CN101620095A - Inorganic phosphorus (phosphate radical) diagnosis/determination kit and method for determining inorganic phosphorus (phosphate radical) concentration-831 - Google Patents

Abstract

The invention relates to an inorganic phosphorus (phosphate radical) diagnosis/determination kit using the technology of an enzyme multiplication method, an enzyme colorimetric method and an ELISA method, a method for determining inorganic phosphorus (phosphate radical) 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, pyruvic acid, adenosine triphosphate, pyruvate oxidase, pyruvate carboxylase, malic 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 inorganic phosphorus (phosphate radical) concentration.

Description

Phos (phosphate radical) diagnosis/determination kit and Phos (phosphate radical) method for measurement of concentration-831

Technical field

The present invention relates to a kind of Phos (phosphate radical) diagnosis/determination kit, the invention still further relates to the method for measuring Phos (phosphate radical) concentration simultaneously, belong to medical science/food/environmental test determination techniques field.

Background technology

87% of phosphorus all is present in the bone in the human body, and phosphorus in the blood and the phosphorus in the bone keep mobile equilibrium, and certain relation is arranged between calcium, the phosphorus concentration in the blood, and normal person's blood calcium raises then that serium inorganic phosphorus reduces, and vice versa.The product of [calcium], [phosphorus] concentration remains on certain limit in the blood plasma; Approximately the product of per 100 milliliters of plasma calcium phosphorus concentrations is 35---40.When the two product greater than 40 the time, calcium and phosphorus are deposited on bone tissue with the bone salts form,, metastatic calcification can take place at＞70 o'clock.When product＜35, then will hinder the calcification of bone tissue, even bone salts is dissolved again, influence osteogenic action, cause rickets or osteomalacia.Blood plasma [Ca], [Pi's] is constant, mainly is subjected to vitamin D, the adjusting of parathyroid hormone and calcitonin.

Because the phosphorus of human body can't directly be measured at present,, the detection of Phos analyzes phosphate anion (H so being actually ₂PO ₄ ^1-, HPO ₄ ^2-).Method commonly used has the phosphomolybdic acid reducing process, non-reduced method, dye binding method, enzyme process, isotope dilution mass spectrometry, atomic absorption spectrophotometry and flow injection analysis etc.Decisive method is an isotope dilution mass spectrometry, and the conventional method of the medium experimental determination Phos that WHO recommends is a colourimetry.

The phosphomolybdic acid reducing process has or not proteinology filtrate method and non-deproteinate method again, and the latter needs to add non-ionics to avoid muddy in reagent.Used reductive agent and kinds of surfactants are a lot, and the more stable reductive agent of performance has ferrous sulphate, iron ammonium sulfate, hydrazine sulfate, Mitouer etc.Surfactant is then the most desirable with polyethylene glycol groups phenyl ether (TritonX-100).

The non-reduced method of phosphomolybdic acid (direct ultraviolet method) is directly measured the phosphomolybdic acid complex poly compounds at 340nm or 325nm, and method is easy, is convenient to robotization.But jaundice, haemolysis, the turbid serum of fat has light absorption at 340nm, must do the sample blank, otherwise the result is higher.

The enzymatic assays Phos is a development trend, and its advantage is to be stable in the neutral range of inorganic phosphate compounds under the enzyme effect, can be used for the automated analysis of conventional sample.

Summary of the invention

The technical problem to be solved in the present invention is: propose a kind of enzyme cycle amplification method (EnzymaticRecycling Method) of utilizing, enzymic colorimetric (Enzymatic Colorimetric Method) and enzyme (even) united method (Couple Reaction) technology, monitoring reduced form nicotinamide coenzyme (reduced coenzyme) is in the variation of 340nm wavelength place absorbance, measured the method for Phos (phosphate radical) concentration, simultaneously, the present invention also will provide Phos (phosphate radical) diagnosis/determination kit in order to realize this method, adopt this reagent not only can be ultraviolet analyser or half, carry out Phos (phosphate radical) concentration determination on the automatic clinical chemistry analyzer, and finding speed is fast, the accuracy height, thereby can obtain practical applying.

Phos of the present invention (phosphate radical) method for measurement of concentration principle is as follows:

Phosphate radical+pyruvic acid+oxygen+water Pyruvate oxidaseAcetyl phosphate+carbon dioxide+hydrogen peroxide

Carbon dioxide+adenosine triphosphate+pyruvic acid Pyruvate carboxylaseAdenosine diphosphate+phosphate radical+oxaloacetic acid

Oxaloacetic acid+reduced coenzyme Malic dehydrogenaseMalic acid+coenzyme

This method is used pyruvate oxidase (pyruvate oxidase; EC 1.2.3.3) enzyme (idol) connection pyruvate carboxylase (pyruvate carboxylase; EC 6.4.1.1), malic dehydrogenase (malate dehydrogenase; EC 1.1.1.37) enzymatic reaction continuous monitoring method.Pyruvate oxidase is as the effect enzyme, and function is that Phos (phosphate radical) enzymolysis is produced carbon dioxide.Pyruvate carboxylase is as cyclophorase: the enzymatic catalysis of pyruvate carboxylase becomes carbon dioxide again Phos (phosphate radical) once more, and it is recycling that Phos (phosphate radical) constantly is repeated, and constantly circulation produces oxaloacetic acid simultaneously.Malic dehydrogenase is as the colour developing enzyme, in the presence of oxaloacetic acid, reduced coenzyme (absorption peak being arranged at the 340nm place) is oxidized into oxidized coenzyme (not having absorption peak 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 Phos (phosphate radical).

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 Phos of the present invention (phosphate radical) diagnosis/determination kit of following composition relation is comparatively desirable:

Damping fluid 100mmol/L

Stabilizing agent 500mmol/L

Reduced coenzyme 0.25mmol/L

Pyruvate oxidase 6000U/L

Pyruvate carboxylase 8000U/L

Malic dehydrogenase 10000U/L

Pyruvic acid 18mmol/L

Adenosine triphosphate 12mmol/L

Phos of the present invention (phosphate radical) diagnosis/determination kit can be single agent, comprising:

Damping fluid, stabilizing agent, reduced coenzyme, pyruvate oxidase, pyruvate carboxylase, malic dehydrogenase, pyruvic acid, adenosine triphosphate.

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, pyruvic acid, adenosine triphosphate.

Reagent 2

Damping fluid, stabilizing agent, pyruvate oxidase, pyruvate carboxylase, malic dehydrogenase.

Reduced coenzyme, pyruvate oxidase, pyruvate carboxylase, malic dehydrogenase, pyruvic acid, the position of adenosine triphosphate 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, pyruvic acid, adenosine triphosphate.

Reagent 2

Damping fluid, stabilizing agent, pyruvate carboxylase, malic dehydrogenase.

Reagent 3

Damping fluid, stabilizing agent, pyruvate oxidase.

Reduced coenzyme, pyruvate oxidase, pyruvate carboxylase, malic dehydrogenase, pyruvic acid, the position of adenosine triphosphate 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 Phos (phosphate radical) 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 Phos of present embodiment (phosphate radical) diagnosing/determining reagent is single reagent, comprising:

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

Stabilizing agent 500mmol/L

Reduced coenzyme 0.25mmol/L

Pyruvate oxidase 6000U/L

Pyruvate carboxylase 8000U/L

Malic dehydrogenase 10000U/L

Pyruvic acid 18mmol/L

Adenosine triphosphate 12mmol/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 Phos (phosphate radical) 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 Phos (phosphate radical).

Embodiment two

The Phos of present embodiment (phosphate radical) diagnosing/determining reagent is double reagent, comprising:

Reagent 1

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

Stabilizing agent 50mmol/L

Reduced coenzyme 0.25mmol/L

Pyruvic acid 18mmol/L

Adenosine triphosphate 12mmol/L

Reagent 2

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

Stabilizing agent 500mmol/L

Pyruvate oxidase 6000U/L

Pyruvate carboxylase 8000U/L

Malic 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 Phos (phosphate radical) 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 Phos (phosphate radical).

Embodiment three

The Phos of present embodiment (phosphate radical) diagnosing/determining reagent is three reagent, comprising:

Reagent 1

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

Stabilizing agent 50mmol/L

Reduced coenzyme 0.25mmol/L

Pyruvic acid 18mmol/L

Adenosine triphosphate 12mmol/L

Reagent 2

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

Stabilizing agent 500mmol/L

Pyruvate carboxylase 8000U/L

Malic dehydrogenase 10000U/L

Reagent 3

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

Stabilizing agent 500mmol/L

Pyruvate oxidase 6000U/L

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

When measuring Phos (phosphate radical) 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 Phos (phosphate radical) 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 Phos (phosphate radical).

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)≤0.0025; Absorbance time response curve should be decline curve until terminal point; Reagent can be surveyed effectively, and (R 〉=0.99) linear range can reach 16mmol/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 0.012 ± 0.006 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. Phos (phosphate radical) method for measurement of concentration that utilizes enzyme cycle amplification method, enzymic colorimetric and enzyme-linked method technology, its method principle is as follows:

Phosphate radical+pyruvic acid+oxygen+water Pyruvate oxidaseAcetyl phosphate+carbon dioxide

+ hydrogen peroxide

Carbon dioxide+adenosine triphosphate+pyruvic acid Pyruvate carboxylaseAdenosine diphosphate+

Phosphate radical+oxaloacetic acid

Oxaloacetic acid+reduced coenzyme Malic dehydrogenaseMalic 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 Phos (phosphate radical).

2. a Phos (phosphate radical) diagnosis/determination kit, principal ingredient comprises:

Damping fluid 20---500mmol/L

Stabilizing agent 1---4000mmol/L

Reduced coenzyme 0.1---0.35mmol/L

Pyruvate oxidase 1000---80000U/L

Pyruvate carboxylase 1000---80000U/L

Malic dehydrogenase 1000---80000U/L

Pyruvic acid 1---50mmol/L

Adenosine triphosphate 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 Phos of claim 2 (phosphate radical) diagnosis/determination kit, it is characterized in that:

Form single agent reagent by damping fluid, stabilizing agent, reduced coenzyme, pyruvate oxidase, pyruvate carboxylase, malic dehydrogenase, pyruvic acid, adenosine triphosphate.

4. according to the described Phos of claim 2 (phosphate radical) diagnosis/determination kit, it is characterized in that:

Form two agent reagent by damping fluid, stabilizing agent, reduced coenzyme, pyruvate oxidase, pyruvate carboxylase, malic dehydrogenase, pyruvic acid, adenosine triphosphate; Reagent 1 is made up of damping fluid, stabilizing agent, reduced coenzyme, pyruvic acid, adenosine triphosphate; Reagent 2 is made up of damping fluid, stabilizing agent, pyruvate oxidase, pyruvate carboxylase, malic dehydrogenase.Reduced coenzyme, pyruvate oxidase, pyruvate carboxylase, malic dehydrogenase, pyruvic acid, the position of adenosine triphosphate in reagent 1 or reagent 2 can not limit.

5. according to the described Phos of claim 2 (phosphate radical) diagnosis/determination kit, it is characterized in that:

Form multi-agent reagent by damping fluid, stabilizing agent, reduced coenzyme, pyruvate oxidase, pyruvate carboxylase, malic dehydrogenase, pyruvic acid, adenosine triphosphate; Reagent 1 is made up of damping fluid, stabilizing agent, reduced coenzyme, pyruvic acid, adenosine triphosphate; Reagent 2 is made up of damping fluid, stabilizing agent, pyruvate carboxylase, malic dehydrogenase; Reagent 3 is made up of damping fluid, stabilizing agent, pyruvate oxidase.Reduced coenzyme, pyruvate oxidase, pyruvate carboxylase, malic dehydrogenase, pyruvic acid, the position of adenosine triphosphate in reagent 1, reagent 2 or reagent 3 can not limit.

6. according to the described Phos of claim 2 (phosphate radical) diagnosis/determination 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 (Propylene Glycol), ethylene glycol (Ethylene glycol) and at least one of the preservatives.