CN103063848B - Kit for determination of apolipoprotein C2 by using immunoturbidimetry - Google Patents

Abstract

The invention provides a kit for determination of apolipoprotein C2 by using immunoturbidimetry. A reagent R1 contains 10 to 500 mM of a buffer solution, 1 to 100 g/L of a surfactant, 5 to 50 g/L of an electrolyte, 2 to 100 g/L of a high-molecular accelerator accelerator, 1 to 50 g/L of a stabilizing agent and 1 to 10 g/L of an antiseptic; a reagent R2 comprises 10 to 500 mM of the buffer solution, 150 to 300 g/L of an anti-human APOC2 antibody, 5 to 50 g/L of the electrolyte, and 1 to 10 g/L of the antiseptic; and a calibrator comprises 10 to 500 mM of the buffer solution, 0 to 100 g/L of an APOC2 antigen, 1 to 50 g/L of the stabilizing agent, 1 to 10 g/L of the antiseptic and 0.01 to 10 g/L of an anti-oxidant. The kit provided by the invention is easy and fast to use, can satisfy clinical requirements for rapid high flux detection of a sample and has the advantages of obviously improved detection efficiency, small batch difference and stable reagents.

Description

A kind of immunoturbidimetry measures the kit of APOC2

Technical field

The present invention relates to medical immunology external diagnosis reagent field, be specifically related to the kit that one immunoturbidimetry measures APOC2 (APOC2).

Background technology

People Apo CII is the single chain polypeptide containing 79 amino acid residues, and molecular weight is 9.1kD.The Apo CII first synthesized is residual containing 101 amino acid, wherein 22 Amino acid profile signal peptides, then changes ripe Apo CII into after removing signal peptide.Carry out Chuo-Fasman analysis to the amino acid sequence of Apo CII to show: the 13-22 of Apo CII, 29-40,43-52 amino acid residue is two property α spirals, there is the function with lipid binding, 9-12,23-26,53-56 amino acid residue is β-bend structure, 61-74AA residue is α-bis-property spiral, and be the region with lipid binding, 1-39,54-69 amino acid residue is β-corner structure.The research of Apo CII cyanogen bromide proteolytie fragrnent or synthesis fragment is shown: the peptide section that Apo CII activates low-density lipoprotein is c-terminus 56-79 amino acid residue, and its activation can be strengthened by the 44-55 amino acid residue section had in conjunction with phospholipid activity; Excision Apo CII carboxyl terminal 3 amino acid residues, the activity that Apo CII activates low-density lipoprotein completely loses.

Low-density lipoprotein is the key enzyme of chylomicron and very low density lipoprotein (VLDL) hydrolysis.Research shows: ApoCII is the indispensable activator of low-density lipoprotein, and when Apo CII lacks, low-density lipoprotein activity is extremely low; When Apo CII exists, low-density lipoprotein activity can increase 10-50 doubly, and therefore Apo CII has the effect promoting chylomicron and very low density lipoprotein (VLDL) degraded.Apo CII activates: Apo CII accepts fatty acyl group from " enzyme-substrate " intermediate, is then transferred on albumin.Apo CII also has the effect suppressing liver to absorb chylomicron and very low density lipoprotein (VLDL).Find in test in vitro, Apo CII also can suppress liver triglyceride lipase active, suppress degree and Apo CII concentration linear.Due to hepatic lipase can catalysis through by remnant lipoprotein after low-density lipoprotein effect triacylglycerol hydrolysis, therefore Apo CII also may participate in the reset procedure of remnant lipoprotein in blood plasma.Therefore, Apo CII is most important to the structure of lipoprotein, function and metabolism, can provide valuable index for diagnosis lipid-metabolism disease.

There is many deficiencies in existing APOC2 detection technique: as needed special equipment, sample needs pre-service, can not go up automatic clinical chemistry analyzer and carry out batch detection analysis etc.Euzymelinked immunosorbent assay (ELISA) Application comparison is general, and its principle is connected with solid phase carrier by specific antibody, forms insolubilized antibody; The washing unconjugated antibody of removing and impurity; Add testing sample, make it to react a period of time with insolubilized antibody, allow the antibody of antigen on solid phase carrier in sample be combined; The unconjugated material of washing removing; Add enzyme labelled antibody, the antigen on solid-phase immunity compound is combined with enzyme labelled antibody; The unconjugated enzyme labelled antibody of washing removing; The now amount positive correlation of the enzyme amount on solid phase carrier and the test substance in sample; Add substrate reactions colour developing.Degree according to color reaction qualitatively or quantitatively determines mensuration material.The method is heterogeneous immune detection system, measures process comparatively loaded down with trivial details, length consuming time, lacks unit testing working time; Automaticity is not high, difference between batch and repeatability relatively large; Need to be equipped with multiple Special Equipment, adds somewhat to cost.

Summary of the invention

Technical matters to be solved by this invention is to provide a kind of immunoturbidimetry and measures the kit of APOC2, it is simple to operate, accuracy is high, reproducible, the full-automation of batch sample can be carried out detect, thus defect in elimination above-mentioned background technology.

For solving the problems of the technologies described above, technical scheme of the present invention is:

Immunoturbidimetry measures a kit for APOC2, and this kit comprises reagent R1, reagent R2 and calibration object, wherein,

Reagent R1 comprises damping fluid 10 ~ 500mM, surfactant 1 ~ 100g/L, electrolyte 5 ~ 50g/L, macromolecule accelerator 2 ~ 100g/L, stabilizing agent 1 ~ 50g/L, antiseptic 1 ~ 10g/L;

Reagent R2 comprises damping fluid 10 ~ 500mM, anti-human APOC2 antibody 15% ~ 30%(w/v), electrolyte 5 ~ 50g/L, antiseptic 1 ~ 10g/L;

Calibration object comprises damping fluid 10 ~ 500mM, APOC2 antigen 0 ~ 100mg/L, stabilizing agent 1 ~ 50g/L, antiseptic 1 ~ 10g/L, antioxidant 0.01 ~ 10g/L.

Above-mentioned damping fluid 10 ~ 500mM, is 10 ~ 500mmol/L; Anti-human APOC2 antibody 15% ~ 30%(w/v), being anti-human APOC2 antibody concentration is 150 ~ 300g/L.

Improve as one, in mentioned reagent box,

Described damping fluid is one or more in phosphate, boric acid, glycocoll, TRIS, PBS, CAPSO, HEPES damping fluid;

Anti-human APOC2 antibody comprises that mouse-anti people, rabbit are anti-human, goat-anti people, chicken are anti-human, one or both in duck anti-human antibody;

The kind of anti-human APOC2 antibody comprises the one in monoclonal antibody or polyclonal antibody;

Described electrolyte comprises one or more in sodium chloride, magnesium chloride, potassium chloride, magnesium sulfate;

Described macromolecule accelerator comprise in Macrogol 2000, Macrogol 4000, Macrogol 6000, PEG 8000 one or more;

Described stabilizing agent comprises one or more in bovine serum albumin, disodium ethylene diamine tetraacetate, glucose, trehalose, mannitol;

Described surfactant comprises the one in Triton series, Tween series, bay ether, polyoxyethylene alkyl phenyl ether, ethylene nonyl phenyl ether, polyoxethylene octylphenyl phenylate;

Described antioxidant comprises one or more in butylated hydroxy anisole, Licorice root antioxidant, dibutyl hydroxy toluene, benzene polyphenol, dilauryl thiodipropionate series;

Described antiseptic comprises one or more in Sodium azide, phenol, P-hydroxybenzoic acid, ethyl-para-hydroxybenzoate, ethyl mercury sodium thiosulfate.

In order to ensure the consistance of buffer system, as the preferred technical scheme of one, described reagent R1 is identical with the damping fluid kind in reagent R2.

As the preferred technical scheme of one, in this kit,

Reagent R1 comprises phosphate buffer 10 ~ 100mM, polyoxyethylene laurel ether 1 ~ 50g/L, sodium chloride 5 ~ 15g/L, Macrogol 6000 50 ~ 100g/L, disodium ethylene diamine tetraacetate 5 ~ 15g/L, Sodium azide 1 ~ 5g/L;

Reagent R2 comprises phosphate buffer 10 ~ 100mM, goat-anti people APOC2 polyclonal antibody, sodium chloride 5 ~ 15g/L, Sodium azide 1 ~ 5g/L;

Calibration object comprises glycine buffer 10 ~ 500mM, APOC2 antigen 0 ~ 80mg/L, disodium ethylene diamine tetraacetate 5 ~ 50g/L, Sodium azide 1 ~ 10g/L, butylated hydroxy anisole 0.01 ~ 10g/L.

Described calibration object can be mixed with high concentration single-point calibration product, becomes the reference calibrations product of 5 variable concentrations in use with normal saline dilution, also directly can be prepared into the reference calibrations product of 5 variable concentrations.

Immunoturbidimetry of the present invention is that antigen-antibody is in conjunction with dynamic measurement method, its ultimate principle is: corresponding antigen-antibody reaction occurs the anti-human APOC2 antibody in the APOC2 antigen in sample and reagent R2, form insoluble antigen-antibody complex, make reactant liquor produce certain turbidity.In reactant liquor during anti-APOC2 antibody excess, the amount of the immune complex of formation increases, the turbidity also corresponding increase of reactant liquor along with the increase of APOC2 antigen amount in sample.Measure the light absorption value of this compound at a particular wavelength, the calibration curve demarcated with calibration object compares, and can calculate the content of the APOC2 in sample.

Owing to have employed technique scheme, the invention has the beneficial effects as follows:

Compared with prior art, kit provided by the invention is fast easy to use, and can directly adopt automatic clinical chemistry analyzer to detect, method of operating and step be simplified, meet the requirement that clinical fast high-flux detects sample, detection efficiency significantly improves; Kit, owing to employing the optimization agent combination of screening, makes the stability of reagent greatly improve, and can stablize for 2-8 DEG C and deposit more than 1 year.Therefore kit of the present invention has very high clinical value, is applicable to clinical expansion.

Accompanying drawing explanation

Fig. 1 is the method flow schematic diagram that the embodiment of the present invention 3,4 and 5 detects APOC2 content;

Fig. 2 is the typical curve of the APOC2 calibration object of the embodiment of the present invention 3;

Fig. 3 is the embodiment of the present invention 4 and contrast agent correlative trend figure.

Embodiment

The technological means realized to make the present invention, creation characteristic, reaching object and effect is easy to understand, below in conjunction with specific embodiment, setting forth the present invention further.

Embodiment 1

1. reagent preparation:

Reagent R1:

Reagent R2:

The anti-human APOC2 polyclonal antibody 15% (w/v) of rabbit

Glycocoll (damping fluid) 500mM

Potassium chloride (electrolyte) 50g/L

Ethyl-para-hydroxybenzoate (antiseptic) 1g/L

Calibration object:

Phosphate (damping fluid) 500mM

Disodium ethylene diamine tetraacetate (stabilizing agent) 50g/L

Sodium azide (antiseptic) 1g/L

Butylated hydroxy anisole (antioxidant) 0.01g/L

The APOC2 antigen of respective amount adds in described solution by calibration object concentration as required, prepares APOC2 calibration object.This calibration object can be high concentration single-point calibration product, becomes the reference calibrations product of 5 variable concentrations in use with normal saline dilution, also directly can be prepared into the reference calibrations product of 5 variable concentrations.The present embodiment prepares the reference calibrations product of 5 variable concentrations, is respectively 0mg/L, 9.4mg/L, 18.8mg/L, 37.5mg/L, 75mg/L.Then use the membrane filtration of 0.22 μm degerming, place 2 ~ 8 DEG C of preservations.

Embodiment 2

Reagent R1:

Reagent R2:

The anti-human APOC2 monoclonal antibody 30% (w/v) of chicken

Phosphate (damping fluid) 10mM

Sodium chloride (electrolyte) 5g/L

Sodium azide (antiseptic) 10g/L

Calibration object:

What the present embodiment was selected is high concentration single-point reference calibrations product, and APOC2 antigen concentration is 60mg/L, then uses the membrane filtration of 0.22 μm degerming, places 2 ~ 8 DEG C of preservations.Become the reference calibrations product of 5 variable concentrations during use with normal saline dilution, be respectively 0.1mg/L, 7.5mg/L, 15mg/L, 30mg/L, 60mg/L.

Embodiment 3

1. reagent preparation:

Reagent R1:

Reagent R2:

Goat-anti people APOC2 polyclonal antibody 22% (w/v)

Phosphate (damping fluid) 60mM

Sodium chloride (electrolyte) 8.5g/L

Sodium azide (antiseptic) 2g/L

Calibration object:

Glycocoll (damping fluid) 20mM

Disodium ethylene diamine tetraacetate (stabilizing agent) 8g/L

Sodium azide (antiseptic) 4g/L

Butylated hydroxy anisole (antioxidant) 0.4g/L

The APOC2 antigen of respective amount adds in described solution by calibration object concentration as required, prepares APOC2 calibration object.This calibration object can be high concentration single-point calibration product, becomes the reference calibrations product of 5 variable concentrations in use with normal saline dilution, also directly can be prepared into the reference calibrations product of 5 variable concentrations.The present embodiment prepares the reference calibrations product of 5 variable concentrations, is respectively 0mg/L, 11.3mg/L, 22.5mg/L, 45mg/L, 90mg/L.Then use the membrane filtration of 0.22 μm degerming, place 2 ~ 8 DEG C of preservations.

2. detection method:

As shown in Figure 1, analytical approach is Two point end assay to APOC2 detection method, specifically: sample application amount is that 6 μ l, reagent R1 and R2 application of sample amount are respectively 210 μ l and 70 μ l; 210 μ l reagent R1 add 6 μ l samples 37 DEG C of insulations 5 minutes, after record absorbance OD1, add 70 μ l reagent R2, and 37 DEG C are continued insulation and record absorbance OD2 after 5 minutes; Determined wavelength is respectively: predominant wavelength 340nm, commplementary wave length 800nm.

3. Specification Curve of Increasing:

Adopt the present embodiment reagent and described assay method, the typical curve of 5 the variable concentrations APOC2 standard items using Hitachi's automatic clinical chemistry analyzer to record, as shown in Figure 2.X-axis represents APOC2 content, and Y-axis represents absorbance.This typical curve may be used for detecting sample APOC2 content.

4, variable concentrations sample accuracy detects

Get 3 parts of clinical samples at random, use Hitachi automatic clinical chemistry analyzer to 3 this difference of increment duplicate detection 10 times, testing result is as shown in table 1.

The APOC2 detectable concentration (10 times) of table 13 increment basis and the coefficient of variation

Result shows, and the coefficient of variation that three increments originally record is respectively 1.8%, 2.1%, 2.2%, is all less than 3%, shows that kit of the present invention has comparatively high precision.

Embodiment 4

1. reagent preparation:

Reagent R1:

Reagent R2:

Goat-anti people APOC2 polyclonal antibody 20% (w/v)

PBS (damping fluid) 30mM

Sodium chloride (electrolyte) 9g/L

Sodium azide (antiseptic) 3g/L

Calibration object:

CAPSO (damping fluid) 50mM

Bovine serum albumin (stabilizing agent) 7g/L

Sodium azide (antiseptic) 5g/L

Butylated hydroxy anisole (antioxidant) 0.6g/L

The APOC2 antigen of respective amount adds in described solution by calibration object concentration as required, prepares APOC2 calibration object.This calibration object can be high concentration single-point calibration product, becomes the reference calibrations product of 5 variable concentrations in use with normal saline dilution, also directly can be prepared into the reference calibrations product of 5 variable concentrations.The present embodiment prepares the reference calibrations product of 5 variable concentrations, is respectively 0mg/L, 9mg/L, 18mg/L, 36mg/L, 72mg/L.Then use the membrane filtration of 0.22 μm degerming, place 2 ~ 8 DEG C of preservations.

2. detection method:

As shown in Figure 1, analytical approach is Two point end assay to APOC2 detection method, specifically: sample application amount is that 6 μ l, reagent R1 and R2 application of sample amount are respectively 210 μ l and 70 μ l; 210 μ l reagent R1 add 6 μ l samples 37 DEG C of insulations 5 minutes, after record absorbance OD1, add 70 μ l reagent R2, and 37 DEG C are continued insulation and record absorbance OD2 after 5 minutes; Determined wavelength is respectively: predominant wavelength 340nm, commplementary wave length 800nm.

3. different reagent detects contrast:

According to the method drawing standard curve of embodiment 3.Use reagent of the present invention and import contrast agent (Japanese Sekisui Medical Treatment Co., Ltd reagent) respectively, use Hitachi's automatic clinical chemistry analyzer to measure by each autoregressive parameter 50 parts of clinical samples simultaneously, linear regression is carried out to measured value.The regression equation that linear regression obtains is Y=1.0116X-0.4418, coefficient R 2=0.9951, as shown in Figure 3.Wherein X is reagent of the present invention, and Y is contrast agent.Result shows reagent of the present invention and contrast agent correlativity very well, illustrates that reagent of the present invention has good specificity and accuracy.Reagent of the present invention goes for the full-automatic of the series such as Hitachi, Olympus, Beckman and semi-automatic biochemical analyzer, and analytical approach and parameter suitably can adjust according to different type of machines.

Embodiment 5

1. reagent preparation:

Reagent R1:

Reagent R2:

Goat-anti people APOC2 polyclonal antibody 18% (w/v)

Phosphate (damping fluid) 60mM

Sodium chloride (electrolyte) 9g/L

Sodium azide (antiseptic) 4g/L

Calibration object:

PBS (damping fluid) 20mM

Disodium ethylene diamine tetraacetate (stabilizing agent) 7g/L

Sodium azide (antiseptic) 5g/L

Butylated hydroxy anisole (antioxidant) 0.5g/L

The APOC2 antigen of respective amount adds in described solution by calibration object concentration as required, prepares APOC2 calibration object.This calibration object can be high concentration single-point calibration product, becomes the reference calibrations product of 5 variable concentrations in use with normal saline dilution, also directly can be prepared into the reference calibrations product of 5 variable concentrations.What the present embodiment was selected is high concentration single-point reference calibrations product, and APOC2 antigen concentration is 80mg/L, then uses the membrane filtration of 0.22 μm degerming, places 2 ~ 8 DEG C of preservations.Become the reference calibrations product of 5 variable concentrations during use with normal saline dilution, be respectively 0mg/L, 10mg/L, 20mg/L, 40mg/L, 80mg/L.

2. detection method:

As shown in Figure 1, analytical approach is Two point end assay to APOC2 detection method, specifically: sample application amount is that 6 μ l, reagent R1 and R2 application of sample amount are respectively 210 μ l and 70 μ l; 210 μ l reagent R1 add 6 μ l samples 37 DEG C of insulations 5 minutes, after record absorbance OD1, add 70 μ l reagent R2, and 37 DEG C are continued insulation and record absorbance OD2 after 5 minutes; Determined wavelength is respectively: predominant wavelength 340nm, commplementary wave length 800nm.

3. different embodiment kit detects contrast:

According to the method drawing standard curve of embodiment 3.Preparing a APOC2 concentration is the sample of 27.5mg/L, and use the kit of embodiment 3,4,5 to sample duplicate detection 10 times respectively, testing result is as shown in table 2.

The APOC2 Concentration Testing contrast of table 23 kind of embodiment kit

From table 2, the APOC2 concentration that three kinds of embodiment kits record is all close to actual value, and the coefficient of variation that three kinds of kits record all is less than 3%, kit stable performance of the present invention is described, measures accurately.

The present invention is not limited to above-mentioned embodiment, and all are based on technical conceive of the present invention, and done structural improvement, all falls among protection scope of the present invention.

Claims (1)

1. measure a kit for APOC2 with immunoturbidimetry, it is characterized in that: described kit comprises reagent R1, reagent R2 and calibration object, wherein,

Reagent R1 comprises phosphate buffer 10 ~ 100mM, polyoxyethylene laurel ether 1 ~ 50g/L, sodium chloride 5 ~ 15g/L, Macrogol 6000 50 ~ 100g/L, disodium ethylene diamine tetraacetate 5 ~ 15g/L, Sodium azide 1 ~ 5g/L;

Reagent R2 comprises phosphate buffer 10 ~ 100mM, goat-anti people APOC2 polyclonal antibody, sodium chloride 5 ~ 15g/L, Sodium azide 1 ~ 5g/L;

Calibration object comprises glycine buffer 10 ~ 500mM, APOC2 antigen 0 ~ 80mg/L, disodium ethylene diamine tetraacetate 5 ~ 50g/L, Sodium azide 1 ~ 10g/L, butylated hydroxy anisole 0.01 ~ 10g/L.