CN101324595A - Manganese ion diagnosis/determination reagent kit and method for determining manganese ion concentration - Google Patents

Abstract

The invention relates to a kit for diagnosing/mensurating manganese 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 manganese 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, oxaloacetic acid, acetyl coenzyme A, oxaloacetic decarboxylase, pyruvate dehydrogenase and a stabilizer. Through mixing a sample and the reagent by a certain volume ratio, a series of enzymatic reactions occur, 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 manganese ions.

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 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 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 dehydrogenase

Pyruvic acid+coacetylase+coenzyme

Oxaloacetic decarboxylase (oxaloacetatedecarboxylase EC 4.1.1.3) enzyme (idol) connection pyruvic dehydrogenase (pyruvatedehydrogenase that this method application need is manganese ion activated; EC 1.2.1.51) enzymatic reaction continuous monitoring method/speed ratio color method.Oxaloacetic decarboxylase, under the activation of manganese ion, the reaction of enzymolysis oxaloacetic acid produces carbon dioxide, the effect of uniting pyruvic 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,, can be calculated the concentration of manganese ion by measuring the speed that 340nm place absorbance descends.

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 acid 20mmol/L

Acetyl coenzyme A 20mmol/L

Oxaloacetic decarboxylase 10000U/L

Pyruvic dehydrogenase 10000U/L

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

Damping fluid, stabilizing agent, reduced coenzyme, oxaloacetic acid, acetyl coenzyme A, oxaloacetic decarboxylase, pyruvic 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, oxaloacetic acid, acetyl coenzyme A.

Reagent 2

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

Reduced coenzyme, oxaloacetic acid, acetyl coenzyme A, oxaloacetic decarboxylase, the position of pyruvic 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, oxaloacetic acid, acetyl coenzyme A.

Reagent 2

Damping fluid, stabilizing agent, pyruvic dehydrogenase.

Reagent 3

Damping fluid, stabilizing agent, oxaloacetic decarboxylase.

Reduced coenzyme, oxaloacetic acid, acetyl coenzyme A, oxaloacetic decarboxylase, the position of pyruvic 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 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 acid 20mmol/L

Acetyl coenzyme A 20mmol/L

Oxaloacetic decarboxylase 10000U/L

Pyruvic dehydrogenase 10000U/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 5 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 20mmol/L

Acetyl coenzyme A 20mmol/L

Reagent 2

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

Stabilizing agent 500mmol/L

Oxaloacetic decarboxylase 10000U/L

Pyruvic 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 5 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 20mmol/L

Acetyl coenzyme A 20mmol/L

Reagent 2

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

Stabilizing agent 500mmol/L

Pyruvic dehydrogenase 10000U/L

Reagent 3

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

Stabilizing agent 500mmol/L

Oxaloacetic decarboxylase 10000U/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 5 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 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. manganese ion concentration assay method that utilizes 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 dehydrogenase

Pyruvic acid+coacetylase+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 acid 1---50mmol/L

Acetyl coenzyme A 1---50mmol/L

Oxaloacetic decarboxylase 1000---80000U/L

Pyruvic 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 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 acid, acetyl coenzyme A, oxaloacetic decarboxylase, pyruvic dehydrogenase.

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 acid, acetyl coenzyme A, oxaloacetic decarboxylase, pyruvic dehydrogenase; Reagent 1 is made up of damping fluid, stabilizing agent, reduced coenzyme, oxaloacetic acid, acetyl coenzyme A; Reagent 2 is made up of damping fluid, stabilizing agent, oxaloacetic decarboxylase, pyruvic dehydrogenase.Reduced coenzyme, oxaloacetic acid, acetyl coenzyme A, oxaloacetic decarboxylase, the position of pyruvic dehydrogenase 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 acid, acetyl coenzyme A, oxaloacetic decarboxylase, pyruvic dehydrogenase; Reagent 1 is made up of damping fluid, stabilizing agent, reduced coenzyme, oxaloacetic acid, acetyl coenzyme A; Reagent 2 is made up of damping fluid, stabilizing agent, pyruvic dehydrogenase; Reagent 3 is made up of damping fluid, stabilizing agent oxaloacetic decarboxylase.Reduced coenzyme, oxaloacetic acid, acetyl coenzyme A, oxaloacetic decarboxylase, the position of pyruvic dehydrogenase 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 (Ammonia Sulfate), glycerine (Glycerol), propylene glycol (PropyleneGlycol), ethylene glycol (Ethylene glycol) and at least one of the preservatives.