CN104634965A - Angiotensin I detection reagent kit as well as preparation method and application thereof - Google Patents

Angiotensin I detection reagent kit as well as preparation method and application thereof Download PDF

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Publication number
CN104634965A
CN104634965A CN201510069923.7A CN201510069923A CN104634965A CN 104634965 A CN104634965 A CN 104634965A CN 201510069923 A CN201510069923 A CN 201510069923A CN 104634965 A CN104634965 A CN 104634965A
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component
antigen
antibody
kit
angiotensini
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饶微
李钦
徐红
彭露
罗凯
李婷华
袁锦云
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Shenzhen New Industries Biomedical Engineering Co Ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/76—Chemiluminescence; Bioluminescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2410/00—Assays, e.g. immunoassays or enzyme assays, involving peptides of less than 20 animo acids
    • G01N2410/02—Angiotensins; Related peptides

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Abstract

The invention relates to a chemiluminescence immunity detection reagent kit for detecting angiotensin I and a preparation method thereof. The reagent kit comprises a component A and a component B, wherein the component A is an angiotensin I antigen or an adapter of an angiotensin I antigen and a protein carrier, the component B is an anti-angiotensin I antibody, one of the component A and the component B is marked with a trace marker, and the other one is coated with magnetic spheres. The invention further relates to a method for detecting concentration of the angiotensin I by utilizing the reagent kit. According to the angiotensin I detection reagent kit, the concentration of the angiotensin I is determined by utilizing the reagent kit, so that the sensitivity and accuracy in detection are high.

Description

AngiotensinⅠ detection kit and its preparation method and application
Technical field
The present invention relates to a kind of detection kit, being specifically related to a kind of chemiluminescence immune detection reagent kit for detecting angiotensinⅠ.The invention still further relates to the preparation method of this kit, and use this kit to detect the method for angiotensinⅠ concentration.
Background technology
AngiotensinⅠ (Angiotensin I, A I) acts on the proangiotensin of hepatic secretion by feritin and produces, and the speed that A I produces is one of important indicator representing RA (PRA).
Renin-angiotensin-aldosterone system (renin-angiotensin-aldosterone system, RAAS) be a Hormone system, when massive blood loss or blood pressure drops, renal secretion feritin, the hydrolysis of renin catalyzes proangiotensin produces A I, A I does not have biologic activity substantially, but intravascular angiotensin-converting enzyme (Angiotensin Converting Emzyme, ACE) is sheared C-end two amino acid residues and forms A II.A II has efficient vasoconstriction effect, thus blood pressure is raised.A II also can stimulate adrenocortical secretion aldosterone, and aldosterone can promote that kidney is to the heavily absorption of water and sodion, increases body fluid capacity, raising blood pressure then.
At present, the method measuring A I clinically mainly contains radioimmunoassays, euzymelinked immunosorbent assay (ELISA) etc.Such as, Beijing North Institute of Biological Technology produce iodine [ 125i] A I radioimmunoassay kit adopts competition law principle to measure A I content in blood plasma.Add sample and rabbit anti-A I antibody (blueness) and 125after I-A I label (redness), 125a I competition binding rabbit anti-A I antibody in I-A I label and sample, adds donkey anti-rabbit immune release agent, and after fully shaking up, room temperature places 15min, and 3000 revs/min of centrifugal 15min, remove supernatant, surveys the radiocounting (cpm) of sediment tube.A I concentration in sample is carried out quantitatively according to the Log-Logit mathematical model set up by calibration object concentration and corresponding radiocounting, thus detects A I content in sample.
But there is many deficiencies in radioimmunoassays and euzymelinked immunosorbent assay (ELISA).Such as, above-mentioned radioimmunoassays exist radioactive contamination, label half life period short, to operator, there is radioactive damage, and complex operation, length consuming time, sensitivity is low, and sensing range is narrow, and can not realize full-automatic defect.Traditional radioimmunoassays or euzymelinked immunosorbent assay (ELISA) method long for detection time, simultaneously mainly rely on the serial troublesome operation such as pure manual application of sample, efficiency is low, easily causes experimental result error large; Because enzymatic reaction is thorough not, and be subject to external interference factor impact, as temperature, time and material concentration impact, when therefore detecting, specificity is low, and poor sensitivity, sensing range is narrow.Therefore, this area is needed badly and is a kind ofly improved the safety and stability detecting reagent while highly sensitive, operates easier, can also realize the detection kit of the detection A I of testing process full-automation by analytical instrument.
Summary of the invention
In order to solve above-mentioned problems of the prior art, the object of this invention is to provide a kind of chemiluminescence immune detection reagent kit for detecting A I, to obtain high detection sensitivity and accuracy, the stability detecting reagent can also be improved, extend the holding time detecting reagent.
The present invention is also provided for the preparation method of the chemiluminescence immune detection reagent kit detecting A I.
In addition, the present invention also provides the application according to A I chemiluminescence immune detection reagent kit provided by the invention, especially this kit is adopted, the method of A I detection is carried out by Full-automatic chemiluminescence method, reduce the running time, reduce manual operation error, utilize the specificity of chemical tracing label simultaneously, improve detection sensitivity.
According to the present invention, provide a kind of chemiluminescence immune detection reagent kit for detecting A I, described kit comprises component A and B component, described component A is the connector of A I antigen or A I antigen and protein carrier, B component is anti-A I antibody, one mark trace labelling thing in described component A and B component, another kind of bag is by magnetic ball.
Should be understood that in mentioned reagent box provided by the invention, described anti-A I antibody can be one or more anti-A I monoclonal antibodies and/or anti-A I polyclonal antibody.In fact, mentioned in the present invention antibody can be monoclonal antibody and/or polyclonal antibody.
The protein carrier that the present invention is suitable for can be selected from least one in the protein carrier commonly used this area.Such as, described protein carrier is selected from least one in bovine serum albumin(BSA) (BSA), human serum albumins (HSA), albumin rabbit serum (RSA), hemocyanin (KLH), ox IgG, human IgG, ovalbumin (OVA), myoglobins and thyroglobulin.
According to the present invention, described trace labelling thing can be selected from this area the trace labelling thing being usually used in labelled antigen or antibody, such as be selected from least one in diamantane, luminol and derivant thereof, different luminol and derivant, acridinium ester, alkaline phosphatase and horseradish peroxidase, be preferably N-(4-ammonia butyl) the different luminol of-N-ethyl (ABEI).
According to the present invention, described trace labelling thing directly marks or indirect labelling A I antigen (or connector of itself and protein carrier) or anti-A I antibody.Wherein, indirect labelling includes but not limited to by fluorescein isothiocynate (FITC) and anti-FITC antibody system or Streptavidin (SA) and biotin (Biotin) system indirect labelling.Described " directly mark " refers to that ABEI directly marks with determined antigen or be connected for the antibody of determined antigen; Described " indirect labelling " refers to and makes ABEI mark A I antigen (or its connector) with protein carrier or anti-A I antibody by intermediary link system, and described intermediary links system and includes but not limited to FITC and anti-FITC antibody system or Streptavidin and biotin system.The present inventor finds, indirect labelling is conducive to weakening steric effect, is conducive to the amplification of signal, makes detection sensitiveer.
According to the present invention, A I antigen (or connector of itself and protein carrier) or anti-A I antibody direct coated magnetic ball, or indirectly wrapped by magnetic ball by FITC and anti-FITC antibody system or Streptavidin and biotin system.Described " direct coated " refers to and utilizes A I antigen (or connector of itself and protein carrier) or anti-A I antibody directly to carry out bag quilt to magnetic ball; Described " indirectly wrapping quilt " refers to and links system by intermediary, make A I antigen (or connector of itself and protein carrier) or anti-A I antibody carry out bag quilt to magnetic ball, described intermediary link system includes but not limited to FITC and anti-FITC antibody system or Streptavidin and biotin system.Equally, the advantage of indirect bag quilt is, is conducive to weakening steric effect, is conducive to the amplification of signal, makes detection sensitiveer.
Being applicable to magnetic ball of the present invention also referred to as magnetic bead, can be magnetic microsphere conventional in this area.Preferably, the magnetic ball that the present invention uses is by nano level Fe 2o 3or Fe 3o 4magnetic particle and high-molecular organic material carry out compound, and form the micron-sized solid phase microballoon with superparamagnetism and huge amount protein adsorption capacity, have and can be magnetized rapidly under additional magnetic fields, after withdrawing magnetic field, remanent magnetism is the attribute of zero.Wherein, the kind of described high-molecular organic material is not particularly limited, and can select as required.
It is 0.1-5 μm that magnetic microsphere used in the present invention should be able to meet diameter, and magnetic microsphere with various active functional group, can also include but not limited to-OH ,-COOH ,-NH by surface modification 2.
In a specific embodiment, described magnetic ball is Fe 2o 3or Fe 3o 4the complex of magnetic nano-particle and high-molecular organic material, and the particle diameter with 0.1-5 μm; Further, described magnetic ball optionally by surface modification with one or more activity functional groups.
According to embodiments more of the present invention, described kit comprises any one the component be selected from component A1 and component A2, and is selected from any one the component in B component 1, B component 2 and B component 3; Wherein, component A1 is A I antigen (or connector of itself and protein carrier) that ABEI directly marks; Component A2 is the ABEI of marked by streptavidin and biotinylated A I antigen (or connector of itself and protein carrier); B component 1 is the magnetic ball of anti-A I antibody direct coated; B component 2 is the magnetic ball of biotinylated anti-A I antibody and Streptavidin bag quilt; And B component 3 is the FITC of anti-A I antibody labeling and the magnetic ball of anti-FITC antibody bag quilt.
According to other embodiments of the present invention, described kit comprises any one the component be selected from component C1 and component C2, and is selected from any one the component in component D1, component D2 and component D3; Wherein, component C1 is anti-A I antibody that ABEI directly marks; Component C2 is the ABEI of marked by streptavidin and biotinylated anti-A I antibody; Component D1 is the magnetic ball of A I antigen (or connector of itself and protein carrier) direct coated; Component D2 is the magnetic ball of biotinylated A I antigen (or connector of itself and protein carrier) and Streptavidin bag quilt; And component D3 is the magnetic ball of the FITC that marks of A I antigen (or connector of itself and protein carrier) and anti-FITC antibody bag quilt.
According to the present invention, described kit can also comprise the low spot calibration object of A I antigen (or connector of itself and protein carrier) and high some calibration object, and optionally comprises damping fluid.Low spot calibration object of the present invention and high some calibration object be both comparatively speaking, wherein " low spot calibration object ", refer to that it is the calibration object that 0.2-2ng/ml obtains that A I antigen (or connector of itself and protein carrier) is diluted to concentration with 50% cow's serum goods; And " high some calibration object " refers to that it is the calibration object that 8-24ng/ml obtains that A I antigen (or connector of itself and protein carrier) is diluted to concentration with 50% cow's serum goods.
According to kit provided by the invention, each constituent concentration comprised is preferably as follows: AI antigen is 0.002-0.01mg/ml; Anti-AI antibody is 0.05-1mg/ml; Magnetic ball is 0.05-1mg/ml; FITC is 0.002-0.01mg/ml; Anti-FITC antibody is 0.05-1mg/ml; Streptavidin is 0.05-1mg/ml; Biotin is 0.002-0.01mg/ml; Trace labelling thing is 0.2-1mg/l; And if employ protein carrier, its concentration is 2-10mg/l.The concentration of above-mentioned each composition is all based on the gauge of independent reagent constituents comprising this composition.
In one embodiment of the present invention, in kit of the present invention, A I antigen (or connector of itself and protein carrier) marks ABEI, and with anti-A I antibody bag by magnetic ball.
Such as, in a specific embodiment of the present invention, described kit comprises the ABEI of mark A I antigen (or its connector) with protein carrier, the magnetic ball of anti-A I antibody bag quilt, low spot calibration object and highly puts calibration object.
Such as, in a specific embodiment of the present invention, described kit comprises the ABEI of mark A I antigen (or its connector) with protein carrier, biotinylated anti-A I antibody, the magnetic ball of Streptavidin bag quilt, low spot calibration object and highly puts calibration object.
Such as, in a specific embodiment of the present invention, described kit comprises the ABEI of mark A I antigen (or its connector) with protein carrier, the FITC of anti-A I antibody labeling, the magnetic ball of anti-FITC polyclonal antibody bag quilt, low spot calibration object and highly puts calibration object.
Such as, in a specific embodiment of the present invention, described kit comprises biotinylated A I antigen (or connector of itself and protein carrier), the ABEI of marked by streptavidin, the magnetic ball of anti-A I antibody bag quilt, low spot calibration object and high some calibration object.
In another embodiment of the invention, in kit of the present invention, anti-A I antibody marks ABEI, and wrap by magnetic ball with A I antigen (or connector of itself and protein carrier).
Such as, in a specific embodiment of the present invention, described kit comprises ABEI, A I antigen (or connector of itself and protein carrier) marking anti-A I antibody and wraps the magnetic ball of quilt, low spot calibration object and high some calibration object.
Such as, in a specific embodiment of the present invention, described kit comprises ABEI, the FITC of mark A I antigen (or its connector) with protein carrier, the magnetic ball of anti-FITC polyclonal antibody bag quilt, the low spot calibration object that mark anti-A I antibody and highly puts calibration object.
Such as, in a specific embodiment of the present invention, the magnetic ball that described kit comprises biotinylated anti-A I antibody, ABEI, A I antigen (or connector of itself and protein carrier) of labelled streptavidin wraps quilt, low spot calibration object and high some calibration object.
Such as, in a specific embodiment of the present invention, described kit comprises the ABEI, biotinylated A I antigen (or connector of itself and protein carrier), the magnetic ball of Streptavidin bag quilt, low spot calibration object and the high some calibration object that mark anti-A I antibody.
Present invention also offers a kind of method for the preparation of kit as above, described method comprises: the one in component A and B component is directly or indirectly marked trace labelling thing, directly or indirectly wraps another kind by magnetic ball.
According to method provided by the invention, described indirect labelling comprises trace labelling thing is marked described component A or B component by fluorescein isothiocynate and anti-fluorescein isothiocynate Antibody System or Streptavidin and biotin system.
According to method provided by the invention, described indirect bag is included and described component A or B component is indirectly wrapped by magnetic ball by fluorescein isothiocynate and anti-fluorescein isothiocynate Antibody System or Streptavidin and biotin system.
In some specific embodiments, method for the preparation of described kit comprises the following steps: i) A I antigen (or connector of itself and protein carrier) markers step, utilizes ABEI to mark A I antigen (or connector of itself and protein carrier) directly or indirectly; And ii) anti-A I antibody bag by magnetic ball step, anti-A I antibody is wrapped directly or indirectly by magnetic ball.
In other specific embodiments, the method for the preparation of described kit comprises the following steps: i ') anti-A I antibody labeling step, utilize ABEI to mark anti-A I antibody directly or indirectly; And ii ') A I antigen (or connector of itself and protein carrier) wraps by magnetic ball step, A I antigen (or connector of itself and protein carrier) is wrapped directly or indirectly by magnetic ball.
In some embodiments, in step I) in, by ABEI by FITC and anti-FITC antibody system or Streptavidin and biotin system indirect labelling A I antigen (or connector of itself and protein carrier); And/or at step I i) in, anti-A I antibody is wrapped by magnetic ball indirectly by FITC and anti-FITC antibody system or Streptavidin and biotin system.
In some embodiments, in step I ') in, by ABEI by FITC and anti-FITC antibody system or Streptavidin and biotin system indirect labelling anti-A I antibody; And/or at step I i ') in, A I antigen (or connector of itself and protein carrier) is wrapped by magnetic ball indirectly by FITC and anti-FITC antibody system or Streptavidin and biotin system.
Kit preparation method according to the present invention can also comprise the configuration of low spot calibration object and high some calibration object, can further include the assembling of kit.
According to the present invention, additionally provide a kind of method detecting A I concentration, described method is comprised use kit as above and is detected A I concentration in testing sample by Chemiluminescence immunoassay.
In one embodiment, the method for described detection A I concentration comprises use kit as above, detects A I concentration by chemical illumination immunity analysis instrument.In a preferred embodiment in accordance with this invention, described method is fully automatically carried out.According to the present invention, described chemical illumination immunity analysis instrument is preferably Maglumi sequence of chemical luminescence immunoassay instrument (production of Shenzhen NPD projects biomedical engineering incorporated company).
Particularly, mark ABEI on A I antigen (or connector of itself and protein carrier), utilize anti-A I antibody bag by the situation of magnetic ball, the detecting step carrying out chemiluminescence detection can comprise: 1) obtain sample to be tested.Sample to be tested can be the serum, blood plasma and the whole blood that directly obtain, also can be to carry out separation by extraction Human Blood to obtain, or the sample after enzyme inhibitor process; 2) utilize A I antigen of band ABEI mark (or its connector) with protein carrier and carry out incubation after mixing with sample to be tested with the magnetic ball of anti-A I antibody, obtain reaction product; 3) upper machine testing chemiluminescence signal after cleaning, obtains corresponding optical signal data; 4) analyze corresponding light signal data, obtain A I antigenic content.
Mark ABEI on anti-A I antibody, utilize A I antigen (or connector of itself and protein carrier) to wrap by the situation of magnetic ball, the detecting step carrying out chemiluminescence detection can comprise: 1) obtain sample to be tested.Sample to be tested can be the serum, blood plasma and the whole blood that directly obtain, also can be to carry out separation by extraction Human Blood to obtain, or the sample after enzyme inhibitor process; 2) carry out incubation by anti-A I antibody of band ABEI mark with after mixing with sample to be tested with the magnetic ball of A I antigen (or its connector) with protein carrier, obtain reaction product; 3) upper machine testing chemiluminescence signal after cleaning, obtains corresponding optical signal data; 4) analyze corresponding light signal data, obtain A I content.
The present invention still further provides the assay method of RA, uses detection kit provided by the invention to measure A I concentration, and then obtains RA value.
Beneficial effect of the present invention is:
1. specificity is high, and sensitivity is good, and sensing range is wider.
2. operation is more simple and easy to do, kit of the present invention can support the use with chemical illumination immunity analysis instrument (especially Maglumi sequence of chemical luminescence immunoassay instrument) simultaneously, full-automation is achieved in sample mensuration process, the detection of A I concentration simply, easily and fast, in bulk can be carried out, ensure that the systematic error detected is less simultaneously.
3. the present invention detects A I by Chemiluminescence immunoassay, avoids the radioactively labelled substance using contaminated environment, be detrimental to health, safer, environmentally friendly; Meanwhile, label of the present invention not only safety, also very stable, overcome the half life period of the label existed in the art methods such as radioimmunoassays short problem.
Embodiment
Below in conjunction with non-limiting example, the present invention is made further explanation and description.It is to be noted, however, that following detailed description of the present invention does not make any restriction to the present invention.It will be understood by those skilled in the art that and can modify to the details of technical solution of the present invention and form or replace down without departing from the spirit and scope of the present invention, but these amendments and replacement all fall within the scope of protection of the present invention.
In following examples:
A I antigen: be purchased from Sigma company;
Anti-A I antibody: be purchased from Biogenesis company;
Goat-anti FITC polyclonal antibody: be purchased from Jackson company of the U.S.;
FITC: purchased from Shanghai Ji Ning Industrial Co., Ltd.;
Magnetic microsphere is produced by Shenzhen New Industries Biomedical Engineering Co., Ltd., and 80% domain size distribution is 1-5 μm, and when the magnetization is 4000 Gauss, the settling time is 10-15 second, and when BSA is 30mg, protein adsorption concentration is 0.8mg-1.2mg;
Biotin, Streptavidin: all purchased from American Biosources companies;
ABEI: provided by Shenzhen New Industries Biomedical Engineering Co., Ltd.;
Maglumi 2000 chemiluminescent analyzer is provided by Shenzhen New Industries Biomedical Engineering Co., Ltd..
Embodiment 1
(1) mark of A I antigen, concrete steps are as follows:
The preparation of dislysate (F solution): add Na in 5000ml container 2cO 314.31g and NaHCO 326.46g, adds purified water and is diluted to 4500ml, is placed on magnetic stirring apparatus for subsequent use by the F solution prepared.
Select the bag filter of suitable interception (conventional molecular weight 14000), measure the size being large enough to hold F solution, after wetting, tighten one end, purified water leak test 3 times (needing without leakage).
The carbonic acid buffer (F solution) getting 100 μ g A I antigen 0.1mol/L pH9.5 adjusts to 1ml.Put into dislysate, stirring at room temperature is dialysed 2 hours, the solution of having dialysed is added 300 μ g ABEI Acibenzolars, and 37 DEG C are reacted 2 hours.
By the connection product of G-25 gel column purifying A I antigen and ABEI.
D 2the preparation of solution: add the 0.5M phosphate buffer (P001 solution) of 200ml, 20g BSA, 8g NaN in 2000ml beaker 3, 2g MgCl 26H 2o, 600ml glycerine, adds purified water and is diluted to 2000ml, filters.
By connection product D good for purifying 2solution two-fold dilution, namely obtains A I antigen being marked with ABEI.
(2) anti-A I antibody bag is by magnetic ball, and concrete steps are as follows:
The preparation of solution A: take 2.55g sodium acetate trihydrate and put into 5000ml beaker, measures 4500ml purified water with graduated cylinder and pours beaker into, after adding the mixing of 14ml acetic acid again, adds purified water and is diluted to 5000ml (pH is 3.6) after to be dissolved.
In little Bai bottle, adding 5 times to wrapping by the solution A of volume, putting into the ultrasonic one side stirring and washing 2-3 minute of ultrasonic instrument, being then placed on magnet, after supernatant is limpid, pouring out supernatant.This step in triplicate.
Be that 1 μm of magnetic bead adds with bag by the pH3.6 acetate buffer solution of volume equivalent by particle diameter, the suspended concentration of magnetic bead is made to be 20mg/ml, add 1-cyclohexyl-2-morpholine ethyl carbodiimide tosilate (CMC) again, make its concentration be 10mg/ml, add anti-A I antibody of purifying.
Bead suspension is put into isothermal vibration water bath 37 DEG C reaction 24 hours (shaking water bath pot jolting speed: 260rpm).
With P001: purified water=1:9 volume ratio preparation pH 7.4 phosphate buffer (PBS) 500ml, add 2.5g BSA and dissolve, mixing, is magnetic bead cleaning fluid.
The bead suspension of temperature being bathed is poured in beaker, is then placed in after magnet precipitates, outwells supernatant, add the magnetic bead cleaning fluid stirring and washing of 5 times of volumes, be then placed on magnet, after supernatant is limpid, outwells supernatant, repeat this cleaning step four times.
C solution is prepared: take MC (methylcellulose) 160g and pour in 5000ml beaker, add purified water to 4000ml, stirs molten 2 hours as heating in the water baths of 90 DEG C.Separately get 4000ml 0.5M phosphate buffer, add 80g NaN 3(analyzing pure), 80ml Tween-20 (analyzing pure), mixing, filters.Add 200g BSA after fully being mixed by these two parts of solution, add water to 40000ml.
Be suspended in C solution by the magnetic bead after cleaning, suspended concentration is 20mg/ml, and namely obtain the magnetic ball of anti-A I antibody bag quilt, this suspension vol is described in the present embodiment and wraps by volume.
Above-mentioned magnetic ball suspending liquid is diluted to the suspending liquid counting 0.1mg/ml with magnetic ball further, for subsequent use.
(3) preparation of A I low spot calibration object, high some calibration object
A I antigen 50% cow's serum goods are diluted to by different proportion two high and low calibration object scaling points that concentration is respectively 14.386ng/ml and 0.667ng/ml.
(4) assemble
Be assembled into kit by after the packing of mentioned reagent composition, be stored in 2 ~ 8 DEG C.
Embodiment 2
(1) mark of A I antigen, concrete steps are as follows:
The configuration of dislysate (F solution): add Na in 5000ml container 2cO 314.31g, NaHCO 326.46g, adds purified water and is diluted to 4500ml.The F solution prepared is placed on magnetic stirring apparatus for subsequent use.
Select the bag filter of suitable interception (conventional molecular weight 14000), measure the size being large enough to hold F solution, after wetting, tighten one end, purified water leak test 3 times (needing without leakage).
The carbonic acid buffer (F solution) getting 100 μ g A I antigen 0.1mol/L pH9.5 adjusts to 1ml.Put into dislysate, stirring at room temperature is dialysed 2 hours, the solution of having dialysed is added 300 μ g ABEI Acibenzolars, and 37 DEG C are reacted 2 hours.
By the connection product of G-25 gel column purifying A I antigen and ABEI.
D 2the preparation of solution: add 200ml 0.5M phosphate buffer, 20g BSA, 8g NaN in 2000ml beaker 3, 2g MgCl 26H 2o, 600ml glycerine, adds purified water and is diluted to 2000ml, filters.
The connection product body D that purifying is good 2solution two-fold dilution.
(2) goat-anti FITC polyclonal antibody bag is by magnetic ball, and concrete steps are as follows:
Solution A is prepared by method in embodiment 1.
In little Bai bottle, adding 5 times to wrapping by the solution A of volume, putting into the ultrasonic one side stirring and washing 2-3 minute of ultrasonic instrument, being then placed on magnet, after supernatant is limpid, pouring out supernatant.This step in triplicate.
Magnetic bead is added bag by the pH3.6 acetate buffer solution of volume equivalent, the suspended concentration making magnetic bead is 20mg/ml, then adds CMC (concentration is 10mg/ml), adds the goat-anti FITC polyclonal antibody of purifying.
Bead suspension is put into isothermal vibration water bath 37 DEG C reaction 24 hours (shaking water bath pot jolting speed: 260rpm).
With P001: the PBS damping fluid 500ml of purified water=1:9 volume ratio preparation pH7.4, adds 2.5g BSA and mix dissolving, be magnetic bead cleaning fluid.
The bead suspension of temperature being bathed is poured in beaker, is then placed in after magnet precipitates, outwells supernatant, add the magnetic bead cleaning fluid stirring and washing of 5 times of volumes, be then placed on magnet, after supernatant is limpid, outwells supernatant, repeat this cleaning step four times.
C solution is prepared according to the method for embodiment 1.
Be suspended in C solution by the magnetic bead after cleaning, suspension concentration is 20mg/ml, and namely obtain the magnetic ball suspending liquid of goat-anti FITC polyclonal antibody bag quilt, this suspension vol is described in the present embodiment and wraps by volume.
Above-mentioned magnetic ball suspending liquid is diluted to the suspending liquid counting 0.5mg/ml with magnetic ball further, for subsequent use.
(3) anti-A I antibody labeling, concrete steps are as follows:
The carbonic acid buffer (F solution) getting 1mg anti-A I antibody 0.1mol/L pH9.5 adjusts to 1ml.Put into dislysate, stirring at room temperature is dialysed 2 hours, and the solution of having dialysed is added 300 μ g FITC, room temperature is shaken while react 24h.
By the connection product of G-25 gel column purifying anti-A I antibody and FITC.
C 2the preparation of solution: add 200ml 0.5M phosphate buffer, 20g BSA, 8g NaN 3, 2g MgCl 26H 2o, add purified water and be diluted to 2000ml (filtration).
Prepare C 2after solution, with the connection product C that purifying is good 2solution two-fold dilution.
(4) preparation of A I low spot calibration object, high some calibration object
It is the high and low calibration object scaling point of 15.50ng/ml and 0.85ng/ml two that A I antigen is diluted to concentration with 50% cow's serum goods by different proportion.
(5) assemble
Be assembled into kit by after the packing of mentioned reagent composition, be stored in 2 ~ 8 DEG C.
Embodiment 3
(1) mark of anti-A I antibody, concrete steps are as follows:
The configuration of dislysate (F solution): add Na in 5000ml beaker 2cO 314.31g, NaHCO 326.46g, adds purified water and is diluted to 4500ml, is placed on magnetic stirring apparatus for subsequent use by the F solution prepared.
Select the bag filter of suitable interception (conventional molecular weight 14000), measure the size being enough to hold F solution, after wetting, tighten one end, purified water leak test 3 times (needing without leakage).
The carbonic acid buffer (F solution) getting 1mg anti-A I antibody 0.1mol/L pH9.5 adjusts to 1ml.Put into dislysate, stirring at room temperature is dialysed 2 hours, the solution of having dialysed is added 300 μ g ABEI Acibenzolars, and 37 DEG C are reacted 2 hours.
By the connection product of G-25 gel column purifying anti-A I antibody and ABEI.
D is prepared by method in embodiment 1 2solution.
By connection product D good for purifying 2solution two-fold dilution.
(2) the antigen coated magnetic ball of A I, concrete steps are as follows:
Solution A is prepared by the method in embodiment 1.
In little Bai bottle, adding 5 times to wrapping by the solution A of volume, putting into the ultrasonic one side stirring and washing 2-3 minute of ultrasonic instrument, being then placed on magnet, after supernatant is limpid, pouring out supernatant.This step in triplicate.
Magnetic bead is added bag by the pH3.6 acetate buffer solution of volume equivalent, the suspended concentration making magnetic bead is 20mg/ml, then adds CMC (concentration is 10mg/ml), adds A I antigen of purifying by certain ratio
Magnetic bead is put into isothermal vibration water bath 37 DEG C reaction 24 hours (shaking water bath pot jolting speed: 260rpm).
With P001: purified water=1:9 volume ratio preparation pH7.4PBS damping fluid 500ml, adds 2.5g BSA and mix dissolving, be magnetic bead cleaning fluid.
The bead suspension of temperature being bathed is poured in beaker, is then placed in after magnet precipitates, outwells supernatant, add the magnetic bead cleaning fluid stirring and washing of 5 times of volumes, be then placed on magnet, after supernatant is limpid, outwells supernatant, repeat this cleaning step four times.
C solution is prepared according to the method for embodiment 1.Be suspended in C solution by the magnetic bead after cleaning, suspended concentration is 20mg/ml, and namely obtain the magnetic ball suspending liquid of anti-A I antibody bag quilt, this suspension vol is described in the present embodiment and wraps by volume.
Above-mentioned magnetic ball suspending liquid is diluted to the suspending liquid counting 1mg/ml with magnetic ball further, for subsequent use.
(3) A I low spot calibration object, high some calibration object
A I antigen 50% cow's serum goods are diluted to by different proportion two high and low calibration object scaling points that concentration is respectively 14.718ng/ml and 0.780ng/ml.
(4) assemble
Be assembled into kit by after the packing of mentioned reagent composition, be stored in 2 ~ 8 DEG C.
Embodiment 4
(1) mark of anti-A I antibody, concrete steps are as follows:
The configuration of dislysate (F solution): add Na in 5000ml beaker 2cO 314.31g, NaHCO 326.46g, adding water is settled to the F solution that 4500ml. prepares and is placed on magnetic stirring apparatus for subsequent use.
Select the bag filter of suitable interception (conventional molecular weight 14000), measure the size being large enough to hold F solution, after wetting, tighten one end, purified water leak test 3 times (needing without leakage).
The carbonic acid buffer (F solution) getting 1mg anti-A I antibody 0.1mol/L pH9.5 adjusts to 1ml.Put into dislysate, stirring at room temperature is dialysed 2 hours, the solution of having dialysed is added 300 μ g ABEI Acibenzolars, and 37 DEG C are reacted 2 hours.
By the connection product of G-25 gel column purifying anti-A I antibody and ABEI.
D is prepared according to the method for embodiment 1 2solution.
By connection product D good for purifying 2solution two-fold dilution.
(2) goat-anti FITC polyclonal antibody bag is by magnetic ball, and concrete steps are as follows:
Solution A is prepared by method in embodiment 1.
In little Bai bottle, adding 5 times to wrapping by the solution A of volume, putting into the ultrasonic one side stirring and washing 2-3 minute of ultrasonic instrument, being then placed on magnet, after supernatant is limpid, pouring out supernatant.This step in triplicate.
Magnetic bead is added bag by the pH3.6 acetate buffer solution of volume equivalent, make magnetic bead suspended concentration be 20mg/ml, then add CMC (concentration is 10mg/ml), add the goat-anti FITC polyclonal antibody of purifying by certain ratio.
Magnetic bead is put into isothermal vibration water bath 37 DEG C reaction 24 hours (shaking water bath pot jolting speed: 260rpm).
With P001: purified water=1:9 volume ratio preparation pH7.4PBS damping fluid 500ml, adds 2.5g BSA mixing and dissolve, be magnetic bead cleaning fluid.
The bead suspension of temperature being bathed is poured in beaker, is then placed in after magnet precipitates, outwells supernatant, add the magnetic bead cleaning fluid stirring and washing of 5 times of volumes, be then placed on magnet, after supernatant is limpid, outwells supernatant, repeat this cleaning step four times.
C solution is prepared according to the method for embodiment 1.
Be suspended in C solution by the magnetic bead after cleaning, suspended concentration is 20mg/ml, and namely obtain the magnetic ball suspending liquid of goat-anti FITC polyclonal antibody bag quilt, this suspension vol is described in the present embodiment and wraps by volume.
Above-mentioned magnetic ball suspending liquid is diluted to the suspending liquid counting 0.5mg/ml with magnetic ball further, for subsequent use.
(3) A I antigenic mark, concrete steps are as follows:
The configuration of dislysate (F solution), adds Na in 5000ml beaker 2cO 314.31g, NaHCO 326.46g, adds water and is settled to 4500ml, and the F solution prepared is placed on magnetic stirring apparatus for subsequent use.
Select the bag filter of suitable interception (conventional 14000), measure suitable size, after wetting, tighten one end, purified water leak test 3 times (needing without leakage).
The carbonic acid buffer (F solution) getting 100 μ g A I antigen 0.1mol/L pH9.5 adjusts to 1ml.Put into dislysate, stirring at room temperature is dialysed 2 hours, and the solution of having dialysed is added 300 μ g FITC, room temperature is shaken while react 24h.
By the connection product of G-25 gel column purifying A I antigen and FITC.
C 2the preparation of solution: add 200ml P001 solution, 20g BSA, 8g NaN 3, 2g MgCl 26H 2o, constant volume are to 2000ml (filtration).
The connection product body C that purifying is good 2solution two-fold dilution.
(4) preparation of A I low spot calibration object, high some calibration object
A I antigen is diluted to concentration with 50% cow's serum goods by different proportion and is respectively the high and low calibration object scaling point of 16.480ng/ml and 2.213ng/ml two.
(5) assemble
Be assembled into kit by after the packing of mentioned reagent composition, be stored in 2 ~ 8 DEG C.
Embodiment 5
(1) mark of anti-A I antibody, concrete steps are as follows:
The carbonic acid buffer (F solution) getting 1mg anti-A I antibody 0.1mol/L pH9.5 adjusts to 1ml.Put into dislysate, stirring at room temperature is dialysed 2 hours, the solution of having dialysed is added 300 μ g ABEI Acibenzolars, and 37 DEG C are reacted 2 hours.
By the connection product of G-25 gel column purifying anti-A I antibody and ABEI.
According to the method preparation D of embodiment 1 2solution, the connection product D that purifying is good 2solution two-fold dilution.
(2) A I antigen biotinylation
The carbonic acid buffer (F solution) getting 100 μ g biotins and 100 μ g A I antigen 0.1mol/L pH9.5 adjusts to 1ml.Put into dislysate, stirring at room temperature is dialysed 2 hours, and 37 DEG C are reacted 2 hours.
By G-25 gel column purifying.
By connection product D good for purifying 2solution two-fold dilution.
(3) SA bag is by magnetic ball, and concrete steps are as follows:
According to the method preparation solution A of embodiment 1.
In little Bai bottle, adding 5 times to wrapping by the solution A of volume, putting into the ultrasonic one side stirring and washing 2-3 minute of ultrasonic instrument, being then placed on magnet, after supernatant is limpid, pouring out supernatant.This step in triplicate.
Added by magnetic bead with bag by the pH3.6 acetate buffer solution of volume equivalent, the suspended concentration making magnetic bead is 20mg/ml, then adds CMC (concentration is 10mg/ml), adds the SA of purifying.
Bead suspension is put into isothermal vibration water bath 37 DEG C reaction 24 hours (shaking water bath pot jolting speed: 260rpm).
With P001: the PBS damping fluid 500ml of purified water=1:9 volume ratio preparation pH7.4, adds 2.5g BSA and mix dissolving, be magnetic bead cleaning fluid.
The bead suspension of temperature being bathed is poured in beaker, is then placed in after magnet precipitates, outwells supernatant, add the magnetic bead cleaning fluid stirring and washing of 5 times of volumes, be then placed on magnet, after supernatant is limpid, outwells supernatant, repeat this cleaning step four times.
C solution is prepared according to the method for embodiment 1.
Be suspended in C solution by the magnetic bead after cleaning, suspended concentration is 20mg/ml, and namely obtain the magnetic ball suspending liquid of SA bag quilt, this suspension vol is described in the present embodiment and wraps by volume.
Above-mentioned magnetic ball suspending liquid is diluted to the suspending liquid counting 0.1mg/ml with magnetic ball further, for subsequent use.
(4) preparation of A I low spot calibration object, high some calibration object
It is the high and low calibration object scaling point of 16.668ng/ml and 1.667ng/ml two that A I antigen is diluted to concentration with 50% cow's serum goods by different proportion.
(5) assemble
Be assembled into kit by after the packing of mentioned reagent composition, be stored in 2 ~ 8 DEG C.
Embodiment 6
(1) anti-A I antibody biotin, concrete steps are as follows:
The carbonic acid buffer (F solution) getting 100 μ g biotins and 1mg anti-A I antibody 0.1mol/L pH9.5 adjusts to 1ml.Put into dislysate, stirring at room temperature is dialysed 2 hours, and 37 DEG C are reacted 2 hours.
By G-25 gel column purifying.
Preparation D2 solution, the connection product D that purifying is good 2solution two-fold dilution.
(2) mark of SA
The carbonic acid buffer (F solution) getting 100 μ g SA 0.1mol/L pH9.5 adjusts to 1ml.Put into dislysate, stirring at room temperature is dialysed 2 hours, the solution of having dialysed is added 300 μ g ABEI Acibenzolars, and 37 DEG C are reacted 2 hours.
G-25 gel column purifying.
The connection product D that purifying is good 2solution two-fold dilution.
(3) the antigen coated magnetic ball of A I, concrete steps are as follows:
Solution A is prepared according to the method for embodiment 1.
In little Bai bottle, adding 5 times to wrapping by the solution A of volume, putting into the ultrasonic one side stirring and washing 2-3 minute of ultrasonic instrument, being then placed on magnet, after supernatant is limpid, pouring out supernatant.This step in triplicate.
Magnetic bead is added bag by the pH3.6 acetate buffer solution of volume equivalent, the suspended concentration making magnetic bead is 20mg/ml, then adds CMC (concentration is 10mg/ml), adds A I antigen of purifying
Bead suspension is put into isothermal vibration water bath 37 DEG C reaction 24 hours (shaking water bath pot jolting speed: 260rpm).
With P001: purified water=1:9 volume ratio preparation pH7.4PBS damping fluid 500ml, adds 2.5g BSA and mix dissolving, be magnetic bead cleaning fluid.
The bead suspension of temperature being bathed is poured in beaker, is then placed in after magnet precipitates, outwells supernatant, add the magnetic bead cleaning fluid stirring and washing of 5 times of volumes, be then placed on magnet, after supernatant is limpid, outwells supernatant, repeat this cleaning step four times.
According to the method preparation C solution of embodiment 1.
Be suspended in C solution by the magnetic bead after cleaning, suspended concentration is 20mg/ml, and namely obtain the magnetic ball suspending liquid of anti-A I antibody bag quilt, this suspension vol is described in the present embodiment and wraps by volume.
Above-mentioned magnetic ball suspending liquid is diluted to the suspending liquid counting 0.1mg/ml with magnetic ball further, for subsequent use.
(4) A I low spot calibration object, high some calibration object
It is the high and low calibration object scaling point of 15.386ng/ml and 1.367ng/ml two that A I antigen is diluted to concentration with 50% cow's serum goods by different proportion.
(5) assemble
Be assembled into kit by after the packing of mentioned reagent composition, be stored in 2 ~ 8 DEG C.
Embodiment 7
(1) mark of A I antigen-BSA, concrete steps are as follows:
The preparation of dislysate (F solution): add Na in 5000ml container 2cO 314.31g and NaHCO 326.46g, adds purified water and is diluted to 4500ml, is placed on magnetic stirring apparatus for subsequent use by the F solution prepared.
Select the bag filter of suitable interception (conventional molecular weight 14000), measure the size being large enough to hold F solution, after wetting, tighten one end, purified water leak test 3 times (needing without leakage).
The carbonic acid buffer (F solution) getting 100 μ g A I antigen-BSA albumen connector 0.1mol/L pH9.5 adjusts to 1ml.Put into dislysate, stirring at room temperature is dialysed 2 hours, the solution of having dialysed is added 300 μ g ABEI Acibenzolars, and 37 DEG C are reacted 2 hours.
By the connection product of G-25 gel column purifying A I antigen-BSA albumen connector and ABEI.
D 2the preparation of solution: add the 0.5M phosphate buffer (P001 solution) of 200ml, 20g BSA, 8g NaN in 2000ml beaker 3, 2g MgCl 26H 2o, 600ml glycerine, adds purified water and is diluted to 2000ml, filters.
By connection product D good for purifying 2solution two-fold dilution, namely obtains the A I antigen-BSA albumen connector being marked with ABEI.
(2) anti-A I antibody bag is by magnetic ball, and concrete steps are as follows:
The preparation of solution A: take 2.55g sodium acetate trihydrate and put into 5000ml beaker, measures 4500ml purified water with graduated cylinder and pours beaker into, after adding the mixing of 14ml acetic acid again, adds purified water and is diluted to 5000ml (pH is 3.6) after to be dissolved.
In little Bai bottle, adding 5 times to wrapping by the solution A of volume, putting into the ultrasonic one side stirring and washing 2-3 minute of ultrasonic instrument, being then placed on magnet, after supernatant is limpid, pouring out supernatant.This step in triplicate.
Be that 1 μm of magnetic bead adds with bag by the pH3.6 acetate buffer solution of volume equivalent by particle diameter, the suspended concentration making magnetic bead is 20mg/ml, then adds CMC (concentration is 10mg/ml), adds anti-A I antibody of purifying.
Bead suspension is put into isothermal vibration water bath 37 DEG C reaction 24 hours (shaking water bath pot jolting speed: 260rpm).
With P001: purified water=1:9 volume ratio preparation pH7.4 phosphate buffer (PBS) 500ml, add 2.5g BSA and dissolve, mixing, is magnetic bead cleaning fluid.
The bead suspension of temperature being bathed is poured in beaker, is then placed in after magnet precipitates, outwells supernatant, add the magnetic bead cleaning fluid stirring and washing of 5 times of volumes, be then placed on magnet, after supernatant is limpid, outwells supernatant, repeat this cleaning step four times.
C solution is prepared according to the method for embodiment 1.
Be suspended in C solution by the magnetic bead after cleaning, suspended concentration is 20mg/ml, and namely obtain the magnetic ball of anti-A I antibody bag quilt, this suspension vol is described in the present embodiment and wraps by volume.
Above-mentioned magnetic ball suspending liquid is diluted to the suspending liquid counting 0.1mg/ml with magnetic ball further, for subsequent use.
(3) preparation of A I low spot calibration object, high some calibration object
A I antigen 50% cow's serum goods are diluted to by different proportion two high and low calibration object scaling points that concentration is respectively 14.386ng/ml and 0.667ng/ml.
(4) assemble
Be assembled into kit by after the packing of mentioned reagent composition, be stored in 2 ~ 8 DEG C.
Embodiment 8
(1) mark of anti-A I antibody, concrete steps are as follows:
The configuration of dislysate (F solution): add Na in 5000ml beaker 2cO 314.31g, NaHCO 326.46g, adding water is settled to the F solution that 4500ml. prepares and is placed on magnetic stirring apparatus for subsequent use.
Select the bag filter of suitable interception (conventional molecular weight 14000), measure the size being large enough to hold F solution, after wetting, tighten one end, purified water leak test 3 times (needing without leakage).
The carbonic acid buffer (F solution) getting 1mg anti-A I antibody 0.1mol/L pH9.5 adjusts to 1ml.Put into dislysate, stirring at room temperature is dialysed 2 hours, the solution of having dialysed is added 300 μ μ g ABEI Acibenzolars, and 37 DEG C are reacted 2 hours.
By the connection product of G-25 gel column purifying anti-A I antibody and ABEI.
D is prepared according to the method for embodiment 1 2solution.
By connection product D good for purifying 2solution two-fold dilution.
(2) goat-anti FITC polyclonal antibody bag is by magnetic ball, and concrete steps are as follows:
Solution A is prepared by method in embodiment 1.
In little Bai bottle, adding 5 times to wrapping by the solution A of volume, putting into the ultrasonic one side stirring and washing 2-3 minute of ultrasonic instrument, being then placed on magnet, after supernatant is limpid, pouring out supernatant.This step in triplicate.
Magnetic bead is added bag by the pH3.6 acetate buffer solution of volume equivalent, make magnetic bead suspended concentration be 20mg/ml, then add CMC (concentration is 10mg/ml), add the goat-anti FITC polyclonal antibody of purifying by certain ratio.
Magnetic bead is put into isothermal vibration water bath 37 DEG C reaction 24 hours (shaking water bath pot jolting speed: 260rpm).
With P001: purified water=1:9 volume ratio preparation pH7.4PBS damping fluid 500ml, adds 2.5g BSA mixing and dissolve, be magnetic bead cleaning fluid.
The bead suspension of temperature being bathed is poured in beaker, is then placed in after magnet precipitates, outwells supernatant, add the magnetic bead cleaning fluid stirring and washing of 5 times of volumes, be then placed on magnet, after supernatant is limpid, outwells supernatant, repeat this cleaning step four times.
C solution is prepared according to the method for embodiment 1.
Be suspended in C solution by the magnetic bead after cleaning, suspended concentration is 20mg/ml, and namely obtain the magnetic ball suspending liquid of goat-anti FITC polyclonal antibody bag quilt, this suspension vol is described in the present embodiment and wraps by volume.
Above-mentioned magnetic ball suspending liquid is diluted to the suspending liquid counting 0.1mg/ml with magnetic ball further, for subsequent use.
(3) A I antigen-BSA albumen connector mark, concrete steps are as follows:
The configuration of dislysate (F solution), adds Na in 5000ml beaker 2cO 314.31g, NaHCO 326.46g, adds water and is settled to 4500ml, and the F solution prepared is placed on magnetic stirring apparatus for subsequent use.
Select the bag filter of suitable interception (conventional 14000), measure suitable size, after wetting, tighten one end, purified water leak test 3 times (needing without leakage).
The carbonic acid buffer (F solution) getting 100 μ g A I antigen-BSA albumen connector 0.1mol/L pH9.5 adjusts to 1ml.Put into dislysate, stirring at room temperature is dialysed 2 hours, and the solution of having dialysed is added 300 μ gFITC, room temperature is shaken while react 24h.
By the connection product of G-25 gel column purifying A I antigen-BSA albumen connector and FITC.
C 2the preparation of solution: add 200ml P001 solution, 20g BSA, 8g NaN 38g, 2g MgCl 26H 2o, constant volume are to 2000ml (filtration).
The connection product body C that purifying is good 2solution two-fold dilution.
(4) preparation of A I low spot calibration object, high some calibration object
A I antigen is diluted to concentration with 50% cow's serum goods by different proportion and is respectively the high and low calibration object scaling point of 16.480ng/ml and 2.213ng/ml two.
(5) assemble
Be assembled into kit by after the packing of mentioned reagent composition, be stored in 2 ~ 8 DEG C.
Embodiment 9: utilize detection kit to carry out chemiluminescence detection A I
A I detection kit using above-described embodiment 1-8 to prepare and Maglumi 2000 chemiluminescent analyzer, detected A I concentration of sample by chemiluminescence immunoassay competition law, sample to be tested is 160 routine clinical samples.A I concentration and relative light intensity (Relative Light Unit, RLU) become certain proportionate relationship, can use analyzer the Fitting Calculation A I concentration automatically.
The concrete steps that the kit choosing gained in embodiment 1 carries out chemoluminescence method detection are described below:
1, on sample rack, calibration object or sample to be tested is loaded successively.Sample to be tested is the sample after enzyme inhibitor process, and this sample is divided into two parts, and portion is placed in 2-8 DEG C, and portion is placed in 37 DEG C, and the sample of two kinds of disposal routes, after one hour, is placed in sample rack by accurate timing.
2, sample rack is inserted the sample storehouse of Maglumi 2000 chemiluminescent analyzer, editing sample number brings into operation test, concrete load procedure is: calibration object or sample to be tested add 100 μ l, then the A I antigenic solution 50 μ l of trace labelling substance markers is added, add the magnetic microsphere suspending liquid 20 μ l of anti-A I antibody bag quilt, mixing, 37 DEG C of incubations 15 minutes, instrument automatically cleans after twice and directly enters the light intensity signal that measuring chamber obtains each sample, is gone out the A I antigen concentration value of sample to be tested by ten point curves and two-point calibration automatic Fitting.Thus same sample obtains different concentration values under two kinds of Temperature Treatment, the concentration of specimens value that 37 DEG C process is deducted the concentration of specimens value of 2-8 DEG C of process, namely obtain RA (PRA).Testing result is in table 1.
The concrete steps that the kit choosing gained in embodiment 4 carries out chemoluminescence method detection are described below:
1, on sample rack, calibration object or sample to be tested is loaded successively.Sample to be tested is the sample after enzyme inhibitor process, and this sample is divided into two parts, and portion is placed in 2-8 DEG C, and portion is placed in 37 DEG C, and the sample of two kinds of disposal routes, after one hour, is placed in sample rack by accurate timing.
2, sample rack is inserted the sample storehouse of Maglumi sequence of chemical luminescence analyzer, editing sample number brings into operation test, concrete load procedure is: calibration object or sample to be tested add 100 μ l, then label the anti-A I antibody-solutions 50 μ l of trace labelling thing, add the FITC 50 μ l of A I antigenic mark, add the magnetic microsphere suspending liquid 20 μ l of anti-FITC antibody bag quilt, mixing, 37 DEG C of incubations 15 minutes, instrument automatically cleans after twice and directly enters the light intensity signal that measuring chamber obtains each sample, the A I antigen concentration value of sample to be tested is gone out by ten point curves and two-point calibration automatic Fitting.Thus same sample obtains different concentration values under two kinds of Temperature Treatment, the concentration of specimens value that 37 DEG C process is deducted the concentration of specimens value of 2-8 DEG C of process, namely obtain RA (PRA).Testing result is in table 1.
The concrete steps that the kit choosing gained in embodiment 7 carries out chemoluminescence method detection are described below:
1, on sample rack, calibration object or sample to be tested is loaded successively.Sample to be tested is the sample after enzyme inhibitor process, and this sample is divided into two parts, and portion is placed in 2-8 DEG C, and portion is placed in 37 DEG C, and the sample of two kinds of disposal routes, after one hour, is placed in sample rack by accurate timing.
2, sample rack is inserted the sample storehouse of Maglumi sequence of chemical luminescence analyzer, editing sample number brings into operation test, concrete load procedure is: calibration object or sample to be tested add 100 μ l, then label the anti-A I antibody-solutions 50 μ l of trace labelling thing, add the FITC 50 μ l of A I antigen-BSA albumen connector mark, add the magnetic microsphere suspending liquid 20 μ l of anti-FITC antibody bag quilt, mixing, 37 DEG C of incubations 15 minutes, instrument automatically cleans after twice and directly enters the light intensity signal that measuring chamber obtains each sample, the A I antigen concentration value of sample to be tested is gone out by ten point curves and two-point calibration automatic Fitting.Thus same sample obtains different concentration values under two kinds of Temperature Treatment, the concentration of specimens value that 37 DEG C process is deducted the concentration of specimens value of 2-8 DEG C of process, namely obtain RA (PRA).Testing result is in table 1.
Comparative example 1
Adopt certain main flow commercialization radioimmunoassay kits existing on the market to detect the routine clinical sample in 120 in embodiment 9, result is as shown in table 1.
Table 1
This clinical comparison is tested in the 160 routine clinical samples chosen, No. 145-160 totally 16 routine samples be primary aldosteronism patient diagnosed, all the other samples are then for the normal sample of health check-up.In primary aldosteronism patient, the activity of renin-angiotensin system is suppressed, because Aldosterone Secretion increases, cause sodium, water retention, and then cause extracellular fluid and blood volume to increase, make glomerular arteriole,afferent pressure increase, cause juxtaglomerular cell and macula densecell suppressed, thus renin secretion is reduced.This kind of patient not only under base state (namely under clinostatism condition) plasma renin activity low, and the force method that application of stimulus feritin is released, as adopted vertical position, low-sodium diet or to diuretics etc., plasma renin activity does not all increase or slightly increases.Detect healthy individuals plasma renin activity sample by embodiment of the present invention kit, 95% fiducial interval is vertical position: 0.1-6.56ng/ml/hr, clinostatism: 0-2.33ng/ml/hr.1.5ng/ml/hr should be less than according to clinical statistics primary aldosteronism patients blood plasma RA.
As shown by the data in table 1, No. 148 samples are only had to demonstrate embodiment of the present invention kit and radioimmunoassay kits testing result is inconsistent; Remove No. 148 testing results, embodiment 1, embodiment 4 and embodiment 7 kit are carried out fitting a straight line to the testing result of all the other samples respectively with radioimmunoassay kits to the testing result of all the other samples, be y=1.004x+0.031 for embodiment 1 linear equation, coefficient R=0.9989; Be y=0.999x+0.040 for embodiment 4 linear equation, coefficient R=0.9989; Be y=0.997x+0.034 coefficient R=0.9985 for embodiment 7 linear equation.Visible, detection kit provided by the invention has good consistance substantially with the testing result of the main flow commercialization radioimmunological kit circulated on the market.
But, add that to the measurement result of No. 148 samples in general kit provided by the invention and detection method have higher accuracy.For No. 148 samples, take from primary aldosteronism patient, its vertical clinostatism renin level generally all can be on the low side.Adopt the kit of embodiment 1, embodiment 4 and embodiment 8 preparation to detect this sample acquired results close to lower limit of confidence interval, represent that its vertical clinostatism RA level is extremely low, the clinical diagnosis of sample is consistent therewith.But use vertical, the clinostatism testing result of radioimmunological kit all close to the fiducial interval upper limit, this does not obviously conform to clinical practice situation.Illustrate that the Detection results of kit provided by the invention and detection method thereof is better than the Detection results of contrasted radioimmunological kit thus, more accurately, truly can react clinical setting.
For the kit prepared in other embodiments above-mentioned, all through clinical examination, effect is consistent with embodiment 1, embodiment 4 and embodiment 7, considers, no longer list check data at this for saving length.
To sum up, compared with certain radioimmunoassay kits commercial, according to the measured value of kit provided by the invention and actual value matching degree better, clinical coincidence rate is higher, illustrates that the diagnosis capability of kit is stronger.In addition, compared to radioimmunoassay kits, kit provided by the invention has higher stability, safety in utilization and the feature of environmental protection.
Although the present invention is described in detail, for a person skilled in the art, the amendment in spirit and scope of the invention will be apparent.In addition, should be understood that, each side that the present invention records, each several part of different embodiment and the various features enumerated can be combined or all or part of exchange.In each above-mentioned embodiment, those embodiments with reference to another embodiment can suitably combine with other embodiment, and this is by understand by those skilled in the art.In addition, the description that it will be understood to those of skill in the art that above is only the mode of example, is not intended to limit the present invention.

Claims (14)

1. one kind for detecting the chemiluminescence immune detection reagent kit of angiotensinⅠ, described kit comprises component A and B component, described component A is the connector of angiotensinⅠ antigen or angiotensinⅠ antigen and protein carrier, B component is antiangiotensin I antibody, one in described component A and B component is marked with trace labelling thing, and another kind of bag is by magnetic ball.
2. kit according to claim 1, it is characterized in that, described protein carrier is selected from least one in bovine serum albumin(BSA), human serum albumins, albumin rabbit serum, hemocyanin, ox IgG, human IgG, ovalbumin, myoglobins and thyroglobulin.
3. kit according to claim 1, it is characterized in that, described trace labelling thing is selected from least one in diamantane, luminol and derivant thereof, different luminol and derivant, acridinium ester, alkaline phosphatase and horseradish peroxidase.
4. kit according to claim 3, it is characterized in that, described trace labelling thing is N-(4-ammonia butyl) the different luminol of-N-ethyl.
5. kit according to claim 1, is characterized in that, described magnetic ball is Fe 2o 3or Fe 3o 4the complex of magnetic nano-particle and high-molecular organic material, and the particle diameter with 0.1-5 μm; Further, described magnetic ball optionally by surface modification with one or more activity functional groups.
6. kit according to claim 1, is characterized in that, in described kit, the concentration of angiotensinⅠ antigen is 0.002-0.01mg/ml; The concentration of antiangiotensin I antibody is 0.05-1mg/ml; The concentration of magnetic ball is 0.05-1mg/ml; The concentration of trace labelling thing is 0.2-1mg/l.
7. kit according to claim 1, is characterized in that,
The direct or indirect marker components A of described trace labelling thing or B component, the mode of described indirect labelling comprises by fluorescein isothiocynate and anti-fluorescein isothiocynate Antibody System or Streptavidin and biotin system indirect labelling component A or B component;
Component A or B component are directly or indirectly wrapped by magnetic ball, and the mode of described indirect bag quilt comprises is wrapped by magnetic ball indirectly by fluorescein isothiocynate and anti-fluorescein isothiocynate Antibody System or Streptavidin and biotin system.
8. kit according to claim 7, is characterized in that, described kit comprises any one the component be selected from component A1 and component A2, and is selected from any one the component in B component 1, B component 2 and B component 3; Wherein
Component A1 is the connector of N-(4-ammonia butyl) the angiotensinⅠ antigen that the different luminol of-N-ethyl directly marks or angiotensinⅠ antigen and protein carrier;
Component A2 is N-(4-ammonia butyl) the different luminol of-N-ethyl of marked by streptavidin and the connector of biotinylated angiotensinⅠ antigen or angiotensinⅠ antigen and protein carrier;
B component 1 is the magnetic ball of antiangiotensin I antibody direct coated;
B component 2 is the magnetic ball of biotinylated antiangiotensin I antibody and Streptavidin bag quilt; And
B component 3 is the fluorescein isothiocynate of antiangiotensin I antibody labeling and the magnetic ball of anti-fluorescein isothiocynate antibody bag quilt.
9. kit according to claim 7, is characterized in that, described kit comprises any one the component be selected from component C1 and component C2, and is selected from any one the component in component D1, component D2 and component D3; Wherein
Component C1 is antiangiotensin I antibody that N-(4-ammonia butyl) the different luminol of-N-ethyl directly marks;
Component C2 is N-(4-ammonia butyl) the different luminol of-N-ethyl and biotinylated antiangiotensin I antibody of marked by streptavidin;
Component D1 is the magnetic ball of the connector direct coated of angiotensinⅠ antigen or angiotensinⅠ antigen and protein carrier;
Component D2 is the connector of biotinylated angiotensinⅠ antigen or angiotensinⅠ antigen and protein carrier and the magnetic ball of Streptavidin bag quilt; And
Component D3 is the magnetic ball of the fluorescein isothiocynate that marks of the connector of angiotensinⅠ antigen or angiotensinⅠ antigen and protein carrier and anti-fluorescein isothiocynate antibody bag quilt.
10. according to the kit in claim 1-9 described in any one, it is characterized in that, described kit also comprises the low spot calibration object of angiotensinⅠ antigen and high some calibration object, and optionally comprises damping fluid.
11. 1 kinds for the preparation of the method as the kit in claim 1-10 as described in any one, described method comprises: the one in component A and B component is directly or indirectly marked trace labelling thing, directly or indirectly wraps another kind by magnetic ball.
12. methods according to claim 11, is characterized in that,
Described indirect labelling comprises trace labelling thing is marked described component A or B component by fluorescein isothiocynate and anti-fluorescein isothiocynate Antibody System or Streptavidin and biotin system;
Described indirect bag is included and described component A or B component is indirectly wrapped by magnetic ball by fluorescein isothiocynate and anti-fluorescein isothiocynate Antibody System or Streptavidin and biotin system.
13. 1 kinds of methods detecting angiotensinⅠ concentration, is characterized in that, described method comprises and using as the kit in claim 1-10 as described in any one, is detected the angiotensinⅠ concentration in testing sample by Chemiluminescence immunoassay.
14. methods according to claim 13, is characterized in that, described method comprises use as the kit in claim 1-10 as described in any one, detects angiotensinⅠ concentration by chemical illumination immunity analysis instrument.
CN201510069923.7A 2015-02-10 2015-02-10 Angiotensin I detection reagent kit as well as preparation method and application thereof Pending CN104634965A (en)

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CN105646668A (en) * 2016-01-22 2016-06-08 深圳市新产业生物医学工程股份有限公司 Derivative of angiotensin I amino acid segment, angiotensin I antigen and preparation and application of angiotensin I antigen
CN106053785A (en) * 2016-05-18 2016-10-26 北京北方生物技术研究所有限公司 Solid antibody pre-coating method for competitive chemiluminescent immunoreactions
CN108700584B (en) * 2017-01-20 2021-11-30 深圳市新产业生物医学工程股份有限公司 Marker complex, preparation method thereof, kit, application and detection system
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CN113985036A (en) * 2021-11-04 2022-01-28 南京岚煜生物科技有限公司 Angiotensin I detection kit and preparation method thereof
CN115074315A (en) * 2022-06-08 2022-09-20 四川省畜牧科学研究院 High-quality pig ear tissue sampling and long-term efficient storage method
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