CN105717307B - The kit and its application method of a kind of sperm quality assessment - Google Patents
The kit and its application method of a kind of sperm quality assessment Download PDFInfo
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Abstract
The invention provides the kit and its application method of a kind of assessment of sperm quality.Described kit includes the cell pyrolysis liquid without inhibitors of enzymes, detection buffer solution 1, protease inhibitors cocktail, reagent A liquid, reagent B liquid, bovine serum albumin(BSA), fluorometric reagent, sialidase standard stock solution and detection buffer solution 2；Described detection buffer solution 1 is phosphate buffer or BWW nutrient solutions；Described reagent A liquid is：PH value to 11.2 11.3 is adjusted in 1%BCA disodium salts, 2% natrium carbonicum calcinatum, 0.16% sodium tartrate, 0.4% NaOH, 0.95% sodium acid carbonate, mixing；Described reagent B liquid is：4% copper sulphate；Described detection buffer solution 2 is the phosphate buffer of sodium acetate containing 0.05mmol/l and 0.25 μ g/ μ l bovine serum albumin(BSA)s.By the detection of sialidase activity in sperm, for the male sterility patient that clinical reason is failed to understand provides diagnosis basis and on.
Description
Technical field
The invention belongs to medical detection technology, and in particular to a kind of kit of sperm quality assessment and its user
Method.
Background technology
With continuing to develop for social industry, the total incidence of infertile couples is significantly raised.European genesiology meeting in 2004
Statistics：In the couple at child-bearing age 1 year can not the person of pregnancy account for 25%, wherein 15% seeks treatment, bridegroom's or husband's side factor causes infertility to account for 50%.Male
The cause of disease sexual disorders (1.7%) of infertility, varicocele (12. 3%), RTI (6.6%), congenital hair
Exception (2.1%) acquired disease day after tomorrow (2.6%) is educated, endocrine disturbance (0.6%) is immunized sexual factor (3.1%), other exceptions
(3%), but it is up to 60% ~ 75% patient and can not find reason, referred to as idiopathic male infertility, they only shows as few smart, weak essence
Or the sperm quality exception such as teratozoospermia.The male sterility of unknown cause may be caused due to many factors, such as long-term stress
Environmental factor causes endocrine disturbance, active oxygen and gene defect etc..
For many years, traditional semen routine analysis are always the most basic clinical indices for judging male fertility.Face
Real sterile reason on bed to the male sterility patient of the overwhelming majority is still unclear.Especially clinically about three/
One infertile patients, the Sperm routine analysis result of male is normal and close to normally.This kind of patient is clinically divided into not clear original
Because of sterility.Therefore, for a long time, there is very big difficulty in the clinical diagnosis to male sterility.Topmost reason is conventional
Semen analysis only determines 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, and the maturation and DNA damage of sperm nucleus, 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
Determine the above function.Capacitation is the necessary condition that sperm ovum binding forms embryonated egg, although the correlation of capacitation is ground
Study carefully existing some progress, but still there are problems that many still undefined.
In clinical position, a part of primary or secondary Infertility male patient utilizes existing conventional semen quality check item
Mesh points out sperm and semen quality without exception.Conventional sperm detection means is CASA (computer aided sperm analysis at present
Computer aided of semen analysis), and AsAb etc..Current the most widely used CASA technologies
With some morphologic coherent detections, the mobility and sperm ratio living of sperm can be mainly evaluated, so as to speculate that it enters female
The fertility in sexual reproduction road, and current sperm function appraisement system is relied on, have one to the evaluation of sperm quality and function
Fixed defect, and be difficult to provide corresponding on to patient.In fact, this is only a side for evaluating sperm function
Face, we are specifically contemplated that the more factors not being revealed that may also have an impact sperm fecundity.
A kind of first patent 201310431847.0, it is proposed that sialidase detection method of influence sperm function.First
Step, expression of the sialidase in sperm is intuitively quantified by monosperm Fluorescence Intensity Assays, or using glimmering under microscope
Photoimaging intuitively observes expression of the sialidase in sperm；If sialidase expression high in sperm, carries out second step,
Sperm is influenceed the sialidase detection method capacitation of sperm function using In-vitro Capacitation liquid, and use Fluorometric assay sialidase
Activity.Although the patent discloses the detection of expression and primary activity of the sialidase protein molecule in sperm.But in detection
It is the enzyme activity determination carried out to capacitation liquid on object, detection is that after carrying out capacitation treatment to sperm, sperm takes off in capacitation liquid
The sialidase for falling, it is therefore an objective to assess the sperm quality after capacitation.Due to sperm sialidase activity in itself and capacitation liquid
In the absence of necessarily associating, the method cannot detect sperm sialidase contained in itself to enzyme activity, it is impossible to which to sperm, quality is entered in itself
Row assessment.Meanwhile, also had the following disadvantages in method：1）Techniqueflow is complicated；2）To experimental facilities and operating personnel's technical ability
It is high with skill requirement；3）It is unsuitable for being applied to clinical labororatory and those having ordinary skill in the art uses；4）Use the reagent of abcam companies
Box, it is expensive.
The content of the invention：
For above-mentioned technical problem, the invention provides the kit and its application method of a kind of assessment of sperm quality.It is right
The sperm function that sperm extracts albumen is measured, and using the gross activity of fresh timely seminal fluid assay sperm sialidase, uses
In evaluate male sterility possibility potential cause, in particular for clinically unknown cause infertility whether with sialidase activity
Relevant situation.
In order to realize foregoing invention purpose, the present invention is adopted the following technical scheme that：
A kind of kit of sperm quality assessment, it is characterised in that：Described kit is included without inhibitors of enzymes
Cell pyrolysis liquid, detection buffer solution 1, protease inhibitors cocktail, reagent A liquid, reagent B liquid, bovine serum albumin(BSA), fluorescence
Reagent, sialidase standard stock solution and detection buffer solution 2.
Cell pyrolysis liquid of the present invention is free of inhibitors of enzymes, and inhibitor can destroy the activity of sialidase, can shadow
Ring enzyme activity determination below.
Described detection buffer solution 1 is PBS（Phosphate buffer）Or BWW nutrient solutions.
Described reagent A liquid is：1%BCA disodium salts, 2% natrium carbonicum calcinatum, 0.16% sodium tartrate, 0.4% hydroxide
PH value to 11.2-11.3 is adjusted in sodium, 0.95% sodium acid carbonate, mixing.
Described reagent B liquid is：4% copper sulphate.
Reagent A liquid is used to provide reaction raw materials BCA（That is bicinchoninic acid）And alkaline environment, it is former that reagent B liquid provides reaction
Material bivalent cupric ion.Specifically, B liquid color is sky blue, and after mixing with A liquid, color is shown as apple green, this
Under alkalescence condition, original bivalent cupric ion can be reduced to univalent copper ion by the protein of addition, and a univalent copper ion
Two BCA molecules can be chelated, thus solution colour is changed into purple by apple green.
Described sialidase standard stock solution is C.perfringens sialidase（AUS）, concentration is 5U/ml, can be convenient
Be diluted to these concentration gradients of 5/2.5/1.25/0.625/0.312/0 mU/ml.It is suitable using C.perfringens sialidase
For the detection method of a small amount of sperm of the invention.
Described fluorometric reagent is 2 ' -4-methyl umbelliferone base-α-D-N- n acetylneuraminic acid n sodium, with higher sensitive
Degree.
The concrete structure of 2 ' -4-methyl umbelliferone base-α-D-N- n acetylneuraminic acid n sodium is as follows：
。
Preferably, the concentration of the fluorometric reagent is 10mM, there is provided the concentrate of fluorometric reagent, it is ensured that its stability,
It is not easily decomposed and is quenched, can separately reduce volume, is easy to mounted box.
Described detection buffer solution 2 is the PBS containing 0.05 mmoL/L sodium acetates and 0.25 μ g/ μ l bovine serum albumin(BSA)s.Inspection
The buffer system of buffer solution 2 is surveyed, is directed to sialidase activity and is detected specific preparation.
Preferably, the pH value of the sodium acetate is 5-6, is directed to the optimal working environment of saliva enzymatic activity.
The application method of the kit of sperm quality assessment of the present invention：Comprise the following steps that：
The washing of A, sperm
Collect fresh liquefaction semen sample, the detection washing sperm of buffer solution 1 fully removes the refining composition of residual, then from
The heart isolates sperm.
It is of the invention first to be washed to addition detection buffer solution 1 in semen sample, then it is centrifugally separating to obtain Sperm pellets.This
It is, because refining is sticky in itself, all sperms in can not making seminal fluid to be directly centrifuged to it and fully precipitates, adds detection buffer solution 1 pair
After it is diluted, centrifugally operated effect is more preferable.
Preferably, 3 centrifugations are carried out to seminal fluid, it is ensured that in the case that the no refining of detection is disturbed, centrifugation is reduced as far as possible
The number of times of sperm, it is ensured that sperm motility.
B, extraction sperm protein
To cell pyrolysis liquid and protease inhibitors cocktail is added in the sperm that step A is isolated, mix, treat without bright
Aobvious cell precipitation is fully cracking, and the high speed centrifugation at 4 DEG C, supernatant is protein extract；
The addition of protease inhibitors cocktail is the effect for protease inhibition, and the effect of protease is degraded egg
White matter, and thing sialidase to be checked of the invention is exactly a kind of protein, adds protease inhibitors to prevent sialidase
It is degraded.
Preferably, the addition volume of the lysate is 2-3 times of sperm volume, can so ensure the sensitivity of detection.
Preferably, described high speed centrifugation refers to, with the centrifugation 5min of 12000r/min, cell to be removed as far as possible（Essence
Son）Fragment, otherwise these fragments can influence the specificity of detection.
C, the protein concentration for determining sperm protein extract
（1）The protein standard stoste of bovine serum albumin(BSA) is prepared with cell pyrolysis liquid, then 2/1.0/0.5/ is pressed with lysate
Protein standard stoste is diluted to protein standard solution by concentration gradient 0.25/0.125/0mg/ml, is added successively in detection plate
Enter each protein standard solution；Add sample to be tested in detection plate, add lysate dilution.
Preferably, the concentration that the protein standard stoste of bovine serum albumin(BSA) is prepared with cell pyrolysis liquid is 20mg/ml, can
In -20 DEG C of long term storages, it is easy 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 liquid gradient can be detected according to the expression quantity and this method of the protein concentration of Sperm extract and set
Fixed.
（2）By A:B=50：1 volume ratio reagent preparation A liquid and the mixed liquor of B liquid, is added into above-mentioned each protein standard
In the hole of solution and sample to be tested, gently mix, detection plate is placed in 37 DEG C of reaction 30min, then read each hole with ELIASA
Absorbance, the protein concentration of sample to be tested is calculated according to protein standard curve.
Preferably, described detection plate is 96 hole transparent panels.
Preferably, described ELIASA reads each hole absorbance under 562nm wavelength.
Sample protein concentration should be in the range of 0.1-2mg/ml, if it was exceeded, test sample should will be treated in hole in the hole of detection
This protein concentration is adjusted within this range of linearity.
D. sperm sialidase activity is determined
（1）Sialidase standard stock solution is pressed 5/2.5/1.25/0.625/0.312/0 mU/ml's with detection buffer solution 2
Concentration gradient is diluted to sialidase standard liquid, to sequentially adding each sialidase standard liquid in detection plate；Add
Sample to be tested in detection plate, add detection buffer solution 2 dilute.
Sialidase standard liquid gradient according to the expression quantity and this method of sperm sialidase can be detected it is sensitive
Spend and set.
Preferably, described detection plate is 96 hole blackboards, and the present invention determines the activity of sialidase using fluorescence method, black
The detection plate of color is optimal to sensitivity and specificity, and fluorescent dye can be protected not to be quenched by light, hence it is evident that reduce light
The negative effect being quenched to fluorometric reagent, so that the sensitivity of detection, stability and accuracy are greatly enhanced.
（2）Fluorometric reagent is diluted with detection buffer solution 2, above-mentioned each sialidase standard liquid and sample to be tested is added into
Hole in, then detection plate is placed in after 37 DEG C of lucifuges are incubated 1-2 hour, with ELIASA each hole fluorescence intensity of reading, according to saliva
Phytase activity standard curve calculates the sialidase activity of sample to be tested, finally draws sialidase activity/protein concentration
Value.
Preferably, it is optimal working concentration with detecting that the concentration that buffer solution 2 is diluted to fluorometric reagent is 0.05mM.
Preferably, ELIASA reads each Kong Ying under the wavelength that excitation wavelength and launch wavelength are respectively 365nm and 450nm
Luminous intensity.365nm is maximum excitation wavelength, and 450nm is optimal launch wavelength.
The unit enzyme activity value of sialidase activity/protein concentration（U AUS/g protein）Reflection unit sperm protein
Sialidase activity quantitative values, the data according to unit enzyme activity value can make the critical value that crowd expresses sialidase activity.
According to the critical value, the service quality of sperm can be tentatively judged clinically by unit enzyme activity value, according at present
Some data, for normal fertile men and infertile patients, this critical value is about 0.2；The sperm good for locomitivity and
The poor sperm of locomitivity, this critical value is about 0.24；For the normal sperm of motility rate and the low sperm of motility rate, this is critical
Value about 0.3.
Preferably, should be in the range of 0.3-5mU/ml, if if for sample sialidase activity in the hole of detection
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 are：
1st, the reagent in kit of the present invention selects routine test reagent, and combines the particularity to sperm detection, drop
Low experimental cost, required instrument and equipment, consumptive material species are few, only centrifuge, ELIASA, EP pipes, pipette tips and detection plate etc.,
It is Routine Test Lab configuration；And operating method is simple, only it is centrifuged, sample-adding etc., in the absence of need to repeatedly practise obtaining
The situation of operation skill, can be widely applied to clinical labororatory and those having ordinary skill in the art uses.
2nd, kit of the invention is used to assess sperm quality in itself, the reason for examination is infertile.According to unit enzyme activity
The data of value can make the critical value that crowd expresses sialidase activity, according to the critical value, can be clinically by unit enzyme
The preliminary service quality for judging sperm of value living, the possibility potential cause for evaluating male sterility, in particular for clinically not
Bright reason infertility whether the situation relevant with sialidase activity.
3rd, the present invention is sufficiently washed to sperm, fully removes the refining composition of residual, makes the egg remained in refining
Bletilla sialidase does not interfere with the accuracy of testing result；Optimize the standard items albumen set during survey sperm protein concentration dense
The standard items enzyme activity gradient set when degree gradient and survey enzyme activity, if gradient sets unreasonable, can influence very much the sensitive of result
Degree and accuracy；Enzyme activity is surveyed using black detection plate, the negative effect that light is quenched to fluorometric reagent is can obviously reduce, thus greatly
The big stability for improving experimental result, sensitivity and accuracy；Final testing result is designed as sialic acid enzyme activity/protein concentration
Value, can so reduce the difference between different samples, make to have more comparativity each other, it is ensured that the stability of testing result and can
By property.
Brief description of the drawings
Fig. 1 is to determine eupyrene sperm respectively using kit of the present invention（People）And sterile sperm（People）Sialic acid enzyme activity
The unit enzyme activity value of property/protein concentration.
Specific embodiment
Essentiality content of the invention is described in further detail with reference to specific embodiment.
Embodiment 1
A kind of kit of sperm quality assessment, described kit include the cell pyrolysis liquid without inhibitors of enzymes,
Detection buffer solution 1, protease inhibitors cocktail, reagent A liquid, reagent B liquid, bovine serum albumin(BSA), fluorometric reagent, sialic acid
Enzyme standard stock solution and detection buffer solution 2；
Described detection buffer solution 1 is phosphate buffer；
Described reagent A liquid is：1%BCA disodium salts, 2% natrium carbonicum calcinatum, 0.16% sodium tartrate, 0.4% hydroxide
PH value to 11.2 is adjusted in sodium, 0.95% sodium acid carbonate, mixing；
Described reagent B liquid is：4% copper sulphate；
Described detection buffer solution 2 is the phosphoric acid containing 0.05 mmol/l sodium acetates and 0.25 μ g/ μ l bovine serum albumin(BSA)s
Buffer solution.
Eupyrene sperm is determined using kit of the invention respectively（People）And sterile sperm（People）Sialidase activity, knot
The sialidase activity of fruit display eupyrene sperm is apparently higher than sterile sperm（See Fig. 1）.
Embodiment 2
A kind of kit of sperm quality assessment, described kit include the cell pyrolysis liquid without inhibitors of enzymes,
Detection buffer solution 1, protease inhibitors cocktail, reagent A liquid, reagent B liquid, bovine serum albumin(BSA), fluorometric reagent, sialic acid
Enzyme standard stock solution and detection buffer solution 2；
Described detection buffer solution 1 is phosphate buffer；
Described reagent A liquid is：1%BCA disodium salts, 2% natrium carbonicum calcinatum, 0.16% sodium tartrate, 0.4% hydroxide
PH value to 11.3 is adjusted in sodium, 0.95% sodium acid carbonate, mixing；
Described reagent B liquid is：4% copper sulphate；
Described detection buffer solution 2 is the phosphoric acid containing 0.05 mmol/l sodium acetates and 0.25 μ g/ μ l bovine serum albumin(BSA)s
Buffer solution.
Described sialidase standard stock solution is C.perfringens sialidase, and concentration is 5U/ml.
Embodiment 3
A kind of kit of sperm quality assessment, described kit include the cell pyrolysis liquid without inhibitors of enzymes,
Detection buffer solution 1, protease inhibitors cocktail, reagent A liquid, reagent B liquid, bovine serum albumin(BSA), fluorometric reagent, sialic acid
Enzyme standard stock solution and detection buffer solution 2；
Described detection buffer solution 1 is BWW nutrient solutions；
Described reagent A liquid is：1%BCA disodium salts, 2% natrium carbonicum calcinatum, 0.16% sodium tartrate, 0.4% hydroxide
PH value to 11.25 is adjusted in sodium, 0.95% sodium acid carbonate, mixing；
Described reagent B liquid is：4% copper sulphate；
Described detection buffer solution 2 is the phosphoric acid containing 0.05 mmol/l sodium acetates and 0.25 μ g/ μ l bovine serum albumin(BSA)s
Buffer solution.
Described sialidase standard stock solution is C.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
A kind of kit of sperm quality assessment, described kit include the cell pyrolysis liquid without inhibitors of enzymes,
Detection buffer solution 1, protease inhibitors cocktail, reagent A liquid, reagent B liquid, bovine serum albumin(BSA), fluorometric reagent, sialic acid
Enzyme standard stock solution and detection buffer solution 2；
Described detection buffer solution 1 is BWW nutrient solutions；
Described reagent A liquid is：1%BCA disodium salts, 2% natrium carbonicum calcinatum, 0.16% sodium tartrate, 0.4% hydroxide
PH value to 11.28 is adjusted in sodium, 0.95% sodium acid carbonate, mixing；
Described reagent B liquid is：4% copper sulphate；
Described detection buffer solution 2 is the phosphoric acid containing 0.05 mmol/l sodium acetates and 0.25 μ g/ μ l bovine serum albumin(BSA)s
Buffer solution.
Described sialidase standard stock solution is C.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 the fluorometric reagent is 10mM.
Embodiment 5
A kind of kit of sperm quality assessment, described kit include the cell pyrolysis liquid without inhibitors of enzymes,
Detection buffer solution 1, protease inhibitors cocktail, reagent A liquid, reagent B liquid, bovine serum albumin(BSA), fluorometric reagent, sialic acid
Enzyme standard stock solution and detection buffer solution 2；
Described detection buffer solution 1 is phosphate buffer；
Described reagent A liquid is：1%BCA disodium salts, 2% natrium carbonicum calcinatum, 0.16% sodium tartrate, 0.4% hydroxide
PH value to 11.2 is adjusted in sodium, 0.95% sodium acid carbonate, mixing；
Described reagent B liquid is：4% copper sulphate；
Described detection buffer solution 2 is the phosphoric acid containing 0.05 mmol/l sodium acetates and 0.25 μ g/ μ l bovine serum albumin(BSA)s
Buffer solution.
Described sialidase standard stock solution is C.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 the fluorometric reagent is 10mM.
The pH value of the sodium acetate is 5.
Embodiment 6
A kind of kit of sperm quality assessment, described kit include the cell pyrolysis liquid without inhibitors of enzymes,
Detection buffer solution 1, protease inhibitors cocktail, reagent A liquid, reagent B liquid, bovine serum albumin(BSA), fluorometric reagent, sialic acid
Enzyme standard stock solution and detection buffer solution 2；
Described detection buffer solution 1 is phosphate buffer；
Described reagent A liquid is：1%BCA disodium salts, 2% natrium carbonicum calcinatum, 0.16% sodium tartrate, 0.4% hydroxide
PH value to 11.3 is adjusted in sodium, 0.95% sodium acid carbonate, mixing；
Described reagent B liquid is：4% copper sulphate；
Described detection buffer solution 2 is the phosphoric acid containing 0.05 mmol/l sodium acetates and 0.25 μ g/ μ l bovine serum albumin(BSA)s
Buffer solution.
Described sialidase standard stock solution is C.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 the fluorometric reagent is 10mM.
The pH value of the sodium acetate is 6.
Embodiment 7
A kind of kit of sperm quality assessment, described kit include the cell pyrolysis liquid without inhibitors of enzymes,
Detection buffer solution 1, protease inhibitors cocktail, reagent A liquid, reagent B liquid, bovine serum albumin(BSA), fluorometric reagent, sialic acid
Enzyme standard stock solution and detection buffer solution 2；
Described detection buffer solution 1 is phosphate buffer；
Described reagent A liquid is：1%BCA disodium salts, 2% natrium carbonicum calcinatum, 0.16% sodium tartrate, 0.4% hydroxide
PH value to 11.25 is adjusted in sodium, 0.95% sodium acid carbonate, mixing；
Described reagent B liquid is：4% copper sulphate；
Described detection buffer solution 2 is the phosphoric acid containing 0.05 mmol/l sodium acetates and 0.25 μ g/ μ l bovine serum albumin(BSA)s
Buffer solution.
Described sialidase standard stock solution is C.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 the fluorometric reagent is 10mM.
The pH value of the sodium acetate is 5.5.
Embodiment 8
A kind of application method of the kit of sperm quality assessment of the present invention, comprises the following steps that：
The washing of A, sperm
Collect fresh liquefaction semen sample, the detection washing sperm of buffer solution 1 fully removes the refining composition of residual, then from
The heart isolates sperm；
B, extraction sperm protein
To cell pyrolysis liquid and protease inhibitors cocktail is added in the sperm that step A is isolated, mix, treat without bright
Aobvious cell precipitation is fully cracking, and the high speed centrifugation at 4 DEG C, supernatant is protein extract；
C, the protein concentration for determining sperm protein extract
（1）The protein standard stoste of bovine serum albumin(BSA) is prepared with cell pyrolysis liquid, then 2/1.0/0.5/ is pressed with lysate
Protein standard stoste is diluted to protein standard solution by concentration gradient 0.25/0.125/0mg/ml, is added successively in detection plate
Enter each protein standard solution；Add sample to be tested in detection plate, add lysate dilution.
（2）By A:B=50：1 volume ratio reagent preparation A liquid and the mixed liquor of B liquid, is added into above-mentioned each protein standard
In the hole of solution and sample to be tested, gently mix, detection plate is placed in 37 DEG C of reaction 30min, then read each hole with ELIASA
Absorbance, the protein concentration of sample to be tested is calculated according to protein standard curve.
D, measure sperm sialidase activity
（1）Sialidase standard stock solution is pressed 5/2.5/1.25/0.625/0.312/0 mU/ml's with detection buffer solution 2
Concentration gradient is diluted to sialidase standard liquid, to sequentially adding each sialidase standard liquid in detection plate；Add
Sample to be tested in detection plate, add detection buffer solution 2 dilute.
（2）Fluorometric reagent is diluted with detection buffer solution 2, above-mentioned each sialidase standard liquid and sample to be tested is added into
Hole in, then detection plate is placed in after 37 DEG C of lucifuges are incubated 1 hour, each hole fluorescence intensity is read with ELIASA, 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
A kind of application method of the kit of sperm quality assessment of the present invention, comprises the following steps that：
The washing of A, sperm
Collect fresh liquefaction semen sample, the detection washing sperm of buffer solution 1 fully removes the refining composition of residual, then from
The heart isolates sperm；
B, extraction sperm protein
To cell pyrolysis liquid and protease inhibitors cocktail is added in the sperm that step A is isolated, mix, treat without bright
Aobvious cell precipitation is fully cracking, and the high speed centrifugation at 4 DEG C, supernatant is protein extract；
C, the protein concentration for determining sperm protein extract
（1）The protein standard stoste of bovine serum albumin(BSA) is prepared with cell pyrolysis liquid, then 2/1.0/0.5/ is pressed with lysate
Protein standard stoste is diluted to protein standard solution by concentration gradient 0.25/0.125/0mg/ml, is added successively in detection plate
Enter each protein standard solution；Add sample to be tested in detection plate, add lysate dilution.
（2）By A:B=50：1 volume ratio reagent preparation A liquid and the mixed liquor of B liquid, is added into above-mentioned each protein standard
In the hole of solution and sample to be tested, gently mix, detection plate is placed in 37 DEG C of reaction 30min, then read each hole with ELIASA
Absorbance, the protein concentration of sample to be tested is calculated according to protein standard curve.
D, measure sperm sialidase activity
（1）Sialidase standard stock solution is pressed 5/2.5/1.25/0.625/0.312/0 mU/ml's with detection buffer solution 2
Concentration gradient is diluted to sialidase standard liquid, to sequentially adding each sialidase standard liquid in detection plate；Add
Sample to be tested in detection plate, add detection buffer solution 2 dilute.
（2）Fluorometric reagent is diluted with detection buffer solution 2, above-mentioned each sialidase standard liquid and sample to be tested is added into
Hole in, then detection plate is placed in after 37 DEG C of lucifuges are incubated 2 hours, each hole fluorescence intensity is read with ELIASA, 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
.
Should be in the range of 0.3-5mU/ml for sample sialidase activity in the hole of detection, should be by hole if exceeding
The sialidase activity value of sample to be tested is adjusted within this range of linearity.
Embodiment 10
A kind of application method of the kit of sperm quality assessment of the present invention, comprises the following steps that：
The washing of A, sperm
Collect fresh liquefaction semen sample, the detection washing sperm of buffer solution 1 fully removes the refining composition of residual, then from
The heart isolates sperm；
B, extraction sperm protein
To cell pyrolysis liquid and protease inhibitors cocktail is added in the sperm that step A is isolated, mix, treat without bright
Aobvious cell precipitation is fully cracking, and the high speed centrifugation at 4 DEG C, supernatant is protein extract；
C, the protein concentration for determining sperm protein extract
（1）The protein standard stoste of bovine serum albumin(BSA) is prepared with cell pyrolysis liquid, then 2/1.0/0.5/ is pressed with lysate
Protein standard stoste is diluted to protein standard solution by concentration gradient 0.25/0.125/0mg/ml, is added successively in detection plate
Enter each protein standard solution；Add sample to be tested in detection plate, add lysate dilution.
（2）By A:B=50：1 volume ratio reagent preparation A liquid and the mixed liquor of B liquid, is added into above-mentioned each protein standard
In the hole of solution and sample to be tested, gently mix, detection plate is placed in 37 DEG C of reaction 30min, then read each hole with ELIASA
Absorbance, the protein concentration of sample to be tested is calculated according to protein standard curve.
D, measure sperm sialidase activity
（1）Sialidase standard stock solution is pressed 5/2.5/1.25/0.625/0.312/0 mU/ml's with detection buffer solution 2
Concentration gradient is diluted to sialidase standard liquid, to sequentially adding each sialidase standard liquid in detection plate；Add
Sample to be tested in detection plate, add detection buffer solution 2 dilute.
（2）Fluorometric reagent is diluted with detection buffer solution 2, above-mentioned each sialidase standard liquid and sample to be tested is added into
Hole in, then detection plate is placed in after 37 DEG C of lucifuges are incubated 1.5 hours, each hole fluorescence intensity is read with ELIASA, according to saliva
Phytase activity standard curve calculates the sialidase activity of sample to be tested, finally draws sialidase activity/protein concentration
Value.
Should be in the range of 0.3-5mU/ml for sample sialidase activity in the hole of detection, should be by hole if exceeding
The sialidase activity value of sample to be tested is adjusted within this range of linearity.
Embodiment 11
The present embodiment is on the basis of embodiment 8：
Described step B, the addition volume of cell pyrolysis liquid is 2 times of sperm volume.
Embodiment 12
The present embodiment is on the basis of embodiment 8：
Described step B, the addition volume of cell pyrolysis liquid is 3 times of sperm volume.
Described step C, the concentration that the protein standard stoste of bovine serum albumin(BSA) is prepared with cell pyrolysis liquid is 20mg/
ml。
Embodiment 13
The present embodiment is on the basis of embodiment 8：
Described step B, the addition volume of cell pyrolysis liquid is 2.5 times of sperm volume.
Described step C, the concentration that the protein standard stoste of bovine serum albumin(BSA) is prepared with cell pyrolysis liquid is 20mg/
ml。
Described step C, ELIASA reads each hole absorbance under 562nm wavelength.
Embodiment 14
The present embodiment is on the basis of embodiment 9：
Described step B, the addition volume of cell pyrolysis liquid is 2.3 times of sperm volume.
Described step C, the concentration that the protein standard stoste of bovine serum albumin(BSA) is prepared with cell pyrolysis liquid is 20mg/
ml。
Described step C, ELIASA reads each hole absorbance under 562nm wavelength.
The detection plate of the step C is 96 hole transparent panels, and the detection plate of the D steps is 96 hole blackboards.
Described D steps, ELIASA reads under the wavelength that excitation wavelength and launch wavelength are respectively 365nm and 450nm
Each hole fluorescence intensity.
Embodiment 15
The present embodiment is on the basis of embodiment 9：
Described step B, the addition volume of cell pyrolysis liquid is 2 times of sperm volume.
Described step C, the concentration that the protein standard stoste of bovine serum albumin(BSA) is prepared with cell pyrolysis liquid is 20mg/
ml。
Described step C, ELIASA reads each hole absorbance under 562nm wavelength.
The detection plate of the step C is 96 hole transparent panels, and the detection plate of the D steps is 96 hole blackboards.
Described D steps, the concentration that fluorometric reagent is diluted to detection buffer solution 2 is 0.05mM.
Described D steps, ELIASA reads under the wavelength that excitation wavelength and launch wavelength are respectively 365nm and 450nm
Each hole fluorescence intensity.
Embodiment 16
The present embodiment is on the basis of embodiment 10：
Described step B, the addition volume of cell pyrolysis liquid is 3 times of sperm volume.
Described step C, the concentration that the protein standard stoste of bovine serum albumin(BSA) is prepared with cell pyrolysis liquid is 20mg/
ml。
Described step C, ELIASA reads each hole absorbance under 562nm wavelength.
The detection plate of the step C is 96 hole transparent panels, and the detection plate of the D steps is 96 hole blackboards.
Described D steps, the concentration that fluorometric reagent is diluted to detection buffer solution 2 is 0.05mM.
Described D steps, ELIASA reads under the wavelength that excitation wavelength and launch wavelength are respectively 365nm and 450nm
Each hole fluorescence intensity.
Embodiment 17
Consumptive material（Sterilize）：15mlEP is managed, 1.5mlEP pipes, 3 kinds of specification pipette tips（1ml/200μl/10μl）, the culture of 96 holes
Plate, 96 hole detection plates
Instrument：High speed low temperature centrifugal machine, ELIASA
A kind of application method of the kit of sperm quality assessment of the present invention, comprises the following steps that：
The washing of A, sperm
1. fresh liquefaction semen sample collects 4-5ml（Adjusted according to patient's sperm quantity, preserved on ice and transport）.
2. isometric PBS is added, and piping and druming is uniform.
3. low-speed centrifugal（1500rcf/5min）, abandoning supernatant.
4. PBS to 3-4ml is added, and piping and druming is uniform.
5. low-speed centrifugal（1500rcf/5min）, abandoning supernatant, supernatant is remained to 300 μ l or so, piping and druming is uniform.
6. PBS to 1ml, high speed centrifugation are added（4 degree, 12000rpm/5min）, abandoning supernatant as far as possible.
B, extraction sperm protein
7. to 2-3 times of lysate and protease inhibitors cocktail of volume of addition in Sperm pellets（It is final concentration of
1X）, blow and beat and mix, treat without obvious cell precipitation i.e. fully cracking, the high speed centrifugation at 4 DEG C（12000rpm/5min）, supernatant
Liquid is protein extract；
C, the protein concentration for determining sperm protein extract
8. BSA protein standard stostes are prepared with lysate（20mg/ml）, the protein standard stoste of appropriate 20mg/ml is taken,
Protein standard solution is diluted to by the concentration gradient of 2/1.0/0.5/0.25/0.125/0mg/ml with lysate, to 96 holes
Each protein standard solution of 20 μ l is sequentially added in transparent panel.
Plus 2 microlitres of samples to be tested are in 96 orifice plates, plus lysate is to 20 microlitres 9..
10. A is pressed:B=50：1 volume ratio reagent preparation A liquid and the mixed liquor of B liquid（Room temperature is stablized in 24 hours）, by its
Add above-mentioned each hole, 200 microlitres/hole.
11. gently mix 30s, 96 hole transparent panels are placed in into 37 degree of reaction 30min, then with ELIASA in 562nm wavelength
It is lower to read each hole absorbance, the protein concentration of sample to be tested is calculated according to protein standard curve.
Should be in the range of 0.1-2mg/ml, if should will be to be measured in hole if for sample protein concentration in the hole detected
The protein concentration of sample is adjusted within this range of linearity.
D. sperm sialidase activity is determined
1. sialidase standard stock solution is pressed 5/2.5/1.25/0.625/0.312/0 mU/ml's with detection buffer solution 2
Concentration gradient is diluted to sialidase standard liquid, and to being sequentially added in 96 hole blackboards, each sialidase standards of 50 μ l are molten
Liquid.
Plus 25 microlitres of samples to be tested are in 96 hole blackboards, plus 2 to 50 microlitres of buffer solution of detection 2..
3. 1 is pressed with detection buffer solution 2:200 dilution fluorometric reagents, are added into above-mentioned each hole, 50 microlitres/hole.
4. 96 hole blackboards are placed in into 37 degree of lucifuges to be incubated 1-2 hours, then with ELIASA in excitation wavelength/launch wavelength
To read each hole fluorescence intensity under 365/450nm wavelength, the saliva of sample to be tested is calculated according to sialidase activity standard curve
Liquid phytase activity.
5. the last sialidase activity/total protein concentration that will be obtained, calculates unit enzyme activity value (U AUS/g
Protein).
Should be in the range of 0.3-5mU/ml for sample sialidase activity in the hole of detection, should be by hole if exceeding
The sialidase activity value of sample to be tested is adjusted within this range of linearity.
Claims (12)
1. the kit that a kind of sperm quality is assessed, it is characterised in that：Described kit includes thin without inhibitors of enzymes
Cellular lysate liquid, detection buffer solution 1, protease inhibitors cocktail, reagent A liquid, reagent B liquid, bovine serum albumin(BSA), fluorescence examination
Agent, sialidase standard stock solution and detection buffer solution 2；
Described detection buffer solution 1 is phosphate buffer or BWW nutrient solutions；
Described reagent A liquid is：1%BCA disodium salts, 2% natrium carbonicum calcinatum, 0.16% sodium tartrate, 0.4% NaOH,
PH value to 11.2-11.3 is adjusted in 0.95% sodium acid carbonate, mixing；
Described reagent B liquid is：4% copper sulphate；
Described detection buffer solution 2 is the phosphoric acid buffer containing 0.05 mmol/l sodium acetates and 0.25 μ g/ μ l bovine serum albumin(BSA)s
Liquid.
2. the kit that a kind of sperm quality according to claim 1 is assessed, it is characterised in that：Described sialidase mark
Quasi- stoste is C.perfringens sialidase, and concentration is 5U/ml.
3. the kit that a kind of sperm quality according to claim 1 is assessed, it is characterised in that：Described fluorometric reagent is
2 ' -4-methyl umbelliferone base-α-D-N- n acetylneuraminic acid n sodium.
4. the kit that a kind of sperm quality according to claim 3 is assessed, it is characterised in that：The fluorometric reagent it is dense
It is 10mM to spend.
5. the kit that a kind of sperm quality according to claim 1 is assessed, it is characterised in that：The pH value of the sodium acetate
It is 5-6.
6. the application method of the kit of a kind of sperm quality assessment according to claim 1, it is characterised in that：Specific step
It is rapid as follows：
The washing of A, sperm
Collect fresh liquefaction semen sample, the detection washing sperm of buffer solution 1 fully removes the refining composition of residual, then be centrifuged point
Separate out sperm；
B, extraction sperm protein
To cell pyrolysis liquid and protease inhibitors cocktail is added in the sperm that step A is isolated, mix, treat without obvious
Cell precipitation is fully cracking, and the high speed centrifugation at 4 DEG C, supernatant is protein extract；
C, the protein concentration for determining sperm protein extract
（1）The protein standard stoste of bovine serum albumin(BSA) is prepared with cell pyrolysis liquid, then 2/1.0/0.5/0.25/ is pressed with lysate
Protein standard stoste is diluted to protein standard solution by the concentration gradient of 0.125/0mg/ml, to sequentially adding each egg in detection plate
White standard liquid；Add sample to be tested in detection plate, add lysate dilution；
（2）By A:B=50：1 volume ratio reagent preparation A liquid and the mixed liquor of B liquid, is added into above-mentioned each protein standard solution
In the hole of sample to be tested, gently mix, detection plate is placed in 37 DEG C of reaction 30min, then read each hole extinction with ELIASA
Degree, the protein concentration of sample to be tested is calculated according to protein standard curve；
D, measure sperm sialidase activity
（1）Sialidase standard stock solution is pressed the concentration of 5/2.5/1.25/0.625/0.312/0 mU/ml with detection buffer solution 2
Gradient is diluted to sialidase standard liquid, to sequentially adding each sialidase standard liquid in detection plate；Add to be measured
Sample in detection plate, add detection buffer solution 2 dilute；
（2）Fluorometric reagent is diluted with detection buffer solution 2, the hole of above-mentioned each sialidase standard liquid and sample to be tested is added into
It is interior, then detection plate is placed in after 37 DEG C of lucifuges are incubated 1-2 hour, with ELIASA each hole fluorescence intensity of reading, 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.
7. the application method of the kit of a kind of sperm quality assessment according to claim 6, it is characterised in that：Described
Step B, the addition volume of cell pyrolysis liquid is 2-3 times of sperm volume.
8. the application method of the kit of a kind of sperm quality assessment according to claim 6, it is characterised in that：Described
Step C, the concentration that the protein standard stoste of bovine serum albumin(BSA) is prepared with cell pyrolysis liquid is 20mg/ml.
9. the application method of the kit of a kind of sperm quality assessment according to claim 6, it is characterised in that：Described
Step C, ELIASA reads each hole absorbance under 562nm wavelength.
10. the application method of the kit of a kind of sperm quality assessment according to claim 6, it is characterised in that：The C
The detection plate of step is 96 hole transparent panels, and the detection plate of the D steps is 96 hole blackboards.
A kind of 11. application methods of the kit of sperm quality assessment according to claim 6, it is characterised in that：It is described
D steps, it is 0.05mM to be diluted to the concentration of fluorometric reagent with detection buffer solution 2.
A kind of 12. application methods of the kit of sperm quality assessment according to claim 6, it is characterised in that：It is described
D steps, ELIASA reads each hole fluorescence intensity under the wavelength that excitation wavelength and launch wavelength are respectively 365nm and 450nm.
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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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| CN108132213A (en) * | 2017-12-05 | 2018-06-08 | 深圳市博锐德生物科技有限公司 | Spermatozoon activity oxygen class substance quantitative determination reagent kit and application thereof and application method |
| CN110286080A (en) * | 2018-10-25 | 2019-09-27 | 中国科学院苏州生物医学工程技术研究所 | Ejaculated sperm cells rapid typing detection reagent box and detection method |
| CN111122520A (en) * | 2018-10-31 | 2020-05-08 | 四川大学华西第二医院 | Embryo implantation detection kit and application and use method thereof |
| CN110824169B (en) * | 2019-10-14 | 2024-01-02 | 上海创宏生物科技有限公司 | Pig farm biological safety assessment detection kit and detection method |
| 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 |
| CN113416772A (en) * | 2021-05-21 | 2021-09-21 | 赛雷纳（中国）医疗科技有限公司 | Composition formula of sperm DNA fragmentation quality control product |
| 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 |
| CN114574541A (en) * | 2022-04-01 | 2022-06-03 | 钟晓明 | Method for accurately detecting influence of whitening product on melanin content in cells |
| 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 |
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| US6541206B1 (en) * | 2000-06-19 | 2003-04-01 | Gabor B. Huszar | Objective biochemical method for assessment of sperm quality |
| US20030068663A1 (en) * | 2001-09-06 | 2003-04-10 | Kelly Huang | Method and kit for measuring skin inflammation or irritation |
| CN1737572B (en) * | 2004-08-19 | 2010-12-15 | 深圳华康生物医学工程有限公司 | Spermatozoon acrosome enzyme activity quantitative determination reagent and detecting method thereof |
| CN102033129A (en) * | 2009-09-29 | 2011-04-27 | 上海英伯肯医学生物技术有限公司 | Method for detecting pathogenic microorganism by using antigen-stimulated cellular immune response and test pen |
| CN102353672B (en) * | 2011-06-30 | 2013-04-03 | 深圳市博锐德生物科技有限公司 | Quantitative detection method and kit of sperm acrosin activity |
| 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 |
| CN104198248A (en) * | 2014-09-04 | 2014-12-10 | 山西农业大学 | Method for extracting and separating total proteins of mice sperms by using two-dimensional electrophoresis |
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