CN105223348A - The latex enhancing immune turbidimetry detection kit of people's Antithrombin III - Google Patents

Abstract

The present invention relates to a kind of latex enhancing immune turbidimetry detection kit of people's Antithrombin III.Kit provided by the invention comprises the first reagent, the second reagent and calibration object, and wherein the first pack is containing electrolyte, set accelerator, stabilizing agent, surfactant, antiseptic and damping fluid; Second pack is containing the latex particle of polyclonal antibody bag quilt of anti-human Antithrombin III, stabilizing agent, surfactant, antiseptic and damping fluid; Calibration object comprises antiseptic, electrolyte, stabilizing agent, Antithrombin III and damping fluid.This kit high specificity, highly sensitive, stable homogeneous, fast and convenient, can be used for common biochemical analyzer.

Description

The latex enhancing immune turbidimetry detection kit of people's Antithrombin III

Technical field

The present invention relates to clinical examination field.The kit of people's Antithrombin III content in sample is measured than purifying method in particular to a kind of latex enhancing immune that utilizes.

Background technology

Antithrombase (Antithrombin) is called for short AT, and source plasma AT is referred to as pAT.PAT is natural anticoagulant protein important in body, belongs to serine protease inhibitor family member.Its plasma concentration is about 100 to 150 μ g/ml (2 to 3 μm of ol/L).PAT is produced by liver and vascular endothelial cell, is that a kind of vitamin K relies on, the molecular mass that is made up of 432 amino acid is the single chain glycoprotein of 58kDa, containing 4 N glycosylation oligonucleotide chains and 3 disulfide bond.Whether can be divided into the beta isomer of αisomer containing glycosyl and sugar based containing glycosyl according to its asparagine of 135.Glycosyl due to αisomer 135 can stop the combination with heparin, therefore the affinity of itself and heparin is less than beta isomer.Now also obtain restructuring antithrombase by Transgenic Sheep, be called for short rAT.MasaakiHirose etc. have expressed rAT on Chinese hamster ovary cell, make its output reach 1g/L by batch fermentation.The rAT of purifying has the specific activity of pAT, decreases glycosyl amount, enhances the affinity with heparin.

Antithrombin III (AT-III) is the most important hindrance factor as factor Xa in blood, which control solidifying and fibrinous dissolving of blood.In blood, the level of Antithrombin III is according to various disease, symptom and changing.Reduce in disseminated intravascular coagulation (DIC), liver illness, nephrotic syndrome etc.Antithrombin III level reduction in blood may cause heparin therapy effect to present.Therefore, the activity grasping Antithrombin III is as the monitoring of this type of disease, ill-condition analysis, prognosis judgement and heparin therapy or use the index during dispensing of Antithrombin III concentrate formulation significant.

Antithrombin III level increases: can cause hemorrhage.Be found in congenital factor to lack, as hemophilia; Acquired coagulation factor lacks, as oxyhepatitis, kidney transplant, use anticoagulant etc.

Antithrombin III reduced levels: can thrombosis be caused.Be found in congenital antithrombin III to lack and dysfunction, as thrombotic diseases such as lung infraction, dvts; Posteriority lowers, as DIC, chronic liver disease, miocardial infarction etc.

The detection method of Antithrombin III is more, and traditional comprises immune rocket electrophoresis, gel Plaque Technique Detected, Chromogenic assay etc.Wherein chromophoric substrate method (see publication number CN102690862A) is method the most frequently used at present.But the method needs specific apparatus, and required time is longer.Owing to having important clinical meaning when Antithrombin III reduces in human body, therefore develop a kind of highly sensitive detection reagent very meaningful.

Summary of the invention

According to embodiments more of the present invention, provide a kind of latex enhancing immune turbidimetry detection kit of people's Antithrombin III, it comprises the first reagent and the second reagent, wherein:

First pack is containing stabilizing agent, surfactant, antiseptic, electrolyte, set accelerator and damping fluid;

Second pack is containing stabilizing agent, surfactant, antiseptic, electrolyte, the latex particle being coated with anti-human Antithrombin III polyclonal antibody and damping fluid.

In some embodiments, kit of the present invention can also comprise calibration object as required.Calibration object is used for the drafting of typical curve.Therefore, calibration object can be prepared voluntarily, also can use any commercially available calibration object.In some embodiments, calibration object comprises antiseptic, electrolyte, stabilizing agent, people's Antithrombin III and damping fluid.In some embodiments, the content of Antithrombin III in calibration object is between 0 to 1000ng/ml.In some embodiments, Antithrombin III is derived from the body fluid of people and purity >=95%; Such as from human body fluid, natural extraction purifying obtain.

In some embodiments, calibration object comprises the Antithrombin III of four concentration levels, and one of them concentration level is not containing Antithrombin III; The Antithrombin III of different content is added with respectively in all the other calibration objects.In some embodiments, calibration object comprises the stabilizing agent of mass percent 0.1 to 1%, the antiseptic of mass percent 0.01% to 0.1%, the electrolyte of mass percent 0.1 to 5.0%, the damping fluid of the pH6.5 to 8.0 of 10 to 100mmol/L, people's Antithrombin III of concentration known and human serum matrix.Human serum matrix can legal collection from the Healthy Human Serum of medical institutions, after deactivation, remove hepatitis, HIV virus propagate risk; Also can be commercially available human serum matrix.

In some embodiments, anti-human Antithrombin III polyclonal antibody is derived from mammiferous IgG antibody, preferred sheep IgG antibody or rabbit igg antibody.

In some embodiments, the diameter range of latex particle is 50 to 250nm, preferably 100 to 200nm, more preferably 150nm.In some embodiments, the concentration of latex particle in reagent is 0.5 to 5mg/mL.

In some embodiments, latex particle is polystyrene latex particles.Latex particle finishing has functional group, and functional group is selected from the one in carboxyl, amino, hydroxyl, hydrazides, chloromethyl; Preferred carboxyl.By these functional groups, antibody is connected on latex particle.In some embodiments, adopt chemical crosslink technique by antibody bag by latex particle surface, chemical cross-linking agent wherein used is selected from: one or both in carbodiimide (EDAC), N-hydroxy-succinamide (NHS), N-hydroxy thiosuccinimide (Sulfo-NHS), hydrazides, isocyanates; Cross-linking buffer is not containing amino damping fluid, is selected from the one in MES, MOPSO, MOPS, HEPES, phosphate buffer; The pH of cross-linking buffer is between 6.0 to 8.0.

In some embodiments, the electrolyte in the first reagent and the second reagent is selected from one or more in sodium chloride, potassium chloride, magnesium chloride, magnesium sulfate; Preferred sodium chloride.The concentration of electrolyte in reagent is between 0.1 to 5.0%.

In some embodiments, stabilizing agent is selected from one or more in bovine serum albumin(BSA), casein, fishskin gelatin albumen, glucose, fructose, mannose, shitosan, sorbierite, disodium ethylene diamine tetraacetate; Stabilizer concentration is 0.1 to 1%.

In some embodiments, described set accelerator is selected from the one in PEG-4000, PEG-6000, PEG-8000, sodium dextran sulfate, preferred PEG-6000; Set accelerator concentration is between 2 to 4%.

In some embodiments, surfactant is selected from one or more in tween, fatty alcohol polyglycol ether, polyoxyethylene phenyl ether, polyoxyethylene laurel ether; Preferred tween.Surfactant concentration is between mass percent 0.1 to 1.0%.

The invention provides a kind of latex enhancing immune turbidimetry detection kit of people's Antithrombin III, it comprises the first reagent, the second reagent and calibration object, wherein:

First pack contains:

Second pack contains:

Be coated with the latex particle of anti-human Antithrombin III polyclonal antibody, 0.5 to 5mg/mL, preferred 1mg/mL; The wherein preferred 0.6mg/mL of anti-human Antithrombin III polyclonal antibody,

Wherein latex particle is the carboxyl modified polystyrene latex of particle diameter 150nm;

Calibration object comprises:

According to a specific embodiments of the present invention, provide a kind of latex enhancing immune turbidimetry detection kit of people's Antithrombin III, it comprises the first reagent, the second reagent and calibration object, wherein:

First pack contains:

Second pack contains:

Be coated with the latex particle 1mg/mL of anti-human Antithrombin III polyclonal antibody, wherein goat-anti people Antithrombin III polyclonal antibody is 0.6mg/mL,

Wherein latex particle is the carboxyl modified polystyrene latex of particle diameter 150nm;

Calibration object comprises:

Kit of the present invention, adopts latex enhancing immune turbidimetry quantitatively to detect Antithrombin III.Its principle is: utilize chemical crosslink technique by specificity goat-anti people Antithrombin III polyclonal antibody bag by surperficial to latex particle, antigen-antibody immune response is there is when there being Antithrombin III antigen in tested sample, form Ag-Ab-latex particle compound, when keeping antibody excess in reaction system, the compound formed can be connected with contiguous latex particle, finally be woven into the marked change of the netted system turbidity that induces reaction, in the now change of reaction system turbidity and tested sample, the amount of Antithrombin III is proportional, measure the absorbance of this turbidity at 570nm wavelength place, contrast calibration curve can obtain the content of Antithrombin III in sample.

Kit high specificity of the present invention, the feature that highly sensitive, stable homogeneous is easy to use, be applicable to industrialization, be convenient to clinical expansion.

Accompanying drawing explanation

Fig. 1 is the sensitivity of kit of the present invention.

Fig. 2 is the linear graph of kit of the present invention.

◆ average; ■ average+2.6SD; ▲ average-2.6SD.

Fig. 3 is that kit of the present invention compares with the correlativity that chromophoric substrate method kit detects clinical sample.

Embodiment

Following examples are to explanation of the present invention and explain further, instead of limitation of the present invention, and any amendment made within the scope of spirit of the present invention and rights protection, all falls within the scope of protection of the present invention.

Embodiment 1: affinity purification prepares goat-anti people Antithrombin III polyclonal antibody

Concrete steps are as described below:

(1) use 8 to 10 sheep in each immunization experiment, before immunity starts, collect animal blood serum.From these serum, the IgG antibody of purifying in contrast.

(2) highly purified for 0.1mg people's Antithrombin III antigen (from Fitzgerald) is dissolved in the phosphate buffer of pH7.8, uses Freund's complete adjuvant emulsification, be expelled in the muscle of sheep, duplicate injection in every 4 weeks.Injecting initial latter 10 weeks, collect serum, and by chloride precipitation, initial gross separation is from the IgG antibody component of serum.

(3) purifying of the polyclonal antibody of goat-anti people Antithrombin III:

Prepare the separating column that affinity purification is used: according to the description of product, highly purified for 10mg people's Antithrombin III antigen is fixed on active HP post (from AmershamPharmaciaBiotech) of HITRAPNHS-;

Loading and wash-out: the IgG component of initial gross separation is diluted to 2mg/mL in phosphate buffer, the antibody diluent of 200mL is flow through pillar, then rinse pillar with the phosphate buffer of 50mL; Use the 0.1M citrate buffer solution antibody elution of 35mLpH3.2 again;

Dialysis purifying: the antibody phosphate buffer of above-mentioned wash-out is dialysed, and use the AmiconCentircon centrifugal filter device with 50000 daltonian molecular cut offs to be concentrated into 3mg/mL, be the goat-anti people Antithrombin III polyclonal antibody of affinity purification.

Embodiment 2: preparation R2 reagent

(1) it is antibody linked to latex surface: after the polyclonal antibody 5mL0.1M phosphate buffer (pH7.8) of above-mentioned for 0.5mg preparation is diluted, add the polystyrene latex solution (Merck) of finishing carboxyl, diameter 150nm, add 5mgEDAC again, 37 DEG C of reactions 6 hours, the glycine buffer (pH8.5) adding 0.5mL0.1M stirred 1h cessation reaction;

(2) latex cleaning: the centrifugal supernatant that removes is to remove the accessory substance etc. of unnecessary antibody, crosslinking chemical and cross-linking reaction, and bottom settlings is bag by good latex.3 times are washed with the glycine-NaOH buffer of 20mL50mM.

(3) latex suspends: the glycine-NaOH buffer (being wherein added with 0.9% sodium chloride, 20mMEDTA, 0.8%BSA, 0.1% polysorbas20,0.1% Sodium azide) of 20mL150mmol/LpH8.0 is joined above-mentioned precipitation, latex particle concentration is made to be 1mg/mL, wherein antibody concentration 0.6mg/mL; Ultrasound wave process 4min ensures that latex particle is in the suspended state of stable homogeneous, obtains homogeneous milky latex suspension, is R2 reagent.

Embodiment 3: preparation R1 reagent

In the Tris-HCl damping fluid of 50mMpH7.2, add 0.9% sodium chloride, 4%PEG-6000,0.2%BSA, 20mMEDTA, 0.5%Tween-20,0.1% Sodium azide, stir and regulate pH to 8.0, be R1 reagent.

Embodiment 4: prepare calibration object

In the pre-service human serum matrix of pH7.250mMTris-HCl damping fluid, add concentration be respectively 100,200,500, the Antithrombin III sterling (Fitzgerald of 1000ng/mL, purity >=95%), stir, as Antithrombin III multiple spot calibration object.0.9% sodium chloride, 1%BSA, 50mMEDTA, 0.1% Sodium azide is also comprised in pretreated human serum matrix.

Test case: the Performance Detection of kit

Test case 1. kit usage (for Hitachi 7080 Biochemical Analyzer):

Mensuration wavelength is 570nm, first adds 150 μ LR1 reagent, and 37 DEG C are reacted 30 seconds; Thereafter add 10 μ L calibration objects, then react 60 seconds; Thereafter add 750 μ LR2 reagent, the assaying reaction absorbance (A1, A2) of the 10th second, 70 seconds, calculate absorbance difference △ A=A2-A1.Often pipe replication 2 times.The absorbance difference △ A recorded for 2 times by each calibration QC is ordinate, and corresponding concentration is horizontal ordinate, makes " concentration-absorbance difference " calibration curve.Get sample to be tested, be measured in the same method the absorbance difference of sample, substituted into calibration curve, the Antithrombin III content in sample to be tested can be calculated.

Test case 2. sensitivity technique:

Take physiological saline as dummy, dilution human serum sample, compound concentration is respectively 5,10,20,40, the sample of 80ng/mL, with the kit of above-mentioned R1, R2 and calibration object composition, 40 times are measured, computation of mean values and standard deviation (SD) according to the method described in 1.The sensitivity (see Fig. 1) of concentration of specimens as Antithrombin III detection kit of blank+2.6SD is greater than using concentration of specimens-2.6SD.As shown in table 1 below, the sensitivity of kit of the present invention can reach 10ng/mL.

Table 1. kit sensitivity technique of the present invention

	0ng/mL	5ng/mL	10ng/mL	20ng/mL	40ng/mL	80ng/mL
							1	2.5	3.4	14.0	22.8	40.4	82.2
2	1.3	7.4	10.0	21.6	41.4	77.2
							3	0.8	9.4	11.7	17.7	39.3	78.4
4	3.7	3.3	9.9	20.7	38.5	80.5
							5	2.7	6.3	10.8	21.5	40.4	79.2
6	1.5	7.3	9.4	19.8	41.5	78.4
							7	2.6	4.4	12.1	20.6	39.5	82.5
8	0.6	5.3	14.4	22.7	40.7	81.5
							9	1.6	7.1	9.5	22.5	38.4	77.4
10	1.3	8.3	12.9	21.6	39.3	79.2
							Average	1.85	6.20	11.45	21.13	39.94	79.63
SD	0.97	2.06	1.84	1.57	1.11	1.92
							Average+2.6SD	4.38	11.56	16.23	25.22	42.82	84.62
Average-2.6SD	-0.68	0.83	6.66	17.04	37.05	74.63

Test case 3. range of linearity detects:

Antithrombin III concentration is about the sample of 1000ng/mL, the dilution of 100 different proportions is made of physiological saline, each concentration is detected according to the method described in 1, the sample replication of each concentration 3 times, the mean value and theoretical concentration that measure concentration are carried out recovery analysis, result all shows deviation and is less than 40% (see table 2), proves linearly can reach 1000ng/mL, sees Fig. 2.

The table 2. kit range of linearity of the present invention detects

The correlativity that test case 4. kit of the present invention and chromophoric substrate kit detect clinical sample compares

With kit of the present invention and chromophoric substrate kit (deriving from Shanghai Sun Bio-Tech Co., Ltd., CN102690862A, reagent lot 20130912P).Detect after 1000 times are diluted respectively to 120 routine patient's sample simultaneously, testing result is shown in Fig. 3, the sample Antithrombin III result detected with chromophoric substrate method is for horizontal ordinate, with the Antithrombin III result of kit detection of the present invention for ordinate does regretional analysis, dependent equation is Y=0.9804X+8.4869, coefficient R 2=0.987, shows through statistical procedures result, the inventive method is good for the correlativity of clinical sample measured value with chromophoric substrate kit method.

The kit of the application can be applied to automatic clinical chemistry analyzer.Contrast agent box is Chromogenic assay, and reagent is dry powder, needs to redissolve during use, and sample needs through complicated and loaded down with trivial details pretreatment process, manual operations, easyly on automatic clinical chemistry analyzer cannot complete experiment.

Claims (13)

1. a latex enhancing immune turbidimetry detection kit for people's Antithrombin III, it comprises the first reagent and the second reagent, wherein:

First pack is containing stabilizing agent, surfactant, antiseptic, electrolyte, set accelerator and damping fluid;

Second pack is containing stabilizing agent, surfactant, antiseptic, electrolyte, the latex particle being coated with anti-human Antithrombin III polyclonal antibody and damping fluid.

2. the latex enhancing immune turbidimetry detection kit of people's Antithrombin III according to claim 1, also comprises calibration object,

Described calibration object comprises antiseptic, electrolyte, stabilizing agent, people's Antithrombin III and damping fluid.

3. the latex enhancing immune turbidimetry detection kit of people's Antithrombin III according to claim 1, wherein:

Anti-human Antithrombin III polyclonal antibody is derived from mammiferous IgG antibody, preferred sheep IgG antibody or rabbit igg antibody.

4. the latex enhancing immune turbidimetry detection kit of people's Antithrombin III according to claim 1, wherein:

The diameter range of latex particle is 50 to 250nm, preferably 100 to 200nm, more preferably 150nm;

The concentration of latex particle is 0.5 to 5mg/mL;

Latex particle is polystyrene latex particles.

5. the latex enhancing immune turbidimetry detection kit of people's Antithrombin III according to claim 1, wherein:

Latex particle finishing has functional group, and functional group is selected from the one in carboxyl, amino, hydroxyl, hydrazides, chloromethyl, preferred carboxyl.

6. the latex enhancing immune turbidimetry detection kit of people's Antithrombin III according to claim 1 and 2, wherein:

Electrolyte is selected from one or more in sodium chloride, potassium chloride, magnesium chloride, magnesium sulfate, preferred sodium chloride;

Electrolyte concentration is between mass percent 0.1 to 5.0%.

7. the latex enhancing immune turbidimetry detection kit of people's Antithrombin III according to claim 1 and 2, wherein:

Described stabilizing agent is selected from one or more in bovine serum albumin(BSA), casein, fishskin gelatin albumen, glucose, fructose, mannose, shitosan, sorbierite, disodium ethylene diamine tetraacetate;

Stabilizer concentration is mass percent 0.1 to 1%.

8. the latex enhancing immune turbidimetry detection kit of people's Antithrombin III according to claim 1, wherein:

Described set accelerator is selected from the one in PEG-4000, PEG-6000, PEG-8000, sodium dextran sulfate, preferred PEG-6000;

Set accelerator concentration is between mass percent 2 to 4%.

9. the latex enhancing immune turbidimetry detection kit of people's Antithrombin III according to claim 1, wherein:

Described surfactant is selected from one or more in tween, fatty alcohol polyglycol ether, polyoxyethylene phenyl ether, polyoxyethylene laurel ether, preferred tween;

Surfactant concentration is between mass percent 0.1 to 1.0%.

10. the latex enhancing immune turbidimetry detection kit of people's Antithrombin III according to claim 2, wherein:

The content of Antithrombin III in calibration object is between 0 to 1000ng/ml.

The latex enhancing immune turbidimetry detection kit of 11. people's Antithrombin IIIs according to claim 2, wherein:

Antithrombin III is derived from human body fluid; Purity >=95%.

The latex enhancing immune turbidimetry detection kit of 12. 1 kinds of people's Antithrombin IIIs, it comprises the first reagent, the second reagent and calibration object, wherein:

First pack contains:

Second pack contains:

Be coated with the latex particle of anti-human Antithrombin III polyclonal antibody, 0.5 to 5mg/mL, preferred 1mg/mL; The wherein preferred 0.6mg/mL of anti-human Antithrombin III polyclonal antibody,

Wherein latex particle is the polystyrene latex of the carboxyl modified of particle diameter 150nm;

Calibration object comprises:

The latex enhancing immune turbidimetry detection kit of 13. 1 kinds of people's Antithrombin IIIs, it comprises the first reagent, the second reagent and calibration object, wherein:

First pack contains:

Second pack contains:

Be coated with the latex particle 1mg/mL of goat-anti people Antithrombin III polyclonal antibody, wherein goat-anti people Antithrombin III polyclonal antibody is 0.6mg/mL,

Wherein latex particle is the polystyrene latex of the carboxyl modified of particle diameter 150nm;

Calibration object comprises: