CN105717307A - Kit for evaluating semen quality and use method thereof - Google Patents
Kit for evaluating semen quality and use method thereof Download PDFInfo
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Abstract
The invention provides a kit for evaluating semen quality and a use method thereof. The kit comprises a cell lysis buffer containing no enzyme activity inhibitor, a detecting buffer solution 1, a protease inhibitor cocktail, a reagent A solution, a reagent B solution, bovine serum albumin, a fluorescent reagent, a sialidase standard stock solution and a detecting buffer solution 2. The defecting buffer solution 1 is a phosphate buffer solution or BWW culture solution. The reagent A solution is prepared from 1% of BCA disodium salt, 2% of anhydrous sodium carbonate, 0.16% of sodium tartrate, 0.4% of sodium hydroxide and 0.95% of sodium bicarbonate, the raw materials are mixed, and the pH value is adjusted to be 11.2 to 11.3. The reagent B solution is prepared from 0.4% of copper sulfate. The detecting buffering solution 2 is a phosphate buffer solution containing 0.05 mol/l sodium acetate and 0.25 microgram/microlitre bovine serum albumin. The activity of sialidase in semen is detected, and diagnostic bases and clinical consulting are provided for male infertility patients with unclear clinical reasons.
Description
Technical field
The invention belongs to medical detection technology, be specifically related to test kit and the user thereof of the assessment of a kind of sperm quality
Method.
Background technology
Along with the development of society's industry, the total incidence of infertile couples is significantly raised.European genesiology meeting in 2004
Statistics: the couple at child-bearing age pregnancy person can not account for 25% in 1 year, wherein 15% seeks treatment, bridegroom's or husband's side factor causes and sterile accounts for 50%.Male
Sterile cause of disease sexual disorders (1.7%), varicocele (12. 3%), reproductive tract infection (6.6%), congenital
Educating exception (2.1%) the acquired disease day after tomorrow (2.6%), endocrine regulation (0.6%), immunity factor (3.1%), other are abnormal
(3%), but being up to the patient of 60% ~ 75% and can not find reason, referred to as idiopathic male infertility, they only show as few essence, azoospermia
Or the sperm quality such as teratozoospermia is abnormal.The male sterility of unknown cause is likely to be due to many factors and causes, such as long-term stress
Environmental factors causes endocrine regulation, active oxygen and genetic flaw etc..
For many years, traditional semen routine analysis is always for judging the most basic clinical indices of male fertility.Face
On bed, the real sterile reason to the male sterility patient of the overwhelming majority is still unclear.The most about three/
One infertile patients, the Sperm routine analysis result of male is all normal and close normal.Clinically this kind of patient is divided into not clear former
Because of infertility.Therefore, for a long time, there is the biggest difficulty in clinical diagnose male sterility.Topmost reason is conventional
Semen analysis only measures the quantity of sperm, viability, activity ratio and form.These indexs can only reflect most basic seminal fluid matter
Amount, it is impossible to reflect all of sperm function, such as, the maturation of sperm nucleus and DNA damage, sperm and human oocyte zona pellucida association reaction
With penetrate, acrosome situation and reaction and with vitellinae membrana binding ability etc..Accordingly, it would be desirable to set up special sperm function tests ability
Measure the above function.Capacitation is the essential condition that sperm ovum binding forms germ cell, although the relevant of capacitation is ground
Study carefully more existing progress, but still there is the most undefined a lot of problem.
In clinical position, a part of primary or secondary Infertility male patient utilizes existing conventional semen quality check item
Mesh prompting sperm and semen quality are without exception.The most conventional sperm detection means is CASA (computer aided sperm analysis
Computer aided of semen analysis), and antisperm antibody etc..Current the most widely used CASA technology
With some morphologic coherent detections, mainly can evaluate the mobility of sperm and sperm ratio of living, thus speculate that it enters female
The fertility in sexual reproduction road, and rely on current sperm function appraisement system, the evaluation to sperm quality and function exists one
Fixed defect, and be difficult to provide corresponding clinical consultation to patient.It is true that this is only the side evaluating sperm function
Face, we are specifically contemplated that may also have an impact the more of sperm reproductive performance does not has revealed factor.
In first patent 201310431847.0, it is proposed that a kind of sialidase detection method affecting sperm function.First
Step, quantifies sialidase expression in sperm intuitively by monosperm Fluorescence Intensity Assays, or utilizes under microscope glimmering
Photoimaging observes sialidase expression in sperm intuitively；If sialidase is high expressed in sperm, then carry out second step,
Utilize In-vitro Capacitation liquid to make sperm affect the sialidase detection method capacitation of sperm function, and use Fluorometric assay sialidase
Activity.Although this patent discloses the expression in sperm of the sialidase protein molecule and the detection of primary activity.But in detection
On object, be the enzyme activity determination that capacitation liquid is carried out, detection be sperm is carried out capacitation process after, sperm is de-in capacitation liquid
The sialidase fallen, it is therefore an objective to the sperm quality after assessment capacitation.Sialidase activity and capacitation liquid due to sperm itself
Enzyme is lived in not existing and is necessarily associated, and the method cannot detect the sialidase that sperm itself is contained, it is impossible to quality own to sperm is entered
Row assessment.Meanwhile, method there is also following shortcoming: 1) techniqueflow complexity；2) to experimental facilities and operator's technical ability
High with skill requirement；3) it is unsuitable for being applied to clinical laboratory and those having ordinary skill in the art uses；4) reagent of abcam company is used
Box, expensive.
Summary of the invention:
For above-mentioned technical problem, the invention provides test kit and the using method thereof of a kind of sperm quality assessment.To sperm
The sperm function extracting albumen is measured, and uses the gross activity of the fresh sperm sialidase of seminal fluid assay timely, is used for commenting
The possible potential cause of valency male sterility, sterile the most relevant with sialidase activity in particular for unknown cause clinically
Situation.
In order to realize foregoing invention purpose, the present invention adopts the following technical scheme that:
A kind of test kit of sperm quality assessment, it is characterised in that: described test kit includes the cell without inhibitors of enzymes
Lysate, detection buffer 1, protease inhibitor cocktail, reagent A liquid, reagent B liquid, bovine serum albumin, fluorescence examination
Agent, sialidase standard stock solution and detection buffer 2.
Cell pyrolysis liquid of the present invention does not contains inhibitors of enzymes, and inhibitor can destroy the activity of sialidase, can shadow
Ring enzyme activity determination below.
Described detection buffer 1 is PBS(phosphate buffer) or BWW culture fluid.
Described reagent A liquid is: 1%BCA disodium salt, 2% natrium carbonicum calcinatum, 0.16% sodium tartrate, 0.4% hydroxide
Sodium, 0.95% sodium bicarbonate, mixing tune pH value to 11.2-11.3.
Described reagent B liquid is: 4% copper sulfate.
Reagent A liquid is used for providing reaction raw materials BCA(i.e. bicinchoninic acid) and alkaline environment, reagent B liquid provides reaction former
Material bivalent cupric ion.Specifically, B liquid color is sky blue, and after mixing with A liquid, it is green, this that color is shown as Fructus Mali pumilae
Under the conditions of alkalescence, original bivalent cupric ion can be reduced to univalent copper ion by the protein of addition, and a univalent copper ion
Can chelate two BCA molecules, thus solution colour is changed into purple by Fructus Mali pumilae green.
Described sialidase standard stock solution is bacillus perfringens sialidase (AUS), and concentration is 5U/ml, can be convenient
Be diluted to these Concentraton gradient of 5/2.5/1.25/0.625/0.312/0 mU/ml.Bacillus perfringens sialidase is used to fit
Detection method for a small amount of sperm of the present invention.
Described fluorometric reagent is 2 '-4-methyl umbelliferone base-α-D-N-n acetylneuraminic acid n sodium, has higher sensitive
Degree.
The concrete structure of described 2 '-4-methyl umbelliferone base-α-D-N-n acetylneuraminic acid n sodium is as follows:
。
Preferably, the concentration of described fluorometric reagent is 10mM, it is provided that the concentrated solution of fluorometric reagent, it is ensured that its stability,
It is not easily decomposed and cancellation, separately can reduce volume, it is simple to mounted box.
Described detection buffer 2 is containing 0.05 mmoL/L sodium acetate and the PBS of 0.25 μ g/ μ l bovine serum albumin.Inspection
Survey the buffer system of buffer 2, detect specific preparation for sialidase activity.
Preferably, the pH value of described sodium acetate is 5-6, is the working environment optimal for ptyalin activity.
The using method of the test kit of sperm quality of the present invention assessment: specifically comprise the following steps that
A, the washing of sperm
Collecting fresh liquefaction semen sample, detection buffer 1 washs sperm, fully removes the refining composition of residual, more centrifugal point
Separate out sperm.
The present invention first adds detection buffer 1 in semen sample and washs, then is centrifugally separating to obtain Sperm pellets.This
It is owing to refining itself is sticky, directly it is centrifuged and all sperms in seminal fluid can not be made fully to precipitate, add detection buffer 1 right
After it is diluted, centrifugally operated effect is more preferable.
Preferably, seminal fluid carries out 3 times be centrifuged, it is ensured that in the case of detection does not has refining interference, reduce centrifugal as far as possible
The number of times of sperm, it is ensured that motility of sperm.
B, extraction sperm protein
In the isolated sperm of step A, add cell pyrolysis liquid and protease inhibitor cocktail, mixing, treat without significantly
Cell precipitation the most fully cracking, high speed centrifugation at 4 DEG C, supernatant i.e. protein extract；
The addition of protease inhibitor cocktail is used to the effect of protease inhibition, and the effect of protease is protein degradation
Matter, and the thing sialidase to be checked of the present invention is exactly a kind of protein, adds protease inhibitor and is possible to prevent sialidase quilt
Degraded.
Preferably, 2-3 times that addition volume is sperm volume of described lysate, so can ensure the sensitivity of detection.
Preferably, described high speed centrifugation refers to the centrifugation 5min with 12000r/min, removes cell (essence as far as possible
Son) fragment, otherwise these fragments can affect the specificity of detection.
C, the protein concentration of mensuration sperm protein extract
(1) by the protein standard stock solution of cell pyrolysis liquid preparation bovine serum albumin, then 2/1.0/0.5/0.25/ is pressed with lysate
Protein standard stock solution is diluted to protein standard solution by the Concentraton gradient of 0.125/0mg/ml, is sequentially added into each egg in detection plate
White standard solution；Addition sample to be tested, in detection plate, adds lysate dilution.
Preferably, it is 20mg/ml by the concentration of the protein standard stock solution of cell pyrolysis liquid preparation bovine serum albumin, can
In-20 DEG C of long term storages, it is simple to the preparation of these protein concentration gradients of 2/1.0/0.5/0.25/0.125/0mg/ml below.Egg
Sensitivity that white standard solution gradient can be detected by according to expression and this method of the protein concentration of Sperm extract and set
Fixed.
(2) press volume ratio reagent preparation A liquid and the mixed liquor of B liquid of A:B=50:1, be added into above-mentioned each protein standard
In the hole of solution and sample to be tested, mix gently, detection plate is placed in 37 DEG C of reaction 30min, then reads each hole by microplate reader
Absorbance, calculates the protein concentration of sample to be tested according to protein standard curve.
Preferably, described detection plate is 96 hole lamella lucidas.
Preferably, described microplate reader reads each hole absorbance under 562nm wavelength.
In the hole of detection, sample protein concentration should be in the range of 0.1-2mg/ml, if it was exceeded, test sample should be treated by hole
This protein concentration adjusts within this range of linearity.
D. sperm sialidase activity is measured
(1) with detection buffer 2, sialidase standard stock solution is pressed the concentration of 5/2.5/1.25/0.625/0.312/0 mU/ml
Gradient is diluted to sialidase standard solution, is sequentially added into each sialidase standard solution in detection plate；Add to be measured
Sample, in detection plate, adds detection buffer 2 and dilutes.
Sialidase standard solution gradient according to the expression of sperm sialidase and this method can be detected by sensitive
Spend and set.
Preferably, described detection plate is 96 hole blackboards, the present invention use fluorescence method to measure the activity of sialidase, black
The detection plate of color is optimal to sensitivity and specificity, can protect fluorescent dye not by light cancellation, hence it is evident that to reduce light
Negative effect to fluorometric reagent cancellation, so that sensitivity, stability and the accuracy of detection are greatly enhanced.
(2) dilute fluorometric reagent with detection buffer 2, be added into above-mentioned each sialidase standard solution and sample to be tested
Hole in, then detection plate is placed in after 37 DEG C of lucifuges hatch 1-2 hour, reads each hole fluorescence intensity by microplate reader, according to saliva
Phytase activity standard curve calculates the sialidase activity of sample to be tested, finally draws sialidase activity/protein concentration
It is worth.
Preferably, the concentration being diluted to fluorometric reagent with detection buffer 2 is 0.05mM, for optimal working concentration.
Preferably, microplate reader reads each Kong Ying under the wavelength that excitation wavelength and transmitting wavelength are respectively 365nm and 450nm
Light intensity.365nm is maximum excitation wavelength, and 450nm is for most preferably launching wavelength.
The unit enzyme of sialidase activity/protein concentration is lived and is worth (U AUS/g protein) reflection unit sperm protein
Sialidase activity quantitative values, the crowd of making can express the marginal value of sialidase activity according to the live data of value of unit enzyme.
According to this marginal value, can live clinically by unit enzyme and be worth the service quality tentatively judging sperm, according to the most
Some data, for normal fertile men and infertile patients, this marginal value is about 0.2；For the good sperm of motor capacity and
The sperm that motor capacity is poor, this marginal value is about 0.24；Sperm normal for motility rate and the low sperm of motility rate, this is critical
Value is about 0.3.
Preferably, in the hole of detection, sample sialidase activity should be in the range of 0.3-5mU/ml, if exceeded,
The sialidase activity value of sample to be tested in hole should be adjusted within this range of linearity.
The beneficial effects of the present invention is:
1, the reagent in test kit of the present invention all selects routine test reagent, and combines the particularity to sperm detection, reduces
Experimental cost, required instrument and equipment, consumptive material kind are few, and only centrifuge, microplate reader, EP pipe, rifle head and detection plate etc., be
Routine Test Lab configures；And operational approach is simple, it is only centrifugal, sample-adding etc., does not exist and need to repeatedly practise obtaining operation
The situation of skill, can be widely applied to clinical laboratory and those having ordinary skill in the art uses.
2, the test kit of the present invention is used for assessing the quality of sperm own, the reason of examination sterility and infertility.Live according to unit enzyme
The data of value the crowd of making can express the marginal value of sialidase activity, according to this marginal value, and can be clinically by unit enzyme
Value of living tentatively judges the service quality of sperm, for evaluating the possible potential cause of male sterility, in particular for the most not
The sterile situation the most relevant with sialidase activity of bright reason.
3, sperm is sufficiently washed by the present invention, fully removes the refining composition of residual, makes the egg of residual in refining
Pseudobulbus Bletillae (Rhizoma Bletillae) sialidase does not interferes with the accuracy of testing result；Optimize the standard substance albumen arranged when surveying sperm protein concentration dense
The standard substance enzyme stepladder degree that degree gradient and survey enzyme are arranged when living, if gradient arranges unreasonable, all can affect very much the sensitive of result
Degree and accuracy；Use black detection plate to survey enzyme to live, can obviously reduce the light negative effect to fluorometric reagent cancellation, the most greatly
The big raising stability of experimental result, sensitivity and accuracy；Final testing result is designed as sialidase work/protein concentration
Value, so can reduce the difference between different sample, make to have more each other comparability, it is ensured that the stability of testing result and can
By property.
Accompanying drawing explanation
The sialidase that Fig. 1 measures eupyrene sperm (people) and sterile sperm (people) respectively for using test kit of the present invention is lived
Property/protein concentration unit enzyme live value.
Detailed description of the invention
Below in conjunction with detailed description of the invention, the essentiality content of the present invention is described in further detail.
Embodiment 1
The test kit of a kind of sperm quality assessment, described test kit includes the cell pyrolysis liquid without inhibitors of enzymes, detection
Buffer 1, protease inhibitor cocktail, reagent A liquid, reagent B liquid, bovine serum albumin, fluorometric reagent, sialidase mark
Quasi-stock solution and detection buffer 2；
Described detection buffer 1 is phosphate buffer；
Described reagent A liquid is: 1%BCA disodium salt, 2% natrium carbonicum calcinatum, 0.16% sodium tartrate, 0.4% sodium hydroxide,
0.95% sodium bicarbonate, mixing adjusts pH value to 11.2；
Described reagent B liquid is: 4% copper sulfate；
Described detection buffer 2 is containing 0.05 mmol/l sodium acetate and the phosphoric acid buffer of 0.25 μ g/ μ l bovine serum albumin
Liquid.
The test kit using the present invention measures eupyrene sperm (people) and the sialidase activity of sterile sperm (people) respectively, and result shows
Show that the sialidase activity of eupyrene sperm is apparently higher than sterile sperm (see figure 1).
Embodiment 2
The test kit of a kind of sperm quality assessment, described test kit includes the cell pyrolysis liquid without inhibitors of enzymes, detection
Buffer 1, protease inhibitor cocktail, reagent A liquid, reagent B liquid, bovine serum albumin, fluorometric reagent, sialidase mark
Quasi-stock solution and detection buffer 2；
Described detection buffer 1 is phosphate buffer；
Described reagent A liquid is: 1%BCA disodium salt, 2% natrium carbonicum calcinatum, 0.16% sodium tartrate, 0.4% sodium hydroxide,
0.95% sodium bicarbonate, mixing adjusts pH value to 11.3；
Described reagent B liquid is: 4% copper sulfate；
Described detection buffer 2 is containing 0.05 mmol/l sodium acetate and the phosphoric acid buffer of 0.25 μ g/ μ l bovine serum albumin
Liquid.
Described sialidase standard stock solution is bacillus perfringens sialidase, and concentration is 5U/ml.
Embodiment 3
The test kit of a kind of sperm quality assessment, described test kit includes the cell pyrolysis liquid without inhibitors of enzymes, detection
Buffer 1, protease inhibitor cocktail, reagent A liquid, reagent B liquid, bovine serum albumin, fluorometric reagent, sialidase mark
Quasi-stock solution and detection buffer 2；
Described detection buffer 1 is BWW culture fluid；
Described reagent A liquid is: 1%BCA disodium salt, 2% natrium carbonicum calcinatum, 0.16% sodium tartrate, 0.4% sodium hydroxide,
0.95% sodium bicarbonate, mixing adjusts pH value to 11.25；
Described reagent B liquid is: 4% copper sulfate；
Described detection buffer 2 is containing 0.05 mmol/l sodium acetate and the phosphoric acid buffer of 0.25 μ g/ μ l bovine serum albumin
Liquid.
Described sialidase standard stock solution is bacillus perfringens sialidase, and concentration is 5U/ml.
Described fluorometric reagent is 2 '-4-methyl umbelliferone base-α-D-N-n acetylneuraminic acid n sodium.
Embodiment 4
The test kit of a kind of sperm quality assessment, described test kit includes the cell pyrolysis liquid without inhibitors of enzymes, detection
Buffer 1, protease inhibitor cocktail, reagent A liquid, reagent B liquid, bovine serum albumin, fluorometric reagent, sialidase mark
Quasi-stock solution and detection buffer 2；
Described detection buffer 1 is BWW culture fluid；
Described reagent A liquid is: 1%BCA disodium salt, 2% natrium carbonicum calcinatum, 0.16% sodium tartrate, 0.4% sodium hydroxide,
0.95% sodium bicarbonate, mixing adjusts pH value to 11.28；
Described reagent B liquid is: 4% copper sulfate；
Described detection buffer 2 is containing 0.05 mmol/l sodium acetate and the phosphoric acid buffer of 0.25 μ g/ μ l bovine serum albumin
Liquid.
Described sialidase standard stock solution is bacillus perfringens sialidase, and concentration is 5U/ml.
Described fluorometric reagent is 2 '-4-methyl umbelliferone base-α-D-N-n acetylneuraminic acid n sodium.
The concentration of described fluorometric reagent is 10mM.
Embodiment 5
The test kit of a kind of sperm quality assessment, described test kit includes the cell pyrolysis liquid without inhibitors of enzymes, detection
Buffer 1, protease inhibitor cocktail, reagent A liquid, reagent B liquid, bovine serum albumin, fluorometric reagent, sialidase mark
Quasi-stock solution and detection buffer 2；
Described detection buffer 1 is phosphate buffer；
Described reagent A liquid is: 1%BCA disodium salt, 2% natrium carbonicum calcinatum, 0.16% sodium tartrate, 0.4% sodium hydroxide,
0.95% sodium bicarbonate, mixing adjusts pH value to 11.2；
Described reagent B liquid is: 4% copper sulfate；
Described detection buffer 2 is containing 0.05 mmol/l sodium acetate and the phosphoric acid buffer of 0.25 μ g/ μ l bovine serum albumin
Liquid.
Described sialidase standard stock solution is bacillus perfringens sialidase, and concentration is 5U/ml.
Described fluorometric reagent is 2 '-4-methyl umbelliferone base-α-D-N-n acetylneuraminic acid n sodium.
The concentration of described fluorometric reagent is 10mM.
The pH value of described sodium acetate is 5.
Embodiment 6
The test kit of a kind of sperm quality assessment, described test kit includes the cell pyrolysis liquid without inhibitors of enzymes, detection
Buffer 1, protease inhibitor cocktail, reagent A liquid, reagent B liquid, bovine serum albumin, fluorometric reagent, sialidase mark
Quasi-stock solution and detection buffer 2；
Described detection buffer 1 is phosphate buffer；
Described reagent A liquid is: 1%BCA disodium salt, 2% natrium carbonicum calcinatum, 0.16% sodium tartrate, 0.4% sodium hydroxide,
0.95% sodium bicarbonate, mixing adjusts pH value to 11.3；
Described reagent B liquid is: 4% copper sulfate；
Described detection buffer 2 is containing 0.05 mmol/l sodium acetate and the phosphoric acid buffer of 0.25 μ g/ μ l bovine serum albumin
Liquid.
Described sialidase standard stock solution is bacillus perfringens sialidase, and concentration is 5U/ml.
Described fluorometric reagent is 2 '-4-methyl umbelliferone base-α-D-N-n acetylneuraminic acid n sodium.
The concentration of described fluorometric reagent is 10mM.
The pH value of described sodium acetate is 6.
Embodiment 7
The test kit of a kind of sperm quality assessment, described test kit includes the cell pyrolysis liquid without inhibitors of enzymes, detection
Buffer 1, protease inhibitor cocktail, reagent A liquid, reagent B liquid, bovine serum albumin, fluorometric reagent, sialidase mark
Quasi-stock solution and detection buffer 2；
Described detection buffer 1 is phosphate buffer；
Described reagent A liquid is: 1%BCA disodium salt, 2% natrium carbonicum calcinatum, 0.16% sodium tartrate, 0.4% sodium hydroxide,
0.95% sodium bicarbonate, mixing adjusts pH value to 11.25；
Described reagent B liquid is: 4% copper sulfate；
Described detection buffer 2 is containing 0.05 mmol/l sodium acetate and the phosphoric acid buffer of 0.25 μ g/ μ l bovine serum albumin
Liquid.
Described sialidase standard stock solution is bacillus perfringens sialidase, and concentration is 5U/ml.
Described fluorometric reagent is 2 '-4-methyl umbelliferone base-α-D-N-n acetylneuraminic acid n sodium.
The concentration of described fluorometric reagent is 10mM.
The pH value of described sodium acetate is 5.5.
Embodiment 8
The using method of the test kit of a kind of sperm quality of the present invention assessment, specifically comprises the following steps that
A, the washing of sperm
Collecting fresh liquefaction semen sample, detection buffer 1 washs sperm, fully removes the refining composition of residual, more centrifugal point
Separate out sperm；
B, extraction sperm protein
In the isolated sperm of step A, add cell pyrolysis liquid and protease inhibitor cocktail, mixing, treat without significantly
Cell precipitation the most fully cracking, high speed centrifugation at 4 DEG C, supernatant i.e. protein extract；
C, the protein concentration of mensuration sperm protein extract
(1) by the protein standard stock solution of cell pyrolysis liquid preparation bovine serum albumin, then 2/1.0/0.5/0.25/ is pressed with lysate
Protein standard stock solution is diluted to protein standard solution by the Concentraton gradient of 0.125/0mg/ml, is sequentially added into each egg in detection plate
White standard solution；Addition sample to be tested, in detection plate, adds lysate dilution.
(2) press volume ratio reagent preparation A liquid and the mixed liquor of B liquid of A:B=50:1, be added into above-mentioned each protein standard
In the hole of solution and sample to be tested, mix gently, detection plate is placed in 37 DEG C of reaction 30min, then reads each hole by microplate reader
Absorbance, calculates the protein concentration of sample to be tested according to protein standard curve.
D, mensuration sperm sialidase activity
(1) with detection buffer 2, sialidase standard stock solution is pressed the concentration of 5/2.5/1.25/0.625/0.312/0 mU/ml
Gradient is diluted to sialidase standard solution, is sequentially added into each sialidase standard solution in detection plate；Add to be measured
Sample, in detection plate, adds detection buffer 2 and dilutes.
(2) dilute fluorometric reagent with detection buffer 2, be added into above-mentioned each sialidase standard solution and sample to be tested
Hole in, then detection plate is placed in after 37 DEG C of lucifuges hatch 1 hour, reads each hole fluorescence intensity by microplate reader, according to sialic acid
Enzymatic activity standard curve calculates the sialidase activity of sample to be tested, finally draws the value of sialidase activity/protein concentration
?.
Embodiment 9
The using method of the test kit of a kind of sperm quality of the present invention assessment, specifically comprises the following steps that
A, the washing of sperm
Collecting fresh liquefaction semen sample, detection buffer 1 washs sperm, fully removes the refining composition of residual, more centrifugal point
Separate out sperm；
B, extraction sperm protein
In the isolated sperm of step A, add cell pyrolysis liquid and protease inhibitor cocktail, mixing, treat without significantly
Cell precipitation the most fully cracking, high speed centrifugation at 4 DEG C, supernatant i.e. protein extract；
C, the protein concentration of mensuration sperm protein extract
(1) by the protein standard stock solution of cell pyrolysis liquid preparation bovine serum albumin, then 2/1.0/0.5/0.25/ is pressed with lysate
Protein standard stock solution is diluted to protein standard solution by the Concentraton gradient of 0.125/0mg/ml, is sequentially added into each egg in detection plate
White standard solution；Addition sample to be tested, in detection plate, adds lysate dilution.
(2) press volume ratio reagent preparation A liquid and the mixed liquor of B liquid of A:B=50:1, be added into above-mentioned each protein standard
In the hole of solution and sample to be tested, mix gently, detection plate is placed in 37 DEG C of reaction 30min, then reads each hole by microplate reader
Absorbance, calculates the protein concentration of sample to be tested according to protein standard curve.
D, mensuration sperm sialidase activity
(1) with detection buffer 2, sialidase standard stock solution is pressed the concentration of 5/2.5/1.25/0.625/0.312/0 mU/ml
Gradient is diluted to sialidase standard solution, is sequentially added into each sialidase standard solution in detection plate；Add to be measured
Sample, in detection plate, adds detection buffer 2 and dilutes.
(2) dilute fluorometric reagent with detection buffer 2, be added into above-mentioned each sialidase standard solution and sample to be tested
Hole in, then detection plate is placed in after 37 DEG C of lucifuges hatch 2 hours, reads each hole fluorescence intensity by microplate reader, according to sialic acid
Enzymatic activity standard curve calculates the sialidase activity of sample to be tested, finally draws the value of sialidase activity/protein concentration
?.
In the hole of detection, sample sialidase activity should be in the range of 0.3-5mU/ml, if exceeded, and should be by hole
The sialidase activity value of sample to be tested adjusts within this range of linearity.
Embodiment 10
The using method of the test kit of a kind of sperm quality of the present invention assessment, specifically comprises the following steps that
A, the washing of sperm
Collecting fresh liquefaction semen sample, detection buffer 1 washs sperm, fully removes the refining composition of residual, more centrifugal point
Separate out sperm；
B, extraction sperm protein
In the isolated sperm of step A, add cell pyrolysis liquid and protease inhibitor cocktail, mixing, treat without significantly
Cell precipitation the most fully cracking, high speed centrifugation at 4 DEG C, supernatant i.e. protein extract；
C, the protein concentration of mensuration sperm protein extract
(1) by the protein standard stock solution of cell pyrolysis liquid preparation bovine serum albumin, then 2/1.0/0.5/0.25/ is pressed with lysate
Protein standard stock solution is diluted to protein standard solution by the Concentraton gradient of 0.125/0mg/ml, is sequentially added into each egg in detection plate
White standard solution；Addition sample to be tested, in detection plate, adds lysate dilution.
(2) press volume ratio reagent preparation A liquid and the mixed liquor of B liquid of A:B=50:1, be added into above-mentioned each protein standard
In the hole of solution and sample to be tested, mix gently, detection plate is placed in 37 DEG C of reaction 30min, then reads each hole by microplate reader
Absorbance, calculates the protein concentration of sample to be tested according to protein standard curve.
D, mensuration sperm sialidase activity
(1) with detection buffer 2, sialidase standard stock solution is pressed the concentration of 5/2.5/1.25/0.625/0.312/0 mU/ml
Gradient is diluted to sialidase standard solution, is sequentially added into each sialidase standard solution in detection plate；Add to be measured
Sample, in detection plate, adds detection buffer 2 and dilutes.
(2) dilute fluorometric reagent with detection buffer 2, be added into above-mentioned each sialidase standard solution and sample to be tested
Hole in, then detection plate is placed in after 37 DEG C of lucifuges hatch 1.5 hours, reads each hole fluorescence intensity by microplate reader, according to saliva
Phytase activity standard curve calculates the sialidase activity of sample to be tested, finally draws sialidase activity/protein concentration
It is worth.
In the hole of detection, sample sialidase activity should be in the range of 0.3-5mU/ml, if exceeded, and should be by hole
The sialidase activity value of sample to be tested adjusts within this range of linearity.
Embodiment 11
The present embodiment is on the basis of embodiment 8:
Described step B, 2 times that addition volume is sperm volume of cell pyrolysis liquid.
Embodiment 12
The present embodiment is on the basis of embodiment 8:
Described step B, 3 times that addition volume is sperm volume of cell pyrolysis liquid.
Described step C, is 20mg/ by the concentration of the protein standard stock solution of cell pyrolysis liquid preparation bovine serum albumin
ml。
Embodiment 13
The present embodiment is on the basis of embodiment 8:
Described step B, 2.5 times that addition volume is sperm volume of cell pyrolysis liquid.
Described step C, is 20mg/ by the concentration of the protein standard stock solution of cell pyrolysis liquid preparation bovine serum albumin
ml。
Described step C, microplate reader reads each hole absorbance under 562nm wavelength.
Embodiment 14
The present embodiment is on the basis of embodiment 9:
Described step B, 2.3 times that addition volume is sperm volume of cell pyrolysis liquid.
Described step C, is 20mg/ by the concentration of the protein standard stock solution of cell pyrolysis liquid preparation bovine serum albumin
ml。
Described step C, microplate reader reads each hole absorbance under 562nm wavelength.
The detection plate of described step C is 96 hole lamella lucidas, and the detection plate of described D step is 96 hole blackboards.
Described D step, microplate reader is in excitation wavelength and launches reading under the wavelength that wavelength is respectively 365nm and 450nm
Each hole fluorescence intensity.
Embodiment 15
The present embodiment is on the basis of embodiment 9:
Described step B, 2 times that addition volume is sperm volume of cell pyrolysis liquid.
Described step C, is 20mg/ by the concentration of the protein standard stock solution of cell pyrolysis liquid preparation bovine serum albumin
ml。
Described step C, microplate reader reads each hole absorbance under 562nm wavelength.
The detection plate of described step C is 96 hole lamella lucidas, and the detection plate of described D step is 96 hole blackboards.
Described D step, the concentration being diluted to fluorometric reagent with detection buffer 2 is 0.05mM.
Described D step, microplate reader is in excitation wavelength and launches reading under the wavelength that wavelength is respectively 365nm and 450nm
Each hole fluorescence intensity.
Embodiment 16
The present embodiment is on the basis of embodiment 10:
Described step B, 3 times that addition volume is sperm volume of cell pyrolysis liquid.
Described step C, is 20mg/ by the concentration of the protein standard stock solution of cell pyrolysis liquid preparation bovine serum albumin
ml。
Described step C, microplate reader reads each hole absorbance under 562nm wavelength.
The detection plate of described step C is 96 hole lamella lucidas, and the detection plate of described D step is 96 hole blackboards.
Described D step, the concentration being diluted to fluorometric reagent with detection buffer 2 is 0.05mM.
Described D step, microplate reader is in excitation wavelength and launches reading under the wavelength that wavelength is respectively 365nm and 450nm
Each hole fluorescence intensity.
Embodiment 17
Consumptive material (equal sterilizing): 15mlEP manages, and 1.5mlEP manages, 3 kinds of specification rifle heads (1ml/200 μ l/10 μ l), 96 well culture plates, and 96
Hole detection plate
Instrument: high speed low temperature centrifugal machine, microplate reader
The using method of the test kit of a kind of sperm quality of the present invention assessment, specifically comprises the following steps that
A, the washing of sperm
The freshest liquefaction semen sample is collected 4-5ml(and is adjusted according to patient's sperm quantity, preserves on ice and transports).
2. adding equal-volume PBS, piping and druming is uniformly.
3. low-speed centrifugal (1500rcf/5min), abandoning supernatant.
4. adding PBS to 3-4ml, piping and druming is uniformly.
5. low-speed centrifugal (1500rcf/5min), abandoning supernatant, supernatant is remained to 300 μ about l, piping and druming is uniformly.
6. add PBS to 1ml, high speed centrifugation (4 degree, 12000rpm/5min), abandoning supernatant as far as possible.
B, extraction sperm protein
7. in Sperm pellets, add lysate and the final concentration of 1X of protease inhibitor cocktail(of 2-3 times of volume), blow
Play mixing, treat without obvious cell precipitation the most fully cracking, high speed centrifugation (12000rpm/5min) at 4 DEG C, supernatant i.e. egg
White extract；
C, the protein concentration of mensuration sperm protein extract
8., with lysate preparation BSA protein standard stock solution (20mg/ml), take the protein standard stock solution of appropriate 20mg/ml, with splitting
Solve liquid and be diluted to protein standard solution by the Concentraton gradient of 2/1.0/0.5/0.25/0.125/0mg/ml, transparent to 96 holes
20 μ l each protein standard solution it is sequentially added in plate.
9. add 2 microlitre samples to be tested in 96 orifice plates, add lysate to 20 microlitres.
10. press the volume ratio reagent preparation A liquid of A:B=50:1 and the mixed liquor (room temperature is stable in 24 hours) of B liquid, by it
Add above-mentioned each hole, 200 microlitres/hole.
11. mix 30s gently, 96 hole lamella lucidas are placed in 37 degree of reaction 30min, then by microplate reader at 562nm wavelength
Lower reading each hole absorbance, calculates the protein concentration of sample to be tested according to protein standard curve.
In the hole of detection, sample protein concentration should be in the range of 0.1-2mg/ml, if exceeded, and should be by be measured in hole
The protein concentration of sample adjusts within this range of linearity.
D. sperm sialidase activity is measured
1. with detection buffer 2, sialidase standard stock solution is pressed the concentration of 5/2.5/1.25/0.625/0.312/0 mU/ml
Gradient is diluted to sialidase standard solution, is sequentially added into 50 μ l each sialidase standard solution in 96 hole blackboards.
2. add 25 microlitre samples to be tested in 96 hole blackboards, add detection buffer 2 to 50 microlitre.
3. press 1:200 with detection buffer 2 and dilute fluorometric reagent, be added in above-mentioned each hole, 50 microlitres/hole.
4. 96 hole blackboards are placed in 37 degree of lucifuges hatch 1-2 hour, then by microplate reader at excitation wavelength/transmitting wavelength
For reading each hole fluorescence intensity under 365/450nm wavelength, calculate the saliva of sample to be tested according to sialidase activity standard curve
Liquid phytase activity.
5. sialidase activity/the total protein concentration that finally will obtain, calculates unit enzyme value (U AUS/g alive
Protein).
In the hole of detection, sample sialidase activity should be in the range of 0.3-5mU/ml, if exceeded, and should be by hole
The sialidase activity value of sample to be tested adjusts within this range of linearity.
Claims (12)
1. the test kit of sperm quality assessment, it is characterised in that: it is thin that described test kit includes without inhibitors of enzymes
Cellular lysate liquid, detection buffer 1, protease inhibitor cocktail, reagent A liquid, reagent B liquid, bovine serum albumin, fluorescence examination
Agent, sialidase standard stock solution and detection buffer 2；
Described detection buffer 1 is phosphate buffer or BWW culture fluid；
Described reagent A liquid is: 1%BCA disodium salt, 2% natrium carbonicum calcinatum, 0.16% sodium tartrate, 0.4% sodium hydroxide,
0.95% sodium bicarbonate, mixing tune pH value to 11.2-11.3；
Described reagent B liquid is: 4% copper sulfate；
Described detection buffer 2 is containing 0.05 mmol/l sodium acetate and the phosphoric acid buffer of 0.25 μ g/ μ l bovine serum albumin
Liquid.
The test kit of a kind of sperm quality the most according to claim 1 assessment, it is characterised in that: described sialidase mark
Quasi-stock solution is bacillus perfringens sialidase, and concentration is 5U/ml.
The test kit of a kind of sperm quality the most according to claim 1 assessment, it is characterised in that: described fluorometric reagent is
2 '-4-methyl umbelliferone base-α-D-N-n acetylneuraminic acid n sodium.
The test kit of a kind of sperm quality the most according to claim 3 assessment, it is characterised in that: described fluorometric reagent dense
Degree is 10mM.
The test kit of a kind of sperm quality the most according to claim 1 assessment, it is characterised in that: the pH value of described sodium acetate
For 5-6.
The using method of the test kit of a kind of sperm quality the most according to claim 1 assessment, it is characterised in that: specifically walk
Rapid as follows:
A, the washing of sperm
Collecting fresh liquefaction semen sample, detection buffer 1 washs sperm, fully removes the refining composition of residual, more centrifugal point
Separate out sperm；
B, extraction sperm protein
In the isolated sperm of step A, add cell pyrolysis liquid and protease inhibitor cocktail, mixing, treat without significantly
Cell precipitation the most fully cracking, high speed centrifugation at 4 DEG C, supernatant i.e. protein extract；
C, the protein concentration of mensuration sperm protein extract
(1) by the protein standard stock solution of cell pyrolysis liquid preparation bovine serum albumin, then 2/1.0/0.5/0.25/ is pressed with lysate
Protein standard stock solution is diluted to protein standard solution by the Concentraton gradient of 0.125/0mg/ml, is sequentially added into each egg in detection plate
White standard solution；Addition sample to be tested, in detection plate, adds lysate dilution；
(2) press volume ratio reagent preparation A liquid and the mixed liquor of B liquid of A:B=50:1, be added into above-mentioned each protein standard solution
With in the hole of sample to be tested, mix gently, detection plate is placed in 37 DEG C of reaction 30min, then reads each hole extinction by microplate reader
Degree, calculates the protein concentration of sample to be tested according to protein standard curve；
D, mensuration sperm sialidase activity
(1) with detection buffer 2, sialidase standard stock solution is pressed the concentration of 5/2.5/1.25/0.625/0.312/0 mU/ml
Gradient is diluted to sialidase standard solution, is sequentially added into each sialidase standard solution in detection plate；Add to be measured
Sample, in detection plate, adds detection buffer 2 and dilutes；
(2) dilute fluorometric reagent with detection buffer 2, be added into above-mentioned each sialidase standard solution and the hole of sample to be tested
In, then detection plate is placed in after 37 DEG C of lucifuges hatch 1-2 hour, by microplate reader reading each hole fluorescence intensity, according to sialidase
Activity criteria's curve calculates the sialidase activity of sample to be tested, finally draws the value of sialidase activity/protein concentration i.e.
Can.
The using method of the test kit of a kind of sperm quality the most according to claim 6 assessment, it is characterised in that: described
Step B, 2-3 times that addition volume is sperm volume of cell pyrolysis liquid.
The using method of the test kit of a kind of sperm quality the most according to claim 6 assessment, it is characterised in that: described
Step C, is 20mg/ml by the concentration of the protein standard stock solution of cell pyrolysis liquid preparation bovine serum albumin.
The using method of the test kit of a kind of sperm quality the most according to claim 6 assessment, it is characterised in that: described
Step C, microplate reader reads each hole absorbance under 562nm wavelength.
The using method of the test kit of a kind of sperm quality the most according to claim 6 assessment, it is characterised in that: described C
The detection plate of step is 96 hole lamella lucidas, and the detection plate of described D step is 96 hole blackboards.
The using method of the test kit of 11. a kind of sperm quality according to claim 6 assessments, it is characterised in that: described
D step, being diluted to the concentration of fluorometric reagent with detection buffer 2 is 0.05mM.
The using method of the test kit of 12. a kind of sperm quality according to claim 6 assessments, it is characterised in that: described
D step, microplate reader excitation wavelength and launch wavelength be respectively 365nm and 450nm wavelength under read each hole fluorescence intensity.
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| CN201610147268.7A CN105717307B (en) | 2016-03-16 | 2016-03-16 | The kit and its application method of a kind of sperm quality assessment |
| PCT/CN2016/081268 WO2017156843A1 (en) | 2016-03-16 | 2016-05-06 | Kit for evaluating sperm quality and method of use thereof |
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|---|---|---|---|
| CN201610147268.7A CN105717307B (en) | 2016-03-16 | 2016-03-16 | The kit and its application method of a kind of sperm quality assessment |
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| CN105717307B CN105717307B (en) | 2017-07-04 |
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| CN112945883A (en) * | 2021-03-30 | 2021-06-11 | 南京林业大学 | Enzyme residue detection method for preparing medical intermediate by enzyme method |
| CN114164254A (en) * | 2021-12-16 | 2022-03-11 | 浙江省人民医院 | Application of proteasome in improving success rate of in vitro fertilization |
| 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) * | 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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| WO2017156843A1 (en) | 2017-09-21 |
| CN105717307B (en) | 2017-07-04 |
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