CN101620077A - Manganese ion diagnosis/determination kit and method for determining manganese ion concentration - Google Patents

Abstract

The invention relates to a manganese ion diagnosis/determination kit using the technology of an enzyme multiplication method, an enzyme colorimetric method and an ELISA method, a method for determining manganese ion concentration and composition and components of a reagent, and belongs to the technical field of test and determination of medical science, food and environment. The kit comprises the following main components: buffer solution, reduced coenzyme, oxaloacetic acid, acetylcoenzyme A, ammonium chloride, oxaloacetic decarboxylase, pyruvate dehydrogenase, alanine dehydrogenase and a stabilizer. The method comprises the following steps: mixing a sample and the reagent according to a certain volume ratio to perform a series of enzymic reaction, and then placing reactants under a UV/ visible light analyzer to test the descending speed of absorbance at the position where the dominant wave length is 340nm to determine the concentration of manganese ion.

Description

Manganese ion diagnosis/mensuration kit and manganese ion concentration assay method

Technical field

The present invention relates to a kind of manganese ion diagnosis/mensuration kit, the invention still further relates to the method for measuring manganese ion concentration simultaneously, belong to medical science/food/environmental test determination techniques field.

Background technology

Manganese is indispensable essential composition in human body as a kind of trace element, also is the material poisonous to human body simultaneously.Under state of nature, exist with the form of two valencys, trivalent and tetravalence, degree of oxidation is low more, and toxicity is high more.Usually manganese has eight kinds of different states of oxidation.The biological significance of manganese is as accessory factor in the human body diversified function to be arranged.Yet, in many enzyme activating reactions, Mn ²⁺And Mg ²⁺Can substitute mutually.

Sampling Graphite Furnace Atomic Absorption spectrophotometry commonly used, atomic emissions ICP-OES.It is the best way that neutron excites analysis (NAA), but only can could measure in the good laboratory of some conditions.

Summary of the invention

The technical problem to be solved in the present invention is: propose a kind of enzyme multiplication method (Enzymatic DoublingMethod) that utilizes, enzymic colorimetric (Enzymatic Colorimetric Method) and enzyme (even) united method (CoupleReaction) technology, monitoring reduced form nicotinamide coenzyme (reduced coenzyme) is in the variation of 340nm wavelength place absorbance, measured the method for manganese ion concentration, simultaneously, the present invention also will provide in order to realize the manganese ion diagnosis/mensuration kit of this method, adopt this reagent not only can be ultraviolet analyser or half, carrying out manganese ion concentration on the automatic clinical chemistry analyzer measures, and finding speed is fast, the accuracy height, thereby can obtain practical applying.

Manganese ion concentration assay method principle of the present invention is as follows:

Oxaloacetic acid Oxaloacetic decarboxylase/Mn ²⁺ Pyruvic acid+carbon dioxide

Carbon dioxide+acetyl coenzyme A+reduced coenzyme Pyruvic dehydrogenasePyruvic acid+

Coacetylase+coenzyme

Ammonia+pyruvic acid+reduced coenzyme Alanine dehydrogenaseAlanine+water+coenzyme

This method application need manganese ion intensifies active oxaloacetic decarboxylase (oxaloacetatedecarboxylase; EC 4.1.1.3) enzyme (idol) connection pyruvic dehydrogenase (pyruvate dehydrogenase; EC1.2.1.51), alanine dehydrogenase (alanine dehydrogenase; EC 1.4.1.1) enzymatic reaction continuous monitoring method/speed ratio color method.The activation degree of oxaloacetic decarboxylase is directly proportional with manganese ion concentration.The oxaloacetic decarboxylase enzymolysis oxaloacetic acid reaction that manganese ion intensifies under the activity produces pyruvic acid and carbon dioxide, the double action of uniting pyruvic dehydrogenase, alanine dehydrogenase again by (idol), final secondary is oxidized into coenzyme (not having absorption peak at the 340nm place) with reduced coenzyme (absorption peak being arranged at the 340nm place), 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 manganese 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 manganese ion diagnosis/mensuration kit of the present invention of following composition relation is ideal comparatively:

Damping fluid 100mmol/L

Stabilizing agent 500mmol/L

Reduced coenzyme 0.25mmol/L

Oxaloacetic decarboxylase 6000U/L

Pyruvic dehydrogenase 8000U/L

Alanine dehydrogenase 10000U/L

Oxaloacetic acid 12mmol/L

Acetyl coenzyme A 6mmol/L

Ammonium chloride 15mmol/L

Manganese ion diagnosis/mensuration kit of the present invention can be single agent, comprising:

Damping fluid, stabilizing agent, reduced coenzyme, oxaloacetic decarboxylase, pyruvic dehydrogenase, alanine dehydrogenase, oxaloacetic acid, acetyl coenzyme A, ammonium chloride.

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, oxaloacetic acid, acetyl coenzyme A, ammonium chloride.

Reagent 2

Damping fluid, stabilizing agent, oxaloacetic decarboxylase, pyruvic dehydrogenase, alanine dehydrogenase.

Reduced coenzyme, oxaloacetic decarboxylase, pyruvic dehydrogenase, alanine dehydrogenase, oxaloacetic acid, acetyl coenzyme A, the position of ammonium chloride 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, oxaloacetic acid, acetyl coenzyme A, ammonium chloride.

Reagent 2

Damping fluid, stabilizing agent, pyruvic dehydrogenase, alanine dehydrogenase.

Reagent 3

Damping fluid, stabilizing agent, oxaloacetic decarboxylase.

Reduced coenzyme, oxaloacetic decarboxylase, pyruvic dehydrogenase, alanine dehydrogenase, oxaloacetic acid, acetyl coenzyme A, the position of ammonium chloride 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 manganese 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

Manganese ion diagnosis/mensuration the reagent of present embodiment is single reagent, comprising:

Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L

Stabilizing agent 500mmol/L

Reduced coenzyme 0.25mmol/L

Oxaloacetic decarboxylase 6000U/L

Pyruvic dehydrogenase 8000U/L

Alanine dehydrogenase 10000U/L

Oxaloacetic acid 12mmol/L

Acetyl coenzyme A 6mmol/L

Ammonium chloride 15mmol/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 manganese ion sample and reagent is 1/25, the Direction of Reaction is negative reaction (reaction descends), about about 1 minute of time delay is about 2 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 manganese ion.

Embodiment two

Manganese ion diagnosis/mensuration the 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

Oxaloacetic acid 12mmol/L

Acetyl coenzyme A 6mmol/L

Ammonium chloride 15mmol/L

Reagent 2

Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L

Stabilizing agent 500mmol/L

Oxaloacetic decarboxylase 6000U/L

Pyruvic dehydrogenase 8000U/L

Alanine dehydrogenase 10000U/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 manganese ion sample and reagent 1, reagent 2 is 2/20/5, the Direction of Reaction is negative reaction (reaction descends), about about 1 minute of time delay is about 2 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 manganese ion.

Embodiment three

Manganese ion diagnosis/mensuration the 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

Oxaloacetic acid 12mmol/L

Acetyl coenzyme A 6mmol/L

Ammonium chloride 15mmol/L

Reagent 2

Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L

Stabilizing agent 500mmol/L

Pyruvic dehydrogenase 8000U/L

Alanine dehydrogenase 10000U/L

Reagent 3

Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L

Stabilizing agent 500mmol/L

Oxaloacetic decarboxylase 6000U/L

Reagent divides the bottle of packing into after all dissolving and preparing, and makes liquid three reagent, can directly use.

When measuring manganese 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 manganese 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 1 minute of time delay is about 2 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 manganese ion.

The applicant adopts other assay methods 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, experimental results show that: adopt assay method of the present invention can draw required measurement result by general biochemical analyzer fully---the blank reagent absorbance changes (Δ A/min)≤0.002; Absorbance time response curve should be decline curve until terminal point; Reagent can be surveyed effectively, and (R 〉=0.99) linear range can reach 5mmol/L; The inaccuracy of reagent test, its relative deviation be no more than ± and 5%; The coefficient of variation (CV)≤2% of the precision of reagent test (repeatability); The sensitivity of reagent can reach 0.18 ± 0.09 Δ A/mmol/L; Reagent is preserved down at 2-8 ℃, and activity can be stablized 1 year;---the present invention is highly sensitive, degree of accuracy good, the linear range broadness, and stationary phase is long, is enough to easy to utilize.

Claims (6)

1. manganese ion concentration assay method that utilizes enzyme multiplication method, enzymic colorimetric and enzyme-linked method technology, its method principle is as follows:

Oxaloacetic acid Oxaloacetic decarboxylase/Mn ²⁺ Pyruvic acid+carbon dioxide

Carbon dioxide+acetyl coenzyme A+reduced coenzyme Pyruvic dehydrogenasePyruvic acid+

Coacetylase+coenzyme

Ammonia+pyruvic acid+reduced coenzyme Alanine dehydrogenaseAlanine+water+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 manganese ion.

2. manganese ion diagnosis/mensuration kit, principal ingredient comprises:

Damping fluid 20---500mmol/L

Stabilizing agent 1---4000mmol/L

Reduced coenzyme 0.1---0.35mmol/L

Oxaloacetic decarboxylase 1000---80000U/L

Pyruvic dehydrogenase 1000---80000U/L

Alanine dehydrogenase 1000---80000U/L

Oxaloacetic acid 1---50mmol/L

Acetyl coenzyme A 1---50mmol/L

Ammonium chloride 1---50mmol/L

The concentration of reagent composition not necessarily is only limited to above-mentioned scope; Effect is better in this scope, and outside this scope, reagent still can reagentia.

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 manganese ion diagnosis/mensuration of claim 2 kit, it is characterized in that:

Form single agent reagent by damping fluid, stabilizing agent, reduced coenzyme, oxaloacetic decarboxylase, pyruvic dehydrogenase, alanine dehydrogenase, oxaloacetic acid, acetyl coenzyme A, ammonium chloride.

4. according to the described manganese ion diagnosis/mensuration of claim 2 kit, it is characterized in that:

Form two agent reagent by damping fluid, stabilizing agent, reduced coenzyme, oxaloacetic decarboxylase, pyruvic dehydrogenase, alanine dehydrogenase, oxaloacetic acid, acetyl coenzyme A, ammonium chloride; Reagent 1 is made up of damping fluid, stabilizing agent, reduced coenzyme, oxaloacetic acid, acetyl coenzyme A, ammonium chloride; Reagent 2 is made up of damping fluid, stabilizing agent, oxaloacetic decarboxylase, pyruvic dehydrogenase, alanine dehydrogenase.Reduced coenzyme, oxaloacetic decarboxylase, pyruvic dehydrogenase, alanine dehydrogenase, oxaloacetic acid, acetyl coenzyme A, the position of ammonium chloride in reagent 1 or reagent 2 can not limit.

5. according to the described manganese ion diagnosis/mensuration of claim 2 kit, it is characterized in that:

Form multi-agent reagent by damping fluid, stabilizing agent, reduced coenzyme, oxaloacetic decarboxylase, pyruvic dehydrogenase, alanine dehydrogenase, oxaloacetic acid, acetyl coenzyme A, ammonium chloride; Reagent 1 is made up of damping fluid, stabilizing agent, reduced coenzyme, oxaloacetic acid, acetyl coenzyme A, ammonium chloride; Reagent 2 is made up of damping fluid, stabilizing agent, pyruvic dehydrogenase, alanine dehydrogenase; Reagent 3 is made up of damping fluid, stabilizing agent, oxaloacetic decarboxylase.Reduced coenzyme, oxaloacetic decarboxylase, pyruvic dehydrogenase, alanine dehydrogenase, oxaloacetic acid, acetyl coenzyme A, the position of ammonium chloride in reagent 1, reagent 2 or reagent 3 can not limit.

6. according to the described manganese ion diagnosis/mensuration of claim 2 kit, it is characterized in that: also comprise stabilizing agent 1-4000mmol/L or 0.1%-100% volume ratio.Described stabilizing agent is: ammonium sulfate (AmmoniaSulfate), glycerine (Glycerol), propylene glycol (Propylene Glycol), ethylene glycol (Ethylene glycol) and at least one of the preservatives.