CN100504361C - Quantitative detection agent and detection method for seminal plasm acid phosphatase - Google Patents
Quantitative detection agent and detection method for seminal plasm acid phosphatase Download PDFInfo
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- CN100504361C CN100504361C CNB2004100406632A CN200410040663A CN100504361C CN 100504361 C CN100504361 C CN 100504361C CN B2004100406632 A CNB2004100406632 A CN B2004100406632A CN 200410040663 A CN200410040663 A CN 200410040663A CN 100504361 C CN100504361 C CN 100504361C
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- acid phosphatase
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- acp
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Abstract
A reagent for detecting acid phosphatase of seminal plasma quantitatively consists of substrate, termination solution, reference material and quality control solution containing acid phosphatase. Its detecting method includes incubating substrate in standard tube synchronously with quality control tube and analysis tube, reacting on quality control solution with substrate and reacting on semen sample in analysis tube with substrate, terminating reacting in quality control tube and in analysis tube, calculating out concentration value of seminal plasma sample according to absorbance of each tube.
Description
Technical field
The invention belongs to the external diagnosis reagent class, specifically be applicable to the detection by quantitative of seminal acid phosphatase.
Background technology
Male sterility clinical with theoretical research in, acid phosphatase (ACP) occupies very consequence as prostatic its functional attributes, prostatitis patient refining ACP content reduces, hypertrophy of the prostate or early stage its content of prostate malignant tumour person increase.
The World Health Organization (WHO) recommends paranitrophenol phosphate (PNPP) method to measure refining ACP, but its method is because sample and standard are not synchronous reaction, make the accuracy of testing result be subjected to the influence of experimental situation, operator's skill level, thereby the operator can not judge the reliability of testing result.
Refining ACP is as one of prostate common function index, and the accuracy of its testing result influences clinical diagnosis, therefore further improves one's methods and learns the particular importance that seems.
Summary of the invention
But refining ACP detection by quantitative reagent and detection method that fundamental purpose of the present invention is to provide a kind of detectable quality, judges the measurement result confidence level.
Of the present invention time a purpose is to provide a kind of stable, active storage period long refining ACP detection by quantitative reagent and detection method.
Another purpose of the present invention is to provide refining ACP detection by quantitative reagent and the detection method that a kind of testing result error is little, accuracy is high.
For achieving the above object, the present invention proposes a kind of refining ACP detection by quantitative reagent, comprises substrate, stop buffer, standard items and contains the control liquid of ACP.Wherein control liquid is the citrate buffer solution that contains ACP, ammonium sulfate and glycocoll.The activity of ACP is that the concentration of 100～3200U/ml, ammonium sulfate is that the concentration of 0.1～0.5 mol, glycocoll is 0.1～1.0 mol in the described control liquid.
Also comprise dilution, described dilution is the deionized water solution that contains citric acid 0.05～0.5 mol.
Further, comprise that also ACP preserves liquid, described preservation liquid is that weight content is the deionized water solution of 0.5～2.0% sulfuric acid monohydrate hydrogen sodium.
Further, the present invention also proposes a kind of refining ACP quantitative detection method, comprises the steps: to add in the standard pipe substrate, with Quality Control pipe, the synchronous incubation of mensuration pipe; Add substrate and control liquid in the Quality Control pipe, and fully reaction; Measure adding semen sample and substrate in the pipe, and fully reaction; Stop buffer stops Quality Control pipe and the reaction of measuring in the pipe; Absorbance after bioassay standard pipe, Quality Control pipe, mensuration tube reaction stop respectively; Calculate the semen sample concentration value according to the absorbance of measuring.
Described control liquid is the citrate buffer solution that contains ACP, ammonium sulfate and glycocoll.
Described control liquid is that the activity of ACP is that the concentration of 100～3200U/ml, ammonium sulfate is that the concentration of 0.1～0.5 mol, glycocoll is 0.1～1.0 mol.
Semen sample diluted in the described mensuration pipe, described dilution is for containing citric acid 0.05～0.5 mol.
Also add ACP in the described mensuration pipe and preserve liquid, described preservation liquid be weight content be 0.5～2.0%-deionized water solution of water niter cake.
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:
1, in refining ACP detection by quantitative reagent, add control liquid, if the result who detects the control liquid gained in predetermined field of activity or index, then explanation is this time operated credible; If the testing result that detects control liquid is not in predetermined field of activity or index, then expression might misoperation or reagent in some or all lost efficacy, thereby can monitor whether reagent reliable, whether reagent lost efficacy, operating process whether accurately and the confidence level of measurement result;
2, adopt the ammonium sulfate and the glycocoll of suitable concn in the control liquid, can be stabilized under the normal state the very easily ACP of inactivation, thereby make control liquid active no change in the storage period in 1 year;
3, detect the method for refining ACP, in titer, also added substrate, because self decomposing, substrate can produce a part of material in preservation and incubation process, this material is identical with product behind the ACP catalytic substrate, therefore, to carry out the absorbance that colorimetric obtains after the seminal fluid cessation reaction, deduct in the standard pipe substrate self and decompose and carry out the absorbance that colorimetric obtains, can eliminate substrate preserve and the incubation process in because the experimental error that self decomposition causes;
4, because the solution of the paranitrophenol sodium phosphate in the substrate is at room temperature unstable, generally to make freeze-dried powder or, therefore adopt the dry plate agent of paranitrophenol sodium phosphate-20 ℃ of preservations down, can improve the term of validity of substrate greatly.
Embodiment
Embodiment, a kind of refining ACP detection by quantitative reagent comprise that ACP preserves liquid, stop buffer, dilution, 2 above standard items, substrate, control liquid.
Wherein ACP preserves liquid and is mainly used in the ACP that stablizes in the refining sample, if detect after leaving and taking sample at once, does not then need to preserve liquid with ACP; Described ACP preserves liquid and adopts NaHSO in the present invention
4Deionized water solution, be dissolved with in every 1L deionized water 12.0 the gram NaHSO
4H
2O, NaHSO in the deionized water solution
4H
2The weight content of O is 0.5～2.0%, can also adopt the ACP among other substitution of materials the present invention that can stablize ACP to preserve liquid.
Because ACP concentration is very high in the refining sample, dilution be used for diluting refining ACP activity to proper range so that detect; Described dilution adopts citric acid, Sodium azide (NaN in the present invention
3) deionized water solution, be dissolved with citric acid 0.09mol in every 1L deionized water, described dilution is for containing citric acid 0.05～0.5mol/l, NaN
3Weight content be 0.01～0.10% deionized water solution; NaN wherein
3As antiseptic, can also replace with other antiseptic; Can also can dilute dilution among the substitution of materials the present invention of the ACP activity in the refining with other.
Substrate is to make ACP in the refining sample resolve into coloring matter and the activity of calculating ACP; Described substrate adopts in the present invention is the paranitrophenol sodium phosphate dry plate agent of commercially available 5mg/ sheet, can also replace the present invention's substrate with other common substrates that is used for refining ACP detection by quantitative, perhaps adopt the substrate among other substitution of materials the present invention that contain the paranitrophenol sodium phosphate.
Product after stop buffer is used for stopping the active of ACP and substrate is decomposed is at the formed alkaline environment displaing yellow of terminator, described stop buffer adopts the deionized water solution of NaOH (NaOH) in the present invention, contain 4g NaOH in every 1L deionized water, the concentration of NaOH is 0.05～0.50mol/l in the deionized water solution, wherein the NaOH in the stop buffer can replace with sodium carbonate or other alkaline matters, and the stop buffer among the present invention can also be replaced with other alkaline matters that can stop the activity of ACP.
Control liquid and substrate reactions, according to the reaction testing result whether in the scope given in advance, if testing result is in scope then represent that operating result is reliable, if testing result is not in scope then represent in the wrong or reagent of operating process part or all of inefficacy is arranged; Described control liquid adopts the citrate buffer solution of ACP, ammonium sulfate, glycocoll in the present invention, every 1000ml pH6.0, and containing activity in the 0.2mol/L citrate buffer solution is 10
5～3.2 * 10
6The ACP of U, 0.1～0.5 mol sulfuric acid ammonium, 0.1～1.0 moles of glycocoll, the volume content of ACP is 0.2～2.0% or adopts the ACP of active 100～3200U/ml (but to have the branch that the ACP raw material has liquid and solid now in the citrate buffer solution, the contained ACP activity of different material is all variant, therefore also can adopt other active As CP to replace the ACP that is adopted among the present invention) according to the experiment needs, the concentration of ammonium sulfate is 0.1～0.5mol/l, the concentration of glycocoll is 0.1～1.0mol/l, described ammonium sulfate and glycocoll, be used to be stabilized under the normal state the very easily ACP of inactivation, thereby make control liquid active no change in the storage period in 1 year, ammonium sulfate and glycocoll surpass given concentration range, then the effect of stable acidic phosphatase activity is not strong; Control liquid can also with other contain ACP, can catalytic substrate the substitution of materials.
Following standard items stock solution and other standard items more than 1:
1) production standard product stock solution: paranitrophenol (PNP) concentration is the deionized water solution of 4.8～14.4mmol/L;
2) standard items 1:NaOH concentration is the deionized water solution of 0.05～0.5 mol;
3) standard items 2: get 0.125 milliliter of standard items 4, be settled to 100 milliliters with 0.1mol/l NaOH;
4) standard items 3: get 0.5 milliliter of standard items 4, be settled to 100 milliliters with 0.1mol/l NaOH;
5) standard items 4: get 2 milliliters of standard stock solutions, be settled to 100 milliliters with 0.1mol/l NaOH;
Wherein standard items are the working standard product in the refining ACP detection by quantitative, and the pNP in the described standard items is the product after substrate is decomposed by ACP, is used for the concentration of contrast with the detection decomposition product, and calculates the activity of ACP.
Can also be with mentioned reagent generate a reagent box, wherein ACP preserves liquid 50～100ul, dilution 1000～2000ul, control liquid 5～50ul, substrate 0.2～0.8mg, stop buffer 500～2000ul, each standard items 500～2000ul.
The preferred reagent box is that ACP preserves liquid 100ul, sample diluent 2000ul, control liquid 10ul, substrate 0.4mg, stop buffer 1000ul, each standard items 1000ul.
The preparation method of each reagent in the above-mentioned refining ACP detection by quantitative reagent:
A, ACP preserve the process for making of liquid
1) takes by weighing 12.0 gram NaHSO
4H
2O is dissolved in the lL deionized water, 4 ℃ of storages after the stirring and dissolving;
The process for making of B, dilution
2) with deionized water dissolving citric acid 0.09mol, NaN
31g to 800ml;
3) regulate pH to 4.8 with 1mol/1NaOH, be settled to 1000ml, 4 ℃ of storages;
The process for making of C substrate
4) be the paranitrophenol sodium phosphate dry plate agent of commercially available 5mg/ sheet, 4 ℃ of storages;
The process for making of D, stop buffer
5) get NaOH (AR) 100～120 grams and put beaker, add deionized water 200ml, because can solution temperature be raise heat release when NaOH is water-soluble, therefore beaker is put in the cold water behind the stirring and dissolving NaOH, pour in the vinyon bottle room temperature into and leave standstill a few days or fully dissolving, wherein AR represents that the purity of NaOH is pure for analyzing, and can also replace with the NaOH of chemical pure or other purity;
6) get dense NaOH supernatant 4.5ml, add in the 500ml volumetric flask, add deionized water to scale, because this NaOH solution is oversaturated, having small amounts of sodium hydroxide can not dissolve, and therefore need get supernatant;
7) use that to demarcate, proofread and correct its concentration through 3 hours Potassium Hydrogen Phthalate of 105 ℃ of dryings be 0.1mol/l, wherein dry Potassium Hydrogen Phthalate is a method of generally acknowledging, mainly is in order to dry, to make weighing accurate;
The process for making of E, standard stock solution (9.6mmol/L)
8) be ten thousand in scale division value/take by weighing 9.6mmol PNP on the electronic balance of gram to be dissolved in the 1000ml deionized water, place brown bottle-20 ℃ storage;
The process for making of F, control liquid
9) get active 10
5～3.2x10
6The ACP of U is dissolved in 1000ml PH6.0, in the 0.2mol/L citrate buffer solution.Add 0.15mol ammonium sulfate, 0.5mol glycocoll vibration molten back filtration.Put 4 ℃ of preservations.
Detect refining ACP activity with ACP reagent, concrete steps are:
One, reagent is prepared
Every substrate dry powder adds the dissolving of 1.25ml dilution, puts brown bottle and covers the light preservation.
Two, sample is prepared
1, writes down the seminal fluid total amount (ml) that the person under inspection once ejaculates.Be no more than 30 minutes after the seminal fluid liquefaction, with 3000g centrifugal 10 minutes, keep refining;
2, add 100ul refining and 100ulACP in the Eppendorf pipe and preserve liquid, fully mixing obtains detecting sample ,-20 ℃ of preservations; This step mainly is in order to allow the ACP in the sample at low temperatures can long preservation;
Three, method of operating
1, sample to be detected is obtained diluting sample for 2500 times with diluted;
2, further operation sees the following form:
Accurately hatch 30min or 37 ℃ of fully reactions down for 37 ℃
Four, calculate
1, when microplate reader is programmed, because sample is diluted, in order directly to obtain the actual concentrations of sample, import the standard pipe parameter again after need handling standard pipe actual concentrations value, promptly calculate the activity of acid phosphatase of standard pipe concentration correspondence, sample detection of active value is directly exported through microplate reader.The principle that standard pipe actual concentrations value is handled is as follows: if microplate reader is directly carried out colorimetric to each pipe, directly Shu Chu sample concentration is the concentration of the sample after diluting; When the standard pipe concentration that obtains with the sample coefficient corresponding to the sample after the dilution on duty with the actual concentrations of standard pipe, and when being input in the microplate reader program corresponding to the standard pipe concentration of sample after the dilution, be equivalent to the dilution after sample concentration divided by the sample coefficient, so just can directly export the actual concentrations of sample by microplate reader.
Typical curve is with the match of smooth curve mode, as follows on microplate reader the concentration of input standard pipe
ACP international unit (IU): 1 ACP=37 of unit ℃ of following per minute produces 1umol paranitrophenol (PNP)
Sample coefficient=always hatch volume ÷ sample volume ÷ reaction time (min) * refining extension rate
＝1110÷10÷30×5000
＝18500
Standard pipe concentration (U/L)=standard pipe actual concentrations (umol/l) * 18500 * 1/1.11
=standard pipe actual concentrations (umol/l) * 16667
Standard pipe concentration (U/ml)=standard pipe actual concentrations (umol/l) * 16.667
Standard pipe concentration (U/ml) during standard pipe actual concentrations (umol/l) input microplate reader program
0 0
12 200
48 800
192 3200
Five, result's report
The sample final detection result reports in " refining ACP gross activity " mode, promptly
Seminal fluid volume (the ml)=IU/ of gross activity=sample concentration (U/ml) * once ejaculation once ejaculates
In the following formula/be the every meaning of expression
Six, clinical applicability
Detect refining ACP gross activity prostatitis is had diagnostic value.
Claims (8)
1, a kind of seminal acid phosphatase detection by quantitative reagent comprises substrate, stop buffer and standard items, it is characterized in that: also comprise the control liquid that contains acid phosphatase, described control liquid is the citrate buffer solution that contains acid phosphatase, ammonium sulfate and glycocoll; Described stop buffer is the deionized water solution of the alkaline matter of NaOH or other activity that can stop ACP.
2, seminal acid phosphatase detection by quantitative reagent as claimed in claim 1 is characterized in that: acid phosphates activity is that the concentration of 100～3200U/ml, ammonium sulfate is that the concentration of 0.1～0.5 mol, glycocoll is 0.1～1.0 mol in the described control liquid.
3, seminal acid phosphatase detection by quantitative reagent as claimed in claim 1 or 2 is characterized in that: also comprise dilution, described dilution is for containing citric acid 0.05～0.5 mol, NaN
3Weight content be 0.01～0.10% deionized water solution.
4, seminal acid phosphatase detection by quantitative reagent as claimed in claim 3 is characterized in that: comprise that also acid phosphatase preserves liquid, described preservation liquid is that weight content is the deionized water solution of 0.5～2.0% sulfuric acid monohydrate hydrogen sodium.
5, the described seminal acid phosphatase detection by quantitative of claim 1 reagent carries out the seminal acid phosphatase quantitative detection method, it is characterized in that, comprises the steps:
1) adds substrate in the standard pipe, with Quality Control pipe, the synchronous incubation of mensuration pipe;
2) add substrate and control liquid in the Quality Control pipe, and fully reaction;
3) measure adding refining sample and substrate in the pipe, and fully reaction;
4) in Quality Control pipe and mensuration pipe, add stop buffer respectively, be used for stopping Quality Control pipe and the reaction of measuring pipe;
5) absorbance after bioassay standard pipe, Quality Control pipe, mensuration tube reaction stop respectively;
6) calculate refining concentration of specimens value according to the absorbance of measuring.
6, seminal acid phosphatase quantitative detection method as claimed in claim 5 is characterized in that: described control liquid is that acid phosphates activity is that the concentration of 100～3200U/ml, ammonium sulfate is that the concentration of 0.1～0.5 mol, glycocoll is 0.1～1.0 mol.
7, as claim 5 or 6 described seminal acid phosphatase quantitative detection method, it is characterized in that: the refining sample diluted in the described mensuration pipe, described dilution is for containing citric acid 0.05～0.5 mol, NaN
3Weight content be 0.01～0.10% deionized water solution.
8, seminal acid phosphatase quantitative detection method as claimed in claim 7, it is characterized in that: also comprise acid phosphatase preservation liquid, be used for preserving the activity of acid phosphatase of the sample that can not in time detect, described preservation liquid is that weight content is the deionized water solution of 0.5～2.0% sulfuric acid monohydrate hydrogen sodium.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2004100406632A CN100504361C (en) | 2004-09-08 | 2004-09-08 | Quantitative detection agent and detection method for seminal plasm acid phosphatase |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2004100406632A CN100504361C (en) | 2004-09-08 | 2004-09-08 | Quantitative detection agent and detection method for seminal plasm acid phosphatase |
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| CN1746665A CN1746665A (en) | 2006-03-15 |
| CN100504361C true CN100504361C (en) | 2009-06-24 |
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Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101974612A (en) * | 2010-09-29 | 2011-02-16 | 南京欣迪生物药业工程有限责任公司 | Kit for detecting activity of acid phosphatase in seminal plasma and detection method |
| 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 |
| CN103808712B (en) * | 2012-11-05 | 2017-12-19 | 深圳迈瑞生物医疗电子股份有限公司 | A kind of liquid reagent box and detection method for acid phosphatase detection |
| CN103900884B (en) * | 2014-04-11 | 2016-09-14 | 深圳市博锐德生物科技有限公司 | Refining is combined quality-control product and preparation method thereof, test kit |
| CN109975220A (en) * | 2019-02-15 | 2019-07-05 | 深圳华康生物医学工程有限公司 | A kind of refining neutral α-glucosidase assay kit |
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