CN101793800A - Lactose diagnosis/measurement reagent (kit) and method for measuring lactose concentration - Google Patents

Abstract

The invention relates to a lactose diagnosis/measurement reagent (kit) by using the technology of an enzyme colorimetric method and an enzyme coupling method, also relates to a method for measuring lactose concentration, and the composition and components of the reagent, and belongs to the technical field of medicine/food inspection and measurement. The reagent (kit) of the invention comprises the following components: buffer solution, NADH, adenosine triphosphoric acid, lactase, heterophosphatase, aldose-6-phosphate reductase, and a stabilizing agent. A sample and a reagent are mixed in a certain volume ratio to undergo a series of enzymatic reaction, the reactant is placed under an ultraviolet/visible light analytical instrument, and the rising degree of the absorbance of the reactant at the main wavelength of 340nm is measured, so that the lactose concentration is measured.

Description

Lactose diagnosis/mensuration reagent (box) and lactose concn assay method

Technical field

The present invention relates to a kind of lactose diagnosis/mensuration reagent (box), the invention still further relates to the method for measuring lactose concn simultaneously, belong to medical science/Food Inspection determination techniques field.

Background technology

Lactose is the main energy source of infant, and it is interior after glucose and galactose are resolved in the effect of small intestine lactase to enter body, and galactose is the essential material that infant brain is grown, with the close ties that shot up of infant's brain.For preventing that admixture substitutes lactose with other reducing sugars such as glucose in the whey powder, the detection of lactose is very necessary.

Milk is a kind of nutritious food, but improper as drinking, and also can bring harmful effect to health.A lot of people symptoms such as abdominal discomfort, abdominal distension, many wind, stomachache even diarrhoea occur after drink milk, this phenomenon medically is being called lactose intolerance, its reason is that these people's small intestines lack lactases, the lactose in can not pegnin, after lactose enters colon, bacterial fermentation in the per rectum produces due to the materials such as a large amount of gases, acetic acid.Studies confirm that in a large number the people who suffers from lactose intolerance also has most of without any symptom, the not anti-receptor of recessiveness that has only the human body biochemical indicator to change, medically is called lactose malabsorption or alactasia.

According to statistics, all there is the problem of lactose intolerance in the whole world, and wherein Asian's incidence is the highest, is 95%-100%.According to the investigation of Chinese Center for Disease Control, ground 3-13 such as China Beijing, Shanghai, Guangzhou year children's lactose intolerance and the incidence of lactose malabsorption be about 80%.Lactose intolerance also can cause the human nutrition malabsorption except that causing symptom of digestive tract.According to foreign study, thereby lactose intolerance can reduce absorption, minimizing peak bone amount, the increase bone loss of calcium and cause osteoporosis, and lactose intolerance can influence the absorption of iron and zinc, causes asiderosis and zinc deficiency, and can influence children's's brain growth, very big to human health damage.

In the food analysis field, enzyme assay is a kind of method of widely using and extremely recommending such as international standard mechanisms such as ISO.It is full-fledged and generally accepted for the laboratory that enzyme is analysed law theory.The purified enzyme of use high-quality can be realized the detection to specific composition in the complex material.The target metabolin that this method can detect has carbohydrate, acids and its esters, alcohols and other materials etc.Thus, various food samples all can and obtain good result by fast detecting.

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 lactose concn, simultaneously, the present invention also will provide in order to the lactose diagnosis of realizing this method/mensuration reagent (box), adopt this reagent not only can be ultraviolet analyser or half, carrying out lactose concn on the automatic clinical chemistry analyzer measures, and finding speed is fast, the accuracy height, thereby can obtain practical applying.

Lactose concn assay method of the present invention is as follows:

Lactose+water LactaseGalactose+glucose

Glucose+adenosine triphosphate HexokinaseAdenosine diphosphate+glucose 6-phosphoric acid

Glucose 6-phosphoric acid+reduced coenzyme Aldose-6-phosphoric acid reduction enzymeSorbierite 6-phosphoric acid+coenzyme

This method is used lactase (lactase; EC 3.2.1.108) enzyme (idol) connection hexokinase (Hexokinase; EC 2.7.1.1), aldose-6-phosphoric acid reduction enzyme (aldose-6-phosphate reductase; EC 1.1.1.200) enzymatic reaction colorimetric end-point method.The reaction of lactase enzymolysis lactose produces glucose, the effect of uniting hexokinase, aldose-6-phosphoric acid reduction enzyme 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 degree that reduced coenzyme descends in 340nm place absorbance, by measuring the degree that 340nm place absorbance descends, can calculate the concentration of lactose.

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 lactose diagnosis of the present invention/the mensurations reagent (box) of following composition relation is ideal comparatively:

Damping fluid 100mmol/L

Stabilizing agent 500mmol/L

Reduced coenzyme 0.25mmol/L

Lactase 6000U/L

Hexokinase 8000U/L

Aldose-6-phosphoric acid reduction enzyme 10000U/L

Adenosine triphosphate 7mmol/L

Lactose diagnosis of the present invention/mensuration reagent (box) can be single agent, comprising:

Damping fluid, stabilizing agent, reduced coenzyme, lactase, hexokinase, aldose-6-phosphoric acid reduction enzyme, adenosine triphosphate.

Reagent (box) can be 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 triphosphate.

Reagent 2

Damping fluid, stabilizing agent, lactase, hexokinase, aldose-6-phosphoric acid reduction enzyme.

Reduced coenzyme, lactase, hexokinase, aldose-6-phosphoric acid reduction enzyme, the position of adenosine triphosphate in reagent 1 or reagent 2 can not limit.Reagent (box) can be 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 triphosphate.

Reagent 2

Damping fluid, stabilizing agent, hexokinase, aldose-6-phosphoric acid reduction enzyme.

Reagent 3

Damping fluid, stabilizing agent, lactase.

Reduced coenzyme, lactase, hexokinase, aldose-6-phosphoric acid reduction enzyme, the position of adenosine triphosphate in reagent 1, reagent 2 or reagent 3 can not limit.Reagent (box) can be 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 lactose concn, 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 lactose diagnosis of present embodiment/mensuration reagent is single reagent, comprising:

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

Stabilizing agent 500mmol/L

Reduced coenzyme 0.25mmol/L

Lactase 6000U/L

Hexokinase 8000U/L

Aldose-6-phosphoric acid reduction enzyme 10000U/L

Adenosine triphosphate 7mmol/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 milk sugar specimen 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 the degree that predominant wavelength 340nm absorbance descends, thereby calculates the concentration of lactose.

Embodiment two

The lactose diagnosis of present embodiment/mensuration reagent is double reagent, comprising:

Reagent 1

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

Stabilizing agent 50mmol/L

Reduced coenzyme 0.25mmol/L

Adenosine triphosphate 7mmol/L

Reagent 2

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

Stabilizing agent 500mmol/L

Lactase 6000U/L

Hexokinase 8000U/L

Aldose-6-phosphoric acid reduction enzyme 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 milk sugar specimen 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 the degree that predominant wavelength 340nm absorbance descends, thereby calculates the concentration of lactose.

Embodiment three

The lactose diagnosis of present embodiment/mensuration reagent is three reagent, comprising:

Reagent 1

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

Stabilizing agent 50mmol/L

Reduced coenzyme 0.25mmol/L

Adenosine triphosphate 7mmol/L

Reagent 2

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

Stabilizing agent 500mmol/L

Hexokinase 8000U/L

Aldose-6-phosphoric acid reduction enzyme 10000U/L

Reagent 3

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

Stabilizing agent 500mmol/L

Lactase 6000U/L

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

When measuring lactose concn, 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 milk sugar specimen 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 the degree that predominant wavelength 340nm absorbance descends, thereby calculates the concentration of lactose.

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)≤0.0007; Absorbance time response curve should be decline curve until terminal point; Reagent can be surveyed effectively, and (R 〉=0.99) linear range can reach 15mmol/L; The inaccuracy of reagent test, its relative deviation be no more than ± and 3%; The coefficient of variation (CV)≤2% of the precision of reagent test (repeatability); The sensitivity of reagent can reach 0.0024 ± 0.0012 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. lactose concn assay method that utilizes enzymic colorimetric and enzyme-linked method technology, its method is as follows:

Lactose+water LactaseGalactose+glucose

Glucose+adenosine triphosphate HexokinaseAdenosine diphosphate+glucose 6-phosphoric acid

Glucose 6-phosphoric acid+reduced coenzyme Aldose-6-phosphoric acid reduction enzymeSorbierite 6-phosphoric acid+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 descends, calculate the concentration measurement result of lactose.

2. lactose diagnosis/mensuration reagent (box), principal ingredient comprises:

Damping fluid 20---500mmol/L

Stabilizing agent 1---4000mmol/L

Reduced coenzyme 0.1---0.35mmol/L

Lactase 1000---80000U/L

Hexokinase 1 000---80000U/L

Aldose-6-phosphoric acid reduction enzyme 1000---80000U/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: reagent (box) can be 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 lactose diagnosis of claim 2/mensuration reagent (box), it is characterized in that:

Form single agent reagent by damping fluid, stabilizing agent, reduced coenzyme, lactase, hexokinase, aldose-6-phosphoric acid reduction enzyme, adenosine triphosphate.

4. according to the described lactose diagnosis of claim 2/mensuration reagent (box), it is characterized in that:

Form two agent reagent by damping fluid, stabilizing agent, reduced coenzyme, lactase, hexokinase, aldose-6-phosphoric acid reduction enzyme, adenosine triphosphate; Reagent 1 is made up of damping fluid, stabilizing agent, reduced coenzyme, adenosine triphosphate; Reagent 2 is made up of damping fluid, stabilizing agent, lactase, hexokinase, aldose-6-phosphoric acid reduction enzyme.Reduced coenzyme, lactase, hexokinase, aldose-6-phosphoric acid reduction enzyme, the position of adenosine triphosphate in reagent 1 or reagent 2 can not limit.

5. according to the described lactose diagnosis of claim 2/mensuration reagent (box), it is characterized in that:

Form multi-agent reagent by damping fluid, stabilizing agent, reduced coenzyme, lactase, hexokinase, aldose-6-phosphoric acid reduction enzyme, adenosine triphosphate; Reagent 1 is made up of damping fluid, stabilizing agent, reduced coenzyme, adenosine triphosphate; Reagent 2 is made up of damping fluid, stabilizing agent, hexokinase, aldose-6-phosphoric acid reduction enzyme; Reagent 3 is made up of damping fluid, stabilizing agent, lactase.Reduced coenzyme, lactase, hexokinase, aldose-6-phosphoric acid reduction enzyme, the position of adenosine triphosphate in reagent 1, reagent 2 or reagent 3 can not limit.

6. according to the described lactose diagnosis of claim 2/mensuration reagent (box), 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.