquid is obtained through the first incubation treatment.
  • Sample liquid use the cleaning liquid to perform a third washing process on the sixth sample liquid to prepare a seventh sample liquid, add the fluorescent probe liquid to the seventh sample liquid and undergo a second incubation process
  • An eighth sample liquid is prepared, the eighth sample liquid is washed a fourth time with the cleaning liquid to prepare a ninth sample liquid, and a flow cytometer is used to detect the sperm sialidase NEU1 in the ninth sample liquid. or the expression level of NEU3.
  • the blocking treatment includes adding the blocking solution to the fourth sample solution, letting it stand for 30 to 40 minutes, and then centrifuging to remove the supernatant.
  • the first incubation treatment includes adding the antibody solution to the fifth sample solution, incubating at 18-30°C for 2-2.5 hours, and then centrifuging to remove the supernatant.
  • the second incubation process includes adding the fluorescent probe solution to the seventh sample solution, incubating at 18-30°C for 1-1.5 hours, and then centrifuging to remove the supernatant.
  • the present disclosure also provides a method for diagnosing the fertility of a male subject, including:
  • the sample is semen.
  • Figure 1 is a scatter plot of the first flow detection of control group 1 of the present disclosure
  • Figure 2 is a second flow detection scatter plot of control group 1 of the present disclosure
  • Figure 3 is a flow cytometric detection histogram of control group 1 of the present disclosure
  • Figure 4 is a scatter plot of the first flow detection of control group 2 of the present disclosure
  • Figure 5 is a second flow detection scatter plot of control group 2 of the present disclosure.
  • Figure 6 is a flow cytometric detection histogram of control group 2 of the present disclosure.
  • Figure 7 is a scatter plot of the first flow detection in Embodiment 1 of the present disclosure.
  • Figure 8 is a second flow detection scatter plot of Embodiment 1 of the present disclosure.
  • Figure 9 is a flow detection histogram of Embodiment 1 of the present disclosure.
  • Figure 10 is a scatter plot of the first flow detection in Embodiment 2 of the present disclosure.
  • Figure 11 is a second flow detection scatter plot of Embodiment 2 of the present disclosure.
  • Figure 12 is a flow detection histogram of Embodiment 2 of the present disclosure.
  • Figure 13 is a scatter plot of the first flow detection of Comparative Example 1 of the present disclosure.
  • Figure 14 is a second flow detection scatter plot of Comparative Example 1 of the present disclosure.
  • Figure 15 is a flow detection histogram of Comparative Example 1 of the present disclosure.
  • Figure 16 is a scatter plot of the first flow detection of Comparative Example 2 of the present disclosure.
  • Figure 17 is a second flow detection scatter plot of Comparative Example 2 of the present disclosure.
  • Figure 18 is a flow detection histogram of Comparative Example 2 of the present disclosure.
  • Figure 19 is a first flow detection scatter plot of Comparative Example 3 of the present disclosure.
  • Figure 20 is a second flow detection scatter plot of Comparative Example 3 of the present disclosure.
  • Figure 21 is a flow detection histogram of Comparative Example 3 of the present disclosure.
  • Figure 22 is a first flow detection scatter plot of Comparative Example 4 of the present disclosure.
  • Figure 23 is a second flow detection scatter plot of Comparative Example 4 of the present disclosure.
  • Figure 24 is a flow detection histogram of Comparative Example 4 of the present disclosure.
  • Figure 25 is a first flow detection scatter plot of Comparative Example 5 of the present disclosure.
  • Figure 26 is a second flow detection scatter plot of Comparative Example 5 of the present disclosure.
  • Figure 27 is a flow detection histogram of Comparative Example 5 of the present disclosure.
  • Figure 28 is a first flow detection scatter plot of Comparative Example 6 of the present disclosure.
  • Figure 29 is a second flow detection scatter plot of Comparative Example 6 of the present disclosure.
  • Figure 30 is a flow detection histogram of Comparative Example 6 of the present disclosure.
  • Figure 31 is a first flow detection scatter plot of Comparative Example 7 of the present disclosure.
  • Figure 32 is a second flow detection scatter plot of Comparative Example 7 of the present disclosure.
  • Figure 33 is a flow detection histogram of Comparative Example 7 of the present disclosure.
  • Figure 34 is a first flow detection scatter plot of Comparative Example 8 of the present disclosure.
  • Figure 35 is a second flow detection scatter plot of Comparative Example 8 of the present disclosure.
  • Figure 36 is a flow detection histogram of Comparative Example 8 of the present disclosure.
  • sperm sialidase 1/3 detection kit and its preparation method and a method for detecting sperm sialidase 1/3 expression levels according to the embodiments of the present disclosure:
  • One embodiment of the present disclosure provides a sperm sialidase 1/3 detection kit, which includes a fixative solution, a permeabilizing solution, a buffer solution, a blocking solution, a cleaning solution, an antibody solution and a fluorescent probe solution.
  • sperm sialidase 1/3 refers to two types of sialidase found in sperm. They are named NEU1 and NEU1 respectively based on their differences in cleavage substrates and intracellular distribution. NEU3, which can affect sperm quality, is referred to as “sperm sialidase 1/3" in this article.
  • NEU3 which can affect sperm quality
  • sperm sialidase 1/3 is referred to as "sperm sialidase 1/3" in this article.
  • the sialic acid on the sperm membrane is shed from the sperm cell membrane in the form of a single sugar molecule.
  • the sialidase (NEU1 or NEU3) on the sperm membrane is It falls off due to its fluidity, and participates in shearing to cause single sialic acid molecules to fall off.
  • sperm sialidase 1/3 is mainly involved in sperm capacitation, sperm-egg recognition, and sperm-egg binding energy during fertilization, and can be used to evaluate sperm fertilization ability and fertility outcomes.
  • the fixative includes sodium dihydrogen phosphate, sodium hydrogen phosphate, paraformaldehyde (PFA) and water.
  • the concentration of paraformaldehyde in the fixative is 35 to 45 g/L
  • the concentration of sodium dihydrogen phosphate is 0.015 to 0.02 mol/L
  • the concentration of sodium hydrogen phosphate is 0.13 to 0.14 mol/L.
  • the pH value of the fixative solution is 7.0-7.4.
  • Buffers include sodium phosphate dibasic, sodium hydrogen phosphate, sodium chloride, and water.
  • the concentration of sodium hydrogen phosphate in the buffer is 0.15-0.17 mol/L
  • the concentration of sodium dihydrogen phosphate is 0.035-0.04 mol/L
  • the concentration of sodium chloride is 0.15-0.16 mol/L.
  • the pH value of the buffer solution is 7.2-7.4.
  • the permeabilizing fluid includes polyethylene glycol octylphenyl ether (Triton X-100), sodium dihydrogen phosphate, sodium hydrogen phosphate, sodium chloride and water.
  • the concentration of polyethylene glycol octyl phenyl ether in the permeabilization solution is 0.8-1.2 mL/L
  • the concentration of sodium dihydrogen phosphate is 0.005-0.0056 mol/L
  • the concentration of sodium hydrogen phosphate is 0.00117-0.00133 mol/L
  • the concentration of sodium chloride is 0.005 ⁇ 0.0053mol/L.
  • Blocking solutions include bovine serum albumin (BSA), sodium phosphate dibasic, sodium hydrogen phosphate, sodium chloride, and water.
  • BSA bovine serum albumin
  • sodium phosphate dibasic sodium phosphate dibasic
  • sodium hydrogen phosphate sodium chloride
  • water water
  • the concentration of bovine serum albumin in the blocking solution is 10-14g/L
  • the concentration of sodium dihydrogen phosphate is 0.005-0.0056mol/L
  • the concentration of sodium hydrogenphosphate is 0.00117-0.00133mol/L
  • the concentration of sodium chloride The concentration is 0.005 ⁇ 0.0053mol/L.
  • the cleaning solution includes Tween 20, sodium dihydrogen phosphate, sodium hydrogen phosphate, sodium chloride and water.
  • the concentration of Tween 20 in the cleaning solution is 0.8 ⁇ 1.2mL/L
  • the concentration of sodium dihydrogen phosphate is 0.005 ⁇ 0.0056mol/L
  • the concentration of sodium hydrogenphosphate is 0.00117 ⁇ 0.00133mol/L
  • the concentration of sodium chloride The concentration is 0.005 ⁇ 0.0053mol/L.
  • the antibody solution includes bovine serum albumin, water, and biotinylated antibody NEU1 or biotinylated antibody NEU3.
  • the disclosed sperm sialidase 1/3 detection kit can establish an immune flow indirect labeling method, which has strong specificity and high sensitivity, and can detect the expression level of sperm sialidase NEU1 or NEU3 in semen.
  • the sperm sialidase 1/3 detection kit of the present disclosure has high detection accuracy, and has the characteristics of rapidity and large single detection volume.
  • biotinylated antibody NEU1 or biotinylated antibody NEU3 is as follows:
  • Protein NEU1 (a total of 415AAs, (the sequence is shown in SEQ ID NO:2)):
  • NCBI registration number: NM_006656.5 (Website link: https://www.ncbi.nlm.nih.gov/nuccore/NM_006656.5?report fasta), Homo sapiens neuraminidase 3, NEU3, mRNA (sequence shown in SEQ ID NO:3)
  • Protein NEU3 (a total of 461Aas, the sequence is shown in SEQ ID NO:4):
  • SDS-PAGE test generally 8 to 10 mice are immunized, and the protein purity is at least 80%.
  • NEU1 or NEU3 Tail blood collection was performed one week after the third immunization.
  • rat tail blood collection method fix the animal and expose the rat tail, immerse the tail in warm water at 45-50°C for several minutes to make the veins congest, wipe dry, and then wipe and disinfect with an alcohol cotton ball. Cut off about 0.2-0.3cm from the tip of the tail. Wipe away the first drop of blood. Then use a capillary tube to quantitatively draw tail blood, and perform titer testing.
  • mice The titers of 5 mice all reached 1:128000.
  • Fusion Use a fusion method that combines liquid and semi-solid to achieve high-throughput screening.
  • Subclone screening Clone amplification/supernatant collection, screening and sorting of antibody affinity, adding the final antibody application experiment to the screening stage to achieve anchor screening of high-quality antibodies
  • Expansion of positive clones 2-3 rounds of subcloning Screen to obtain hybridoma cell lines that stably secrete positive antibodies, select 3-5 well-verified strains for expanded culture, and freeze the rest for backup.
  • mice sensitization Balb/c mice are sensitized to liquid paraffin, preferably multiparous mice, and the injection volume is 500 ⁇ l/mouse. Hybridization can be injected after 10 days.
  • Inject cells Collect hybridoma cells and wash them twice with PBS. Take 1 million to 1.5 million cells and inject them into the abdominal cavity of mice. One week later, it can be seen that the mice are inactive and the abdominal cavity of the mice is swollen.
  • Protein A affinity purification of the antibody, dialyze and concentrate, measure the concentration, adjust the concentration to an integer, and measure the purity.
  • the concentration of bovine serum albumin in the antibody solution is 10-14g/L
  • the concentration of biotinylated antibody NEU1 or biotinylated antibody NEU3 is 5-10mg/L, such as 5mg/L, 6mg/L, 7mg/L. L, 8 mg/L, 9 mg/L or 10 mg/L, or an interval value between any two of the above endpoint values.
  • the fluorescent probe solution includes bovine serum albumin and R-phycoerythrin-labeled streptavidin (SA-PE).
  • the concentration of bovine serum albumin in the fluorescent probe solution is 10-14g/L
  • the concentration of R-phycoerythrin-labeled streptavidin is 4-8mg/L, such as 4mg/L, 4.5mg/L.
  • L 5mg/L, 5.5mg/L, 6mg/L, 6.5mg/L, 7mg/L, 7.5mg/L or 8mg/L, or an interval value between any two of the above endpoint values.
  • the disclosed sperm sialidase 1/3 detection kit combines biotinylated antibodies with R-phycoerythrin-labeled streptavidin, and utilizes the reaction principle of cascade amplification to enhance reaction sensitivity and reduce non-specific interference.
  • the sperm sialidase 1/3 detection kit includes at least 5 mL fixative solution, at least 5 mL permeabilization solution, at least 25 mL buffer solution, at least 5 mL blocking solution, at least 10 mL cleaning solution, at least 50 ⁇ L antibody solution and at least 2 mL fluorescent probe. Injections.
  • the disclosed sperm sialidase 1/3 detection kit can establish an immune flow indirect labeling method, which has strong specificity and high sensitivity, and can detect the expression level of sperm sialidase NEU1 or NEU3 in semen.
  • the disclosed sperm sialidase 1/3 detection kit uses biotinylated antibodies combined with R-phycoerythrin-labeled streptavidin, and utilizes the reaction principle of cascade amplification to enhance reaction sensitivity and reduce non-specificity. Interference, high detection accuracy, fast speed and large single detection volume.
  • One embodiment of the present disclosure also provides a method for preparing the above-mentioned sperm sialidase 1/3 detection kit, which includes: separately preparing a fixative solution, a permeabilizing solution, a buffer solution, a blocking solution, a cleaning solution, an antibody solution and a fluorescent probe. injection solution, and then divide the fixative solution, permeabilization solution, buffer solution, blocking solution, cleaning solution, antibody solution and fluorescent probe solution into a sperm sialidase 1/3 detection kit.
  • the preparation method of the sperm sialidase 1/3 detection kit disclosed by the present disclosure is simple and simple, and the prepared fixative solution, permeabilizing solution, buffer solution, blocking solution, cleaning solution, antibody solution and fluorescent probe solution are all divided and packaged. During assembly, different reagents can be selected according to different steps.
  • the present disclosure also provides a method for detecting the expression level of sperm sialidase 1/3 using the above-mentioned sperm sialidase 1/3 detection kit, which includes the following steps:
  • the disclosed method for detecting the expression level of sperm sialidase 1/3 is relatively simple, fast and highly accurate.
  • One embodiment of the present disclosure also provides a method for diagnosing the fertility of a male subject, including:
  • A) Contact the sperm sialidase 1/3 detection kit or the sperm sialidase 1/3 detection reagent with a sample from the subject under conditions sufficient for the binding reaction to occur to perform a binding reaction;
  • the sample is semen.
  • the present disclosure provides a sperm sialidase 1/3 detection kit and its preparation method, and a method for detecting the expression level of sperm sialidase 1/3, which can quickly detect the expression level of sperm sialidase NEU1 or NEU3 in semen. .
  • sperm sialidase 1/3 detection kit and its preparation method and the method for detecting the expression level of sperm sialidase 1/3 of the present disclosure will be described in further detail below with reference to the examples.
  • the embodiments of the present disclosure provide a sperm sialidase NEU1 detection kit and its preparation method, and a method for detecting the expression level of sperm sialidase NEU1.
  • the embodiments of the present disclosure provide a sperm sialidase NEU3 detection kit and a preparation method thereof, as well as a method for detecting the expression level of sperm sialidase NEU3.
  • Example 2 is based on Example 1 and changes the biotinylated antibody NEU1 into the biotinylated antibody NEU3, leaving the others unchanged.
  • the comparative example of the present disclosure provides a sperm sialidase NEU1 detection kit and its preparation method, and a method for detecting the expression level of sperm sialidase NEU1.
  • the SA-PE was changed to fluorescein isothiocyanate isomer (FITC) based on Example 1, and the others remained unchanged.
  • FITC fluorescein isothiocyanate isomer
  • the comparative example of the present disclosure provides a sperm sialidase NEU3 detection kit and its preparation method, and a method for detecting the expression level of sperm sialidase NEU3.
  • the SA-PE was changed to FITC based on Example 2, and the others remained unchanged.
  • the comparative example of the present disclosure provides a sperm sialidase NEU1 detection kit and its preparation method, and a method for detecting the expression level of sperm sialidase NEU1.
  • the time of the fixation treatment was changed based on Example 1, from leaving it at 25°C for 10 minutes to leaving it at 25°C for 20 minutes.
  • the comparative example of the present disclosure provides a sperm sialidase NEU1 detection kit and its preparation method, and a method for detecting the expression level of sperm sialidase NEU1.
  • the time of the sealing treatment was changed based on Example 1, from leaving it at 25°C for 30 minutes to leaving it at 25°C for 20 minutes.
  • the comparative example of the present disclosure provides a sperm sialidase NEU1 detection kit and its preparation method, and a method for detecting the expression level of sperm sialidase NEU1.
  • the time of the first incubation treatment was changed based on Example 1, from incubation at 25°C for 2 hours to incubation at 25°C for 1 hour.
  • the comparative example of the present disclosure provides a sperm sialidase NEU3 detection kit and its preparation method, and a method for detecting the expression level of sperm sialidase NEU3.
  • the time of the first incubation treatment was changed based on Example 2, from incubation at 25°C for 2 hours to incubation at 25°C for 1 hour.
  • the comparative example of the present disclosure provides a sperm sialidase NEU1 detection kit and its preparation method, and a method for detecting the expression level of sperm sialidase NEU1.
  • the time of the second incubation treatment was changed based on Example 1, from incubation at 25°C for 1 h to incubation at 25°C for 0.5h.
  • the comparative example of the present disclosure provides a sperm sialidase NEU3 detection kit and its preparation method, and a method for detecting the expression level of sperm sialidase NEU3.
  • the time of the second incubation treatment was changed based on Example 2, from incubation at 25°C for 1 hour to incubation at 25°C for 0.5 hour.
  • Control group 1 is based on Example 1, and no antibody solution is added in the first incubation treatment.
  • the control group 2 is based on Example 2, and in the first incubation treatment. No antibody solution is added.
  • the flow detection scatter plots and flow detection histograms of Control Groups 1 to 2 are shown in Figures 1 to 6 respectively.
  • the flow detection scatter plots and flow detection histograms of Examples 1 to 2 and Comparative Examples 1 to 8 As shown in Figures 7 to 36, the positive ratios of Examples 1 to 2 and Comparative Examples 1 to 8 are as shown in Table 1.
  • the disclosed sperm sialidase 1/3 detection kit can establish an immune flow indirect labeling method, which has strong specificity and high sensitivity, and can detect the expression level of sperm sialidase NEU1 or NEU3 in semen. At the same time, the disclosed sperm sialidase 1/3 detection kit has high detection accuracy, and has the characteristics of rapidity and large single detection volume, so it has excellent practical performance and broad market prospects.

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Abstract

精子唾液酸酶1/3检测试剂盒及其制备方法、检测精子唾液酸酶1/3表达水平的方法，其中，精子唾液酸酶1/3检测试剂盒包括：固定液、通透液、缓冲液、封闭液、清洗液、抗体液和荧光探针液，抗体液包括生物素化抗体NEU1或生物素化抗体NEU3。精子唾液酸酶1/3检测试剂盒能够建立免疫流式间接标记法，特异性强，具有较高的灵敏度，能对精液中精子唾液酸酶NEU1或NEU3的表达水平进行检测。同时，精子唾液酸酶1/3检测试剂盒检测准确性高，且具有快速和单次检测量大的特点。

Description

精子唾液酸酶1/3检测试剂盒及其制备方法、检测精子唾液酸酶1/3表达水平的方法
相关申请的交叉引用
本公开要求于2022年06月21日提交中国专利局的申请号为CN202210703448.4、名称为“精子唾液酸酶1/3检测试剂盒及其制备方法、检测精子唾液酸酶1/3表达水平的方法”的中国专利申请的优先权，其全部内容通过引用结合在本公开中。
技术领域
本公开涉及检测技术领域，具体而言，涉及一种精子唾液酸酶1/3检测试剂盒及其制备方法、检测精子唾液酸酶1/3表达水平的方法。
背景技术
随着社会的不断发展，不育夫妇越来越多，全球不育夫妇占已婚夫妇10％-15％。据统计，男性生殖能力异常的比例不低于50％。传统的精液常规分析一直是用于判断男性生育力的最基本临床指标。多年来，临床上对绝大部分的男性不育患者的真正不育原因仍然不清楚。尤其是临床上大约有三分之一不育患者，男性的常规精液分析结果均正常和接近正常。临床上把这类患者划为不明原因不育症。常规精液分析只测定精子的数量，存活力，活动率和形态。这些指标只能反映最基本的精液质量，不能反映所有的精子功能。
发明内容
本公开提供一种精子唾液酸酶1/3检测试剂盒，其特征在于，所述精子唾液酸酶1/3检测试剂盒包括：固定液、通透液、缓冲液、封闭液、清洗液、抗体液和荧光探针液；
所述抗体液包括生物素化抗体NEU1或生物素化抗体NEU3。
可选地，所述抗体液中生物素化抗体NEU1或生物素化抗体NEU3浓度为5～10mg/L。
可选地，所述荧光探针液包括R-藻红蛋白标记链霉亲和素。
可选地，所述荧光探针液中R-藻红蛋白标记链霉亲和素的浓度为4～8mg/L。
可选地，所述固定液包括磷酸二氢钠、磷酸氢钠、多聚甲醛和水。
可选地，所述缓冲液包括磷酸二氢钠、磷酸氢钠、氯化钠和水。
可选地，所述通透液包括聚乙二醇辛基苯基醚、磷酸二氢钠、磷酸氢钠、氯化钠和水。
可选地，所述封闭液包括牛血清白蛋白、磷酸二氢钠、磷酸氢钠、氯化钠和水。
可选地，所述清洗液包括吐温20、磷酸二氢钠、磷酸氢钠、氯化钠和水。
可选地，所述抗体液包括牛血清白蛋白、水以及生物素化抗体NEU1或生物素化抗体NEU3。
可选地，所述荧光探针液包括牛血清白蛋白、R-藻红蛋白标记链霉亲和素和水。
可选地，固定液中多聚甲醛的浓度为35～45g/L，磷酸二氢钠的浓度为0.015～0.02mol/L，磷酸氢钠的浓度为0.13～0.14mol/L。
可选地，缓冲液中磷酸氢钠的浓度为0.15～0.17mol/L，磷酸二氢钠的浓度为0.035～0.04mol/L，氯化钠的浓度为0.15～0.16mol/L。
可选地，通透液中聚乙二醇辛基苯基醚的浓度为0.8～1.2mL/L，磷酸二氢钠的浓度为0.005～0.0056mol/L，磷酸氢钠的浓度为0.00117～0.00133mol/L，氯化钠的浓度为0.005～0.0053mol/L。
可选地，封闭液中牛血清白蛋白的浓度为10～14g/L，磷酸二氢钠的浓度为0.005～0.0056mol/L，磷酸氢钠的浓度为0.00117～0.00133mol/L，氯化钠的浓度为0.005～0.0053mol/L。
可选地，清洗液中吐温20的浓度为0.8～1.2mL/L，磷酸二氢钠的浓度为0.005～0.0056mol/L，磷酸氢钠的浓度为0.00117～0.00133mol/L，氯化钠的浓度为0.005～0.0053mol/L。
本公开还提供一种精子唾液酸酶1/3检测试剂，所述精子唾液酸酶1/3检测试剂盒包括：固定液、通透液、缓冲液、封闭液、清洗液、抗体液和荧光探针液；
所述抗体液包括生物素化抗体NEU1或生物素化抗体NEU3。
可选地，所述抗体液中生物素化抗体NEU1或生物素化抗体NEU3浓度为5～10mg/L。
可选地，所述荧光探针液包括R-藻红蛋白标记链霉亲和素。
可选地，所述荧光探针液中R-藻红蛋白标记链霉亲和素的浓度为4～8mg/L。
可选地，所述固定液包括磷酸二氢钠、磷酸氢钠、多聚甲醛和水。
可选地，所述缓冲液包括磷酸二氢钠、磷酸氢钠、氯化钠和水。
可选地，所述通透液包括聚乙二醇辛基苯基醚、磷酸二氢钠、磷酸氢钠、氯化钠和水。
可选地，所述封闭液包括牛血清白蛋白、磷酸二氢钠、磷酸氢钠、氯化钠和水。
可选地，所述清洗液包括吐温20、磷酸二氢钠、磷酸氢钠、氯化钠和水。
可选地，所述抗体液包括牛血清白蛋白、水以及生物素化抗体NEU1或生物素化抗体NEU3。
可选地，所述荧光探针液包括牛血清白蛋白、R-藻红蛋白标记链霉亲和素和水。
本公开还提供制备上文任一项所述的精子唾液酸酶1/3检测试剂盒的方法，所述制备精子唾液酸酶1/3检测试剂盒的方法包括：分别制备所述固定液、所述通透液、所述缓冲液、所述封闭液、所述清洗液、所述抗体液和所述荧光探针液，然后将所述固定液、所述通透液、所述缓冲液、所述封闭液、所述清洗液、所述抗体液和所述荧光探针 液分装后组装成所述精子唾液酸酶1/3检测试剂盒。
本公开还提供一种利用上文任一项所述的精子唾液酸酶1/3检测试剂盒检测精子唾液酸酶1/3表达水平的方法，所述检测精子唾液酸酶1/3表达水平的方法包括：取精液样品，采用所述清洗液对所述精液样品进行第一次洗涤处理制得第一样品液，采用所述固定液对所述第一样品液进行固定处理制得第二样品液，采用所述通透液对所述第一样品进行通透处理制得第三样品液，采用所述清洗液对所述第三样品液进行第二次洗涤处理制得第四样品液，采用所述封闭液对所述第四样品液进行封闭处理制得第五样品液，在所述第五样品液中加入所述抗体液并经第一次孵育处理制得第六样品液，采用所述清洗液对所述第六样品液进行第三次洗涤处理制得第七样品液，在所述第七样品液中加入所述荧光探针液并经第二次孵育处理制得第八样品液，采用所述清洗液对所述第八样品液进行第四次洗涤处理制得第九样品液，采用流式细胞仪检测所述第九样品液中精子唾液酸酶NEU1或NEU3的表达水平。
可选地，所述封闭处理包括在所述第四样品液中加入所述封闭液，静止30～40min，然后离心去除上清液。
可选地，所述第一次孵育处理包括在所述第五样品液中加入所述抗体液，在18～30℃下孵育2～2.5h，然后离心去除上清液。
可选地，所述第二次孵育处理包括在所述第七样品液中加入所述荧光探针液，在18～30℃下孵育1～1.5h，然后离心去除上清液。
可选地，在上机检测前，在所述第九样品液中加入所述缓冲液。
本公开还提供一种诊断男性受试者生育能力的方法，包括：
A)在足以发生结合反应的条件下，使上文任一项所述的精子唾液酸酶1/3检测试剂盒或上文任一项所述的精子唾液酸酶1/3检测试剂与来自所述受试者的样品接触以进行结合反应；以及
B)检测结合反应产生的免疫复合物。
可选地，所述样品为精液。
附图说明
为了更清楚地说明本公开实施例的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，应当理解，以下附图仅示出了本公开的某些实施例，因此不应被看作是对范围的限定，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他相关的附图。
图1为本公开对照组1的第一流式检测散点图；
图2为本公开对照组1的第二流式检测散点图；
图3为本公开对照组1的流式检测直方图；
图4为本公开对照组2的第一流式检测散点图；
图5为本公开对照组2的第二流式检测散点图；
图6为本公开对照组2的流式检测直方图；
图7为本公开实施例1的第一流式检测散点图；
图8为本公开实施例1的第二流式检测散点图；
图9为本公开实施例1的流式检测直方图；
图10为本公开实施例2的第一流式检测散点图；
图11为本公开实施例2的第二流式检测散点图；
图12为本公开实施例2的流式检测直方图；
图13为本公开对比例1的第一流式检测散点图；
图14为本公开对比例1的第二流式检测散点图；
图15为本公开对比例1的流式检测直方图；
图16为本公开对比例2的第一流式检测散点图；
图17为本公开对比例2的第二流式检测散点图；
图18为本公开对比例2的流式检测直方图；
图19为本公开对比例3的第一流式检测散点图；
图20为本公开对比例3的第二流式检测散点图；
图21为本公开对比例3的流式检测直方图；
图22为本公开对比例4的第一流式检测散点图；
图23为本公开对比例4的第二流式检测散点图；
图24为本公开对比例4的流式检测直方图；
图25为本公开对比例5的第一流式检测散点图；
图26为本公开对比例5的第二流式检测散点图；
图27为本公开对比例5的流式检测直方图；
图28为本公开对比例6的第一流式检测散点图；
图29为本公开对比例6的第二流式检测散点图；
图30为本公开对比例6的流式检测直方图；
图31为本公开对比例7的第一流式检测散点图；
图32为本公开对比例7的第二流式检测散点图；
图33为本公开对比例7的流式检测直方图；
图34为本公开对比例8的第一流式检测散点图；
图35为本公开对比例8的第二流式检测散点图；
图36为本公开对比例8的流式检测直方图。
具体实施方式
下面将结合实施例对本公开的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本公开，而不应视为限制本公开的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
以下针对本公开实施方式的一种精子唾液酸酶1/3检测试剂盒及其制备方法、检测精子唾液酸酶1/3表达水平的方法进行说明：
本公开一实施方式提供一种精子唾液酸酶1/3检测试剂盒，其包括固定液、通透液、缓冲液、封闭液、清洗液、抗体液和荧光探针液。
如本文所用，术语“精子唾液酸酶1/3”是指在精子上共发现了2种唾液酸酶，根据剪切底物的不同以及在细胞内分布的不同，将其分别命名为NEU1与NEU3，并且能够影响精子质量，在本文中简称为“精子唾液酸酶1/3”。唾液酸在人的体外获能过程中，精子膜上的唾液酸以单个糖分子的方式从精子的细胞膜上脱落，而与此同时，精子膜上的唾液酸酶(NEU1或NEU3)由于精子膜的流动性而脱落，并参与剪切致单个唾液酸分子脱落。因此，对精液样品中NEU1或NEU3进行检测，精子唾液酸酶1/3在受精过程中主要参与精子获能、精卵识别和精卵结合能，可用于对精子受精能力和生育结局的评估。
可选地，固定液包括磷酸二氢钠、磷酸氢钠、多聚甲醛(PFA)和水。
可选地，固定液中多聚甲醛的浓度为35～45g/L，磷酸二氢钠的浓度为0.015～0.02mol/L，磷酸氢钠的浓度为0.13～0.14mol/L。
可选地，固定液的pH值为7.0～7.4。
缓冲液包括磷酸二氢钠、磷酸氢钠、氯化钠和水。
可选地，缓冲液中磷酸氢钠的浓度为0.15～0.17mol/L，磷酸二氢钠的浓度为0.035～0.04mol/L，氯化钠的浓度为0.15～0.16mol/L。
可选地，缓冲液的pH值为7.2～7.4。
通透液包括聚乙二醇辛基苯基醚(Triton X-100)、磷酸二氢钠、磷酸氢钠、氯化钠和水。
可选地，通透液中聚乙二醇辛基苯基醚的浓度为0.8～1.2mL/L，磷酸二氢钠的浓度为0.005～0.0056mol/L，磷酸氢钠的浓度为0.00117～0.00133mol/L，氯化钠的浓度为0.005～0.0053mol/L。
封闭液包括牛血清白蛋白(BSA)、磷酸二氢钠、磷酸氢钠、氯化钠和水。
可选地，封闭液中牛血清白蛋白的浓度为10～14g/L，磷酸二氢钠的浓度为0.005～0.0056mol/L，磷酸氢钠的浓度为0.00117～0.00133mol/L，氯化钠的浓度为0.005～0.0053mol/L。
清洗液包括吐温20(Tween 20)、磷酸二氢钠、磷酸氢钠、氯化钠和水。
可选地，清洗液中吐温20的浓度为0.8～1.2mL/L，磷酸二氢钠的浓度为0.005～0.0056mol/L，磷酸氢钠的浓度为0.00117～0.00133mol/L，氯化钠的浓度为0.005～0.0053mol/L。
抗体液包括牛血清白蛋白、水以及生物素化抗体NEU1或生物素化抗体NEU3。
本公开的精子唾液酸酶1/3检测试剂盒能够建立免疫流式间接标记法，其具有特异性强及较高的灵敏度，能对精液中精子唾液酸酶NEU1或NEU3的表达水平进行检测。同时，本公开的精子唾液酸酶1/3检测试剂盒检测准确性高，且具有快速和单次检测量大的特点。
可选地，生物素化抗体NEU1或生物素化抗体NEU3的制备方法如下：
S1、抗原制备
NEU1基因的基因序列：
NCBI登录号：NM_000434.3(网址链接：https://www.ncbi.nlm.nih.gov/nuccore/NM_000434.3？report＝fasta)，智人唾液酸酶(Homo sapiens neuraminidase 1,NEU1),mRNA(序列如SEQ ID NO:1所示)

蛋白NEU1(一共415AAs，(序列如SEQ ID NO:2所示))：
>sp|Q99519|NEUR1_HUMAN Sialidase-1OS＝Homo sapiens OX＝9606GN＝NEU1PE＝1SV＝1
(该序列可以通过以下网址链接获得：https://rest.uniprot.org/uniprotkb/Q99519.fasta)
NEU3基因的基因序列：
NCBI登录号：NM_006656.5(网址链接：https://www.ncbi.nlm.nih.gov/nuccore/NM_006656.5？report＝fasta)，智人唾液酸酶(Homo sapiens neuraminidase 3,NEU3),mRNA(序列如SEQ ID NO:3所示)
蛋白NEU3(一共461Aas，序列如SEQ ID NO:4所示)：
>sp|Q9UQ49-2|NEUR3_HUMAN Isoform 2of Sialidase-3OS＝Homo sapiens OX＝9606GN＝NEU3
(该序列可以通过以下网页链接获得：https://rest.uniprot.org/uniprotkb/Q9UQ49-2.fasta)

设计引物，构建pPIC9K的表达载体，构建完成后，测序结果100％正确无突变，进入小试验证，尽量通过助溶标签和温度诱导的方案实现分泌表达，采用金属螯合镍柱进行亲和层析，纯化蛋白。
S2、抗原质控
SDS-PAGE检测，一般免疫8～10只小鼠，蛋白纯度至少在80％以上。
S3、小鼠免疫
表1小鼠免疫
S4、尾血采集及效价检测
NEU1或NEU3：三免疫一周后进行尾血采集。
少量取血：鼠尾采血法——固定动物并露出鼠尾，将尾部侵入45-50℃温水中数分钟，使静脉充血，擦干，再用酒精棉球擦拭消毒。剪掉尾尖约0.2-0.3cm。拭去第一滴血。然后用毛细管定量吸取尾血，并进行效价检测。
表2效价检测数据
5只小鼠效价均达到1：128000。
S5、细胞融合及阳性克隆筛选
融合：采用液体和半固体相结合的融合方式实现高通量筛选。
亚克隆筛选：克隆扩增/上清收集，并对抗体亲和力做筛选排序，将最终抗体应用实验增加到筛选阶段，实现高质量抗体的锚定筛选阳性克隆扩大培养：经2-3轮亚克隆筛选获取稳定分泌阳性抗体的杂交瘤细胞株，选取验证较好的3-5株进行扩大培养，其余冻存备份。
S6、腹水制备及抗体纯化
小鼠致敏：液体石蜡致敏Balb/c小鼠，以经产鼠为宜，注射体积500μl/只。10天后可注射杂交。
瘤细胞制备腹水。
注射细胞：收集杂交瘤细胞并用PBS洗涤两遍，取100到150万细胞注射于小鼠腹腔，一周后可见小鼠状态不活跃并且小鼠的腹腔肿大。
腹水采集：注射细胞一周后，小鼠腹腔肿大时，用无菌注射器于小鼠腹腔采集腹水，每隔一到两天采集一次，多次反复采集直到小鼠自然死亡。采集到的腹水分装归类最后于-20℃冰箱保存。
采用蛋白A(Protein A)亲和纯化抗体，透析浓缩，浓度测定，浓度调整为整数，纯度测定。
对纯化的抗体进行效价检测
S7、抗体配对筛选
使用表达抗原及阳性样本，选择最合适的抗体配对。
可选地，抗体液中牛血清白蛋白的浓度为10～14g/L，生物素化抗体NEU1或生物素化抗体NEU3浓度为5～10mg/L，诸如5mg/L、6mg/L、7mg/L、8mg/L、9mg/L或10mg/L，或者上述任意两个端点值之间的区间值。
荧光探针液包括牛血清白蛋白和R-藻红蛋白标记链霉亲和素(SA-PE)。
可选地，荧光探针液中牛血清白蛋白的浓度为10～14g/L，R-藻红蛋白标记链霉亲和素的浓度为4～8mg/L，诸如4mg/L、4.5mg/L、5mg/L、5.5mg/L、6mg/L、6.5mg/L、7mg/L、7.5mg/L或8mg/L，或者上述任意两个端点值之间的区间值。
本公开的精子唾液酸酶1/3检测试剂盒采用生物素化抗体与R-藻红蛋白标记链霉亲和素结合，利用级联放大的反应原理，增强了反应灵敏度，降低非特异性干扰。
可选地，精子唾液酸酶1/3检测试剂盒包括至少5mL固定液、至少5mL通透液、至少25mL缓冲液、至少5mL封闭液、至少10mL清洗液、至少50μL抗体液和至少2mL荧光探针液。
本公开的精子唾液酸酶1/3检测试剂盒能够建立免疫流式间接标记法，其具有特异性强及较高的灵敏度，能对精液中精子唾液酸酶NEU1或NEU3的表达水平进行检测。同时，本公开的精子唾液酸酶1/3检测试剂盒采用生物素化抗体与R-藻红蛋白标记链霉亲和素结合，利用级联放大的反应原理，增强了反应灵敏度，降低非特异性干扰，检测准确性高，且具有快速和单次检测量大的特点。
本公开一实施方式还提供一种上述精子唾液酸酶1/3检测试剂盒的制备方法，其包括：分别制备固定液、通透液、缓冲液、封闭液、清洗液、抗体液和荧光探针液，然后将固定液、通透液、缓冲液、封闭液、清洗液、抗体液和荧光探针液分装后组装成精子唾液酸酶1/3检测试剂盒。
本公开的精子唾液酸酶1/3检测试剂盒的制备方法简便，且制得的固定液、通透液、缓冲液、封闭液、清洗液、抗体液和荧光探针液均分装后再组装，在使用时可以根据不同的步骤选取不同的试剂。
本公开还提供一种利用上述的精子唾液酸酶1/3检测试剂盒检测精子唾液酸酶1/3表达水平的方法，其包括以下步骤：
S1、第一次洗涤处理
取0.5mL精液样本，加入1～2mL缓冲液，在3000～3500rpm的转速下离心5～8min，去除上清液，重复上述操作3～5次后，加入1～2mL缓冲液，制得第一样品液。
S2、固定处理
取200μL第一样品液，在3000～3500rpm的转速下离心5～8min，去除上清液，加入200～300μL固定液，5～30℃下静置10～15min，在3000～3500rpm的转速下离心5～8min，去除上清液，加入200～300μL缓冲液，在3000～3500rpm的转速下离心5～8min，去除上清液，制得第二样品液。
S3、通透处理
取制得的第二样品液，加入200～300μL通透液，5～30℃下静置15～20min，在3000～3500rpm的转速下离心5～8min，去除上清液，制得第三样品液。
S4、第二次洗涤处理
取制得的第三样品液，加入200～300μL缓冲液，在3000～3500rpm的转速下离心5～8min，去除上清液，制得第四样品液。
S5、封闭处理
取制得的第四样品液，加入200～300μL封闭液，5～30℃下静置30～40min，在3000～3500rpm的转速下离心2～5min，去除上清液，制得第五样品液。
S6、第一次孵育处理
取制得的第五样品液，加入100μL抗体液，在18～30℃下孵育2～2.5h，在3000～3500rpm的转速下离心2～5min，去除上清液，制得第六样品液。
S7、第三次洗涤处理
取制得的第六样品液，加入200～300μL清洗液，在3000～3500rpm的转速下离心2～5min，去除上清液，制得第七样品液。
S8、第二次孵育处理
取制得的第七样品液，加入100μL荧光探针液，在18～30℃下孵育1～1.5h，在3000～3500rpm的转速下离心2～5min，去除上清液，制得第八样品液。
S9、第四次洗涤处理
取制得的第八样品液，加入200～300μL清洗液，在3000～3500rpm的转速下离心2～5min，去除上清液，制得第九样品液。
S10、检测
避光条件下，向制得的第九样品液中加入300～400μL缓冲液，制成精子悬液，采用流式细胞仪检测精子悬液中精子唾液酸酶NEU1或NEU3的表达水平。
本公开检测精子唾液酸酶1/3表达水平的方法比较简便且快速，准确性高。
本公开一实施方式还提供一种诊断男性受试者生育能力的方法，包括：
A)在足以发生结合反应的条件下，使精子唾液酸酶1/3检测试剂盒或精子唾液酸酶1/3检测试剂与来自所述受试者的样品接触以进行结合反应；以及
B)检测结合反应产生的免疫复合物。
可选地，所述样品为精液。
本公开提供了一种精子唾液酸酶1/3检测试剂盒及其制备方法、检测精子唾液酸酶1/3表达水平的方法，其能够快速检测精液中精子唾液酸酶NEU1或NEU3的表达水平。
实施例
以下结合实施例对本公开的一种精子唾液酸酶1/3检测试剂盒及其制备方法、检测精子唾液酸酶1/3表达水平的方法作进一步的详细描述。
实施例1
本公开实施例提供一种精子唾液酸酶NEU1检测试剂盒及其制备方法、检测精子唾液酸酶NEU1表达水平的方法。
1、精子唾液酸酶NEU1检测试剂盒及其制备方法
S1、配制溶液
配制固定液：称取2.9g NaH2PO4·2H2O、19.24g Na2HPO4和40gPFA，用700mL纯化水充分溶解后，用纯化水定容至1000mL，固定液的pH值为7.4。
配制缓冲液：称取174.09g Na2HPO4·12H2O、17.79g NaH2PO4·2H2O和27g NaCl，用2700mL纯化水充分溶解后，用纯化水定容至3000mL，缓冲液的pH值为7.4。
配制通透液：称取1mL Triton X-100和100mL缓冲液，用700mL纯化水混合均匀后，用纯化水定容至1000mL。
配制封闭液：称取10g BSA和100mL缓冲液，用700mL纯化水充分溶解以及混合均匀后，用纯化水定容至1000mL。
配制清洗液：称取1mL Tween 20和100mL缓冲液，用700mL纯化水混合均匀后，用纯化水定容至1000mL。
配制抗体液：称取10g BSA和5mg生物素化抗体NEU1，用700mL纯化水充分溶解以及混合均匀后，用纯化水定容至1000mL。
配制荧光探针液：称取10g BSA和4mg SA-PE，用700mL纯化水充分溶解以及混合均匀后，用纯化水定容至1000mL。
S2、组装
分别取5mL固定液、5mL通透液、25mL缓冲液、5mL封闭液、10mL清洗液、50μL抗体液和2mL荧光探针液分装后组装成精子唾液酸酶NEU1检测试剂盒。
2、检测精子唾液酸酶NEU1表达水平的方法
S1、第一次洗涤处理
取0.5mL精液样本，加入1mL缓冲液，在3000rpm的转速下离心5min，去除上清液，重复上述操作3次后，加入1mL缓冲液，制得第一样品液。
S2、固定处理
取200μL第一样品液，在3000rpm的转速下离心5min，去除上清液，加入200μL固定液，25℃下静置10min，在3000rpm的转速下离心5min，去除上清液，加入200μL缓冲液，在3000rpm的转速下离心5min，去除上清液，制得第二样品液。
S3、通透处理
取制得的第二样品液，加入200μL通透液，25℃下静置15min，在3000rpm的转速下离心5min，去除上清液，制得第三样品液。
S4、第二次洗涤处理
取制得的第三样品液，加入200μL缓冲液，在3000rpm的转速下离心5min，去除上清液，制得第四样品液。
S5、封闭处理
取制得的第四样品液，加入200μL封闭液，25℃下静置30min，在3000rpm的转速下离心2min，去除上清液，制得第五样品液。
S6、第一次孵育处理
取制得的第五样品液，加入100μL抗体液，在25℃下孵育2h，在3000rpm的转速下离心2min，去除上清液，制得第六样品液。
S7、第三次洗涤处理
取制得的第六样品液，加入200μL清洗液，在3000rpm的转速下离心2min，去除上清液，制得第七样品液。
S8、第二次孵育处理
取制得的第七样品液，加入100μL荧光探针液，在25℃下孵育1h，在3000rpm的转速下离心2min，去除上清液，制得第八样品液。
S9、第四次洗涤处理
取制得的第八样品液，加入200μL清洗液，在3000rpm的转速下离心2min，去除上清液，制得第九样品液。
S10、检测
避光条件下，向制得的第九样品液中加入300μL缓冲液，制成精子悬液，采用流式细胞仪检测精子悬液中精子唾液酸酶NEU1的表达水平。
实施例2
本公开实施例提供一种精子唾液酸酶NEU3检测试剂盒及其制备方法、检测精子唾液酸酶NEU3表达水平的方法。实施例2在实施例1的基础上改变生物素化抗体NEU1为生物素化抗体NEU3，其他不变。
对比例1
本公开对比例提供一种精子唾液酸酶NEU1检测试剂盒及其制备方法、检测精子唾液酸酶NEU1表达水平的方法。对比例1在实施例1的基础上改变SA-PE为异硫氰酸荧光素(Fluorescein isothiocyanate isomer，FITC)，其他不变。
对比例2
本公开对比例提供一种精子唾液酸酶NEU3检测试剂盒及其制备方法、检测精子唾液酸酶NEU3表达水平的方法。对比例2在实施例2的基础上改变SA-PE为FITC，其他不变。
对比例3
本公开对比例提供一种精子唾液酸酶NEU1检测试剂盒及其制备方法、检测精子唾液酸酶NEU1表达水平的方法。对比例3在实施例1的基础上改变固定处理的时间，由25℃下静置10min改为25℃下静置20min。
对比例4
本公开对比例提供一种精子唾液酸酶NEU1检测试剂盒及其制备方法、检测精子唾液酸酶NEU1表达水平的方法。对比例4在实施例1的基础上改变封闭处理的时间，由25℃下静置30min改为25℃下静置20min。
对比例5
本公开对比例提供一种精子唾液酸酶NEU1检测试剂盒及其制备方法、检测精子唾液酸酶NEU1表达水平的方法。对比例5在实施例1的基础上改变第一次孵育处理的时间，由25℃下孵育2h改为25℃下孵育1h。
对比例6
本公开对比例提供一种精子唾液酸酶NEU3检测试剂盒及其制备方法、检测精子唾液酸酶NEU3表达水平的方法。对比例6在实施例2的基础上改变第一次孵育处理的时间，由25℃下孵育2h改为25℃下孵育1h。
对比例7
本公开对比例提供一种精子唾液酸酶NEU1检测试剂盒及其制备方法、检测精子唾液酸酶NEU1表达水平的方法。对比例7在实施例1的基础上改变第二次孵育处理的时间，由25℃下孵育1h改为25℃下孵育0.5h。
对比例8
本公开对比例提供一种精子唾液酸酶NEU3检测试剂盒及其制备方法、检测精子唾液酸酶NEU3表达水平的方法。对比例8在实施例2的基础上改变第二次孵育处理的时间，由25℃下孵育1h改为25℃下孵育0.5h。
试验例1
设置对照组1和对照组2，对照组1在实施例1的基础上，在第一次孵育处理中不加入抗体液，对照组2在实施例2的基础上，在第一次孵育处理中不加入抗体液。对照组1～2的流式检测散点图、流式检测直方图分别如图1～6所示，实施例1～2和对比例1～8的流式检测散点图、流式检测直方图，如图7～36所示，实施例1～2和对比例1～8的阳性比例如表1所示。
表1实施例1～2和对比例1～8的阳性比例
由对比例1和实施例1对比、对比例2和实施例2对比可知，将SA-PE换成FITC，检测得到的阳性比例降低。
由对比例3和实施例1对比可知，将固定处理的时间增加至20min，检测得到的阳性比例降低。
由对比例4和实施例1对比可知，将封闭处理的时间减少至20min，检测得到的阳性比例降低。
由对比例5和实施例1对比、对比例6和实施例2对比可知，将第一次孵育处理的时间减少至1h，检测得到的阳性比例降低。
由对比例7和实施例1对比、对比例8和实施例2对比可知，将第二次孵育处理的时间减少至0.5h，检测得到的阳性比例降低。
以上所述仅为本公开的实施例而已，并不用于限制本公开，对于本领域的技术人员来说，本公开可以有各种更改和变化。凡在本公开的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本公开的保护范围之内。
工业实用性
本公开的精子唾液酸酶1/3检测试剂盒能够建立免疫流式间接标记法，其具有特异性强及较高的灵敏度，能对精液中精子唾液酸酶NEU1或NEU3的表达水平进行检测。同时，本公开的精子唾液酸酶1/3检测试剂盒检测准确性高，且具有快速和单次检测量大的特点，因此具有优异的实用性能和广阔的市场前景。

Claims (15)

  1. 一种精子唾液酸酶1/3检测试剂盒，其特征在于，所述精子唾液酸酶1/3检测试剂盒包括：固定液、通透液、缓冲液、封闭液、清洗液、抗体液和荧光探针液；
    所述抗体液包括生物素化抗体NEU1或生物素化抗体NEU3。
  2. 根据权利要求1所述的精子唾液酸酶1/3检测试剂盒，其特征在于，所述抗体液中生物素化抗体NEU1或生物素化抗体NEU3浓度为5～10mg/L。
  3. 根据权利要求1或2所述的精子唾液酸酶1/3检测试剂盒，其特征在于，所述荧光探针液包括R-藻红蛋白标记链霉亲和素；
    可选地，所述荧光探针液中R-藻红蛋白标记链霉亲和素的浓度为4～8mg/L。
  4. 根据权利要求1-3中任一项所述的精子唾液酸酶1/3检测试剂盒，其特征在于，满足以下条件a～f中的至少一项：
    a、所述固定液包括磷酸二氢钠、磷酸氢钠、多聚甲醛和水；
    b、所述缓冲液包括磷酸二氢钠、磷酸氢钠、氯化钠和水；
    c、所述通透液包括聚乙二醇辛基苯基醚、磷酸二氢钠、磷酸氢钠、氯化钠和水；
    d、所述封闭液包括牛血清白蛋白、磷酸二氢钠、磷酸氢钠、氯化钠和水；
    e、所述清洗液包括吐温20、磷酸二氢钠、磷酸氢钠、氯化钠和水；
    f、所述抗体液包括牛血清白蛋白、水以及生物素化抗体NEU1或生物素化抗体NEU3；
    g、所述荧光探针液包括牛血清白蛋白、R-藻红蛋白标记链霉亲和素和水。
  5. 根据权利要求4所述的精子唾液酸酶1/3检测试剂盒，其特征在于，满足以下条件I～V中的至少一项：
    I.固定液中多聚甲醛的浓度为35～45g/L，磷酸二氢钠的浓度为0.015～0.02mol/L，磷酸氢钠的浓度为0.13～0.14mol/L；
    II.缓冲液中磷酸氢钠的浓度为0.15～0.17mol/L，磷酸二氢钠的浓度为0.035～0.04mol/L，氯化钠的浓度为0.15～0.16mol/L；
    III.通透液中聚乙二醇辛基苯基醚的浓度为0.8～1.2mL/L，磷酸二氢钠的浓度为0.005～0.0056mol/L，磷酸氢钠的浓度为0.00117～0.00133mol/L，氯化钠的浓度为0.005～0.0053mol/L；
    IV.封闭液中牛血清白蛋白的浓度为10～14g/L，磷酸二氢钠的浓度为0.005～0.0056mol/L，磷酸氢钠的浓度为0.00117～0.00133mol/L，氯化钠的浓度为0.005～0.0053mol/L；
    V.清洗液中吐温20的浓度为0.8～1.2mL/L，磷酸二氢钠的浓度为0.005～0.0056mol/L，磷酸氢钠的浓度为0.00117～0.00133mol/L，氯化钠的浓度为0.005～0.0053mol/L。
  6. 一种精子唾液酸酶1/3检测试剂，其特征在于，所述精子唾液酸酶1/3检测试剂盒包括：固定液、通透液、缓冲液、封闭液、清洗液、抗体液和荧光探针液；
    所述抗体液包括生物素化抗体NEU1或生物素化抗体NEU3。
  7. 根据权利要求6所述的精子唾液酸酶1/3检测试剂，其特征在于，所述抗体液中生物素化抗体NEU1或生物素化抗体NEU3浓度为5～10mg/L。
  8. 根据权利要求6或7所述的精子唾液酸酶1/3检测试剂，其特征在于，满足以下条件A～I中的至少一项：
    A.所述荧光探针液包括R-藻红蛋白标记链霉亲和素；
    B.所述荧光探针液中R-藻红蛋白标记链霉亲和素的浓度为4～8mg/L；
    C.所述固定液包括磷酸二氢钠、磷酸氢钠、多聚甲醛和水；
    D.所述缓冲液包括磷酸二氢钠、磷酸氢钠、氯化钠和水；
    E.所述通透液包括聚乙二醇辛基苯基醚、磷酸二氢钠、磷酸氢钠、氯化钠和水；
    F.所述封闭液包括牛血清白蛋白、磷酸二氢钠、磷酸氢钠、氯化钠和水；
    G.所述清洗液包括吐温20、磷酸二氢钠、磷酸氢钠、氯化钠和水；
    H.所述抗体液包括牛血清白蛋白、水以及生物素化抗体NEU1或生物素化抗体NEU3；
    I.所述荧光探针液包括牛血清白蛋白、R-藻红蛋白标记链霉亲和素和水。
  9. 一种制备权利要求1～5任一项所述的精子唾液酸酶1/3检测试剂盒的方法，其特征在于，所述制备精子唾液酸酶1/3检测试剂盒的方法包括：分别制备所述固定液、所述通透液、所述缓冲液、所述封闭液、所述清洗液、所述抗体液和所述荧光探针液，然后将所述固定液、所述通透液、所述缓冲液、所述封闭液、所述清洗液、所述抗体液和所述荧光探针液分装后组装成所述精子唾液酸酶1/3检测试剂盒。
  10. 一种利用权利要求1～5任一项所述的精子唾液酸酶1/3检测试剂盒检测精子唾液酸酶1/3表达水平的方法，其特征在于，所述检测精子唾液酸酶1/3表达水平的方法包括：取精液样品，采用所述清洗液对所述精液样品进行第一次洗涤处理制得第一样品液，采用所述固定液对所述第一样品液进行固定处理制得第二样品液，采用所述通透液对所述第一样品进行通透处理制得第三样品液，采用所述清洗液对所述第三样品液进行第二次洗涤处理制得第四样品液，采用所述封闭液对所述第四样品液进行封闭处理制得第五样品液，在所述第五样品液中加入所述抗体液并经第一次孵育处理制得第六样品液，采用所述清洗液对所述第六样品液进行第三次洗涤处理制得第七样品液，在所述第七样品液中加入所述荧光探针液并经第二次孵育处理制得第八样品液，采用所述清洗液对所述第八样品液进行第四次洗涤处理制得第九样品液，采用流式细胞仪检测所述第九样品液中精子唾液酸酶NEU1或NEU3的表达水平。
  11. 根据权利要求10所述的检测精子唾液酸酶1/3表达水平的方法，其特征在于，所述封闭处理包括在所述第四样品液中加入所述封闭液，静止30～40min，然后离心去除上清液。
  12. 根据权利要求10或11所述的检测精子唾液酸酶1/3表达水平的方法，其特征在于，所述第一次孵育处理包 括在所述第五样品液中加入所述抗体液，在18～30℃下孵育2～2.5h，然后离心去除上清液。
  13. 根据权利要求10或11所述的检测精子唾液酸酶1/3表达水平的方法，其特征在于，所述第二次孵育处理包括在所述第七样品液中加入所述荧光探针液，在18～30℃下孵育1～1.5h，然后离心去除上清液。
  14. 根据权利要求10或11所述的检测精子唾液酸酶1/3表达水平的方法，其特征在于，在上机检测前，在所述第九样品液中加入所述缓冲液。
  15. 一种诊断男性受试者生育能力的方法，包括：
    A)在足以发生结合反应的条件下，使权利要求1～5任一项所述的精子唾液酸酶1/3检测试剂盒或权利要求6-8任一项所述的精子唾液酸酶1/3检测试剂与来自所述受试者的样品接触以进行结合反应；以及
    B)检测结合反应产生的免疫复合物；
    可选地，所述样品为精液。
PCT/CN2023/088755 2022-06-21 2023-04-17 精子唾液酸酶1/3检测试剂盒及其制备方法、检测精子唾液酸酶1/3表达水平的方法 WO2023246254A1 (zh)

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