CN115032391A - Sperm sialidase 1/3 detection kit, preparation method thereof and method for detecting sperm sialidase 1/3 expression level - Google Patents
Sperm sialidase 1/3 detection kit, preparation method thereof and method for detecting sperm sialidase 1/3 expression level Download PDFInfo
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- 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
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- G—PHYSICS
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- 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
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Abstract
The application provides a sperm sialidase 1/3 detection kit, a preparation method thereof and a method for detecting sperm sialidase 1/3 expression level, belonging to the technical field of detection. The sperm sialidase 1/3 detection kit comprises: fixing liquid, permeable liquid, buffer liquid, confining liquid, cleaning liquid, antibody liquid and fluorescent probe liquid. The antibody fluid includes biotinylated antibody NEU1 or biotinylated antibody NEU 3. The sperm sialidase 1/3 detection kit can establish an immune flow type indirect labeling method, has strong specificity and higher sensitivity, and can detect the expression level of sperm sialidase NEU1 or NEU3 in semen. Meanwhile, the sperm sialidase 1/3 detection kit has high detection accuracy and has the characteristics of rapidness and large single detection amount.
Description
Technical Field
The application relates to the technical field of detection, in particular to a sperm sialidase 1/3 detection kit, a preparation method thereof and a method for detecting sperm sialidase 1/3 expression level.
Background
With the continuous development of society, sterile couples are more and more, and global sterile couples account for 10% -15% of married couples. According to statistics, the proportion of the male reproductive capacity abnormality is not less than 50%. Conventional semen routine analysis has been the most basic clinical indicator for determining male fertility. For many years, the actual cause of infertility in the clinic for the vast majority of male infertility patients remains unclear. Especially, about one third of patients with infertility clinically have normal and nearly normal results of conventional semen analysis of males. Clinically, these patients are classified as infertility of unknown origin. Conventional semen analysis measures only sperm count, viability, motility and morphology. These indices reflect only the most basic semen quality and not all sperm functions.
Disclosure of Invention
The application provides a sperm sialidase 1/3 detection kit, a preparation method thereof and a method for detecting expression level of sperm sialidase 1/3, which can rapidly detect expression level of sperm sialidase NEU1 or NEU3 in semen.
The embodiment of the application is realized as follows:
in a first aspect, the present application illustrates a sperm sialidase 1/3 detection kit comprising: fixing liquid, permeable liquid, buffer liquid, confining liquid, cleaning liquid, antibody liquid and fluorescent probe liquid.
The antibody fluid includes biotinylated antibody NEU1 or biotinylated antibody NEU 3.
In the technical scheme, the sperm sialidase 1/3 detection kit can establish an immune flow indirect labeling method, has strong specificity and high sensitivity, and can detect the expression level of sperm sialidase NEU1 or NEU3 in semen. Meanwhile, the sperm sialidase 1/3 detection kit has high detection accuracy and has the characteristics of high speed and large single detection amount.
In a first possible example of the first aspect of the present application in combination with the first aspect, the concentration of the biotinylated antibody NEU1 or biotinylated antibody NEU3 in the antibody fluid is 5-10 mg/L.
In a second possible example of the first aspect of the present application in combination with the first aspect, the fluorescent probe solution includes R-phycoerythrin-labeled streptavidin.
Optionally, the concentration of the R-phycoerythrin labeled streptavidin in the fluorescent probe solution is 4-8 mg.
In the above example, the sperm sialidase 1/3 detection kit of the present application employs a combination of a biotinylated antibody and R-phycoerythrin labeled streptavidin, and utilizes a cascade amplification reaction principle to enhance reaction sensitivity and reduce non-specific interference.
In combination with the first aspect, in a third possible example of the first aspect of the present application, at least one of the following conditions a to f is satisfied:
a. the fixing liquid comprises sodium dihydrogen phosphate, sodium hydrogen phosphate, paraformaldehyde and water.
b. The buffer solution comprises sodium dihydrogen phosphate, sodium hydrogen phosphate, sodium chloride and water.
c. The penetrating fluid comprises polyethylene glycol octyl phenyl ether, sodium dihydrogen phosphate, sodium hydrogen phosphate, sodium chloride and water.
d. The sealing liquid comprises bovine serum albumin, sodium dihydrogen phosphate, sodium hydrogen phosphate, sodium chloride and water.
e. The cleaning solution comprises tween 20, sodium dihydrogen phosphate, sodium hydrogen phosphate, sodium chloride and water.
f. The antibody fluid comprises bovine serum albumin, water and a biotinylated antibody NEU1 or biotinylated antibody NEU 3;
g. the fluorescent probe solution comprises bovine serum albumin, R-phycoerythrin labeled streptavidin and water.
In a second aspect, the present application provides a method for preparing the sperm sialidase 1/3 detection kit, comprising: respectively preparing a fixing solution, a permeable solution, a buffer solution, a confining solution, a cleaning solution, an antibody solution and a fluorescent probe solution, and then subpackaging the fixing solution, the permeable solution, the buffer solution, the confining solution, the cleaning solution, the antibody solution and the fluorescent probe solution to assemble the sperm sialidase 1/3 detection kit.
In the technical scheme, the preparation method of the sperm sialidase 1/3 detection kit is simple and convenient, the prepared fixing solution, the prepared permeable solution, the prepared buffer solution, the prepared confining solution, the prepared cleaning solution, the prepared antibody solution and the prepared fluorescent probe solution are subpackaged and then assembled, and different reagents can be selected according to different steps when the kit is used.
In a third aspect, the present application provides, as an example, a method for detecting the expression level of sperm sialidase 1/3 using the sperm sialidase 1/3 test kit described above, comprising: taking a semen sample, carrying out first washing treatment on the semen sample by adopting a cleaning solution to prepare a first sample solution, carrying out fixing treatment on the first sample solution by adopting a fixing solution to prepare a second sample solution, carrying out penetrating treatment on the first sample by adopting a penetrating solution to prepare a third sample solution, carrying out second washing treatment on the third sample solution by adopting the cleaning solution to prepare a fourth sample solution, carrying out sealing treatment on the fourth sample solution by adopting a sealing solution to prepare a fifth sample solution, adding antibody liquid into the fifth sample liquid, performing primary incubation treatment to obtain a sixth sample liquid, performing tertiary washing treatment on the sixth sample liquid by using a cleaning solution to obtain a seventh sample liquid, and adding a fluorescent probe solution into the seventh sample solution, carrying out secondary incubation treatment to obtain an eighth sample solution, carrying out fourth washing treatment on the eighth sample solution by using a cleaning solution to obtain a ninth sample solution, and detecting the expression level of sperm sialidase NEU1 or NEU3 in the ninth sample solution by using a flow cytometer.
In the technical scheme, the method for detecting the expression level of the sperm sialidase 1/3 is simple, convenient and rapid, and has high accuracy.
With reference to the third aspect, in a first possible example of the third aspect of the present application, the blocking treatment includes adding a blocking solution to the fourth sample solution, standing for 30-40 min, and then centrifuging to remove the supernatant.
In a second possible example of the third aspect of the present application, in combination with the third aspect, the first incubation treatment includes adding an antibody solution to the fifth sample solution, incubating at 18-30 ℃ for 2-2.5 h, and centrifuging to remove the supernatant.
In a third possible example of the third aspect of the present application, in combination with the third aspect, the second incubation treatment includes adding a fluorescent probe solution to the seventh sample solution, incubating at 18 to 30 ℃ for 1 to 1.5 hours, and centrifuging to remove the supernatant.
With reference to the third aspect, in a fourth possible example of the third aspect of the present application, a buffer is added to the ninth sample solution before the on-machine detection.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are required to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present application and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained from the drawings without inventive effort.
FIG. 1 is a first flow chart of the present application, control 1;
FIG. 2 is a second flow-through test scattergram of control 1 of the present application;
FIG. 3 is a flow detection histogram of the control group 1 of the present application;
FIG. 4 is a first flow chart of control 2 of the present application;
FIG. 5 is a second flow-through test scattergram of control 2 of the present application;
FIG. 6 is a flow detection histogram of control group 2 of the present application;
FIG. 7 is a first flow chart for detection of scattergrams according to example 1 of the present application;
fig. 8 is a second flow detection scattergram according to embodiment 1 of the present application;
FIG. 9 is a flow detection histogram of embodiment 1 of the present application;
FIG. 10 is a first flow chart for detection of scattergrams in example 2 of the present application;
FIG. 11 is a second flow chart for examining scattergrams in example 2 of the present application;
FIG. 12 is a flow detection histogram of embodiment 2 of the present application;
FIG. 13 is a first flow-through scattergram of comparative example 1 of the present application;
FIG. 14 is a second flow-through assay scatterplot of comparative example 1 of the present application;
FIG. 15 is a flow detection histogram of comparative example 1 of the present application;
FIG. 16 is a first flow-through detection scatterplot of comparative example 2 of the present application;
FIG. 17 is a second flow-through assay scatterplot of comparative example 2 of the present application;
FIG. 18 is a flow detection histogram of comparative example 2 of the present application;
FIG. 19 is a first flow-through scattergram of comparative example 3 of the present application;
FIG. 20 is a second flow-through assay scatterplot of comparative example 3 of the present application;
FIG. 21 is a flow detection histogram of comparative example 3 of the present application;
FIG. 22 is a first flow-through scattergram of comparative example 4 of the present application;
FIG. 23 is a second flow-through assay scatterplot of comparative example 4 of the present application;
FIG. 24 is a flow detection histogram of comparative example 4 of the present application;
FIG. 25 is a first flow-through detection scatterplot of comparative example 5 of the present application;
FIG. 26 is a second flow-through assay scatterplot of comparative example 5 of the present application;
FIG. 27 is a flow detection histogram of comparative example 5 of the present application;
FIG. 28 is a first flow-through scattergram of comparative example 6 of the present application;
FIG. 29 is a second flow-through assay scatterplot of comparative example 6 of the present application;
FIG. 30 is a flow detection histogram of comparative example 6 of the present application;
FIG. 31 is a first flow-through scattergram of comparative example 7 of the present application;
FIG. 32 is a second flow-through assay scatterplot of comparative example 7 of the present application;
FIG. 33 is a flow detection histogram of comparative example 7 of the present application;
FIG. 34 is a first flow-through scattergram of comparative example 8 of the present application;
FIG. 35 is a second flow-through assay scatterplot of comparative example 8 of the present application;
FIG. 36 is a flow detection histogram of comparative example 8 of the present application.
Detailed Description
Embodiments of the present application will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present application and should not be construed as limiting the scope of the present application. The examples, in which specific conditions are not specified, were carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The following detailed description is provided for a sperm sialidase 1/3 detection kit, a preparation method thereof, and a method for detecting sperm sialidase 1/3 expression level in the embodiments of the present application:
the embodiment of the application provides a sperm sialidase 1/3 detection kit, which comprises a fixing solution, a permeable solution, a buffer solution, a confining solution, a cleaning solution, an antibody solution and a fluorescent probe solution.
The fixing liquid comprises sodium dihydrogen phosphate, sodium hydrogen phosphate, Paraformaldehyde (PFA) and water.
Optionally, the concentration of paraformaldehyde in the fixing solution is 35-45 g/L, the concentration of sodium dihydrogen phosphate is 0.015-0.02 mol/L, and the concentration of sodium hydrogen phosphate is 0.13-0.14 mol/L.
Optionally, the pH value of the stationary liquid is 7.0-7.4.
The buffer solution comprises sodium dihydrogen phosphate, sodium hydrogen phosphate, sodium chloride and water.
Optionally, the concentration of sodium hydrogen phosphate in the buffer solution is 0.15-0.17 mol/L, the concentration of sodium dihydrogen phosphate is 0.035-0.04 mol/L, and the concentration of sodium chloride is 0.15-0.16 mol/L.
Optionally, the pH value of the buffer solution is 7.2-7.4.
The permeate liquid comprises polyethylene glycol octyl phenyl ether (Triton X-100), sodium dihydrogen phosphate, sodium hydrogen phosphate, sodium chloride and water.
Optionally, the concentration of polyethylene glycol octyl phenyl ether in the permeation 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, and the concentration of sodium chloride is 0.005-0.0053 mol/L.
The blocking solution comprises Bovine Serum Albumin (BSA), sodium dihydrogen phosphate, sodium hydrogen phosphate, sodium chloride and water.
Optionally, the concentration of bovine serum albumin in the sealing solution is 10-14 g/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, and the concentration of sodium chloride is 0.005-0.0053 mol/L.
The cleaning solution comprises Tween 20(Tween 20), sodium dihydrogen phosphate, sodium hydrogen phosphate, sodium chloride and water.
Optionally, the concentration of Tween 20 in the cleaning 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, and the concentration of sodium chloride is 0.005-0.0053 mol/L.
The antibody fluid comprises bovine serum albumin, water and biotinylated antibody NEU1 or biotinylated antibody NEU 3.
Biotinylated antibody NEU1 or biotinylated antibody NEU3 was prepared as follows:
s1, antigen preparation
Gene sequence of NEU1 gene:
NM_000434.3Homo sapiens neuraminidase 1(NEU1),mRNA
GAGCTACTTGAAGACCAATTAGAGTCCGGGAAGCGCGGCGGGGCCTCCAGACCGGGGCGGGCTTAAGGGTGACATCTGCGCTTTAAAGGGTCCGGGTCAGCTGACTCCCGACTCTGTGGAGTCTAGCTGCCAGGGTCGCGGCAGCTGCGGGGAGAGATGACTGGGGAGCGACCCAGCACGGCGCTCCCGGACAGACGCTGGGGGCCGCGGATTCTGGGCTTCTGGGGAGGCTGTAGGGTTTGGGTGTTTGCCGCGATCTTCCTGCTGCTGTCTCTGGCAGCCTCCTGGTCCAAGGCTGAGAACGACTTCGGTCTGGTGCAGCCGCTGGTGACCATGGAGCAACTGCTGTGGGTGAGCGGGAGACAGATCGGCTCAGTGGACACCTTCCGCATCCCGCTCATCACAGCCACTCCGCGGGGCACTCTTCTCGCCTTTGCTGAGGCGAGGAAAATGTCCTCATCCGATGAGGGGGCCAAGTTCATCGCCCTGCGGAGGTCCATGGACCAGGGCAGCACATGGTCTCCTACAGCGTTCATTGTCAATGATGGGGATGTCCCCGATGGGCTGAACCTTGGGGCAGTAGTGAGCGATGTTGAGACAGGAGTAGTATTTCTTTTCTACTCCCTTTGTGCTCACAAGGCCGGCTGCCAGGTGGCCTCTACCATGTTGGTATGGAGCAAGGATGATGGTGTTTCCTGGAGCACACCCCGGAATCTCTCCCTGGATATTGGCACTGAAGTGTTTGCCCCTGGACCGGGCTCTGGTATTCAGAAACAGCGGGAGCCACGGAAGGGCCGCCTCATCGTGTGTGGCCATGGGACGCTGGAGCGGGACGGAGTCTTCTGTCTCCTCAGCGATGATCATGGTGCCTCCTGGCGCTACGGAAGTGGGGTCAGCGGCATCCCCTACGGTCAGCCCAAGCAGGAAAATGATTTCAATCCTGATGAATGCCAGCCCTATGAGCTCCCAGATGGCTCAGTCGTCATCAATGCCCGAAACCAGAACAACTACCACTGCCACTGCCGAATTGTCCTCCGCAGCTATGATGCCTGTGATACACTAAGGCCCCGTGATGTGACCTTCGACCCTGAGCTCGTGGACCCTGTGGTAGCTGCAGGAGCTGTAGTCACCAGCTCCGGCATTGTCTTCTTCTCCAACCCAGCACATCCAGAGTTCCGAGTGAACCTGAC
CCTGCGATGGAGCTTCAGCAATGGTACCTCATGGCGGAAAGAGACAGTCCAGCTATGGCCAGGCCCCAGT
GGCTATTCATCCCTGGCAACCCTGGAGGGCAGCATGGATGGAGAGGAGCAGGCCCCCCAGCTCTACGTCC
TGTATGAGAAAGGCCGGAACCACTACACAGAGAGCATCTCCGTGGCCAAAATCAGTGTCTATGGGACACT
CTGAGCTGTGCCACTGCCACAGGGGTATTCTGCCTTCAGGACTCTGCCTTCAGGAACACGGGTCTGTAGA
GGGTCTGCTGGAGACGCCTGAAAGACAGTTCCATCTTCCTTTAGACTCCAGCCTTGGCAAAATCACCTTC
CCTTTACCAGGGAAATCACTTCCTTTAGGACTGAAAGCTAGGCGTCCTCTCCCACAAAAAAGTCCTGCCC
TCATCTGAGAATACTGTCTTTCCATATGGCTAAGTGTGGCCCCACCACCCTCTCTGCCCTCCCGGGACAT
TGATTGGTCCTGTCTTGGGCAGGTCTAGTGAGCTGTAGAATTGAATCAATGTGAACTCAGGGAACTGGGG
AAGGCTGAGCCTCCTCTTTGGTGTTGCGGTAAGATAACCGACAGGGCTGGTGAAAGTCCCCAGATGGCAGGATATTTGGTTTCAGAGTAAGGACTAGGTGCACCACCATGACTGACTATCAATCAAAATGTTTGTAACTT
AAAATTTTTAATGAAGGATAATGAATATTTGTAGAGTCTCTATGGTTCTGTCAATGCACATCTTCGTGTC
TGTTTTCCTCATGTATCCTTGTGAGCCTGGGTGAGTTCTGGGGAGAGACCTGATGTGCGTACTGCCTGTG
AAAATCTGACTTTGGCAAATCAAATCCTCTTTTCCTTTTGAAAAAAAAAAAAAAAAAA
protein NEU1 (total 415 AAs):
>sp|Q99519|NEUR1_HUMAN Sialidase-1OS＝Homo sapiens OX＝9606GN＝NEU1PE＝1SV＝1
MTGERPSTALPDRRWGPRILGFWGGCRVWVFAAIFLLLSLAASWSKAENDFGLVQPLVTMEQLLWVSG
RQIGSVDTFRIPLITATPRGTLLAFAEARKMSSSDEGAKFIALRRSMDQGSTWSPTAFIVNDGDVPDGLNL
GAVVSDVETGVVFLFYSLCAHKAGCQVASTMLVWSKDDGVSWSTPRNLSLDIGTEVFAPGPGSGIQKQR
EPRKGRLIVCGHGTLERDGVFCLLSDDHGASWRYGSGVSGIPYGQPKQENDFNPDECQPYELPDGSVVIN
ARNQNNYHCHCRIVLRSYDACDTLRPRDVTFDPELVDPVVAAGAVVTSSGIVFFSNPAHPEFRVNLTLR
WSFSNGTSWRKETVQLWPGPSGYSSLATLEGSMDGEEQAPQLYVLYEKGRNHYTESISVAKISVYGTL
gene sequence of NEU3 gene:
>NM_006656.5Homo sapiens neuraminidase 3(NEU3),mRNA
CTTTTCGTTGCCGTTACCTGTTTCCGGCAGTCGACACGCTCTTCGCTTCTCGGGGCTTGTCTCCGTGTCC
TCCGTCTCAGTTGTTTCTCCCTCTCTATCCTCCTCTGTCTCAGTCTCCCCAGCCTTGGGGCCGGTGCCTC
TTCCGGGCTTCGGCGAATGAGACCTGCGGACCTGCCCCCGCGCCCCATGGAAGAATCCCCGGCGTCCAGC
TCTGCCCCGACAGAGACGGAGGAGCCGGGGTCCAGTGCAGAGGTCATGGAAGAAGTGACAACATGCTCCTTCAACAGCCCTCTGTTCCGGCAGGAAGATGACAGAGGGATTACCTACCGGATCCCAGCCCTGCTCTACATACCCCCCACCCACACCTTCCTGGCCTTTGCAGAGAAGCGTTCTACGAGGAGAGATGAGGATGCTCTCCAC
CTGGTGCTGAGGCGAGGGTTGAGGATTGGGCAGTTGGTACAGTGGGGGCCCCTGAAGCCACTGATGGAAGCCACACTACCGGGGCATCGGACCATGAACCCCTGTCCTGTATGGGAGCAGAAGAGTGGTTGTGTGTTCCTGTTCTTCATCTGTGTGCGGGGCCATGTCACAGAGCGTCAACAGATTGTGTCAGGCAGGAATGCTGCCCGCCTTTGCTTCATCTACAGTCAGGATGCTGGATGTTCATGGAGTGAGGTGAGGGACTTGACTGAGGAGGTCATTGGCTCAGAGCTGAAGCACTGGGCCACATTTGCTGTGGGCCCAGGTCATGGCATCCAGCTGCAGTCAGGGAGACTGGTCATCCCTGCGTATACCTACTACATCCCTTCCTGGTTCTTTTGCTTCCAGCTACCATGTAAA
ACCAGGCCTCATTCTCTGATGATCTACAGTGATGACCTAGGGGTCACATGGCACCATGGTAGACTCATTA
GGCCCATGGTTACAGTAGAATGTGAAGTGGCAGAGGTGACTGGGAGGGCTGGCCACCCTGTGCTATATTG
CAGTGCCCGGACACCAAACAGGTGCCGGGCAGAGGCGCTCAGCACTGACCATGGTGAAGGCTTTCAGAGACTGGCCCTGAGTCGACAGCTCTGTGAGCCCCCACATGGTTGCCAAGGGAGTGTGGTAAGTTTCCGGCCCCTGGAGATCCCACATAGGTGCCAGGACTCTAGCAGCAAAGATGCACCCACCATTCAGCAGAGCTCTCCAGGCAGTTCACTGAGGCTGGAGGAGGAAGCTGGAACACCGTCAGAATCATGGCTCTTGTACTCACACCCAACCAGTAGGAAACAGAGGGTTGACCTAGGTATCTATCTCAACCAGACCCCCTTGGAGGCTGCCTGCTGGTCCCGCCCCTGGATCTTGCACTGTGGGCCCTGTGGCTACTCTGATCTGGCTGCTCTGGAGGAGGAGGGCTTGTTTGGGTGTTTGTTTGAATGTGGGACCAAGCAAGAGTGTGAGCAGATTGCCTTCCGCCTGTTTACACACCGGGAGATCCTGAGTCACCTGCAGGGGGACTGCACCAGCCCTGGTAGGAACCCAAGCCAATTCAAAAGCAATTAATTGGCTTAGGACCCAATTTCCATAGATGCAAATGGCAGTTACAGACAGGTTAACAGAAGCTACTGAAGTCTACAGATAATCAAAAAACTTAATATTCTGTTCCCTACCTTTTTTCACTTTTCCTCCTCCAAAGAGCAA
AATGAAAATTTTGCCTTAGCTACTGCAGTGGAAAGAGCACTGAACTAGGAGTTGGAAGACAAGGATGTGGTCCTGGCTCTGCCACTGGCTTGCTTTTGGACCTTGGATGTGTCACCTGAACTCTCTGGACCTCAGGTTTC
CATCTGTAAAATGAGAGTATTGGTTCTAAGATTTCTCATCTTCTCATCCCTAGGACAAGCATAGTGCCTG
CATGCTTCATGATCAGTAAGTCCTGGCTGCATAAAGGACTCTGATGTCAAAATGGAAACCAGGGGACTTA
CCTTTTCACATGACTTACCCCTCATCCGAGTGTGAGGTTACAAGCAGGTGTCATGGCAGGAAGGAAGACC
AGATCTGTATGATTTGTTCCATTTTTAATAACAAAAATATCCACACCCTTTTAATAATGCTCAGTTCTGT
AGGCTCTCTATCCTAGAGGAATTGAGCAAAACAGAAGAATCATGAAGTCTCCTACCTTCTACAGCCTTGT
AGTTCTGCTTACCTTCTCTTCCTCATCCAGAAAGCATCATTTTCTAGGGAGAACAATGAGAATCTCAATG
CCAGTAGTACTGGATAATAGTGCGTATTGCTTCTGGTGGCATTACCCTGATGATGGGCTGAAGTTCATTT
ATTAGGGTGGTTCCTGATGGGAAAAGGACATGGATTAGGACTTTAAAACACTGGACAGAATTTCCCACAG
TCTTTGCCCTCAAGGAGTTCACCAGTTTATGGGGCTAGAAGAGCGAGAAAATTCAAGAAAATAAATGTAG
CTGGTGGGAGACTTTGTAGATGTTGGGCTATATGTTGGGGTGATGGTAGCTCCTGATGTAATTTTCTTAG
TTGCATCTTCAATATGCCTGGAGTCGTCTGTCCAAGGCTTGTCCAGGCTTCTGGGTTTCTCTCAAGTTTG
TTTTTCTCAGGATATTGTCCTGGCCCAGCTACTCCTTTACCTGTGAGAAGATCTTCACCATTAGGAAGAT
CTCTAGACCCCCAGATCTCAGAATCAGGCCTATTTGTGTAGGCCCATGGAAATCACTACTTGTGAAGTAG
AGATGCCTTTTTGTCTAA
protein NEU3 (total 461 AAs):
>sp|Q9UQ49-2|NEUR3_HUMAN Isoform 2of Sialidase-3 OS＝Homo sapiens OX＝9606 GN＝NEU3
MRPADLPPRPMEESPASSSAPTETEEPGSSAEVMEEVTTCSFNSPLFRQEDDRGITYRIPALLYIPPTHTFLA
FAEKRSTRRDEDALHLVLRRGLRIGQLVQWGPLKPLMEATLPGHRTMNPCPVWEQKSGCVFLFFICVRG
HVTERQQIVSGRNAARLCFIYSQDAGCSWSEVRDLTEEVIGSELKHWATFAVGPGHGIQLQSGRLVIPAY
TYYIPSWFFCFQLPCKTRPHSLMIYSDDLGVTWHHGRLIRPMVTVECEVAEVTGRAGHPVLYCSARTPNR
CRAEALSTDHGEGFQRLALSRQLCEPPHGCQGSVVSFRPLEIPHRCQDSSSKDAPTIQQSSPGSSLRLEEEA
GTPSESWLLYSHPTSRKQRVDLGIYLNQTPLEAACWSRPWILHCGPCGYSDLAALEEEGLFGCLFECGTK
QECEQIAFRLFTHREILSHLQGDCTSPGRNPSQFKSN
designing a primer, constructing an expression vector of the expression vector of pPIC9K, after the construction is finished, determining that the sequencing result is 100% correct and has no mutation, performing small-scale test verification, realizing secretory expression by a scheme of a solubilizing-aid label and temperature induction as much as possible, performing affinity chromatography by adopting a metal chelating nickel column, and purifying protein.
S2, antigen quality control
Through SDS-PAGE detection, 8-10 mice are generally immunized, and the protein purity is at least more than 80%.
S3, mouse immunization
TABLE 1 mouse immunization
| Date | Content providing method and apparatus | Immunization dose | Number of mice immunized per antigen | Adjuvant type | Immune site |
| Exempt from NEU1 or NEU3 | One need not | 100 ug/piece | 5 | Freund's adjuvant | Under the skin |
| After two weeks | Two exempt from | 50 ug/piece | 5 | Freund's adjuvant | Under the skin |
| After three weeks | Sanwu | 50 ug/piece | 5 | Freund's adjuvant | Under the skin |
S4, tail blood collection and titer detection
NEU1 or NEU 3: tail blood collection was performed one week after the three immunizations.
Taking blood in a small amount: blood sampling from rat tail-fixing animal and exposing rat tail, immersing tail in 45-50 deg.C warm water for several minutes to make vein become congested, wiping dry, and wiping with alcohol cotton ball for disinfection. The tail tip is cut off by about 0.2-0.3 cm. The first drop of blood was wiped off. Then, tail blood was quantitatively aspirated by a capillary tube, and titer detection was performed.
TABLE 2 potency assay data
| Dilution factor | Mouse 1 | |
|
|
Mouse 5 |
| 500 | 3.449 | 3.553 | 3.704 | 3.57 | 3.281 |
| 2000 | 2.94 | 2.818 | 3.061 | 2.717 | 2.575 |
| 8000 | 2.238 | 1.685 | 2.096 | 1.588 | 1.764 |
| 32000 | 1.194 | 0.705 | 0.861 | 0.629 | 0.838 |
| 128000 | 0.331 | 0.205 | 0.258 | 0.19 | 0.262 |
| 512000 | 0.118 | 0.089 | 0.087 | 0.079 | 0.089 |
| PBS | 0.081 | 0.084 | 0.084 | 0.085 | 0.073 |
The titer of 5 mice reaches 1: 128000.
s5, cell fusion and positive clone screening
Fusing: the high-throughput screening is realized by adopting a fusion mode combining liquid and semisolid.
Subclone screening: cloning, amplifying and collecting supernatant, screening and sequencing the affinity of the antibody, adding the final antibody application experiment to a screening stage, and realizing the amplification culture of the anchoring screening positive clone of the high-quality antibody: obtaining hybridoma cell strains capable of stably secreting positive antibodies through 2-3 rounds of subcloning and screening, selecting 3-5 strains with better verification for amplification culture, and freezing and storing the rest for backup.
S6, ascites preparation and antibody purification
Mouse sensitization: balb/c mice were sensitized with liquid paraffin, preferably with a multiparous mouse, and the injection volume was 500 ul/mouse. Hybridization can be injected after 10 days.
Ascites is prepared from the tumor cells.
Cell injection: hybridoma cells were collected and washed twice with PBS, and 100 to 150 ten thousand cells were injected into the abdominal cavity of the mouse, and after one week, the mouse was inactive and the abdominal cavity of the mouse was enlarged.
Ascites collection: after injecting cells for one week and when the abdominal cavity of the mouse is swollen, ascites is collected in the abdominal cavity of the mouse by a sterile syringe, and the ascites is collected once every one to two days and repeatedly collected for a plurality of times until the mouse dies naturally. The collected ascites is divided, classified and stored in a refrigerator at the temperature of 20 ℃ below zero.
Adopting Protein A affinity purified antibody, dialyzing, concentrating, measuring concentration, adjusting concentration to be integer, and measuring purity.
Performing titer detection on the purified antibody
S7, antibody pairing screening
Using the expressed antigen and the positive sample, the most appropriate antibody pairing is selected.
Optionally, the concentration of bovine serum albumin in the antibody fluid is 10-14 g/L, and the concentration of the biotinylated antibody NEU1 or the biotinylated antibody NEU3 is 5-10 mg/L.
The fluorescent probe solution comprises bovine serum albumin and R-phycoerythrin labeled streptavidin (SA-PE).
Optionally, the concentration of bovine serum albumin in the fluorescent probe solution is 10-14 g/L, and the concentration of R-phycoerythrin-labeled streptavidin is 4-8 mg.
Optionally, the sperm sialidase 1/3 test kit comprises at least 5mL of a fixative solution, at least 5mL of a permeation solution, at least 25mL of a buffer solution, at least 5mL of a blocking solution, at least 10mL of a wash solution, at least 50 μ L of an antibody solution, and at least 2mL of a fluorescent probe solution.
The sperm sialidase 1/3 detection kit can establish an immune flow type indirect labeling method, has strong specificity and higher sensitivity, and can detect the expression level of sperm sialidase NEU1 or NEU3 in semen. Meanwhile, the sperm sialidase 1/3 detection kit disclosed by the application adopts the combination of a biotinylated antibody and R-phycoerythrin labeled streptavidin, and utilizes the cascade amplification reaction principle, so that the reaction sensitivity is enhanced, the non-specific interference is reduced, the detection accuracy is high, and the sperm sialidase 1/3 detection kit has the characteristics of rapidness and large single detection amount.
The application also provides a preparation method of the sperm sialidase 1/3 detection kit, which comprises the following steps: respectively preparing a fixing solution, a permeable solution, a buffer solution, a confining solution, a cleaning solution, an antibody solution and a fluorescent probe solution, and then subpackaging the fixing solution, the permeable solution, the buffer solution, the confining solution, the cleaning solution, the antibody solution and the fluorescent probe solution to assemble the sperm sialidase 1/3 detection kit.
The preparation method of the sperm sialidase 1/3 detection kit is simple and convenient, the prepared fixing solution, permeable solution, buffer solution, confining solution, cleaning solution, antibody solution and fluorescent probe solution are subpackaged and then assembled, and different reagents can be selected according to different steps when the kit is used.
The application also provides a method for detecting the expression level of sperm sialidase 1/3 by using the sperm sialidase 1/3 detection kit, which comprises the following steps:
s1, first washing treatment
And taking 0.5mL of semen sample, adding 1-2 mL of buffer solution, centrifuging for 5-8 min at the rotating speed of 3000-3500 rpm, removing the supernatant, repeating the operation for 3-5 times, and adding 1-2 mL of buffer solution to prepare the first sample solution.
S2, fixing processing
And (3) centrifuging 200 mu L of the first sample liquid for 5-8 min at the rotating speed of 3000-3500 rpm, removing the supernatant, adding 200-300 mu L of stationary liquid, standing for 10-15 min at 5-30 ℃, centrifuging for 5-8 min at the rotating speed of 3000-3500 rpm, removing the supernatant, adding 200-300 mu L of buffer solution, centrifuging for 5-8 min at the rotating speed of 3000-3500 rpm, removing the supernatant, and preparing the second sample liquid.
S3, through processing
And adding 200-300 mu L of penetrating liquid into the prepared second sample liquid, standing for 15-20 min at 5-30 ℃, centrifuging for 5-8 min at the rotating speed of 3000-3500 rpm, and removing supernatant to prepare a third sample liquid.
S4, second washing treatment
And adding 200-300 mu L of buffer solution into the prepared third sample solution, centrifuging for 5-8 min at the rotating speed of 3000-3500 rpm, and removing supernatant to prepare a fourth sample solution.
S5, sealing treatment
And adding 200-300 mu L of confining liquid into the prepared fourth sample liquid, standing for 30-40 min at 5-30 ℃, centrifuging for 2-5 min at the rotating speed of 3000-3500 rpm, and removing the supernatant to prepare a fifth sample liquid.
S6, first incubation treatment
And adding 100 mu L of antibody liquid into the prepared fifth sample liquid, incubating for 2-2.5 h at 18-30 ℃, centrifuging for 2-5 min at the rotating speed of 3000-3500 rpm, and removing the supernatant to prepare a sixth sample liquid.
S7, third washing treatment
And adding 200-300 mu L of cleaning solution into the prepared sixth sample solution, centrifuging for 2-5 min at the rotating speed of 3000-3500 rpm, and removing the supernatant to prepare a seventh sample solution.
S8, second incubation treatment
And adding 100 mu L of fluorescent probe solution into the prepared seventh sample solution, incubating for 1-1.5 h at 18-30 ℃, centrifuging for 2-5 min at the rotating speed of 3000-3500 rpm, and removing the supernatant to prepare an eighth sample solution.
S9, fourth washing treatment
And adding 200-300 mu L of cleaning solution into the prepared eighth sample solution, centrifuging for 2-5 min at the rotating speed of 3000-3500 rpm, and removing the supernatant to prepare a ninth sample solution.
S10, detection
And adding 300-400 mu L of buffer solution into the prepared ninth sample solution under the condition of keeping out of the sun to prepare sperm suspension, and detecting the expression level of sperm sialidase NEU1 or NEU3 in the sperm suspension by adopting a flow cytometer.
The method for detecting the expression level of the sperm sialidase 1/3 is simple, convenient and rapid, and has high accuracy.
The sperm sialidase 1/3 test kit and the preparation method thereof, and the method for detecting expression level of sperm sialidase 1/3 of the present application are further described in detail with reference to the following examples.
Example 1
The embodiment of the application provides a sperm sialidase NEU1 detection kit, a preparation method thereof and a method for detecting expression level of sperm sialidase NEU 1.
1. Sperm sialidase NEU1 detection kit and preparation method thereof
S1 preparation solution
Preparing a stationary liquid: 2.9g NaH was weighed 2 PO 4 · 2 H2O、19.24g Na 2 HPO 4 And 40g of PFA, which was dissolved sufficiently in 700mL of purified water, and then the volume was adjusted to 1000mL with purified water, and the pH of the fixative was 7.4.
Preparing a buffer solution: 174.09g of Na are weighed out 2 HPO 4 ·12H 2 O、17.79g NaH 2 PO 4 ·2H 2 O and 27g NaCl were dissolved in 2700mL of purified water sufficiently, and the volume was adjusted to 3000mL with purified water, and the pH of the buffer was 7.4.
Preparing a liquid permeable agent: 1mL of Triton X-100 and 100mL of buffer were weighed, mixed well with 700mL of purified water, and then the volume was adjusted to 1000mL with purified water.
Preparing a sealing liquid: 10g BSA and 100mL buffer were weighed, dissolved well in 700mL purified water and mixed well, and then made up to 1000mL with purified water.
Preparing a cleaning solution: weighing 1mL of Tween 20 and 100mL of buffer solution, uniformly mixing with 700mL of purified water, and then diluting to 1000mL with purified water.
Preparing an antibody liquid: 10g BSA and 5mg biotinylated antibody NEU1 were weighed, dissolved thoroughly in 700mL purified water and mixed well, and then brought to 1000mL volume with purified water.
Preparing a fluorescent probe solution: 10g BSA and 4mg SA-PE were weighed, dissolved thoroughly in 700mL purified water and mixed well, and then the volume was adjusted to 1000mL with purified water.
S2, assembling
5mL of stationary liquid, 5mL of permeable liquid, 25mL of buffer solution, 5mL of confining liquid, 10mL of cleaning liquid, 50 μ L of antibody liquid and 2mL of fluorescent probe liquid are respectively subpackaged to assemble the sperm sialidase NEU1 detection kit.
2. Method for detecting sperm sialidase NEU1 expression level
S1, first washing treatment
Taking 0.5mL of semen sample, adding 1mL of buffer solution, centrifuging at 3000rpm for 5min, removing supernatant, repeating the above operation for 3 times, and adding 1mL of buffer solution to obtain the first sample solution.
S2, fixing processing
And (3) centrifuging 200 mu L of the first sample solution at the rotating speed of 3000rpm for 5min, removing the supernatant, adding 200 mu L of the stationary solution, standing at 25 ℃ for 10min, centrifuging at the rotating speed of 3000rpm for 5min, removing the supernatant, adding 200 mu L of the buffer solution, centrifuging at the rotating speed of 3000rpm for 5min, and removing the supernatant to obtain a second sample solution.
S3 permeation treatment
Adding 200 μ L of the second sample solution, standing at 25 deg.C for 15min, centrifuging at 3000rpm for 5min, and removing the supernatant to obtain a third sample solution.
S4, second washing treatment
And adding 200 mu L of buffer solution into the prepared third sample solution, centrifuging for 5min at the rotating speed of 3000rpm, and removing the supernatant to prepare a fourth sample solution.
S5, sealing treatment
Adding 200 μ L of confining liquid into the fourth sample solution, standing at 25 deg.C for 30min, centrifuging at 3000rpm for 2min, and removing supernatant to obtain fifth sample solution.
S6, first incubation treatment
And adding 100 mu L of antibody liquid into the prepared fifth sample liquid, incubating for 2h at 25 ℃, centrifuging for 2min at the rotating speed of 3000rpm, and removing the supernatant to prepare a sixth sample liquid.
S7, third washing treatment
And adding 200 mu L of cleaning solution into the prepared sixth sample solution, centrifuging for 2min at the rotating speed of 3000rpm, and removing the supernatant to obtain a seventh sample solution.
S8, second incubation treatment
Adding 100 μ L fluorescent probe solution into the prepared seventh sample solution, incubating at 25 deg.C for 1h, centrifuging at 3000rpm for 2min, and removing supernatant to obtain eighth sample solution.
S9, fourth washing treatment
And adding 200 mu L of cleaning solution into the prepared eighth sample solution, centrifuging for 2min at the rotating speed of 3000rpm, and removing the supernatant to prepare a ninth sample solution.
S10, detection
And adding 300 mu L of buffer solution into the prepared ninth sample solution under the condition of keeping out of the light to prepare sperm suspension, and detecting the expression level of sperm sialidase NEU1 in the sperm suspension by adopting a flow cytometer.
Example 2
The embodiment of the application provides a sperm sialidase NEU3 detection kit, a preparation method thereof and a method for detecting expression level of sperm sialidase NEU 3. Example 2 on the basis of example 1, the biotinylated antibody NEU1 was changed to biotinylated antibody NEU3, and the others were not changed.
Comparative example 1
The comparative example of the application provides a sperm sialidase NEU1 detection kit, a preparation method thereof and a method for detecting expression level of sperm sialidase NEU 1. Comparative example 1 SA-PE was changed to Fluorescein Isothiocyanate (FITC) on the basis of example 1, and the others were unchanged.
Comparative example 2
The comparative example of the application provides a sperm sialidase NEU3 detection kit, a preparation method thereof and a method for detecting expression level of sperm sialidase NEU 3. Comparative example 2 SA-PE was changed to FITC on the basis of example 2, and the others were unchanged.
Comparative example 3
The comparative example of the application provides a sperm sialidase NEU1 detection kit, a preparation method thereof and a method for detecting expression level of sperm sialidase NEU 1. Comparative example 3 the time of the fixing treatment was changed from 10min at 25 ℃ to 20min at 25 ℃ based on example 1.
Comparative example 4
The comparative example of the application provides a sperm sialidase NEU1 detection kit, a preparation method thereof and a method for detecting expression level of sperm sialidase NEU 1. Comparative example 4 the time of the blocking treatment was changed from 30min at 25 ℃ to 20min at 25 ℃ on the basis of example 1.
Comparative example 5
The comparative example of the application provides a sperm sialidase NEU1 detection kit, a preparation method thereof and a method for detecting expression level of sperm sialidase NEU 1. Comparative example 5 the time of the first incubation treatment was changed from 2h at 25 ℃ to 1h at 25 ℃ based on example 1.
Comparative example 6
The comparative example of the application provides a sperm sialidase NEU3 detection kit, a preparation method thereof and a method for detecting expression level of sperm sialidase NEU 3. Comparative example 6 the time of the first incubation treatment was changed on the basis of example 2 from 2h at 25 ℃ to 1h at 25 ℃.
Comparative example 7
The comparative example of the application provides a sperm sialidase NEU1 detection kit, a preparation method thereof and a method for detecting expression level of sperm sialidase NEU 1. Comparative example 7 the time of the second incubation treatment was changed from 1h at 25 ℃ to 0.5h at 25 ℃ based on example 1.
Comparative example 8
The comparative example of the application provides a sperm sialidase NEU3 detection kit, a preparation method thereof and a method for detecting expression level of sperm sialidase NEU 3. Comparative example 8 the time of the second incubation treatment was changed from 1h at 25 ℃ to 0.5h at 25 ℃ based on example 2.
Test example 1
Control group 1 and control group 2 were set, control group 1 was based on example 1 without adding antibody liquid in the first incubation treatment, and control group 2 was based on example 2 without adding antibody liquid in the first incubation treatment. The flow-through scattergrams and flow-through histograms of the control groups 1 to 2 are shown in FIGS. 1 to 6, the flow-through scattergrams and flow-through histograms of the examples 1 to 2 and the comparative examples 1 to 8 are shown in FIGS. 7 to 36, and the positive ratios of the examples 1 to 2 and the comparative examples 1 to 8 are shown in Table 1.
TABLE 1 Positive proportion of examples 1 to 2 and comparative examples 1 to 8
As can be seen from comparison of comparative example 1 with example 1 and comparison of comparative example 2 with example 2, the SA-PE was changed to FITC, and the rate of positive detection was decreased.
As is clear from comparison between comparative example 3 and example 1, the positive ratio was decreased when the time for the fixing treatment was increased to 20 min.
As is clear from comparison of comparative example 4 and example 1, the positive rate was decreased by reducing the blocking treatment time to 20 min.
As is clear from comparison of comparative example 5 with example 1 and comparison of comparative example 6 with example 2, the positive rate was reduced by reducing the time of the first incubation treatment to 1 hour.
As can be seen from comparison between comparative example 7 and example 1 and comparison between comparative example 8 and example 2, the positive rate was decreased when the time for the second incubation treatment was reduced to 0.5 h.
The foregoing is illustrative of the present application and is not to be construed as limiting thereof, as numerous modifications and variations will be apparent to those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.
Claims (10)
1. A sperm sialidase 1/3 detection kit, wherein the sperm sialidase 1/3 detection kit comprises: fixing liquid, permeable liquid, buffer liquid, confining liquid, cleaning liquid, antibody liquid and fluorescent probe liquid;
the antibody fluid includes biotinylated antibody NEU1 or biotinylated antibody NEU 3.
2. The sperm sialidase 1/3 detection kit of claim 1, wherein the concentration of biotinylated antibody NEU1 or biotinylated antibody NEU3 in the antibody fluid is 5-10 mg/L.
3. The sperm sialidase 1/3 detection kit of claim 1, wherein the fluorescent probe fluid comprises R-phycoerythrin labeled streptavidin;
optionally, the concentration of the R-phycoerythrin labeled streptavidin in the fluorescent probe solution is 4-8 mg/L.
4. The method of preparing a sperm sialidase 1/3 detection kit according to claim 1, wherein at least one of the following conditions a to f is satisfied:
a. the fixing liquid comprises sodium dihydrogen phosphate, sodium hydrogen phosphate, paraformaldehyde and water;
b. the buffer solution comprises sodium dihydrogen phosphate, sodium hydrogen phosphate, sodium chloride and water;
c. the penetrating liquid comprises polyethylene glycol octyl phenyl ether, sodium dihydrogen phosphate, sodium hydrogen phosphate, sodium chloride and water;
d. the sealing liquid comprises bovine serum albumin, sodium dihydrogen phosphate, sodium hydrogen phosphate, sodium chloride and water;
e. the cleaning solution comprises tween 20, sodium dihydrogen phosphate, sodium hydrogen phosphate, sodium chloride and water;
f. the antibody fluid comprises bovine serum albumin, water and a biotinylated antibody NEU1 or biotinylated antibody NEU 3;
g. the fluorescent probe solution comprises bovine serum albumin, R-phycoerythrin labeled streptavidin and water.
5. A method of making a sperm sialidase 1/3 detection kit of any one of claims 1-4, wherein the method of making the sperm sialidase 1/3 detection kit comprises: and respectively preparing the fixing liquid, the permeable liquid, the buffer liquid, the confining liquid, the cleaning liquid, the antibody liquid and the fluorescent probe liquid, and subpackaging the fixing liquid, the permeable liquid, the buffer liquid, the confining liquid, the cleaning liquid, the antibody liquid and the fluorescent probe liquid to assemble the sperm sialidase 1/3 detection kit.
6. A method for detecting the expression level of sperm sialidase 1/3 by using the sperm sialidase 1/3 detection kit of any one of claims 1-4, wherein the method for detecting the expression level of sperm sialidase 1/3 comprises the following steps: taking a semen sample, carrying out first washing treatment on the semen sample by adopting the cleaning solution to prepare a first sample solution, carrying out fixed treatment on the first sample solution by adopting the fixed solution to prepare a second sample solution, carrying out penetrating treatment on the first sample by adopting the penetrating solution to prepare a third sample solution, carrying out second washing treatment on the third sample solution by adopting the cleaning solution to prepare a fourth sample solution, carrying out sealing treatment on the fourth sample solution by adopting the sealing solution to prepare a fifth sample solution, adding the antibody solution into the fifth sample solution and carrying out first incubation treatment to prepare a sixth sample solution, carrying out third washing treatment on the sixth sample solution by adopting the cleaning solution to prepare a seventh sample solution, adding the fluorescent probe solution into the seventh sample solution and carrying out second incubation treatment to prepare an eighth sample solution, and carrying out fourth washing treatment on the eighth sample solution by adopting the cleaning solution to prepare a ninth sample solution, detecting the expression level of sperm sialidase NEU1 or NEU3 in the ninth sample liquid by using a flow cytometer.
7. The method for detecting the expression level of sperm sialidase 1/3 of claim 6, wherein the blocking treatment comprises adding the blocking solution to the fourth sample fluid, standing for 30-40 min, and centrifuging to remove the supernatant.
8. The method for detecting the expression level of sperm sialidase 1/3 of claim 6, wherein the first incubation treatment comprises adding the antibody fluid to the fifth sample fluid, incubating at 18-30 ℃ for 2-2.5 h, and centrifuging to remove the supernatant.
9. The method for detecting the expression level of sperm sialidase 1/3 of claim 6, wherein the second incubation treatment comprises adding the fluorescent probe solution to the seventh sample solution, incubating at 18-30 ℃ for 1-1.5 h, and centrifuging to remove the supernatant.
10. The method of detecting the expression level of sperm sialidase 1/3 of claim 6, wherein the buffer is added to the ninth sample fluid prior to on-machine testing.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210703448.4A CN115032391A (en) | 2022-06-21 | 2022-06-21 | Sperm sialidase 1/3 detection kit, preparation method thereof and method for detecting sperm sialidase 1/3 expression level |
| PCT/CN2023/088755 WO2023246254A1 (en) | 2022-06-21 | 2023-04-17 | Detection kit of sperm sialidase 1/3 and preparation method therefor, and method for detecting expression level of sperm sialidase 1/3 |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210703448.4A CN115032391A (en) | 2022-06-21 | 2022-06-21 | Sperm sialidase 1/3 detection kit, preparation method thereof and method for detecting sperm sialidase 1/3 expression level |
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| Publication Number | Publication Date |
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| CN115032391A true CN115032391A (en) | 2022-09-09 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210703448.4A Pending CN115032391A (en) | 2022-06-21 | 2022-06-21 | Sperm sialidase 1/3 detection kit, preparation method thereof and method for detecting sperm sialidase 1/3 expression level |
Country Status (2)
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| CN (1) | CN115032391A (en) |
| WO (1) | WO2023246254A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023246254A1 (en) * | 2022-06-21 | 2023-12-28 | 成都思瑞多医疗科技有限公司 | Detection kit of sperm sialidase 1/3 and preparation method therefor, and method for detecting expression level of sperm sialidase 1/3 |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009146043A1 (en) * | 2008-03-31 | 2009-12-03 | Cohen B Arial | Assay for semen optimization |
| GR1008033B (en) * | 2012-03-29 | 2013-11-18 | Βασιλειος Τσιλιβακος | Method for the exploration of the presence of endocellular infectious agents in sperm cells |
| AU2013306098A1 (en) * | 2012-08-18 | 2015-02-12 | Academia Sinica | Cell-permeable probes for identification and imaging of sialidases |
| CN103454416B (en) * | 2013-09-22 | 2016-04-13 | 四川大学华西第二医院 | Affect the sialidase detection method of sperm function |
| CN105717307B (en) * | 2016-03-16 | 2017-07-04 | 四川大学华西第二医院 | The kit and its application method of a kind of sperm quality assessment |
| CN115032391A (en) * | 2022-06-21 | 2022-09-09 | 成都思瑞多医疗科技有限公司 | Sperm sialidase 1/3 detection kit, preparation method thereof and method for detecting sperm sialidase 1/3 expression level |
-
2022
- 2022-06-21 CN CN202210703448.4A patent/CN115032391A/en active Pending
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2023
- 2023-04-17 WO PCT/CN2023/088755 patent/WO2023246254A1/en unknown
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023246254A1 (en) * | 2022-06-21 | 2023-12-28 | 成都思瑞多医疗科技有限公司 | Detection kit of sperm sialidase 1/3 and preparation method therefor, and method for detecting expression level of sperm sialidase 1/3 |
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|---|---|
| WO2023246254A1 (en) | 2023-12-28 |
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