CN1737572B - Spermatozoon acrosome enzyme activity quantitative determination reagent and detecting method thereof - Google Patents
Spermatozoon acrosome enzyme activity quantitative determination reagent and detecting method thereof Download PDFInfo
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- CN1737572B CN1737572B CN 200410040527 CN200410040527A CN1737572B CN 1737572 B CN1737572 B CN 1737572B CN 200410040527 CN200410040527 CN 200410040527 CN 200410040527 A CN200410040527 A CN 200410040527A CN 1737572 B CN1737572 B CN 1737572B
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- acrosin
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Abstract
This invention discloses one sperm acrosin activity quantifying testing agent, which comprises depressor, buffer liquid, terminal liquid and bottom liquid, wherein the said bottom liquid comprises N- Alpha- benzoyl -DL-spermine-Rho- nitroaniline of dimethyl formamide solution; The buffer liquid comprises cattle blood serum albumin and deionized water solution. The invention also discloses the method, which computes the acrosin activity according to the mole proportion of N-Alpha- benzoyl -DL-spermine-Rho- nitroaniline analyzed products to realize the measurement by quantifying.
Description
Technical field
The present invention relates to a kind of external diagnosis reagent, be applicable to that specifically the acrosin active level detects.
Background technology
The lysosome that perforatorium is a kind of specialization includes multiple hydrolytic enzyme, and acrosin activity wherein can reflect sperm quality, and acrosin vigor deficiency may cause sterile, is difficult to differentiate sterile reason person by the seminal fluid routine, and often acrosin activity is low.Therefore, acrosin activity is an important evaluation index of judging mankind spermatozoon function and fertility power, also can be used as the foundation that clinical supplementary reproduction (ART) method is selected.
Acrosin is measured immunologic assay and enzyme assay two big classes, immunologic assay complicated operation and reflection be total enzyme content, rather than total enzyme activity; Enzyme assay is representative with the Kennedy method, it is the people's sperm arginine amide enzymatic activity that detects the benzenecarboximidamide sensitivity, it will reflect all almost whole in other words perforatorium enzymatic activitys, but the Kennedy method is owing to himself methodological reason, reaction efficiency is low, reach three hours detection time, be unfavorable for clinically extensively carrying out.
Summary of the invention
The invention is intended to overcome the deficiencies in the prior art, provide a kind of and can realize the acrosin activity detection by quantitative, be swift in response, detection time weak point, stable performance, storage period long acrosin active level detectable and detection method.
Realize the technical scheme of above-mentioned purpose:
A kind of acrosin active level detectable; comprise inhibitor, damping fluid, stop buffer and substrate solution; described substrate solution is the dimethyl formamide solution that contains N-α-benzoyl-DL-spermine acid-ρ-nitroanilide (BAPNA), and the content of BAPNA is 0.2～2.0% in the described substrate solution.
The content of BAPNA preferred 0.5～1.0% in the described substrate solution.。
Described damping fluid is the deionized water solution that contains bovine serum albumin(BSA) (BAS) and polysorbas20 (Tween-20).
Also comprise antiseptic in the described damping fluid.
Described inhibitor is the deionized water solution that contains inorganic salts, N-2-hydroxyethyl piperazine-N ' 2-ethanesulfonic acid (Hepes) and 400 type ficolls (Ficoll 400).
Also comprise antiseptic in the described inhibitor.
A kind of method that detects with acrosin active level detectable comprises the steps: to prepare reactant, each reactant and reacts, calculates acrosin activity according to reaction result;
The step of described preparation reactant is:
A) preparation reactant liquor, with the substrate solution and the damping fluid of weighing, abundant mixing;
B) preparation semen sample after the fresh semen liquefaction, is counted after fully putting upside down mixing, obtains sample sperm concentration M;
The step that described each reactant reacts is:
C) mensuration pipe and blank pipe are set, with the sperm count A that sets divided by the sample sperm concentration M described in the step b), obtain measuring pipe and the required semen volume v of the every pipe of blank pipe, be v=A/M, add seminal fluid v in pipe, the blank pipe respectively at measuring, and carry out centrifugally to measuring pipe, blank pipe seminal fluid, and inhale the refining that goes in the pipe of centrifugal back partly, keep lower floor's sperm precipitation;
D) in the described mensuration pipe of step c), blank pipe, add the inhibitor of equivalent respectively, the precipitation spermatium is evenly suspended;
E) in the described mensuration pipe of step d), blank pipe, add the reactant liquor of equivalent respectively, be used for reacting with seminal fluid;
F) add stop buffer at the blank pipe described in the step e), be used for the reaction of cessation reaction liquid and seminal fluid;
G) after reactant liquor in the fixed tube to be measured and seminal fluid fully react,, be used for the reaction of cessation reaction liquid and seminal fluid measuring pipe adding stop buffer;
H) the described mensuration pipe of step g), blank pipe are carried out centrifugal treating after, in microplate reader, carry out colorimetric, record the absorbance OD1, the OD2 that measure pipe, blank pipe respectively;
The step of described calculating acrosin activity is: according to step h) the sperm count A of mensuration pipe absorbance OD1, blank pipe absorbance OD2, setting and the molar absorptivity C of the product nitroanilide of BAPNA after being decomposed by acrosin calculate acrosin activity.
Above-mentioned steps h) measuring absorbance in is to carry out colorimetric under the 410nm wavelength.
Because technique scheme, in conjunction with the following embodiment that will describe in detail, the technique effect that the present invention gives prominence to is: the molar absorptivity of the product nitroanilide after 1, being decomposed by acrosin according to BAPNA calculates the activity of acrosin, has realized the detection by quantitative of acrosin activity; 2, adopted BAPNA and Tween-20,, can accelerate the reaction velocity of enzyme because BAPNA is the suitableeest substrate of perforatorium enzymic catalytic reaction, Tween-20 destroys sperm membrane, be convenient to the exposure of acrosin,, in 1 hour, can finish reaction by the synergy of above-mentioned two kinds of materials; 3, owing to adopted BAS, can temporarily stablize acrosin, make that the stability of reaction is better, reaction result is more accurate; 4, owing to adopted inhibitor, need not to go the refining mortifier to handle, simplified running program, help the routine clinical acrosin activity detection by quantitative of carrying out the seminal fluid that detects; 5, owing to the employing antiseptic, the reagent stable performance, storage period is long, can store more than 1 year.
Embodiment
Embodiment, a kind of acrosin active level detectable, comprise that (1) inhibitor is the deionized water solution of sodium chloride-containing, Hepes, Ficoll400 and Sodium azide, sodium chloride-containing 0.7 gram, Hepes0.3 gram, Ficoll400 are 5.5 grams, Sodium azide 0.05 gram in every 100ml inhibitor; (2) damping fluid is the deionized water solution of sodium chloride-containing, Hepes, BSA, Sodium azide and Tween-2, sodium chloride-containing 0.35 gram, Hepes0.6 gram, BSA0.5 gram, Sodium azide 0.05 gram, Tween-2035 microlitre in every 100ml damping fluid; (3) stop buffer is the deionized water solution that contains benzenecarboximidamide, contains benzenecarboximidamide 8.73 grams in every 100ml stop buffer; (4) substrate solution is the dimethyl formamide solution that contains BAPNA, contains the BAPNA0.75 gram in every 100ml substrate solution.
The process for making of above-mentioned perforatorium enzymatic activity detectable:
The process for making of A, inhibitor
1) with analytical balance weighing sodium chloride 0.7 gram, Hepes0.3 gram, Ficoll400 are 5.5 grams, and Sodium azide 0.05 restrains, and put to stir in the beaker to be dissolved in 80 ml deionized water.
2) transfer pH to 7.4 with 1N NaOH, move in 100 milliliters of dressing measuring bottles, be settled to 100 milliliters with deionized water, 4 ℃ of preservations.
The process for making of B, damping fluid
3) with analytical balance weighing sodium chloride 0.35 gram, the Hepes0.6 gram, the BSA0.5 gram, Sodium azide 0.05 restrains, and puts to be dissolved in the beaker in 80 ml deionized water.
4) transfer pH to 8.0 with 1N NaOH, move in 100 milliliters of dressing measuring bottles, be settled to 100 milliliters with deionized water, measuring Tween-20 is 35 microlitres, is dissolved in the above-mentioned solution, filters back 4 ℃ of preservations.
The process for making of C, stop buffer
5) take by weighing benzenecarboximidamide 8.73 gram, put and be dissolved in the beaker in 80 ml deionized water.
6) move in 100 milliliters of dressing measuring bottles, be settled to 100 milliliters with deionized water, 4 ℃ of preservations.
The process for making of D, substrate solution
7) take by weighing 0.75 gram BAPNA, put and be dissolved in the beaker in 80 milliliters of dimethyl formamides.
8) move in 100 milliliters of dressing measuring bottles, be settled to 100 milliliters with dimethyl formamide ,-20 ℃ of preservations.
As required, above-mentioned perforatorium enzymatic activity detectable can be made into kit, wherein inhibitor 50～200ul, damping fluid 500～2000ul, stop buffer 50～200ul, substrate solution 100～400ul.
The preferred version of mentioned reagent box is: inhibitor 100ul, damping fluid 800ul, stop buffer 100ul, substrate solution 200ul.
Further, inhibitor can be that sodium chloride content is 0.2～2.0%, Hepes content is 0.1～1.0%, Ficoll400 content is 1～10%, Sodium azide content is 0.01～0.1% deionized water solution; Damping fluid can be that sodium chloride content is 0.1～1.0%, Hepes content is 0.2～1.0%, BSA content is 0.1～1.0%, Sodium azide content is 0.01～0.1%, Tween-20 content is 0.01～0.1% deionized water solution, wherein BSA is used for temporarily stablizing acrosin, Tween-20 is used for destroying the sperm membrane of sample, make acrosin wherein expose, be convenient to detect.Actual detected shows, and is incomplete to the destruction of sperm membrane when the content of Tween-20 is lower than 0.01%, when the content of Tween-20 is higher than 0.1%, is unfavorable for the stable of acrosin; Stop buffer is that benzenecarboximidamide content is 2～20% deionized water solution; Substrate solution is that BAPNA content is 0.2～2.0% dimethyl formamide solution, the content of preferred BAPNA is 0.5～1.0%, 0.2～2.0% content is to set according to the predictable mxm. of people's acrosin, when the content of BAPNA is lower than 0.2%, the reaction velocity of acrosin is slack-off, when the content of BAPNA is higher than 2.0%, the reaction velocity of acrosin no longer increases, excessive BAPNA causes waste, and consider that from practicality and economy the preferred content of BAPNA is 0.5～1.0%.
The test example, the concrete steps that detect the activity of acrosin with perforatorium enzymatic activity detectable are:
One, reagent is prepared
Face with preceding preparation reactant liquor, get 1 part of substrate solution by volume, with 4 parts of damping fluids, abundant mixing.
Two, sample is prepared
1, preparation semen sample after the fresh semen liquefaction, is fully put upside down counting (M * 10 behind the mixing
6/ ml).
2, every part of sample is established simultaneously and is measured pipe and blank pipe, and the sperm count that every pipe is participated in reaction is 7.5 * 10
6Individual.According to sample sperm concentration (M * 10
6/ ml), calculate the required semen volume vml (computing method: v=7.5/M) of every pipe.With the capacity is Eppendorf pipe conduct mensuration pipe and the blank pipe of 1.5ml.After seminal fluid fully put upside down mixing, respectively at measure pipe, the blank pipe respectively adds seminal fluid vml, the centrifugal 20min of 2000g.
3, topple over and discard refining, Eppendorf is managed back-off on thieving paper, inhale and remove to flow to the spout part refining.
Three, determination step
Four, calculate
495 is the molar absorptivity of the product nitroanilide after BAPNA is decomposed by acrosin in the following formula; Because the optical path of cuvette is 0.5cm, according to the definition of molar absorptivity: at the wavelength place of regulation, the absorbance of the solution of 1mol concentration during by optical path 1cm cuvette is so multiply by 2 in the molecule of equation the right.
Five, normal reference value
48.2～218.7 μ IU/10
6Sperm
Six, clinical applicability
1, acrosin activity can reflect sperm quality, is the important indicator of estimating mankind spermatozoon function and fertility power.
2, can be used as the foundation that the supplementary reproduction method is selected.
Claims (9)
1. acrosin active level detectable; comprise inhibitor, damping fluid, stop buffer and substrate solution; it is characterized in that: described substrate solution is the dimethyl formamide solution that contains N-α-benzoyl-DL-spermine acid-ρ-nitroanilide; described damping fluid is the deionized water solution that contains bovine serum albumin(BSA) and polysorbas20; the content of N-α-benzoyl in the described substrate solution-DL-spermine acid-ρ-nitroanilide is 0.2～2.0%; the content of polysorbas20 is 0.01～0.1% in the described damping fluid, and bovine serum albumin content is 0.1～1.0%.
2. acrosin active level detectable as claimed in claim 1 is characterized in that: the content preferred 0.5～1.0% of N-α-benzoyl in the described substrate solution-DL-spermine acid-ρ-nitroanilide.
3. acrosin active level detectable as claimed in claim 2 is characterized in that: also comprise inorganic salts and N-2-hydroxyethyl piperazine-N '-2-ethanesulfonic acid in the described damping fluid.
4. acrosin active level detectable as claimed in claim 3 is characterized in that: also comprise antiseptic in the described damping fluid.
5. acrosin active level detectable as claimed in claim 4 is characterized in that: described inhibitor is the deionized water solution that contains inorganic salts, N-2-hydroxyethyl piperazine-N '-2-ethanesulfonic acid and 400 type ficolls.
6. acrosin active level detectable as claimed in claim 5 is characterized in that: also comprise antiseptic in the described inhibitor.
7. acrosin active level detectable as claimed in claim 6 is characterized in that: described stop buffer is the deionized water solution that contains benzenecarboximidamide.
8. a method that detects with the described acrosin active level of claim 1 detectable comprises that preparation reactant, each reactant react, calculate according to reaction result steps such as acrosin activity, it is characterized in that,
The step of described preparation reactant is:
A) preparation reactant liquor, with the substrate solution and the damping fluid of weighing, abundant mixing;
B) preparation semen sample after the fresh semen liquefaction, is fully counted behind the mixing, obtains sample sperm concentration M;
The step that described each reactant reacts is:
C) mensuration pipe and blank pipe are set, set sperm count A, with the sperm count A that sets divided by the sample sperm concentration M described in the step b), obtain measuring pipe and the required semen volume v of the every pipe of blank pipe, be v=A/M, add semen volume v in pipe, the blank pipe respectively at measuring, and carry out centrifugal mensuration pipe, blank pipe seminal fluid, the refining part in the pipe of centrifugal back is gone in suction, keeps lower floor's sperm precipitation;
D) in the described mensuration pipe of step c), blank pipe, add the inhibitor of equivalent respectively, the precipitation spermatium is evenly suspended;
E) in the described mensuration pipe of step d), blank pipe, add the reactant liquor of equivalent respectively, be used for reacting with seminal fluid;
F) add stop buffer at the blank pipe described in the step e), be used for the reaction of cessation reaction liquid and seminal fluid;
G) after reactant liquor in the fixed tube to be measured and seminal fluid fully react,, be used for the reaction of cessation reaction liquid and seminal fluid measuring pipe adding stop buffer;
H) the described mensuration pipe of step g), blank pipe are carried out centrifugal treating after, in microplate reader, carry out colorimetric, record the absorbance OD1, the OD2 that measure pipe, blank pipe respectively;
The step of described calculating acrosin activity is: according to step h) the sperm count A of mensuration pipe absorbance OD1, blank pipe absorbance OD2, setting and the molar absorptivity C of the product nitroanilide of N-α-benzoyl-DL-spermine acid-ρ-nitroanilide after being decomposed by acrosin calculate acrosin activity.
9. the method as detecting with acrosin active level detectable as described in the claim 8, it is characterized in that: measuring absorbance described step h) is to carry out colorimetric under the 410nm wavelength.
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| CN 200410040527 CN1737572B (en) | 2004-08-19 | 2004-08-19 | Spermatozoon acrosome enzyme activity quantitative determination reagent and detecting method thereof |
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| CN 200410040527 CN1737572B (en) | 2004-08-19 | 2004-08-19 | Spermatozoon acrosome enzyme activity quantitative determination reagent and detecting method thereof |
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| CN1737572A CN1737572A (en) | 2006-02-22 |
| CN1737572B true CN1737572B (en) | 2010-12-15 |
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Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101865844B (en) * | 2010-06-04 | 2012-06-13 | 深圳市博锐德生物科技有限公司 | Acrosome reaction kit, method for inducing acrosome reaction and , and method for evaluating acrosome reaction condition |
| 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 |
| CN106153685B (en) * | 2015-04-27 | 2019-04-05 | 中国科学院宁波材料技术与工程研究所 | Method and kit based on Electrochemical Detection sperm acrosin activities in assessing |
| CN105717307B (en) * | 2016-03-16 | 2017-07-04 | 四川大学华西第二医院 | The kit and its application method of a kind of sperm quality assessment |
| CN106146642A (en) * | 2016-06-27 | 2016-11-23 | 浙江星博生物科技股份有限公司 | A kind of for protein microsphere conjugate detecting sperm acrosin activities in assessing and its preparation method and application |
| CN106896016B (en) * | 2017-05-02 | 2020-04-21 | 浙江星博生物科技股份有限公司 | Rapid extraction method of sperm acrosome enzyme and flow detection method of activity thereof |
| CN109609483A (en) * | 2019-02-15 | 2019-04-12 | 深圳华康生物医学工程有限公司 | A kind of extracting method of acrosin |
| CN113358868A (en) * | 2021-05-24 | 2021-09-07 | 迪瑞医疗科技股份有限公司 | Sperm acrosome enzyme chemiluminescence immunoassay kit and preparation method thereof |
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