CN101620117A

CN101620117A - Inorganic phosphorus (phosphate radical) diagnosis/determination kit and method for determining inorganic phosphorus (phosphate radical) concentration

Info

Publication number: CN101620117A
Application number: CN200810122951A
Authority: CN
Inventors: 王尔中
Original assignee: Suzhou ANJ Biotech Co Ltd
Current assignee: Suzhou ANJ Biotech Co Ltd
Priority date: 2008-06-30
Filing date: 2008-06-30
Publication date: 2010-01-06

Abstract

The invention relates to an inorganic phosphorus (phosphate radical) diagnosis/determination kit using the technology of 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, coenzyme, oxaloacetic acid, acetic acid, ferrocytochrome B1, coenzyme A, phosphoenolpyruvate carboxykinase, two different pyruvate dehydrogenases 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 ascending speed of absorbance at the position where the dominant wave length is 340nm to determine the inorganic phosphorus (phosphate radical) concentration.

Description

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

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 enzymic colorimetric (Enzymatic ColorimetricMethod) and enzyme (even) united method (Couple Reaction) technology utilized, metering 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+oxaloacetic acid Phosphoric acid enol pyruvic acid carboxylaseCarbon dioxide+

Phosphoenolpyruvic acid+water

Acetate+carbon dioxide+ferricytochrome b1 Pyruvic dehydrogenasePyruvic acid+

Ferricytochrome b1+ water

Pyruvic acid+coacetylase+coenzyme Pyruvic dehydrogenaseCarbon dioxide+acetyl coenzyme A+

Reduced coenzyme

This method is used phosphoric acid enol pyruvic acid carboxylase (phosphoenolpyruvate carboxylase; EC4.1.1.31) enzyme (idol) connection pyruvic dehydrogenase (pyruvate dehydrogenase; EC 1.2.2.2), pyruvic dehydrogenase (pyruvate dehydrogenase; EC 1.2.1.51) enzymatic reaction end-point method.Phosphoric acid enol pyruvic acid carboxylase enzymolysis Phos (phosphate radical) reaction produces carbon dioxide, the effect of uniting two different pyruvic dehydrogenases again by (idol), coenzyme (not having absorption peak at the 340nm place) reduces the most at last becomes reduced coenzyme (absorption peak being arranged at the 340nm place), thereby measured the degree that reduced coenzyme rises in 340nm place absorbance, by measuring the degree that 340nm place absorbance rises, 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

Coenzyme 3mmol/L

Phosphoric acid enol pyruvic acid carboxylase 10000U/L

Pyruvic dehydrogenase 12000U/L

Oxaloacetic acid 12mmol/L

Acetate 10mmol/L

Ferricytochrome B1 8mmol/L

Coacetylase 8mmol/L

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

Damping fluid, stabilizing agent, coenzyme, phosphoric acid enol pyruvic acid carboxylase, two different pyruvic dehydrogenases, oxaloacetic acid, acetate, ferricytochrome B1, coacetylase.

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, coenzyme, oxaloacetic acid, acetate, ferricytochrome B1, coacetylase.

Reagent 2

Damping fluid, stabilizing agent, phosphoric acid enol pyruvic acid carboxylase, two different pyruvic dehydrogenases.

Coenzyme, phosphoric acid enol pyruvic acid carboxylase, two different pyruvic dehydrogenases, oxaloacetic acid, acetate, ferricytochrome B1, the positions of coacetylase 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

Reagent 2

Damping fluid, stabilizing agent, two different pyruvic dehydrogenases.

Reagent 3

Damping fluid, stabilizing agent, phosphoric acid enol pyruvic acid carboxylase.

Coenzyme, phosphoric acid enol pyruvic acid carboxylase, two different pyruvic dehydrogenases, oxaloacetic acid, acetate, ferricytochrome B1, the positions of coacetylase 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 coenzyme can be NADP ⁺, NAD ⁺Or thio-NAD ⁺In a kind of.

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

Coenzyme 3mmol/L

Phosphoric acid enol pyruvic acid carboxylase 10000U/L

Pyruvic dehydrogenase 12000U/L

Oxaloacetic acid 12mmol/L

Acetate 10mmol/L

Ferricytochrome B1 8mmol/L

Coacetylase 8mmol/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≤0.1, 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 positive reaction (reaction of rising), about about 0 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 degree that predominant wavelength 340nm absorbance rises, 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

Coenzyme 3mmol/L

Oxaloacetic acid 12mmol/L

Acetate 10mmol/L

Ferricytochrome B1 8mmol/L

Coacetylase 8mmol/L

Reagent 2

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

Stabilizing agent 50mmol/L

Phosphoric acid enol pyruvic acid carboxylase 10000U/L

Pyruvic dehydrogenase 12000U/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≤0.1, 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 positive reaction (reaction of rising), about about 0 minute of time delay is about 5 minutes detection times.

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

Coenzyme 3mmol/L

Oxaloacetic acid 12mmol/L

Acetate 10mmol/L

Ferricytochrome B1 8mmol/L

Coacetylase 8mmol/L

Reagent 2

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

Stabilizing agent 500mmol/L

Pyruvic dehydrogenase 12000U/L

Reagent 3

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

Stabilizing agent 500mmol/L

Phosphoric acid enol pyruvic acid carboxylase 10000U/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≤0.1, 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 positive reaction (reaction of rising), and about about 0 minute of time delay is about 5 minutes detection times.

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)≤0.0012; Absorbance time response curve should be the rising 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

1. the method for measurement of concentration of the Phos (phosphate radical) of enzymic colorimetric and enzyme-linked method, its method principle is as follows:

Phosphoenolpyruvic acid+water

Acetate+carbon dioxide+ferricytochrome b1 Pyruvic dehydrogenasePyruvic acid+

Ferricytochrome b1+ water

Reduced coenzyme

The end reaction thing is placed under ultraviolet analyser or half, the automatic clinical chemistry analyzer, detect the degree that predominant wavelength 340nm absorbance rises, 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

DPN diphosphopyridine nucleotide---6mmol/L

Phosphoric acid enol pyruvic acid carboxylase 1000---80000U/L

Pyruvic dehydrogenase 1000---80000U/L

Oxaloacetic acid 1---50mmol/L

Acetate 1---50mmol/L

Ferricytochrome B1 1---50mmol/L

Coacetylase 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, coenzyme, phosphoric acid enol pyruvic acid carboxylase, two different pyruvic dehydrogenases, oxaloacetic acid, acetate, ferricytochrome B1, coacetylases.

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, coenzyme, phosphoric acid enol pyruvic acid carboxylase, two different pyruvic dehydrogenases, oxaloacetic acid, acetate, ferricytochrome B1, coacetylases; Reagent 1 is made up of damping fluid, stabilizing agent, coenzyme, oxaloacetic acid, acetate, ferricytochrome B1, coacetylase; Reagent 2 is made up of damping fluid, stabilizing agent, phosphoric acid enol pyruvic acid carboxylase, two different pyruvic dehydrogenases.Coenzyme, phosphoric acid enol pyruvic acid carboxylase, two different pyruvic dehydrogenases, oxaloacetic acid, acetate, ferricytochrome B1, the positions of coacetylase 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, coenzyme, phosphoric acid enol pyruvic acid carboxylase, two different pyruvic dehydrogenases, oxaloacetic acid, acetate, ferricytochrome B1, coacetylases; Reagent 1 is made up of damping fluid, stabilizing agent, coenzyme, oxaloacetic acid, acetate, ferricytochrome B1, coacetylase; Reagent 2 is made up of damping fluid, stabilizing agent, two different pyruvic dehydrogenases; Reagent 3 is made up of damping fluid, stabilizing agent, phosphoric acid enol pyruvic acid carboxylase.Coenzyme, phosphoric acid enol pyruvic acid carboxylase, two different pyruvic dehydrogenases, oxaloacetic acid, acetate, ferricytochrome B1, the positions of coacetylase 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.