CN1763220A - Chlorine ion content determination method and chlorine ion diagnosis kit - Google Patents
Chlorine ion content determination method and chlorine ion diagnosis kit Download PDFInfo
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- CN1763220A CN1763220A CN 200410065049 CN200410065049A CN1763220A CN 1763220 A CN1763220 A CN 1763220A CN 200410065049 CN200410065049 CN 200410065049 CN 200410065049 A CN200410065049 A CN 200410065049A CN 1763220 A CN1763220 A CN 1763220A
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- nitrophenol
- chlorine
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- glucosidase
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Abstract
The present invention relates to one chlorine ion content determining method and chlorine ion diagnosis kit, and belongs to the field of medical detection technology. The kit includes buffering solution, deactivated alpha-amylase, 3-chloro-4-nitrophenol-beta-heptyl glucose, alpha beta-glucosidate and stabilizer. Through mixing the sample and reagent in certain volume ratio to generate enzyme coupling reaction, detecting the main wavelength absorbance variation of the reaction product under biochemical analyzer and calculation, ionic chlorine content is obtained. The present invention has the advantages of high sensitivity, high precision and no contamination of inner and outer matters.
Description
Technical field
The present invention relates to a kind of chlorine ion content determination method, the invention still further relates to simultaneously, belong to medical test determination techniques field in order to realize the chlorine ion diagnosis kit of this method.
Background technology
Chlorine in the body fluid exists with ionic species, all almost increases and decreases with the sodium ion balance.It is significant for medical verification to measure chloride ion content, and present known measuring method has multiple:
Chemical assay-mercuric nitrate complexometry method, pharaoh's serpents colorimetry
Method of coulometric analysis
Ion selective electrode method
Enzyme process
Sell sour mercury volumetry and judge relatively difficulty of titration end point, different sample protein contents do not wait, and the depth of its terminal point and intensity are Protean, and are subjected to the interference of bilirubin, oxyphorase and blood fat, so this law precision is obviously not good, and inefficiency.The pharaoh's serpents colorimetry is when measuring, and other factors such as F.Br and I also can react in the serum, and its amount is less than 1mmol/L, so can ignore.Some drugs and bilirubin are all influential to it.This colorimetry is eliminated the influence of subjective factor, and can carry out batch at automatic biochemistry analyzer and measure, and belongs to clinical a kind of method commonly used.Volumetry needs skilled operation, and terminal point is wanted accurately get rid of the interference of subjective factor as far as possible, otherwise error to be very big.Too much bilirubin in the serum.Blood fat and oxyphorase (haemolysis) disturb very big to the result.Volumetry has superseded trend.
Method of coulometric analysis: belong to physics method.Under the constant electric current, place sample with silver electrode, from the Ag of electrode release
+With Cl
-Reaction begins to generate not dissociated AgCl precipitation and picks up counting, and works as Cl
-All and Ag
+Effect finishes, free Ag
+Occur, its solution conductivity obviously increases, and makes Instrument sensor and timing register cut off electric current immediately and calculates the required time of titration as finishing the mensuration process.Br in the sample
-And I
-It is had certain interference, and it is measured seldom, can ignore.This method is easy, fast, need control electric current during mensuration well.
Ion selective electrode method is to measure the best method of chlorion at present, because measure the equal and K of the electrode of chlorion
+, Na
+Electrode is supporting, only needs 100 μ l whole bloods can measure K in the sample
+, Na
+And Cl
-Content and fast, accurately, easy and simple to handle, be the method for clinical use widely at present.But the chlorine ion electrode life-span is shorter, general validity period only 4-6 month.And the Instrument measuring result of different model has certain error.
In recent years, work out novel method with the enzymatic assays chlorion, this law good stability, the result is fit to large-scale Biochemical Analyzer and uses accurately and reliably.In the clinical chemistry analysis, must replace additive method and become the routine analysis project.Yet the research of this respect at present is deep not enough, especially domestic, and retrieval comprises the prior art data of patent documentation, does not find to be fit to the practical technique scheme of national conditions as yet.
The retrieval Chinese patent only finds that application number is 94112025.2, the applying date is the patent that 1994.01.21 and so on measures industrial chlorine, and does not find the mensuration patent of medical field chlorion, and this is from a side illustration, and China's research in this respect is shortcoming relatively also.
Summary of the invention
The technical problem to be solved in the present invention is: propose a kind of chlorine ion content determination method that can overcome above prior art shortcoming, the present invention simultaneously gives corresponding chlorine ion diagnosis kit, adopt the reagent in this test kit not only can on ultraviolet analyser or half, automatic clinical chemistry analyzer, carry out chlorine ion content determination, and finding speed is fast, accuracy is high, thereby can obtain practical applying.
The step that the present invention measures chloride ion content is as follows:
1), with sample with mainly by α-Dian Fenmei (inactivation), 3-chlorine 4-nitrophenol-β-glucoheptose (reagent mix that 3-CNP-β-G7), alpha-glucosidase, beta-glucosidase are formed makes it to take place the reaction of following principle:
α-Dian Fenmei (inactivation)
Clα-Dian Fenmei (must be alive)
3-chlorine 4-nitrophenol-β-glucoheptose (water of CNP-β-G7)+3
α-Dian Fenmei (must be alive)
3-chlorine 4-nitrophenol-β-glucobiose (CNP-β-G2)+3-chlorine 4-nitrophenol-β-grape trisaccharide+3-chlorine 4-nitrophenol-β-grape tetrose+grape trisaccharide (G3)+grape tetrose (G4)
+ grape pentasaccharides (G5)
3-chlorine 4-nitrophenol-β-glucobiose+3-chlorine 4-nitrophenol-β-grape trisaccharide+2 water
Alpha-glucosidase2 chloro-nitrophenol-β-glucose (CNP-β-G1)
Chloro-nitrophenol-β-glucose+water
Beta-glucosidaseChloro-nitrophenol+glucose
2), detect the end reaction thing in the speed that predominant wavelength 405nm absorbancy rises, calculate the size of chloride ion content.
Common step 2) the end reaction thing is placed under ultraviolet analyser or half, the automatic clinical chemistry analyzer device, detect the speed that predominant wavelength 405nm absorbancy rises, calculate the size of chloride ion content.
The blending ratio of above sample and reagent by volume is 1/10 to 1/250, and the mensuration temperature of above process is controlled at 20 ℃ to 50 ℃ conventional scopes, and the reaction times was controlled at conventional 2-30 minute.
The present invention uses chlorine and activates amylase coupling glucoside enzyme reaction colorimetry, after the deactivated amylase of application chlorion activation makes it to recapture activity, coupling glucuroide reaction hydrolysis 3-chlorine 4-nitrophenol-β-glucoheptose produces the chloro-nitrophenol, the chloro-nitrophenol has absorption peak at the 405nm place, raise thereby at the 405nm place absorbancy takes place, the content of chlorion is directly proportional in absorbancy variation that reaction is produced and the sample.At the 405nm place, in Fixed Time Interval, measure the absorbancy lift velocity, the content of chlorion can be calculated.Enzymatic assays inorganic chlorine ion test kit has characteristics such as easy and simple to handle, that specificity is high, sensitivity is good.
Be used to realize that the chlorine ion diagnosis kit of the inventive method can be single agent, comprise:
Damping fluid 40--200mmol/l
α-Dian Fenmei (inactivation) 500--50000U/L
3-chlorine 4-nitrophenol-β-glucoheptose 0.2--3mmol/L
Alpha-glucosidase 500--20000U/L
Beta-glucosidase 500--20000U/L
Stablizer 10--80% (cumulative volume)
Also above single agent can be made into following pair of agent:
Reagent I
Damping fluid 40--200mmol/l
α-Dian Fenmei (inactivation) 500--50000U/L
Alpha-glucosidase 500--20000U/L
Beta-glucosidase 500--20000U/L
Stablizer 10--80% (cumulative volume)
Reagent II
Damping fluid 40--200mmol/l
3-chlorine 4-nitrophenol-β-glucoheptose 0.2--3mmol/L
Stablizer 10--80% (cumulative volume)
The prescription of two agent is not limited only to above-mentioned prescription, the composition of reagent I wherein, 3-chlorine 4-nitrophenol-β-glucoheptose can be placed on reagent II, reagent II composition wherein, alpha-glucosidase or beta-glucosidase etc. also can be placed on reagent I, so can form multiple formulations, not describe in detail one by one at this.
Reagent can also be made into following three reagent, more help the stable of reagent:
Reagent I
Damping fluid 40--200mmol/l
α-Dian Fenmei (inactivation) 500--50000U/L
Stablizer 10--80% (cumulative volume)
Reagent II
Damping fluid 40--200mmol/l
Alpha-glucosidase 500--20000U/L
Beta-glucosidase 500--20000U/L
Stablizer 10--80% (cumulative volume)
Reagent III
Damping fluid 40--200mmol/l
3-chlorine 4-nitrophenol-β-glucoheptose 0.2--3mmol/L
Stablizer 10--80% (cumulative volume)
Three doses prescription is not limited only to above-mentioned prescription, the composition of reagent I wherein, and α-Dian Fenmei (inactivation) can be placed among the reagent II.Reagent II composition wherein, alpha-glucosidase, beta-glucosidase etc. also can be placed among the reagent I, so can form multiple formulations, does not describe in detail one by one.
The β H scope of buffer reagent can be 6.0～11.0.Buffer reagent can be phosphoric acid salt (Phosphate) damping fluid or glycylglycine (Glycylglycine) damping fluid etc., but is not limited only to these damping fluids.
In addition, in order to reduce the cross influence between each reagent composition, the stability of maintenance reagent, so that standing storage, usually add stablizer 10～80% or 10～50mmol/l among reagent I, the reagent II of the reagent I of above single agent, two agent, reagent II or three doses, the reagent III, stablizer can be one or more in ethylene glycol, propylene glycol, glycerine, polyalcohols, sulfuric acid amine or the salt etc.
Experiment shows, takes all factors into consideration from the accuracy of measurement result and economy two aspects of preparation cost, no matter is single agent, two agent or three doses, and the chlorine ion diagnosis kit of the present invention of following system component relation is comparatively desirable:
Damping fluid 80--120mmol/l
α-Dian Fenmei (inactivation) 10000--40000U/L
3-chlorine 4-nitrophenol-β-glucoheptose 0.5--2mmol/L
Alpha-glucosidase 5000--13000U/L
Beta-glucosidase 5000--13000U/L
Stablizer 20--50% (cumulative volume)
Because the present invention utilizes Enzymology method, enzyme digestion reaction to have the high characteristics of specificity fully, is not vulnerable to the interference of other materials of inside and outside source.Easy, the easy handling of enzyme process method.The specificity of enzyme digestion reaction impels test result accurate.Enzyme digestion reaction all is to carry out under buffer conditions, does not have environmental pollution problem.The enzyme process method does not need special, extra instrument, and testing cost is cheap.Therefore can guarantee to have higher test accuracy, be more convenient for applying.
Embodiment
The present invention is further illustrated below in conjunction with examples of implementation.
Embodiment one (single agent)
The chlorine ion diagnosis kit of present embodiment comprises:
Damping fluid 80mmol/l
α-Dian Fenmei (inactivation) 10000U/L
3-chlorine 4-nitrophenol-β-glucoheptose 0.5mmol/L
Alpha-glucosidase 5000U/L
Beta-glucosidase 5000U/L
Stablizer 50% (cumulative volume)
On automatic clinical chemistry analyzer, set: 37 ℃ of temperature, 10 minutes reaction times, test predominant wavelength 405nm, more than the test commplementary wave length 460nm, the volume ratio of tested chlorion sample and reagent is 1/25, the Direction of Reaction is positive reaction.
After adding sample and reagent, make it to mix, following reaction take place:
α-Dian Fenmei (inactivation)
Clα-Dian Fenmei (must be alive)
3-chlorine 4-nitrophenol-β-glucoheptose (water of CNP-β-G7)+3
α-Dian Fenmei (must be alive)
3-chlorine 4-nitrophenol-β-glucobiose (CNP-β-G2)+3-chlorine 4-nitrophenol-β-grape trisaccharide+3-chlorine 4-nitrophenol-β-grape tetrose+grape trisaccharide (G3)+grape tetrose (G4)
+ grape pentasaccharides (G5)
3-chlorine 4-nitrophenol-β-glucobiose+3-chlorine 4-nitrophenol-β-grape trisaccharide+2 water
Alpha-glucosidase2 chloro-nitrophenol-β-glucose (CNP-β-G1)
Chloro-nitrophenol-β-glucose+water
Beta-glucosidaseChloro-nitrophenol+glucose
The end reaction thing is placed under the biochemical analyzer, detect the amplitude that predominant wavelength 405nm absorbancy rises, thereby calculate the content of chlorion.
Present embodiment is used chlorine and is activated amylase coupling glucoside enzyme reaction colorimetry, after the deactivated amylase of application chlorion activation makes it to recapture activity, coupling glucuroide reaction hydrolysis 3-chlorine 4-nitrophenol-β-glucoheptose produces the chloro-nitrophenol, the chloro-nitrophenol has absorption peak at the 405nm place, raise thereby at the 405nm place absorbancy takes place, the content of chlorion is directly proportional in absorbancy variation that reaction is produced and the sample.At the 405nm place, in Fixed Time Interval, measure the absorbancy lift velocity, the content of chlorion can be calculated.
Embodiment two (two agent)
The chlorion diagnostic reagent of present embodiment has:
Reagent I
Damping fluid 100mmol/l
α-Dian Fenmei (inactivation) 25000U/L
Alpha-glucosidase 9000U/L
Beta-glucosidase 9000U/L
Stablizer 50% (cumulative volume)
Reagent II
Damping fluid 100mmol/l
3-chlorine 4-nitrophenol-β-glucoheptose 1mmol/L
Stablizer 20mmol/L
When measuring chloride ion content, temperature is controlled at 30 ℃, 15 minutes reaction times, and test predominant wavelength 405nm, more than the test commplementary wave length 460nm, the volume ratio of tested chlorion sample and reagent is 1/25, the Direction of Reaction is positive reaction.
Concrete determination step is:
α-Dian Fenmei (inactivation) Cl α-Dian Fenmei (must be alive)
3-chlorine 4-nitrophenol-β-glucoheptose (water α-Dian Fenmei (must be alive) of CNP-β-G7)+3
3-chlorine 4-nitrophenol-β-glucobiose (CNP-β-G2)+3-chlorine 4-nitrophenol-β-grape trisaccharide+3-chlorine 4-nitrophenol-β-grape tetrose+grape trisaccharide (G3)+grape tetrose (G4)
+ grape pentasaccharides (G5)
3-chlorine 4-nitrophenol-β-glucobiose+3-chlorine 4-nitrophenol-β-grape trisaccharide+2 water
Alpha-glucosidase 2 chloro-nitrophenol-β-glucose (CNP-β-G1)
Chloro-nitrophenol-β-glucose+water beta-glucosidase chloro-nitrophenol+glucose
The end reaction thing is placed under the biochemical analyzer, detect the amplitude that predominant wavelength 405nm absorbancy rises, thereby calculate the content of chlorion.
The reaction times of each reactions steps was controlled at 15 minutes.
Embodiment three (three doses)
The chlorion diagnostic reagent of present embodiment is three doses, has:
Reagent I
Damping fluid 120mmol/l
α-Dian Fenmei (inactivation) 40000U/L
Stablizer 50% (cumulative volume)
Reagent II
Damping fluid 120mmol/l
Alpha-glucosidase 13000U/L
Beta-glucosidase 13000U/L
Stablizer 50% (cumulative volume)
Reagent III
Damping fluid 120mmol/l
3-chlorine 4-nitrophenol-β-glucoheptose 2mmol/L
Stablizer 20mmol/L
On automatic clinical chemistry analyzer, set: 25 ℃ of temperature, 20 minutes reaction times, test predominant wavelength 405nm, more than the test commplementary wave length 460nm, the volume ratio of tested chlorion sample and reagent is 1/25, the Direction of Reaction is positive reaction.
Concrete determination step is:
α-Dian Fenmei (inactivation) Cl α-Dian Fenmei (must be alive)
3-chlorine 4-nitrophenol-β-glucoheptose (water α-Dian Fenmei (must be alive) of CNP-β-G7)+3
3-chlorine 4-nitrophenol-β-glucobiose (CNP-β-G2)+3-chlorine 4-nitrophenol-β-grape trisaccharide+3-chlorine 4-nitrophenol-β-grape tetrose+grape trisaccharide (G3)+grape tetrose (G4)
+ grape pentasaccharides (G5)
3-chlorine 4-nitrophenol-β-glucobiose+3-chlorine 4-nitrophenol-β-grape trisaccharide+2 water
Alpha-glucosidase 2 chloro-nitrophenol-β-glucose (CNP-β-G1)
Chloro-nitrophenol-β-glucose+water beta-glucosidase chloro-nitrophenol+glucose
The end reaction thing is placed under the biochemical analyzer, detect the amplitude that predominant wavelength 405nm absorbancy rises, thereby calculate the content of chlorion.
The reaction times of each reactions steps was controlled at 20 minutes.
Embodiment four
The chlorion diagnostic reagent of present embodiment has:
Reagent I
Damping fluid 100mmol/l
α-Dian Fenmei (inactivation) 20000U/L
Beta-glucosidase 10000U/L
Stablizer 50% (cumulative volume)
Reagent II
Damping fluid 100mmol/l
Alpha-glucosidase 10000U/L
Stablizer 50% (cumulative volume)
Reagent III
Damping fluid 100mmol/l
3-chlorine 4-nitrophenol-β-glucoheptose 2mmol/L
Stablizer 50% (cumulative volume)
On Biochemical Analyzer, set: 37 ℃ of temperature, 10 minutes reaction times, test predominant wavelength 405nm, more than the test commplementary wave length 460nm, the volume ratio of tested chlorion sample and reagent is 1/25, the Direction of Reaction is positive reaction.
After adding sample and reagent, make it to mix, following reaction take place:
α-Dian Fenmei (inactivation) Cl α-Dian Fenmei (must be alive)
3-chlorine 4-nitrophenol-β-glucoheptose (water α-Dian Fenmei (must be alive) of CNP-β-G7)+3
3-chlorine 4-nitrophenol-β-glucobiose (CNP-β-G2)+3-chlorine 4-nitrophenol-β-grape trisaccharide+3-chlorine 4-nitrophenol-β-grape tetrose+grape trisaccharide (G3)+grape tetrose (G4)
+ grape pentasaccharides (G5)
3-chlorine 4-nitrophenol-β-glucobiose+3-chlorine 4-nitrophenol-β-grape trisaccharide+2 water
Alpha-glucosidase 2 chloro-nitrophenol-β-glucose (CNP-β-G1)
Chloro-nitrophenol-β-glucose+water beta-glucosidase chloro-nitrophenol+glucose
The end reaction thing is placed under the biochemical analyzer, detect the amplitude that predominant wavelength 405nm absorbancy rises, thereby calculate the content of chlorion.
In a word, experiment showed, and adopt measuring method of the present invention can draw required measurement result by ultraviolet analyser or half, automatic clinical chemistry analyzer device fully, and highly sensitive, tolerance range good, is not subjected to the pollution of inside and outside source material.
Claims (8)
1. chlorine ion content determination method, step is as follows:
1), with sample and the reagent mix mainly formed by α-Dian Fenmei (inactivation), 3-chlorine 4-nitrophenol-β-glucoheptose, alpha-glucosidase, beta-glucosidase, make it to take place following reaction:
α-Dian Fenmei (inactivation)
Clα-Dian Fenmei (must be alive)
3-chlorine 4-nitrophenol-β-glucoheptose+3 water
α-Dian Fenmei (must be alive)
3-chlorine 4-nitrophenol-β-glucobiose+3-chlorine 4-nitrophenol-β-grape trisaccharide+
3-chlorine 4-nitrophenol-β-grape tetrose+grape trisaccharide+grape tetrose+grape pentasaccharides
3-chlorine 4-nitrophenol-β-glucobiose+3-chlorine 4-nitrophenol-β-grape trisaccharide+2 water
Alpha-glucosidase2 chloro-nitrophenol-β-glucose
Chloro-nitrophenol-β-glucose+water
Beta-glucosidaseChloro-nitrophenol+glucose
2), detect the end reaction thing in the speed that predominant wavelength 405nm absorbancy rises, calculate the size of chloride ion content.
2. according to the described chlorine ion content determination method of claim 1, it is characterized in that: described step 2) be: the end reaction thing is placed under ultraviolet analyser or half, the automatic clinical chemistry analyzer, detect speed (journey) degree that predominant wavelength 405nm absorbancy rises, calculate the content of chlorion.
3, according to claim 1 or 2 described chlorine ion content determination methods, it is characterized in that: temperature is controlled at 20 ℃ to 50 ℃ scopes, and the reaction times was controlled at 2-30 minute.
4, according to claim 1 or 2 described chlorine ion content determination methods, it is characterized in that: the ratio control of tested chlorion sample and reagent is 1/10 to 1/500.
5. chlorine ion diagnosis kit is grouped into by following one-tenth:
Damping fluid 40---200mmol/l
α-Dian Fenmei (inactivation) 500---50000U/L
3-chlorine 4-nitrophenol-β-glucoheptose 0.2---3mmol/L
Alpha-glucosidase 500---20000U/L
Beta-glucosidase 500---20000U/L
Stablizer 10---80% (cumulative volume)
6. according to the described chlorine ion diagnosis kit of claim 5, it is characterized in that: described reagent is made into single agent, two agent or three doses.
7. according to claim 5 or 6 described chlorine ion diagnosis kits, it is characterized in that: described stablizer is at least a in ethylene glycol, propylene glycol, glycerine, polyalcohols, ammonium sulfate or the salt.
8. according to claim 5 or 6 described chlorine ion diagnosis kits, it is characterized in that: the pH scope of described buffer reagent is 6.0～11.0, and described buffer reagent is a kind of in phosphate buffered saline buffer or the glycylglycine damping fluid.
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|---|---|---|---|
| CN 200410065049 CN1763220A (en) | 2004-10-20 | 2004-10-20 | Chlorine ion content determination method and chlorine ion diagnosis kit |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410065049 CN1763220A (en) | 2004-10-20 | 2004-10-20 | Chlorine ion content determination method and chlorine ion diagnosis kit |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101122606B (en) * | 2006-08-09 | 2012-09-05 | 株式会社神户制钢所 | Device for decomposition reaction of medicament for chemical weapon and monitoring method thereof |
| CN104198691A (en) * | 2014-08-14 | 2014-12-10 | 上海睿康生物科技有限公司 | Stable alpha-amylase detection kit |
| CN113189028A (en) * | 2021-04-19 | 2021-07-30 | 深圳市锦瑞生物科技有限公司 | Serum chlorine detection reagent ball and serum chlorine detection chip |
-
2004
- 2004-10-20 CN CN 200410065049 patent/CN1763220A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101122606B (en) * | 2006-08-09 | 2012-09-05 | 株式会社神户制钢所 | Device for decomposition reaction of medicament for chemical weapon and monitoring method thereof |
| CN104198691A (en) * | 2014-08-14 | 2014-12-10 | 上海睿康生物科技有限公司 | Stable alpha-amylase detection kit |
| CN104198691B (en) * | 2014-08-14 | 2016-08-17 | 上海睿康生物科技有限公司 | A kind of stable α-amylase detection kit |
| CN113189028A (en) * | 2021-04-19 | 2021-07-30 | 深圳市锦瑞生物科技有限公司 | Serum chlorine detection reagent ball and serum chlorine detection chip |
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