CN103063848A - 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 kit of measuring APOC2 with immunoturbidimetry

Technical field

The present invention relates to medical immunology external diagnosis reagent field, be specifically related to a kind of kit of measuring APOC2 (APOC2) with immunoturbidimetry.

Background technology

People Apo CII is the single chain polypeptide that contains 79 amino acid residues, and molecular weight is 9.1kD.To contain 101 amino acid residual for synthetic Apo CII first, and 22 Amino acid profile signal peptides wherein then change ripe Apo CII into after removing signal peptide.The amino acid sequence of Apo CII is carried out Chuo-Fasman be the analysis showed that: the 13-22 of Apo CII, 29-40, the 43-52 amino acid residue is two property α spirals, has the function with lipid binding, 9-12,23-26,53-56 amino acid residue are the β-bend structure, the 61-74AA residue is α-two property spirals, is and the zone of lipid binding, and 1-39,54-69 amino acid residue are β-corner structure.Apo CII cyanogen bromide is hydrolyzed studies show that of fragment or synthetic fragment: the peptide section that Apo CII activates low-density lipoprotein is c-terminus 56-79 amino acid residue, and the 44-55 amino acid residue section that its activation can be had in conjunction with the phosphatide activity is strengthened; 3 amino acid residues of excision Apo CII carboxyl terminal, 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.Studies show that: ApoCII is the indispensable activator of low-density lipoprotein, and when Apo CII lacked, the low-density lipoprotein activity was extremely low; When Apo CII existed, the low-density lipoprotein activity can increase 10-50 doubly, so Apo CII has and promotes chylomicron and with the effect of very low density lipoprotein (VLDL) degraded.The mechanism that Apo CII activates low-density lipoprotein may be: Apo CII accepts fatty acyl group from " enzyme-substrate " intermediate, then it is transferred on the albumin.Apo CII also has the inhibition liver to the effect of chylomicron and very low density lipoprotein (VLDL) picked-up.Find that in vitro test it is active that Apo CII also can suppress the liver triglyceride lipase, inhibition degree and Apo CII concentration are linear.But because hepatic lipase catalysis triacylglycerol hydrolysis in the remnant lipoprotein after with the low-density lipoprotein effect, so Apo CII also may participate in the reset procedure of remnant lipoprotein in the blood plasma.Therefore, Apo CII is most important to structure, function and the metabolism of lipoprotein, can provide valuable index for diagnosis lipid-metabolism disease.

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

Summary of the invention

Technical matters to be solved by this invention provides a kind ofly measures the kit of APOC2 with immunoturbidimetry, and it is simple to operate, accuracy is high, good reproducibility, can carry out that batch sample is full-automatic to be detected, thus defective in the elimination above-mentioned background technology.

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

A kind of kit with immunoturbidimetry mensuration APOC2, 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.

As a kind of improvement, in the mentioned reagent box,

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

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

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

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

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

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

Described surfactant comprises that Triton series, Tween are serial, a kind of in the bay ether, polyoxyethylene alkyl phenyl ether, polyoxyethylene nonylplenyl ether, polyoxyethylene octyl group phenylate;

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

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

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

As a kind of preferred technical scheme, in this kit,

Reagent R1 comprises phosphate buffer 1 0～100mM, 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 1 0～100mM, 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 in use the reference calibrations product of 5 variable concentrations with normal saline dilution, also can directly be prepared into the reference calibrations product of 5 variable concentrations.

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

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

Compared with prior art, kit provided by the invention is easy to use can directly to adopt automatic clinical chemistry analyzer to detect fast, and method of operating and step are simplified, satisfy the requirement that clinical fast high-flux detects sample, detection efficiency obviously improves; Kit, can be stablized for 2-8 ℃ and deposit more than 1 year so that the stability of reagent improves greatly owing to used the optimization agent combination of screening.Therefore kit of the present invention has very high clinical value, is fit to clinical expansion.

Description of drawings

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

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

Fig. 3 is the embodiment of the invention 4 and contrast agent correlativity trend map.

Embodiment

For technological means, creation characteristic that the present invention is realized, reach purpose and effect is easy to understand, below in conjunction with specific embodiment, further set forth the present invention.

Embodiment 1

1. reagent preparation:

Reagent R1:

Reagent R2:

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

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

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

Embodiment 2

Reagent R1:

Reagent R2:

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

Phosphate (damping fluid) 10mM

Sodium chloride (electrolyte) 5g/L

Sodium azide (antiseptic) 10g/L

Calibration object:

What present embodiment was selected is high concentration single-point reference calibrations product, and the APOC2 antigen concentration is 60mg/L, then uses the membrane filtration degerming of 0.22 μ m, places 2～8 ℃ 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

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

2. detection method:

The APOC2 detection method as shown in Figure 1, analytical approach is Two point end assay, specifically: the sample application amount is 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 ℃ of insulations 5 minutes, behind the record absorbance OD1, add 70 μ l reagent R2, and 37 ℃ are continued insulation and record absorbance OD2 after 5 minutes; The detection wavelength is respectively: predominant wavelength 340nm, commplementary wave length 800nm.

3. Specification Curve of Increasing:

Adopt present embodiment reagent and described assay method, the typical curve of 5 variable concentrations APOC2 standard items that use Hitachi automatic clinical chemistry analyzer records, as shown in Figure 2.X-axis represents APOC2 content, and Y-axis represents absorbance.This typical curve can be for detection of sample APOC2 content.

4, variable concentrations sample accuracy detects

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

Table 13 increment APOC2 detectable concentration (10 times) and the coefficient of variation originally

The result shows that the coefficient of variation that three increments originally record is respectively 1.8%, 2.1%, 2.2%, all less than 3%, shows that kit of the present invention has than 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

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

2. detection method:

The APOC2 detection method as shown in Figure 1, analytical approach is Two point end assay, specifically: the sample application amount is 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 ℃ of insulations 5 minutes, behind the record absorbance OD1, add 70 μ l reagent R2, and 37 ℃ are continued insulation and record absorbance OD2 after 5 minutes; The detection wavelength is respectively: predominant wavelength 340nm, commplementary wave length 800nm.

3. different reagent detect contrast:

Method drawing standard curve according to embodiment 3.Use respectively reagent of the present invention and import contrast agent (Japanese Sekisui Medical Treatment Co., Ltd reagent), use Hitachi's automatic clinical chemistry analyzer that 50 parts of clinical samples are measured simultaneously by each autoregressive parameter, measured value is carried out linear regression.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.The result shows that reagent of the present invention and contrast agent correlativity are fine, illustrates that reagent of the present invention has good specificity and accuracy.Reagent of the present invention goes for the full-automatic and semi-automatic biochemical analyzer of the series such as Hitachi, Olympus, Beckman, and analytical approach and parameter can suitably be adjusted 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

Calibration object concentration is as required added the APOC2 antigen of respective amount in the described solution to, prepares the APOC2 calibration object.This calibration object can be high concentration single-point calibration product, becomes in use the reference calibrations product of 5 variable concentrations with normal saline dilution, also can directly be prepared into the reference calibrations product of 5 variable concentrations.What present embodiment was selected is high concentration single-point reference calibrations product, and the APOC2 antigen concentration is 80mg/L, then uses the membrane filtration degerming of 0.22 μ m, places 2～8 ℃ 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:

The APOC2 detection method as shown in Figure 1, analytical approach is Two point end assay, specifically: the sample application amount is 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 ℃ of insulations 5 minutes, behind the record absorbance OD1, add 70 μ l reagent R2, and 37 ℃ are continued insulation and record absorbance OD2 after 5 minutes; The detection wavelength is respectively: predominant wavelength 340nm, commplementary wave length 800nm.

3. different embodiment kits detect contrast:

Method drawing standard curve according to embodiment 3.The sample that to prepare a APOC2 concentration be 27.5mg/L uses respectively embodiment 3,4,5 kit to sample duplicate detection 10 times, and testing result is as shown in table 2.

The APOC2 concentration of table 23 kind of embodiment kit detects contrast

By as seen from Table 2, the APOC2 concentration that three kinds of embodiment kits record is all near actual value, and the variation lines number average that three kinds of kits record illustrates kit stable performance of the present invention less than 3%, measures accurately.

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

Claims (10)

1. measure the kit of APOC2 with immunoturbidimetry for one kind, it is characterized in that: described 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 150～300g/L, 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.

2. as claimed in claim 1ly a kind ofly measure the kit of APOC2 with immunoturbidimetry, it is characterized in that: described damping fluid is one or more in phosphate, boric acid, glycocoll, TRIS, PBS, CAPSO, the HEPES damping fluid.

3. as claimed in claim 1ly a kind ofly measure the kit of APOC2 with immunoturbidimetry, it is characterized in that: anti-human APOC2 antibody comprises that mouse-anti people, rabbit are anti-human, goat-anti people, chicken are anti-human, in the anti-human antibody of duck one or both; The kind of anti-human APOC2 antibody comprises a kind of in monoclonal antibody or the polyclonal antibody.

4. as claimed in claim 1ly a kind ofly measure the kit of APOC2 with immunoturbidimetry, it is characterized in that: described macromolecule accelerator comprises one or more in Macrogol 2000, Macrogol 4000, Macrogol 6000, the PEG 8000.

5. as claimed in claim 1ly a kind ofly measure the kit of APOC2 with immunoturbidimetry, it is characterized in that: described stabilizing agent comprises one or more in bovine serum albumin, disodium ethylene diamine tetraacetate, glucose, trehalose, the mannitol.

6. as claimed in claim 1ly a kind ofly measure the kit of APOC2 with immunoturbidimetry, it is characterized in that: described surfactant comprises that Triton series, Tween are serial, a kind of in the bay ether, polyoxyethylene alkyl phenyl ether, polyoxyethylene nonylplenyl ether, polyoxyethylene octyl group phenylate.

7. as claimed in claim 1ly a kind ofly measure the kit of APOC2 with immunoturbidimetry, it is characterized in that: described antioxidant comprises one or more in butylated hydroxy anisole, Licorice root antioxidant, dibutyl hydroxy toluene, benzene polyphenol, the dilauryl thiodipropionate series.

8. as claimed in claim 1ly a kind ofly measure the kit of APOC2 with immunoturbidimetry, it is characterized in that: described antiseptic comprises one or more in Sodium azide, phenol, P-hydroxybenzoic acid, ethyl-para-hydroxybenzoate, the ethyl mercury sodium thiosulfate.

9. each describedly a kind ofly measures kit of APOC2 with immunoturbidimetry such as claim 1～8, it is characterized in that: described reagent R1 is identical with damping fluid kind among the reagent R2.

10. as claimed in claim 9ly a kind ofly measure the kit of APOC2 with immunoturbidimetry, it is characterized in that: in the described kit,

Reagent R1 comprises phosphate buffer 1 0～100mM, 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 1 0～100mM, 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.

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