CN1595153B - Refined fructose enzyme method quantitative determination reagent - Google Patents
Refined fructose enzyme method quantitative determination reagent Download PDFInfo
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- CN1595153B CN1595153B CN 200410040245 CN200410040245A CN1595153B CN 1595153 B CN1595153 B CN 1595153B CN 200410040245 CN200410040245 CN 200410040245 CN 200410040245 A CN200410040245 A CN 200410040245A CN 1595153 B CN1595153 B CN 1595153B
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- enzyme
- fructose
- protection liquid
- enzyme protection
- liquid
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Abstract
This invention discloses a quantitative measurement agent box for refining fructose enzyme, which comprises buffer liquid, color-developing agent, standard sample and enzyme protection liquid, wherein, the said enzyme protection liquid is the enzyme protection with fructose dehydrogenase and the said enzyme protection liquid is frozen powder and also comprises frozen powder dissolved liquid used as dissolved enzyme protection liquid frozen powder. The said buffer liquid is citrate buffer liquid with antiseptic and comprises terminal liquid used to terminate the color-developing reaction. The terminal liquid has diluted acid. This sample in this invention needn't protein process and is of simple operation. The whole reaction process needs only forty minutes and the agent is stable and can be stored for one year.
Description
Technical field
The invention belongs to the external diagnosis reagent class, specifically be applicable to the detection by quantitative of seminal plasma fructose.
Background technology
In the clinical and theoretical research of male sterility; Seminal plasma fructose is as the specific secretion index of seminal vesicle; Occupy very consequence always, be widely used in differentiating the azoospermatism that azoospermatism and vas deferens, seminal vesicle depauperation due to the simple property obstruction of vas deferen cause; It also can reflect the secretion level of male sex hormone indirectly in addition.
Domestic in the past mainly is to detect seminal plasma fructose with the resorcinol method, but its method exists that accuracy as a result is not high, to want deproteinized, heated culture temperature be 90 ℃, the weakness such as concentrated hydrochloric acid of needs adding severe corrosive to sample.The World Health Organization (WHO) recommends the indoles standard measure to detect seminal plasma fructose, and sample will be handled through deproteinized, heated culture temperature is 50 ℃ but its method also exists, add severe corrosive concentrated hydrochloric acid, need be in deficiencies such as glass test tube internal reactions.
Summary of the invention
The invention is intended to overcome the deficiency of prior art, provide a kind of simple to operate, be swift in response, treatment temperature is low, the seminal plasma fructose enzyme process detection by quantitative kit of safety and environmental protection.
For realizing above-mentioned purpose; The present invention proposes a kind of seminal plasma fructose enzyme process detection by quantitative kit; Comprise damping fluid, developer, standard items and enzyme protection liquid, said enzyme protection liquid is the enzyme protection liquid that contains Fructose dehydrogenase, the preferred 3000～8000U/L of the content of Fructose dehydrogenase in the enzyme protection liquid.
The said enzyme protection liquid that contains Fructose dehydrogenase is freeze-dried powder, also comprises the freeze-dried powder lysate, and the freeze-dried powder lysate is an enzyme protection liquid.
Said damping fluid is the citrate buffer solution that contains antiseptic.
Said developer is for containing iodine azoles four nitroblues (4-Fluoro-3-nitrobenzotrifluoride is called for short NBT) or iodine nitro tetrazole purple (p-Iodonitrotetrazolium violet is called for short INT), the phenol piperazine dimethyl ester sulfate deionized water solution of (4-Amidinophenylmethanesulfonyl fluoride hydrochloride is called for short PMS).
Comprise stop buffer, be used to stop enzymatic reaction.
Said stop buffer is the solution that contains diluted acid.
Because technique scheme, the embodiment that will detail below combining, the technique effect that the present invention gives prominence to is: sample need not to carry out the deproteinized processing, operates simplyr, detects more conveniently, is more suitable for clinical practice; Adopt Fructose dehydrogenase, fast reaction speed, entire reaction course only need 40 minutes; Adopt diluted acid as stop buffer,, environmental protection safer than the concentrated hydrochloric acid of severe corrosive; Heated culture temperature is 37 ℃, and is all lower, safer in operation than the heated culture temperature in resorcinol method and the indoles method; With the freeze-drying of enzyme protection liquid, and in damping fluid, adopted antiseptic, made reagent more stable, can reach 1 year storage period.
Embodiment
Through concrete embodiment the present invention is described in further detail below.
Embodiment, a kind of seminal plasma fructose enzyme process detection by quantitative kit comprises damping fluid, developer, enzyme protection liquid freeze-dried powder, fructose titer (8mM), enzyme protection liquid freeze-dried powder lysate and terminator.Wherein: the 1000ml damping fluid contains Sodium azide (NaN
3) 0.15g, citric acid 9.5g; Contain Fructose dehydrogenase 450U in every 100ml enzyme protection liquid freeze-dried powder; Enzyme protection liquid freeze-dried powder lysate is an enzyme protection liquid; Developer is the deionized water solution that contains NBT and PMS, contains NBT 0.03 gram, PMS0.45 gram in every 100ml developer; Terminator is the deionized water solution that contains the concentrated sulphuric acid, contains 27.5 milliliters of the concentrated sulphuric acids in every 1000ml terminator.
Each composition in the above-mentioned seminal plasma fructose enzyme process detection by quantitative reagent adopts following process to make:
The process for making of A, damping fluid
1) takes by weighing citric acid 9.5g, NaN with analytical balance
30.15g, add deionized water to the 950ml stirring and dissolving.
2) regulate pH (with 1mol/l NaOH) to 4.7～4.9.
3) be settled to 1000ml with deionized water, filter back 4 ℃ of preservations;
The process for making of B, enzyme protection liquid freeze-dried powder
4) get Fructose dehydrogenase 450U, add a small amount of import enzyme protection liquid vibration dissolving after, be settled to 100 milliliters with enzyme protection liquid.
5) freeze-drying after the packing, 4 ℃ of preservations;
The process for making of C, freeze-dried powder lysate
6) import enzyme protection liquid, 4 ℃ of preservations;
The process for making of D, developer
7) with accurate weighing NBT 0.03 gram of analytical balance, place to add 90 milliliters of deionized waters in the beaker.
8) add the PMS0.45 gram again, treat to dissolve fully the back and be settled to cumulative volume 100ml with deionized water;
The process for making of E, fructose titer stock solution (8mM)
9) accurately take by weighing fructose 0.7206g, NaN with analytical balance
30.25g, add the sterilization deionized water and be settled to 500ml.
10) EK, 4 ℃ of preservations;
The process for making of F, terminator
11) accurately measure 27.5 milliliters of the concentrated sulphuric acids, place in the beaker, slowly add in 900 ml deionized water along walls of beaker, the limit edged stirs.
12) treat to move in 1000 milliliters of dressing measuring bottles after solution returns to room temperature, be settled to 1000 milliliters with deionized water.4 ℃ of preservations.
In the foregoing description, damping fluid is NaN
3Content is 0.01～0.1% citrate buffer solution, NaN
3In damping fluid, play antiseptic; Enzyme protection liquid freeze-dried powder is that Fructose dehydrogenase content is the enzyme protection liquid freeze-dried powder of 3000～8000U/L, generally speaking, and in the enzyme protection liquid of seminal plasma fructose enzyme process detection by quantitative reagent; The following 3000U/L that is limited to of Fructose dehydrogenase content; For controlling cost, be limited to 8000U/L on the content of Fructose dehydrogenase, in the scope of 3000～8000U/L; The content of Fructose dehydrogenase is high more, and the speed that catalytic reaction is breasted the tape is fast more; Enzyme protection liquid freeze-dried powder lysate is an enzyme protection liquid; Developer is that NBT content is 0.01～0.1%, PMS content is 0.2～0.8% deionized water solution; The content 0.2～0.8% of the content 0.01～0.1% of NBT and PMS in the developer is with the effective dose of atomic reaction of hydrogen after the Fructose dehydrogenase catalysis seminal plasma fructose dehydrogenation of capacity; NBT can also be with INT or other developer commonly used replacements, and PMS can also use other developer replacement commonly used; Terminator is that concentrated sulphuric acid content is 2～4% deionized water solution, terminator so long as dilute acid soln just, wherein the concentrated sulphuric acid can also use other acid to replace.
Test Example detects seminal plasma fructose content with seminal plasma fructose enzyme process detection by quantitative kit, and concrete steps are:
One, reagent is prepared
1, every bottle of enzyme protection liquid freeze-dried powder adds the dissolving of 0.6ml enzyme protection liquid freeze-dried powder lysate, and room temperature dissolving 30min is for use.
2, preparation working fluid, method is: 9 parts of damping fluids, add 1 part of developer, fully mixing is for use.
Two, sample is prepared
After the fresh semen liquefaction, got part seminal fluid 2000g rapidly centrifugal 10 minutes; Get refining with distilled water dilution in 1: 4.
Three, typical curve preparation
With the downward two-fold dilution of 8mmol/L fructose titer is 4,2 and three concentration of 1mmol/L, with distilled water as zero standard concentration, promptly among standard pipe S1～S5 fructose concentration be respectively 0,1,2,4,8mmol/L.
Four, detect step
1, it is some to get the micropore lath, and each detects positional alignment mode such as the table one of hole on microwell plate:
Respectively detect the arrangement in hole on table one microwell plate
In the table one, S represents gauge orifice, and SB represents the standard blank well, and on behalf of sample, R measure the hole, and RB represents the sample control wells.
2, in micropore, accomplish following operations
The vibration mixing, mounting, 37 ℃ of lucifuge accurate response 40min
Five, colorimetric and calculating
Because the ratio of the concentration of the absorbance of sample (OD) and sample equals the ratio of concentration of OD value and the titer of titer, that is:
Simultaneously the refining sample has been diluted 4 times, therefore when calculating sample concentration, also correspondingly the concentration of standard items multiply by 4 with distilled water.
In sum, calculate fructose content (C in the refining
Sample) formula following:
Wherein, Δ OD
Sample=OD3-OD4, Δ OD
Standard=OD1-OD2
The application concentration (mmol/L) of standard actual concentrations (mmol/L) input ELIASA
0 0
1 4
2 8
4 16
8 32
With Bio-Tek ELx800 ELIASA is example, and it is following that program is provided with major parameter:
MAPPING?DIRECTION：
DOWN；
BLANK?MAP：
P-DOWN；
ENTER?NUMBER?OF?STANDARDS：
05
ENTER?NUMBER?OF?STANDARD?REPLICATES：
01
CONCN.OF?STD1：
0
CONCN.OF?STD2：
4
CONCN.OF?STD3：
8
CONCN.OF?STD4：
16
CONCN.OF?STD5：
32
By " smooth curve (QUAD) " mode match typical curve
ELIASA printout result is and detects sample actual concentration (mmol/L).
Six, result's report
The fructose final detection result is reported with " μ mol/ once ejaculates " mode.
The seminal fluid cumulative volume (ml) of the seminal plasma fructose amount of once ejaculating (μ mol)=seminal plasma fructose concentration (mmol/L) * once ejaculation
Seven, normal reference value
Seminal plasma fructose content >=13 μ the mol that once ejaculate.Wherein 13 μ mol are the reference values of recommending according to WHO.
Eight, clinical applicability
1, can judge the seminal vesicle secreting function; 2, combine the detection of refining neutral alpha-glucosidase, can judge the region of obstruction of obstructive azoospermatism.
Claims (3)
1. seminal plasma fructose enzyme process detection by quantitative kit; Comprise damping fluid, developer, enzyme protection liquid and standard items; It is characterized in that: by mass percentage; Said developer is to contain that iodine azoles four nitroblues or iodine nitro tetrazole are purple, the deionized water solution of phenol piperazine dimethyl ester sulfate, and the content that iodine azoles four nitroblues or iodine nitro tetrazole are purple in the said developer is 0.01～0.1%, the content of phenol piperazine dimethyl ester sulfate is 0.2～0.8%; The content of Fructose dehydrogenase is 3000～8000U/L in the said enzyme protection liquid, and the state that said enzyme protection liquid cooling is frozen into freeze-dried powder is preserved; Said damping fluid is the citrate buffer solution that contains antiseptic; Also comprise the freeze-dried powder lysate, said freeze-dried powder lysate is the enzyme protection liquid that does not contain Fructose dehydrogenase.
2. seminal plasma fructose enzyme process detection by quantitative kit as claimed in claim 1, it is characterized in that: said damping fluid is the citrate buffer solution that contains antiseptic.
3. according to claim 1 or claim 2 seminal plasma fructose enzyme process detection by quantitative kit, it is characterized in that: also comprise stop buffer, be used to stop enzymatic reaction, said stop buffer is the solution that contains diluted acid.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410040245 CN1595153B (en) | 2004-07-14 | 2004-07-14 | Refined fructose enzyme method quantitative determination reagent |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410040245 CN1595153B (en) | 2004-07-14 | 2004-07-14 | Refined fructose enzyme method quantitative determination reagent |
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| Publication Number | Publication Date |
|---|---|
| CN1595153A CN1595153A (en) | 2005-03-16 |
| CN1595153B true CN1595153B (en) | 2012-02-01 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200410040245 Expired - Fee Related CN1595153B (en) | 2004-07-14 | 2004-07-14 | Refined fructose enzyme method quantitative determination reagent |
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Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102012374B (en) * | 2010-09-29 | 2012-08-01 | 南京欣迪生物药业工程有限责任公司 | Kit for detecting concentration of seminal plasma fructose and detection method |
| CN102323230B (en) * | 2011-08-16 | 2013-07-24 | 南京欣迪生物药业工程有限责任公司 | Seminal fructose concentration detection kit and application |
| CN110117791B (en) * | 2019-05-23 | 2023-11-10 | 电子科技大学 | Binding force promoter for brown oxide liquid of high-density interconnection printed circuit board |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1243952A (en) * | 1998-07-03 | 2000-02-09 | 松下电器产业株式会社 | Biological sensor |
-
2004
- 2004-07-14 CN CN 200410040245 patent/CN1595153B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1243952A (en) * | 1998-07-03 | 2000-02-09 | 松下电器产业株式会社 | Biological sensor |
Non-Patent Citations (6)
| Title |
|---|
| Adachi O |
| Ameyama M.Determination of seminal fructose using D-fructosedehydrogenase..Clinica chimica acta15 3.1985,15(3),307-310.. |
| Nakashima K |
| Nakashima K;Takei H;Adachi O;Shinagawa E;Ameyama M.Determination of seminal fructose using D-fructosedehydrogenase..Clinica chimica acta15 3.1985,15(3),307-310.. * |
| Shinagawa E |
| Takei H |
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| CN1595153A (en) | 2005-03-16 |
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