CN101324622A - Ferrous ion diagnosis/determination reagent kit and method for determining ferrous ion concentration - Google Patents
Ferrous ion diagnosis/determination reagent kit and method for determining ferrous ion concentration Download PDFInfo
- Publication number
- CN101324622A CN101324622A CNA200710023382XA CN200710023382A CN101324622A CN 101324622 A CN101324622 A CN 101324622A CN A200710023382X A CNA200710023382X A CN A200710023382XA CN 200710023382 A CN200710023382 A CN 200710023382A CN 101324622 A CN101324622 A CN 101324622A
- Authority
- CN
- China
- Prior art keywords
- reagent
- ferrous ion
- aconitase
- stabilizing agent
- aconitate
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a kit for diagnosing/mensurating ferrous ions by utilizing the technologies of the enzymic colorimetric method and the enzyme linked immunosorbent assay. The invention further relates to a method, a principle and the composition and the components of a reagent for mensurating the concentration of the ferrous ions, and belongs to the technology field of medical/food/environmental inspection and measurement. The main components of the kit include a buffer solution, reduced coenzyme, cis-aconitic acid, aconitase, citrate lyase, malate dehydrogenase and a stabilizer. Through mixing a sample and the reagent by a certain volume ratio, an enzymatic reactions occurs, then the reactant is placed under an ultraviolet/visible light analyzer, and the velocity of the decrease in absorbance at 340nm of the dominant wavelength is detected, thereby mensurating the concentration of the ferrous ions.
Description
Technical field
The present invention relates to a kind of ferrous ion diagnosis/determination reagent kit, the invention still further relates to the method for measuring ferrous ion concentration simultaneously, belong to medical science/food/environmental test determination techniques field.
Background technology
Iron and total iron binding capacity are very important in the body metabolism process.Iron is indispensable material in the human body normal physiological processes.About 3～the 5g of human body iron content, wherein 70% is present in the corpuscular hemoglobin; 25% is distributed in mononuclear phagocyte system (being reticuloendothelial system, ferritin and hemosiderin in liver, spleen, the marrow); 4.9% is distributed in the enzyme of myoglobins, cytochrome, iron content; 0.1% is the iron in the circulating plasma.
Iron in the human body derives from food, mainly is absorbed at duodenum and upper part of small intestine.Iron forms with ferritin and hemosiderin and is stored in marrow, spleen and the liver in the body.There is small amounts of iron to combine, is delivered to each tissue, be utilized or stored by blood plasma with plasma transferrins.
The mensuration of serum levels of iron mainly is to use spectrophotometric method, secondly is atomic absorption method.The latter is because sensitivity is not high and the interference of substrate, than beam split photometry poor reliability.Spectrophotometric method uses a class and contains-the N=C-C=N-structure add lustre to formerly, with ferrous formation five-membered ring, produce aubergine.That early uses has dipyridine and a red shift phenanthroline; Recently the higher ferrous piperazines of sensitivity of recommending more.In addition, the reaction of using chrome azurol S and high ferro is still arranged.
Summary of the invention
The technical problem to be solved in the present invention is: propose a kind of enzymic colorimetric (EnzymaticColorimetric Method) and enzyme (even) united method (Couple Reaction) technology utilized, monitoring reduced form nicotinamide coenzyme (reduced coenzyme) is in the variation of 340nm wavelength place absorbance, measured the method for ferrous ion concentration, simultaneously, the present invention also will provide in order to realize the ferrous ion diagnosis/determination reagent kit of this method, adopt this reagent not only can be ultraviolet analyser or half, carrying out ferrous ion concentration on the automatic clinical chemistry analyzer measures, and finding speed is fast, the accuracy height, thereby can obtain practical applying.
Ferrous ion concentration assay method principle of the present invention is as follows:
Aconitate+water
AconitaseCitric acid
Citric acid
Citrate lyaseAcetate+oxaloacetic acid
Oxaloacetic acid+reduced coenzyme
Malic dehydrogenaseL MALIC ACID+coenzyme
Aconitase (the aconitase that this method application need ferrous ion activates; EC 4.2.1.3) enzyme (idol) connection citrate lyase (citrate lyase; EC 4.1.3.6), malic dehydrogenase (malatedehydrogenase; EC 1.1.1.37) enzyme ' s reaction speeding colourimetry.Aconitase enzymolysis aconitate under ferrous ion activates produces citric acid, the effect of uniting citrate lyase, malic dehydrogenase again by (idol), reduced coenzyme (absorption peak being arranged at the 340nm place) is oxidized into coenzyme (not having absorption peak at the 340nm place) the most at last, thereby measured the speed that reduced coenzyme descends in 340nm place absorbance, by measuring the speed that 340nm place absorbance descends, can calculate the concentration of ferrous ion.
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 ferrous ion diagnosis/determination reagent kit of the present invention of following composition relation is comparatively desirable:
Damping fluid 100mmol/L
Stabilizing agent 500mmol/L
Reduced coenzyme 0.25mmol/L
Aconitate 10mmol/L
Aconitase 6000U/L
Citrate lyase 12000U/L
Malic dehydrogenase 18000U/L
Ferrous ion diagnosis/determination reagent kit of the present invention can be single agent, comprising:
Damping fluid, stabilizing agent, reduced coenzyme, aconitate, aconitase, citrate lyase, malic dehydrogenase.
Kit can be a dry powder, and use the back that is dissolved in water before use; Also can be mixed with liquid reagent, directly use.
Also above-mentioned single agent reagent can be made into following pair of agent reagent:
Reagent 1
Damping fluid, stabilizing agent, reduced coenzyme, aconitate.
Reagent 2
Damping fluid, stabilizing agent, aconitase, citrate lyase, malic dehydrogenase.
Reduced coenzyme, aconitate, aconitase, citrate lyase, the position of malic dehydrogenase in reagent 1 or reagent 2 can not limit.Kit can be a dry powder, and use the back that is dissolved in water before use; Also can be mixed with liquid reagent, directly use.
Above-mentioned single agent reagent can also be made into following three doses of reagent:
Reagent 1
Damping fluid, stabilizing agent, reduced coenzyme, aconitate.
Reagent 2
Damping fluid, stabilizing agent, citrate lyase, malic dehydrogenase.
Reagent 3
Damping fluid, stabilizing agent, aconitase.
Reduced coenzyme, aconitate, aconitase, citrate lyase, the position of malic dehydrogenase in reagent 1, reagent 2 or reagent 3 can not limit.Kit can be a dry powder, and use the back that is dissolved in water before use; Also can be mixed with liquid reagent, directly use.
No matter be single agent, two agent or three doses, the present invention measures the method for ferrous ion concentration, and its reduced coenzyme can be a kind of among NADPH, NADH or the thio-NADH.
Embodiment
The present invention is further illustrated below in conjunction with examples of implementation.
Embodiment one
The ferrous ion diagnosis/determination reagent of present embodiment is single reagent, comprising:
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Reduced coenzyme 0.25mmol/L
Aconitate 10mmol/L
Aconitase 6000U/L
Citrate lyase 12000U/L
Malic dehydrogenase 18000U/L
Reagent divides the bottle of packing into after all dissolving and preparing, and carries out freeze drying, makes powdered reagent; Before the use, add pure water, use after redissolving.
On automatic clinical chemistry analyzer, set: 37 ℃ of temperature, 10 minutes reaction time, initial absorbance 1.8 ± 0.7, test predominant wavelength 340nm, test commplementary wave length 405nm, the volume ratio of tested ferrous ion sample and reagent is 1/25, the Direction of Reaction is negative reaction (reaction descends), about about 2 minutes of time delay is about 3 minutes detection times.
After adding sample and reagent, make them mixed and have reaction, reactant places under the Biochemical Analyzer the most at last, detects the speed that predominant wavelength 340nm absorbance descends, thereby calculates the concentration of ferrous ion.
Embodiment two
The ferrous ion diagnosis/determination reagent of present embodiment is double reagent, comprising:
Reagent 1
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 50mmol/L
Reduced coenzyme 0.25mmol/L
Aconitate 10mmol/L
Reagent 2
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Aconitase 6000U/L
Citrate lyase 12000U/L
Malic dehydrogenase 18000U/L
Reagent divides the bottle of packing into after all dissolving and preparing, and makes liquid double reagent, can directly use.
On automatic clinical chemistry analyzer, set: 37 ℃ of temperature, 10 minutes reaction time, initial absorbance 1.8 ± 0.7, test predominant wavelength 340nm, test commplementary wave length 405nm, the volume ratio of tested ferrous ion sample and reagent 1, reagent 2 is 2/20/5, the Direction of Reaction is negative reaction (reaction descends), about about 2 minutes of time delay is about 3 minutes detection times.
After adding sample and reagent, make them mixed and have reaction, reactant places under the Biochemical Analyzer the most at last, detects the speed that predominant wavelength 340nm absorbance descends, thereby calculates the concentration of ferrous ion.
Embodiment three
The ferrous ion diagnosis/determination reagent of present embodiment is three reagent, comprising:
Reagent 1
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 50mmol/L
Reduced coenzyme 0.25mmol/L
Aconitate 10mmol/L
Reagent 2
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Citrate lyase 12000U/L
Malic dehydrogenase 18000U/L
Reagent 3
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Aconitase 6000U/L
Reagent divides the bottle of packing into after all dissolving and preparing, and makes liquid three reagent, can directly use.
When measuring ferrous ion concentration, on automatic clinical chemistry analyzer, set: 37 ℃ of temperature, 10 minutes reaction time, initial absorbance 1.8 ± 0.7, test predominant wavelength 340nm, test commplementary wave length 405nm, the volume ratio of tested ferrous ion sample and reagent 1, reagent 2, reagent 3 is 4/40/5/5, the Direction of Reaction is negative reaction (reaction descends), and about about 2 minutes of time delay is about 3 minutes detection times.
After adding sample and reagent, make them mixed and have reaction, reactant places under the Biochemical Analyzer the most at last, detects the speed that predominant wavelength 340nm absorbance descends, thereby calculates the concentration of ferrous ion.
The applicant adopts other various reduced form chromogens combinations of putting down in writing in the above summary of the invention all can reach purpose of the present invention through experimental verification, in view of situation such as determination step and above embodiment roughly the same, do not separately enumerate.
In a word, experiment showed, and adopt assay method of the present invention can draw required measurement result by general biochemical analyzer fully, and highly sensitive, degree of accuracy good, and is easy to utilize.
Claims (6)
1. ferrous ion concentration assay method that utilizes enzymic colorimetric and enzyme-linked method technology, its method principle is as follows:
Aconitate+water
AconitaseCitric acid
Citric acid
Citrate lyaseAcetate+oxaloacetic acid
Oxaloacetic acid+reduced coenzyme
Malic dehydrogenaseL MALIC ACID+coenzyme
The end reaction thing is placed under ultraviolet analyser or half, the automatic clinical chemistry analyzer, detect the speed that predominant wavelength 340nm absorbance descends, calculate the concentration measurement result of ferrous ion.
2. ferrous ion diagnosis/determination reagent kit, principal ingredient comprises:
Damping fluid 20-500mmol/L
Stabilizing agent 1-4000mmol/L
Reduced coenzyme 0.1-0.35mmol/L
Aconitate 1-50mmol/L
Aconitase 1000-80000U/L
Citrate lyase 1000-80000U/L
Malic dehydrogenase 1000-80000U/L
It is characterized in that: kit can be a dry powder, and use the back that is dissolved in water before use; Also can be mixed with liquid reagent, directly use.
3. according to the described ferrous ion diagnosis/determination reagent kit of claim 2, it is characterized in that:
Form single agent reagent by damping fluid, stabilizing agent, reduced coenzyme, aconitate, aconitase, citrate lyase, malic dehydrogenase.
4. according to the described ferrous ion diagnosis/determination reagent kit of claim 2, it is characterized in that:
Form two agent reagent by damping fluid, stabilizing agent, reduced coenzyme, aconitate, aconitase, citrate lyase, malic dehydrogenase; Reagent 1 is made up of damping fluid, stabilizing agent, reduced coenzyme, aconitate; Reagent 2 is made up of damping fluid, stabilizing agent, aconitase, citrate lyase, malic dehydrogenase.Reduced coenzyme, aconitate, aconitase, citrate lyase, the position of malic dehydrogenase in reagent 1 or reagent 2 can not limit.
5. according to the described ferrous ion diagnosis/determination reagent kit of claim 2, it is characterized in that:
Form multi-agent reagent by damping fluid, stabilizing agent, reduced coenzyme, aconitate, aconitase, citrate lyase, malic dehydrogenase; Reagent 1 is made up of damping fluid, stabilizing agent, reduced coenzyme, aconitate; Reagent 2 is made up of damping fluid, stabilizing agent, citrate lyase, malic dehydrogenase; Reagent 3 is made up of damping fluid, stabilizing agent, aconitase.Reduced coenzyme, aconitate, aconitase, citrate lyase, the position of malic dehydrogenase in reagent 1, reagent 2 or reagent 3 can not limit.
6. according to the described ferrous ion diagnosis/determination reagent kit of claim 2, it is characterized in that: also comprise stabilizing agent 1-4000mmol/L or 0.1%-100% volume ratio.Described stabilizing agent is: ammonium sulfate (Ammonia Sulfate), glycerine (Glycerol), propylene glycol (Propylene Glycol), ethylene glycol (Ethylene glycol) and at least one of the preservatives.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA200710023382XA CN101324622A (en) | 2007-06-13 | 2007-06-13 | Ferrous ion diagnosis/determination reagent kit and method for determining ferrous ion concentration |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA200710023382XA CN101324622A (en) | 2007-06-13 | 2007-06-13 | Ferrous ion diagnosis/determination reagent kit and method for determining ferrous ion concentration |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101324622A true CN101324622A (en) | 2008-12-17 |
Family
ID=40188245
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA200710023382XA Pending CN101324622A (en) | 2007-06-13 | 2007-06-13 | Ferrous ion diagnosis/determination reagent kit and method for determining ferrous ion concentration |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101324622A (en) |
-
2007
- 2007-06-13 CN CNA200710023382XA patent/CN101324622A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101329345A (en) | Glutathion diagnosis / determination reagent kit and method for determining glutathion concentration | |
| CN101324608A (en) | Ferrous ion diagnosis/determination reagent kit and method for determining ferrous ion concentration | |
| CN101324622A (en) | Ferrous ion diagnosis/determination reagent kit and method for determining ferrous ion concentration | |
| CN101324621A (en) | Ferrous ion diagnosis/determination reagent kit and method for determining ferrous ion concentration | |
| CN101324623A (en) | Ferrous ion diagnosis/determination reagent kit and method for determining ferrous ion concentration | |
| CN101609032A (en) | Phos (phosphate radical) diagnosis/determination kit and Phos (phosphate radical) method for measurement of concentration | |
| CN101324505A (en) | Method for determining citric acid concentration and citric acid determining reagent kit | |
| CN101173951A (en) | Ferrous ion diagnosis/measuring reagent kit and method for measuring ferrous ion concentration | |
| CN101169372A (en) | Glutamate dehydrogenase diagnosis reagent kit and glutamate dehydrogenase activity and concentration determination method | |
| CN101169447A (en) | Ferrous ion diagnosis/determination reagent kit and ferrous ion concentration determination method | |
| CN101750382A (en) | Diagnostic reagent (kit) of adenosine deaminase and method for measuring activity concentration of adenosine deaminase | |
| CN101620142A (en) | Inorganic phosphorus (phosphate radical) diagnosis/determination kit and method for determining inorganic phosphorus (phosphate radical) concentration | |
| CN101609034A (en) | Phos (phosphate radical) diagnosis/determination kit and Phos (phosphate radical) method for measurement of concentration | |
| CN101620160A (en) | Inorganic phosphorus (phosphate radical) diagnosis/determination kit and method for determining inorganic phosphorus (phosphate radical) concentration | |
| CN101750384A (en) | Diagnostic reagent (kit) of adenosine deaminase and method for measuring activity concentration of adenosine deaminase | |
| CN101324617A (en) | Inorganic phosphorus diagnosis/ determination reagent kit and method for determining inorganic phosphorus concentration | |
| CN101324585A (en) | Method for determining amino acid (nitrogen) concentration and amino acid (nitrogen) diagnosis/determination reagent kit | |
| CN101620106A (en) | Inorganic phosphorus (phosphate radical) diagnosis/determination kit and method for determining inorganic phosphorus (phosphate radical) concentration | |
| CN101620108A (en) | Inorganic phosphorus (phosphate radical) diagnosis/determination kit and method for determining inorganic phosphorus (phosphate radical) concentration | |
| CN101609022A (en) | Phos (phosphate radical) diagnosis/determination kit and Phos (phosphate radical) method for measurement of concentration | |
| CN101620109A (en) | Inorganic phosphorus (phosphate radical) diagnosis/determination kit and method for determining inorganic phosphorus (phosphate radical) concentration | |
| CN101620103A (en) | Inorganic phosphorus (phosphate radical) diagnosis/determination kit and method for determining inorganic phosphorus (phosphate radical) concentration | |
| CN101324584A (en) | Method for determining amino acid concentration and amino acid diagnosis/determination reagent kit | |
| CN101620105A (en) | Inorganic phosphorus (phosphate radical) diagnosis/determination kit and method for determining inorganic phosphorus (phosphate radical) concentration | |
| CN101324615A (en) | Inorganic phosphorus diagnosis/ determination reagent kit and method for determining inorganic phosphorus concentration |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Open date: 20081217 |