CN101324615A - Inorganic phosphorus diagnosis/ determination reagent kit and method for determining inorganic phosphorus concentration - Google Patents

Abstract

The invention relates to a kit for diagnosing/mensurating inorganic phosphorus 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 inorganic phosphorus, 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, adenosine diphosphate, oxaloacetic acid, pyruvate carboxylase, lactate 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 degree/velocity of the decrease in absorbance at 340nm of the dominant wavelength is detected, thereby mensurating the concentration of the inorganic phosphorus.

Description

Phos diagnosis/mensuration kit and inorganic phosphorus concentration assay method

Technical field

The present invention relates to a kind of Phos diagnosis/mensuration kit, the invention still further relates to the method for measuring inorganic phosphorus 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 (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 inorganic phosphorus concentration, simultaneously, the present invention also will provide in order to realize the Phos diagnosis/mensuration kit of this method, adopt this reagent not only can be ultraviolet analyser or half, carrying out inorganic phosphorus concentration on the automatic clinical chemistry analyzer measures, and finding speed is fast, the accuracy height, thereby can obtain practical applying.

Inorganic phosphorus concentration assay method principle of the present invention is as follows:

Phosphate radical+adenosine diphosphate+oxaloacetic acid Pyruvate carboxylase

Adenosine triphosphate+pyruvic acid+carbon dioxide

Pyruvic acid+reduced coenzyme Lactic dehydrogenaseL-lactic acid+coenzyme

This method is used pyruvate carboxylase (Pyruvate carboxylase; EC 6.4.1.1) enzyme (idol) connection lactic dehydrogenase (Lactate dehydrogenase; EC 1.1.1.27; EC 1.1.1.28) enzymatic reaction continuous monitoring method/speed ratio color method.Pyruvate carboxylase enzymolysis Phos (phosphate radical) reaction produces pyruvic acid, the effect of uniting lactic 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 degree/speed that reduced coenzyme descends in 340nm place absorbance, by measuring degree/speed that 340nm place absorbance descends, can calculate the concentration of Phos.

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 Phos 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

Adenosine diphosphate 5mmol/L

Oxaloacetic acid 30mmol/L

Pyruvate carboxylase 10000U/L

Lactic dehydrogenase 16000U/L

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

Damping fluid, stabilizing agent, reduced coenzyme, adenosine diphosphate, oxaloacetic acid, pyruvate carboxylase, lactic 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, adenosine diphosphate, oxaloacetic acid.

Reagent 2

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

Reduced coenzyme, adenosine diphosphate, oxaloacetic acid, pyruvate carboxylase, the position of lactic 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, adenosine diphosphate, oxaloacetic acid.

Reagent 2

Damping fluid, stabilizing agent, lactic dehydrogenase.

Reagent 3

Damping fluid, stabilizing agent, pyruvate carboxylase.

Reduced coenzyme, adenosine diphosphate, oxaloacetic acid, pyruvate carboxylase, the position of lactic 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 inorganic phosphorus 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

Phos 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

Adenosine diphosphate 5mmol/L

Oxaloacetic acid 30mmol/L

Pyruvate carboxylase 10000U/L

Lactic dehydrogenase 16000U/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 sample and reagent is 1/25, the Direction of Reaction is negative reaction (reaction descends), 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 degree/speed that predominant wavelength 340nm absorbance descends, thereby calculates the concentration of Phos.

Embodiment two

Phos 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

Adenosine diphosphate 5mmol/L

Oxaloacetic acid 30mmol/L

Reagent 2

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

Stabilizing agent 500mmol/L

Pyruvate carboxylase 10000U/L

Lactic dehydrogenase 16000U/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 sample and reagent 1, reagent 2 is 2/20/5, the Direction of Reaction is negative reaction (reaction descends), 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 degree/speed that predominant wavelength 340nm absorbance descends, thereby calculates the concentration of Phos.

Embodiment three

Phos 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

Adenosine diphosphate 5mmol/L

Oxaloacetic acid 30mmol/L

Reagent 2

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

Stabilizing agent 500mmol/L

Lactic dehydrogenase 16000U/L

Reagent 3

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

Stabilizing agent 500mmol/L

Pyruvate 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 inorganic phosphorus 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 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 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 degree/speed that predominant wavelength 340nm absorbance descends, thereby calculates the concentration of Phos.

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. inorganic phosphorus concentration assay method that utilizes enzymic colorimetric and enzyme-linked method technology, its method principle is as follows:

Phosphate radical+adenosine diphosphate+oxaloacetic acid Pyruvate carboxylase

Adenosine triphosphate+pyruvic acid+carbon dioxide

Pyruvic acid+reduced coenzyme Lactic dehydrogenaseL-lactic acid+coenzyme

The end reaction thing is placed under ultraviolet analyser or half, the automatic clinical chemistry analyzer, detect degree/speed that predominant wavelength 340nm absorbance descends, calculate the concentration measurement result of Phos.

2. Phos diagnosis/mensuration kit, principal ingredient comprises:

Damping fluid 20-500mmol/L

Stabilizing agent 1-4000mmol/L

Reduced coenzyme 0.1-0.35mmol/L

Adenosine diphosphate 1-50mmol/L

Oxaloacetic acid 1-50mmol/L

Pyruvate carboxylase 1000-80000U/L

Lactic dehydrogenase 10 00-80000U/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 Phos diagnosis/mensuration of claim 2 kit, it is characterized in that:

Form single agent reagent by damping fluid, stabilizing agent, reduced coenzyme, adenosine diphosphate, oxaloacetic acid, pyruvate carboxylase, lactic dehydrogenase.

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

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

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

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

6. according to the described Phos 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 (Propylene Glycol), ethylene glycol (Ethylene glycol) and at least one of the preservatives.