CN106053839A - Kit for determining haptoglobin and preparation method thereof - Google Patents

Abstract

The invention discloses a kit for determining haptoglobin and a preparation method thereof. The kit comprises dual-liquid components of a reagent R1 and a reagent R2 which are independent from each other. The reagent R1 is prepared from a buffer solution, an accelerator, a surface active agent, inorganic salt ions and a corrosion remover, and a solvent of the reagent R1 is purified water; the reagent R2 is prepared from a buffer solution, a suspending agent, a surface active agent, inorganic salt ions, a corrosion remover and haptoglobin antibodies, and a solvent of the reagent R2 is purified water. The preparation method comprises the steps that the reagents are prepared according to the component content; a to-be-tested sample is mixed with the reagent R1 and the reagent R2 to be subjected to a sufficient reaction; a fully automatic biochemical analyzer is used for determining an absorptivity difference obtained after the reaction; according to an absorptivity change value, the concentration of haptoglobin in the sample is worked out. The kit has the advantages of being convenient to operate, high in accuracy and the like.

Description

A kind of test kit measuring hoptoglobin and preparation method thereof

Technical field

The present invention relates to medical science and technological field of biochemistry, a kind of test kit measuring hoptoglobin And preparation method thereof.

Background technology

Hoptoglobin, also known as haptoglobin, be a kind of molecular weight be the acidoglycoprotein of 85000, be widely present in people In class and the serum of multiple mammal and other body fluid, in CAM electrophoresis and agarose gel electrophoresis, hoptoglobin is positioned at α 2 zone, has two pairs of peptide chains (α chain and β chain) to be collectively forming the tetramer of α 2 β 2 in molecule.Hoptoglobin mainly closes at liver Becoming, its degraded is also at liver, and the half-life is about 3.5～4 days.

The major function of hoptoglobin is to be combined into stable complex with free hemoglobin, then by monokaryon-huge Macrophage system is disposed, and under normal circumstances, although the erythrocyte of people is the most mechanically damaged in circulating, but still can protect Holding its integrity, this has good plastic cellular morphology and blood microenvironment relative and stablizes relevant with erythrocyte.When certain Kind of reason induction erythrocyte is when Ink vessel transfusing destroys, and a large amount of hemoglobin can be discharged in blood circulation, and hemoglobin can be from The kidney of people filters, and causes kidney damage, even causes irreversible renal dysfunction.When hoptoglobin is blood red with free After protein binding becomes stable complex, owing to its molecule is bigger, it is impossible to from kidney discharge, so can stop hemoglobin from Glomerular filtration, it is to avoid the free hemoglobin infringement to renal tubules, hoptoglobin and free hemoglobin are combined into compound After thing, present the antigenic determinant made new advances, receptor (CD163) can be removed by the hemoglobin of mononuclear cell, Macrophage Surface Identified and combined, degraded by phagocytosis afterwards, thus eliminated hemoglobin free in blood circulation, when detection finds serum knot When closing globin content decline, in conjunction with the clinical manifestation of patient, it may be determined that whether there occurs intravascular hemolytic disease, such as battle array The property sent out nocturnal hemoglobinuria disease and favism, and congenital ahaptoglobinemia disease etc..Also have some other types Intravascular hemolysis and during part-blood extra vascular hemolysis hoptoglobin can reduce, its degree reduced often with characteristic of disease weight phase one Cause.

Hoptoglobin is again a kind of acute stage phase reactive protein, the knot when body is in stress state, in blood Close globin showed increased, during such as pathological states such as myocardial infarction, tumor, inflammation, wound, infection, and apply some hormone, After 17-hydroxy-11-dehydrocorticosterone and androgen, its serum content often has notable rising, and relevant with the order of severity and prognosis, research conclusion Show, serum haptoglobin and the change of Ferritin Levels, patient's tool that acute pulmonary embolism and dvt are formed There is certain diagnostic value.Additionally in the basic research carrying out diabetes with vascular disease patient, it has been found that serum combines Globin content changes with progression of disease generation dependency, and HP genotype is likely to be one of diabetes coronary artery disease change solely Vertical risk factor, result, it is believed that the dynamic monitoring to haptoglobin content, are of value to controlling of diabetic angiopathy patient Treating, especially when setting up diabetics vascular lesion preventative strategies can, tool is of great significance.

Due to the synthesis of hoptoglobin with degraded all at liver, and in the complex formation of hoptoglobin with hemoglobin During degraded, hoptoglobin can not reuse, therefore when liver function goes wrong, and internal combination pearl egg White quantity often occurs significantly to change, and synthesis deficiency then its content reduces, and degraded deficiency then its content increases, now laboratory examination Whether the haptoglobin content in blood is decreased or increased, to diagnosing hepatic diseases, it is judged that the prognosis of disease, helpful.

The defect that the reagent measuring hoptoglobin in the market generally exists operation inconvenience, accuracy of measurement is low, It is thus desirable to improve.

Summary of the invention

The technical problem to be solved is to overcome low the lacking of prior art operation complexity, accuracy of measurement Fall into, and a kind of test kit measuring hoptoglobin and preparation method thereof is provided.

The technical scheme that the present invention solves above-mentioned technical problem and provides is: the invention discloses a kind of mensuration hoptoglobin Test kit, including reagent R1 independent of each other and reagent R2 biliquid component, including composition and corresponding content be:

Reagent R1:

Buffer 15 ~ 185 mmol/L

Accelerator 15 ~ 45 g/L

Surfactant 10.0 ~ 25.0 ml/L

Inorganic ion 10 ~ 300 mmol/L

Preservative 0.1 ~ 0.7 g/L

Its solvent is purified water

Reagent R2:

Buffer 15 ~ 185 mmol/L

Suspending agent 10.0 ~ 30.0 ml/L

Surfactant 10.0 ~ 25.0 ml/L

Inorganic ion 150 ~ 250 mmol/L

Preservative 0.1 ~ 0.7 g/L

Anti-human hoptoglobin antibody 3.0 ~ 18.0 ml/L

Its solvent is purified water.

As preferably, in described reagent R1, described buffer uses Tris buffer, MOPS buffer, PBS buffering The combination of one or more in liquid, MES buffer, glycine buffer, described accelerator use Polyethylene glycol-2000, The combination of one or more in PEG-8 000, polyvinylpyrrolidone, described surfactant use tween 80, One in polyoxyethylene phenyl ether, described inorganic ion uses one or more in sodium chloride, potassium chloride, magnesium chloride Combination, described preservative uses the combination of one or more in phenol, sodium benzoate, sodium azide.

As preferably, in described reagent R2, described buffer uses Tris buffer, MOPS buffer, PBS buffering The combination of one or more in liquid, MES buffer, glycine buffer, described suspending agent uses arabic gum, alginic acid The combination of one or more in sodium, silica sol, glycerol, described surfactant uses tween 80, polyoxyethylene One in phenyl ether, described inorganic ion uses the combination of one or more in sodium chloride, potassium chloride, magnesium chloride, Described preservative uses the combination of one or more in phenol, sodium benzoate, sodium azide.

As preferably, the invention discloses a kind of test kit measuring hoptoglobin, including reagent R1 independent of each other With reagent R2 biliquid component, including composition and corresponding content be:

Reagent R1:

Tris buffer 100 mmol/L

PEG-8 000 30g/L

Tween 80 15 ml/L

Sodium chloride 150 mmol/L

Sodium azide 0.4 g/L

Its solvent is purified water

Reagent R2:

Tris buffer 100 mmol/L

Glycerol 20 ml/L

Tween 80 14 ml/L

Sodium chloride 200 mmol/L

Sodium azide 0.4 g/L

Anti-human hoptoglobin antibody 10.0 ml/L

Its solvent is purified water.

As preferably, the invention also discloses preparation method and the user of the test kit of said determination hoptoglobin Method, comprises the following steps:

A () prepares reagent according to following component content:

Reagent R1:

Buffer 15 ~ 185 mmol/L

Accelerator 15 ~ 45 g/L

Surfactant 10.0 ~ 25.0 ml/L

Inorganic ion 10 ~ 300 mmol/L

Preservative 0.1 ~ 0.7 g/L

Its solvent is purified water

Reagent R2:

Buffer 15 ~ 185 mmol/L

Suspending agent 10.0 ~ 30.0 ml/L

Surfactant 10.0 ~ 25.0 ml/L

Inorganic ion 150 ~ 250 mmol/L

Preservative 0.1 ~ 0.7 g/L

Anti-human hoptoglobin antibody 3.0 ~ 18.0 ml/L

Its solvent is purified water；

B sample to be tested is mixed by () with reagent R1 and reagent R2 so that it is fully react；

C () measures reacted absorbance difference with automatic clinical chemistry analyzer；

D () calculates the concentration of hoptoglobin in sample according to absorbance changing value.

As preferably, in step (b), the volume ratio of described reagent R1 and reagent R2 is 3:1.

As preferably, in step (b), the volume ratio of the cumulative volume of described sample to be tested and reagent R1 and reagent R2 is 1: Between 5 to 1:150.

The Cleaning Principle of the present invention is: the reaction principle of immunoturbidimetry of the present invention is: the combination in sample Globin can occur specific antigen antibody reaction with the anti-human hoptoglobin antibody in reagent R2, forms antigen-antibody and exempts from Epidemic disease complex, forms turbidity, and its turbidity height becomes positive correlation with haptoglobin content in sample, by measure turbidity and with knot Close globin calibration curve to convert, obtain the content of hoptoglobin in sample.

Activity (the g/L)=C of hoptoglobin in sample_S × (g/L)

In formula: Δ A_TThe sample cell absorbance compared with blank tube absorbance

ΔA_SThe calibration pipe absorbance compared with blank tube absorbance

C_SThe concentration of Hp in calibration solution.

Compared with prior art, the present invention has following advantageous benefits: with the addition of suspending agent and accelerator in this reagent, helps Suspension is conducive to the dispersion of antibody and stablizes, and accelerator then can accelerate the reaction of antigen-antibody, even if fainter change is also Can detect, improve reagent analysis sensitivity.

Detailed description of the invention

Further illustrate below in conjunction with specific embodiment, but the present invention is not limited in these embodiments.

Embodiment 1

The test kit of the present invention includes reagent R1 independent of each other and reagent R2 biliquid component, wherein

Reagent R1:

Tris buffer 100 mmol/L

PEG-8 000 30 g/L

Tween 80 15 ml/L

Sodium chloride 150 mmol/L

Sodium azide 0.4 g/L

Its solvent is purified water

Reagent R2:

Tris buffer 100 mmol/L

Glycerol 20 ml/L

Tween 80 14 ml/L

Sodium chloride 200 mmol/L

Sodium azide 0.4 g/L

Anti-human hoptoglobin antibody 10.0 ml/L

Its solvent is purified water.

Embodiment 2

The test kit of the present invention includes reagent R1 independent of each other and reagent R2 biliquid component, wherein

Reagent R1:

Tris buffer 185 mmol/L

Polyethylene glycol-2000 45 g/L

Polyoxyethylene phenyl ether 10.0ml/L

Potassium chloride 300 mmol/L

Sodium benzoate 0.1g/L

Its solvent is purified water

Reagent R2:

Tris buffer 185 mmol/L

Arabic gum 10.0 ml/L

Polyoxyethylene phenyl ether 10.0ml/L

Potassium chloride 250 mmol/L

Sodium benzoate 0.1 g/L

Anti-human hoptoglobin antibody 18.0 ml/L

Its solvent is purified water.

Embodiment 3

The preparation and application of test kit

1, reagent is prepared according to following component content:

Reagent R1:

Tris buffer 100 mmol/L

PEG-8 000 30 g/L

Tween 80 15 ml/L

Sodium chloride 150 mmol/L

Sodium azide 0.4 g/L

Its solvent is purified water

Reagent R2:

Tris buffer 100 mmol/L

Glycerol 20 ml/L

Tween 80 14 ml/L

Sodium chloride 200 mmol/L

Sodium azide 0.4 g/L

Anti-human hoptoglobin antibody 10.0 ml/L

Its solvent is purified water；

2, automatic clinical chemistry analyzer parameter is arranged

(a) detection temperature: 37 DEG C；

(b) detection wavelength: dominant wavelength 340nm, commplementary wave length 700nm；

(c) response time: 10min, wherein, incubation time 5min, measure immediately after adding reagent R2 and read absorbance Read absorbance A 2 after A1,5min, calculate absorbance changes delta A=A2-A1；

(d) the Direction of Reaction: positive reaction；

3, detecting step

A () takes 210 μ l reagent R1 and the mixing of 2 μ l samples to be tested；

B solution after mixing is hatched 5min under conditions of 37 DEG C by ()；

C () adds 70 μ l reagent R2, measure immediately and read absorbance A 1, reads absorbance A 2 after 5min, calculates absorbance change Δ A=A2-A1；

D () is according to activity (the g/L)=C of hoptoglobin in sample_S × (g/L) the combination pearl in sample is calculated The concentration of albumen.

Embodiment 4

The preparation and application of test kit

1, reagent is prepared according to following component content:

Reagent R1:

Tris buffer 185 mmol/L

Polyethylene glycol-2000 45 g/L

Polyoxyethylene phenyl ether 10.0ml/L

Potassium chloride 300 mmol/L

Sodium benzoate 0.1g/L

Its solvent is purified water

Reagent R2:

Tris buffer 185 mmol/L

Arabic gum 10.0 ml/L

Polyoxyethylene phenyl ether 10.0ml/L

Potassium chloride 250 mmol/L

Sodium benzoate 0.1 g/L

Anti-human hoptoglobin antibody 18.0 ml/L

Its solvent is purified water；

2, automatic clinical chemistry analyzer parameter is arranged

(a) detection temperature: 37 DEG C；

(b) detection wavelength: dominant wavelength 340nm, commplementary wave length 700nm；

(c) response time: 10min, wherein, incubation time 5min, measure immediately after adding reagent R2 and read absorbance Read absorbance A 2 after A1,5min, calculate absorbance changes delta A=A2-A1；

(d) the Direction of Reaction: positive reaction；

3, detecting step

A () takes 210 μ l reagent R1 and the mixing of 2 μ l samples to be tested；

B solution after mixing is hatched 5min under conditions of 37 DEG C by ()；

C () adds 70 μ l reagent R2, measure immediately and read absorbance A 1, reads absorbance A 2 after 5min, calculates absorbance change Δ A=A2-A1；

D () is according to activity (the g/L)=C of hoptoglobin in sample_S × (g/L) the combination pearl in sample is calculated The concentration of albumen.

The table 1 test kit measuring hoptoglobin obtained by embodiment 1 and the mensuration obtained by embodiment 2 combine The result that quality-control product 1 is measured by the test kit of globin respectively, wherein the concentration of the hoptoglobin in quality-control product 1 is 0.689 g/L, measurement result is shown in Table 1:

Table 1

	1st time (g/L)	2nd time (g/L)	3rd time (g/L)	Average (g/L)	Deviation (%)
						Embodiment 1	0.677	0.685	0.683	0.682	1.06
Embodiment 2	0.707	0.708	0.700	0.705	2.32

As shown in Table 1, the test kit measuring hoptoglobin obtained by the present invention is to the measurement result deviation of quality-control product 1 relatively Little, therefore accuracy of measurement is high, and embodiment 1 is optimum selection.

The table 2 test kit measuring hoptoglobin obtained by embodiment 1 and the mensuration obtained by embodiment 2 combine The result that quality-control product 2 is measured by the test kit of globin respectively, wherein the concentration of the hoptoglobin in quality-control product 2 is 2.00 g/L, measurement result is shown in Table 2:

Table 2

	1st time (g/L)	2nd time (g/L)	3rd time (g/L)	Average (g/L)	Deviation (%)
						Embodiment 1	1.970	2.024	2.038	2.011	0.53
Embodiment 2	1.946	2.049	1.918	1.971	1.45

As shown in Table 2, the test kit measuring hoptoglobin obtained by the present invention is to the measurement result deviation of quality-control product 2 relatively Little, therefore accuracy of measurement is high, and embodiment 1 is optimum selection.

It is the most anti-that same sample to be tested is carried out by the table 3 test kit measuring hoptoglobin obtained by embodiment 3 The repetition measurement test kit measuring hoptoglobin calmly and obtained by embodiment 4 is surveyed the most repeatedly to what same sample to be tested was carried out Fixed, the result of gained is carried out the calculating of SD and CV, result is as follows:

Table 3

The precision of the test kit measuring hoptoglobin obtained by the present invention is relatively good as shown in Table 3, and can by table 3 Knowing, embodiment 3 is optimum selection.

The principle of above-described embodiment only illustrative present invention and effect thereof, not for limiting the present invention.Any ripe Above-described embodiment all can be modified under the spirit and the scope of the present invention or change by the personage knowing this technology.Cause This, have usually intellectual such as complete with institute under technological thought without departing from disclosed spirit in art All equivalences become are modified or change, and must be contained by the claim of the present invention.

Claims (7)

1. the test kit measuring hoptoglobin, it is characterised in that: include reagent R1 independent of each other and reagent R2 biliquid Body component, including composition and corresponding content be:

Reagent R1:

Buffer 15 ~ 185 mmol/L

Accelerator 15 ~ 45 g/L

Surfactant 10.0 ~ 25.0 ml/L

Inorganic ion 10 ~ 300 mmol/L

Preservative 0.1 ~ 0.7 g/L

Its solvent is purified water

Reagent R2:

Buffer 15 ~ 185 mmol/L

Suspending agent 10.0 ~ 30.0 ml/L

Surfactant 10.0 ~ 25.0 l/L

Inorganic ion 150 ~ 250 mmol/L

Preservative 0.1 ~ 0.7 g/L

Anti-human hoptoglobin antibody 3.0 ~ 18.0 ml/L

Its solvent is purified water.

A kind of test kit measuring hoptoglobin the most according to claim 1, it is characterised in that: described reagent R1 In, described buffer uses in Tris buffer, MOPS buffer, PBS, MES buffer, glycine buffer The combination of one or more, described accelerator uses in Polyethylene glycol-2000, PEG-8 000, polyvinylpyrrolidone The combination of one or more, described surfactant uses the one in tween 80, polyoxyethylene phenyl ether, described Inorganic ion use the combination of one or more in sodium chloride, potassium chloride, magnesium chloride, described preservative use phenol, The combination of one or more in sodium benzoate, sodium azide.

A kind of test kit measuring hoptoglobin the most according to claim 1, it is characterised in that: described reagent R2 In, described buffer uses in Tris buffer, MOPS buffer, PBS, MES buffer, glycine buffer The combination of one or more, described suspending agent uses the one in arabic gum, sodium alginate, silica sol, glycerol Or multiple combination, described surfactant uses the one in tween 80, polyoxyethylene phenyl ether, described inorganic salt Ion uses the combination of one or more in sodium chloride, potassium chloride, magnesium chloride, described preservative to use phenol, benzoic acid The combination of one or more in sodium, sodium azide.

4. according to the arbitrary described a kind of test kit measuring hoptoglobin of claim 1-3, it is characterised in that: include each other Independent reagent R1 and reagent R2 biliquid component, including composition and corresponding content be:

Reagent R1:

Tris buffer 100 mmol/L

PEG-8 000 30 g/L

Tween 80 15 ml/L

Sodium chloride 150 mmol/L

Sodium azide 0.4 g/L

Its solvent is purified water

Reagent R2:

Tris buffer 100 mmol/L

Glycerol 20 ml/L

Tween 80 14 ml/L

Sodium chloride 200 mmol/L

Sodium azide 0.4 g/L

Anti-human hoptoglobin antibody 10.0 ml/L

Its solvent is purified water.

5. according to preparation method and the user of the arbitrary described a kind of test kit measuring hoptoglobin of claim 1-3 Method, it is characterised in that: comprise the following steps:

A () prepares reagent according to following component content:

Reagent R1:

Buffer 15 ~ 185 mmol/L

Accelerator 15 ~ 45 g/L

Surfactant 10.0 ~ 25.0 ml/L

Inorganic ion 10 ~ 300 mmol/L

Preservative 0.1 ~ 0.7 g/L

Its solvent is purified water

Reagent R2:

Buffer 15 ~ 185 mmol/L

Suspending agent 10.0 ~ 30.0 ml/L

Surfactant 10.0 ~ 25.0 ml/L

Inorganic ion 150 ~ 250 mmol/L

Preservative 0.1 ~ 0.7 g/L

Anti-human hoptoglobin antibody 3.0 ~ 18.0 ml/L

Its solvent is purified water；

B sample to be tested is mixed by () with reagent R1 and reagent R2 so that it is fully react；

C () measures reacted absorbance difference with automatic clinical chemistry analyzer；

D () calculates the concentration of hoptoglobin in sample according to absorbance changing value.

The preparation method of a kind of test kit measuring hoptoglobin the most according to claim 5 and using method, it is special Levying and be: in step (b), the volume ratio of described reagent R1 and reagent R2 is 3:1.

The preparation method of a kind of test kit measuring hoptoglobin the most according to claim 5 and using method, it is special Levy and be: in step (b), the volume ratio of the cumulative volume of described sample to be tested and reagent R1 and reagent R2 1:5 to 1:150 it Between.