CN102331502B - Quantitative measurement kit for human S100 protein (S-100) - Google Patents
Quantitative measurement kit for human S100 protein (S-100) Download PDFInfo
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Abstract
The invention discloses a quantitative measurement kit for human S100 protein (S-100). The kit is characterized by comprising an S-100 magnetic separation reagent, an enzyme reactant, a reaction reinforcing agent, a diluent, an S-100 calibrator, an S-100 control, a concentrated cleaning solution and a substrate solution. The invention further discloses a preparation method of the kit. In the kit, a chemoluminescence technology is combined with immune magnetic particles, and an approximately homogeneous reaction system is provided. Compared with the prior art, the kit has higher detection sensitivity and higher specificity. Meanwhile, the result representing time of the kit is shortened by at least 10 minutes in comparison to the prior art, superior performance parameters are achieved, and the product cost is reduced greatly.
Description
Technical field
The present invention relates to measure kit and the method for testing thereof of serum, especially relate to kit and the method for testing thereof of measuring people S100 albumen (S-100) content in serum.
Background technology
People S-100 albumen mainly concentrates in the astroglia and corresponding tumour cell of central nervous system, the S-100 protein molecular is comprised of α subunit and β subunit, its molecular weight is 21000, and biological half-life, 22h, gained the name because energy 100% is dissolved in the saturated ammonium sulfate solution that the pH value is 7.S-100 albumen in cerebrospinal fluid is relevant with the age, along with the increase at age, increases, may be relevant with de-mistake and the cerebrospinal fluid metabolic alterations of the apoptosis of spongiocyte, myelin, but the S-100 protein concentration in blood plasma and age, sex are without definite relation.At present, the clinical S-100 protein monoclonal antibody that used is detected clinical samples, differentiates the tumour origin, has improved the diagnosis of glioma correctness.The distribution of S-100 albumen is comparatively extensive, is mainly used at present the antidiastole of central nerve neuroma, and S-100 can be used as and differentiates whether tumour is the important indicator in ectoderm source.Expression to S-100 albumen in 95 routine astrocytomas is analyzed, and result shows that the S-100 Protein Subtypes all has expression in astrocytoma, and it is expressed and malignancy is negative correlation.Detection angles, mainly contain the method for exempting from of putting, ELISA method, chemoluminescence method etc. for the method for measuring S-100 clinically at present.
Past take to put and exempts to measure kit as the people S100 albumen (S-100) of representative and be subject to methodological restriction, its sensitivity and antijamming capability wretched insufficiency, have very large drawback, basically withdraw from the market, applying at present more is enzyme linked immunosorbent detection technology and chemiluminescence.It is continue Enzyme-multiplied immune technique and the emerging technology grown up after putting immune technology the eighties that chemiluminescence rises in eighties of last century, due to its high sensitivity, high specific, while method is easy, quick, the mark bond is stable, the characteristics such as "dead" isotope damage and pollution, obtained develop rapidly in recent years.
Magnetic particle separation enzyme-linked immunoassay technology is a kind ofly to take magnetic particle as the solid phase carrier of separating, immune magnetic particle isolation technics is combined with the enzyme linked immunosorbent detection technology and a kind of Novel immune detection method of setting up.Traditional E LISA method, the association reaction of antigen, antibody carries out on solid phase (elisa plate reacting hole) surface, and magnetic particle separation enzyme-linked immunoassay, the association reaction of antigen, antibody also carries out under the condition of approximate liquid phase, thereby reaction is fast, thoroughly.Compare with traditional E LISA and have highly sensitively, detect few advantage of used time.
Summary of the invention
The technical issues that need to address of the present invention are to provide a kind of people S100 albumen (S-100) quantitative determination reagent kit and detection method thereof, adopt this kit to carry out the S-100 detection and there is higher sensitivity and specificity, and the time of shorter acquisition testing result and easier mode of operation.
The invention provides the quantitative determination reagent kit of a kind of people S100 albumen (S-100), its reagent comprised has S-100 magnetic separation agent, enzyme reaction thing, increased response agent, dilution, calibration object, quality-control product, cleaning fluid concentrate and substrate solution.
Described magnetic separation agent contains the magnetic microsphere that is marked with anti-S-100 monoclonal antibody.
Described enzyme reaction thing is the anti-S-100 monoclonal antibody that contains alkali phosphatase enzyme mark.
Described increased response agent is the damping fluid that contains Tris.
Described dilution is to contain BSA solution.
Described calibration object and quality-control product are the BSA protein solutions that contains a certain amount of S-100 antigen.
Described cleaning fluid concentrate is the damping fluid that contains TWEEN-20 and Proclin-300.
Described substrate solution is enzyme-catalyzed chemical luminescence substrate solution.
The preparation method of the quantitative determination reagent kit of inventor S100 albumen (S-100), comprise the steps:
The first step: the preparation process of magnetic separation agent
One, magnetic bead buffer solution formulation operations step, preparation 1L:
1, take TRIS 4.58g and NaCl6.81g in the 1L container, after taking 0.96g TWEEN-20 and adding suitable quantity of water in the 20ml container it is dissolved fully, pour in said vesse;
2, with pipettor, Proclin-300 is measured after 0.2ml dissolves fully in the beaker of 10ml purified water, pour in above-mentioned 1L container, then in above-mentioned 1L container, add the 800ml purified water, fully stir;
3, regulate its pH value of PH instrumentation amount, adjust PH, control PH between 7.95-8.05;
4, taking BSA 3g pours in above-mentioned 1L container;
5, finally be settled to 1000ml, filter with the 0.2um filter; Post label after having filtered in 2-8 ℃ of refrigeration house storage.
Two, the preparation process of magnetic separation agent
1, the 1.0mg disuccinimidyl suberate is dissolved in 50ul DMSO, get in the 0.1mol/L PB damping fluid that the anti-S-100 monoclonal antibody of 2mg is dissolved in PH 9.5 to cumulative volume be 1ml;
2, determine the input amount of disuccinimidyl suberate, draw disuccinimidyl suberate with liquid-transfering gun and join in the antibody-solutions of step 1, put room temperature 90min;
3, step 2 antibody-solutions is joined in the Centricon-10 concentration tube then put in high speed freezing centrifuge under 3000g concentrated 30min to volume be 0.5ml;
4, get the 0.5ml magnetic bead, add in the 5ml reaction cup, put into test tube rack special, through magnet adsorption, after 2 minutes, draw supernatant;
5, add 1.5ml PH9.5 0.1mol/L PB at every turn, mix 30 seconds, added, remove supernatant, repetitive operation 3 times; The antibody-solutions that step 4 is obtained joins in above-mentioned magnetic bead, mixes rear room temperature reaction 4 hours;
6,37 ℃ of the TRIS solution 15 minutes that add 0.3ml 1mol/L;
7, add 1.5ml PH 7.2 0.1mol/L PB to clean the magnetic bead of mark at every turn, mix 30 seconds, added, remove supernatant, repetitive operation 3 times;
8, preserve liquid with the 100ml magnetic bead magnetic bead is proceeded to the 125ml vial, be 0.05% S-100 magnetic separation agent; It is 0.1%BSA that magnetic bead is preserved formula of liquid, 0.05% Tween-20,0.02%NaN3,20% ethanol, 4 ℃ of preservations.
9, by magnetic bead buffer solution, the ratio according to 1: 1 mixes magnetic separation agent step 9 obtained, and obtains magnetic separation agent in kit of the present invention.
Second step: enzyme reaction thing preparation process
One, enzyme reaction thing diluent preparing operation steps, preparation 1L:
1, get Tris 6.06g, NaCl 13.0g, Zncl
20.05g, Proclin-300 0.2ml and MgCl
20.05g, in flask, then in flask, add the 800ml purified water, fully stir, reagent is dissolved fully;
2, adjust PH, control PH in the 7.35-7.45 scope;
3, taking BSA 3g pours in above-mentioned beaker;
4, finally be settled to 1000ml, with the 0.2um filter, filter and get final product.
Two, the coupling of alkaline phosphatase and anti-S-100 monoclonal antibody
1, get 10mgALP and add in 5ml physiological saline, join in concentration tube, centrifugal 20 minutes of 3000RPM, be concentrated into 1 milliliter;
2, obtain to step 1 the 0.1M NaIO that adds 0.2ml in concentrate
4Solution, under room temperature, lucifuge stirs 20 minutes, and in the bag filter of then packing into, with the sodium-acetate buffer dialysis of 1mM PH4.4,4 ℃ are spent the night, and collect and retain liquid;
3, obtain in reservation liquid and add 0.2M PH9.5 carbonate buffer solution to step 3, make PH be elevated to 9.0, then add immediately the anti-S-100 monoclonal antibody of 2.5mg, the room temperature lucifuge stirs 2 hours gently, then adds the 4mg/ml NaBH of 0.1ml
4Solution, mix, put 4 ℃ lower 2 hours;
4, above-mentioned liquid is packed in bag filter, to 0.15M PH7.4 PBS dialysis, 4 ℃ are spent the night, and collect and retain liquid;
5, dropwise add the equal-volume saturated ammonium sulfate in step 4 to retain liquid, put 4 ℃ 1 hour, then 3000rpm centrifugal half an hour, abandon supernatant, sediment is washed secondary with semi-saturation ammonium sulfate, finally sediment is dissolved in the PBS of 0.15M PH7.4 of 20ml;
6, solution step 5 obtained is packed in bag filter, within 5 hours, remove ammonium ion with the dialysis of the PB buffer saline of 0.15M PH7.4, collect and retain after liquid 10,000rpm removes precipitation in centrifugal 30 minutes, collect supernatant, the ratio that is 1: 100 according to volume ratio is added 1M MgCl
2Solution, obtain the conjugate of alkaline phosphatase (ALP) and S-100 monoclonal antibody, finally by the conjugate of the alkaline phosphatase (ALP) collected and S-100 monoclonal antibody, with above-mentioned enzyme reaction thing dilution, the volume ratio with 1: 1000 mixes, and obtains the enzyme reaction thing.
The 3rd step:
Increased response agent preparation steps, preparation 1L:
1, take TRIS1.56g and NaCl 4.23g in the 1L container; With pipettor, Proclin-300 is measured after 0.2ml dissolves fully in the beaker of 10ml purified water, pour in above-mentioned 1L container;
2, measure the 800ml purified water in above-mentioned 1L container with graduated cylinder, fully stir, until dissolve fully, adjust PH, control PH between 7.35-7.45;
3, take Mak33 0.9g in above-mentioned 1L container; Last constant volume 1000ml, after dissolving fully, filter with the 0.2um filter.
The 4th step:
The diluent preparing step, preparation 1L:
1, take NaCl9.0g and BSA 60g in the container of 1L;
2, with pipettor, Proclin-300 is measured to 0.5ml and add the dissolving of 10ml purified water, pour in the container of above-mentioned 1L;
3, last constant volume 1000ml, after dissolving fully, filter with the 0.2um filter, posts label in 2-8 ℃ of refrigeration house storage, and the term of validity is 12 months.
The 5th step:
The preparation of calibration object and quality-control product:
Calibration object concentration is respectively 0.1,0.25,1.5,5.0,10ng/ml; Quality-control product concentration is respectively 0.25,5.0ng/ml.
The 6th step:
The cleaning concentrate preparation steps, preparation 1L:
1, take TRIS 12.54g and NaCl 325.6g in the 1L container;
2, after taking 5g Tween-20 and adding 20ml water in the 100ml container it is dissolved fully, pour in above-mentioned 1L container;
3, with pipettor, Proclin-300 is measured after 0.2ml dissolves fully in the beaker that fills the 10ml purified water, pour in above-mentioned 1L container;
4, measure the 800ml purified water in above-mentioned 1L container with graduated cylinder, fully stir, until dissolve fully;
5, adjust PH, control its scope between 7.35-7.45;
6, last constant volume 1000ml, filter and get final product with the 0.2um filter after dissolving fully.
The 7th step:
The substrate solution preparation steps, preparation 1L:
1, take TRIS 2.35g, NaCl 6.41g, Na
2SO
30.002g with Proclin-300 0.2ml is in the 1L beaker;
2, measure the 600ml purified water in the 1l beaker with graduated cylinder, fully stir, until dissolve fully, adjust PH, control its scope between 7.95-8.05;
3, after adding 250ml Lumi-Phos 480, with the 0.2um filter, filter and collect filtrate, by purified water, be settled to 1000ml, after mixing and get final product.
Principle of work of the present invention: this product is a kind of detection method that the sandwich method chemiluminescence immunoassay combines with the magnetic particle isolation technics.In sample, calibration object and quality-control product, add quantitative enzyme mark S-100 monoclonal antibody, in conjunction with S-100 monoclonal antibody and the stabilizing reinforcer of magnetic particle.37 ℃ hatch after, the S-100 monoclonal antibody of combination on magnetic particle is combined with the different epi-positions of S-100 molecule respectively from monoclonal antibody linked with peroxidase, forms " sandwich " structure.Direct precipitation in externally-applied magnetic field, do not need centrifugal separable.Remove supernatant, the compound of washing and precipitating, then add enzyme-catalyzed chemical luminescence substrate.Substrate is catalyzed cracking under the enzyme effect, form unsettled excited state intermediate, just send photon when the excited state intermediate is got back to ground state, form luminescence-producing reaction, the luminous intensity of light-emitting appearance detection reaction can be used, according to typical curve, the S-100 content in sample can be calculated.In sensing range, luminous intensity is directly proportional to the S-100 concentration in sample.
Main innovation part of the present invention is:
1, kit of the present invention combines chemiluminescence with immune magnetic particle, and a kind of reaction system that approaches homogeneous phase is provided, and compared with prior art, kit of the present invention has higher detection sensitivity and specificity, and has reached preferably performance parameter.
2, the invention discloses a kind of new special-purpose stabilizing reinforcer and cleaning fluid concentrate, make course of reaction more reliable and more stable, experimental data is sensitive effectively, when enhancing product performance, and greatly reduces cost of products;
3, the magnetic separation agent in kit, the enzyme reaction thing, stabilizing reinforcer, calibration object, quality-control product, cleaning fluid concentrate, dilution and substrate solution are all the optimization formulas under this reaction system, and giving the use effect phase of this kit and detecting performance provides powerful guarantee.
Embodiment
Embodiment 1,
One, magnetic bead buffer solution formulation operations rules: formula is in Table 1, and the preparation 1L of take is example:
1, take TRIS 4.58g and NaCl6.81g in the 1L container, after taking 0.96g TWEEN-20 and adding suitable quantity of water in the 20ml container it is dissolved fully, pour in said vesse;
2, with pipettor, Proclin-300 is measured after 0.2ml dissolves fully in the beaker of a small amount of purified water, pour in above-mentioned 1L container; Then add the 800ml purified water in the 1L container, fully stir, reagent is dissolved fully;
3, regulate its pH value of PH instrumentation amount; Adjust PH with HCl or NaOH, measure its PH and meet the requirements between 7.95-8.05;
4, taking BSA (bovine serum albumin(BSA)) 3g pours in said vesse;
5, finally be settled to 1000ml, survey pH value, scope meets the requirements between 7.95-8.05, with the 0.2um filter, filters; Post label after having filtered in 28 ℃ of refrigeration house storages, the magnetic bead buffer solution term of validity is 12 months;
Magnetic bead buffer solution table 1
Two, the preparation process of magnetic separation agent
1,1.0mg DSS (disuccinimidyl suberate) is dissolved in 50ul DMSO, concentration is 20mg/ml; Get the anti-S-100 monoclonal antibody of 2mg (day strong bio-pharmaceuticals (day Feng) company limited, concentration is 2.8mg/ml) be dissolved in the 0.1mol/L PB damping fluid of PH 9.5 to cumulative volume be 1ml;
2, calculate the input amount of DSS, calculate according to following formula: (antibody quality/16000) * 10 * 368/C
DSS), C wherein
DSSRefer to the amount of substance concentration mol/L of DSS;
3, join in the antibody-solutions of step 1 with the DSS of liquid-transfering gun absorption respective volume, put room temperature 90min;
4, step 3 antibody-solutions is joined in the Centricon-10 concentration tube then put in the sigma2-16k high speed freezing centrifuge under the centrifugal force of 3000g concentrated about 30min to volume be 0.5ml;
5, get the 0.5ml magnetic bead, magnetic bead concentration is 0.5g/ml, and described magnetic bead diameter, between 0.9 μ m, adds in the 5ml reaction cup, puts into test tube rack special, through magnet adsorption, after 2 minutes, draws supernatant;
6, add 1.5ml PH9.5 0.1mol/L PB at every turn, mix 30 seconds, added, remove supernatant, repetitive operation 3 times; The antibody-solutions that step 4 is obtained joins in magnetic bead, mixes rear room temperature reaction 4 hours, keeps mixing state;
7, add the TRIS solution 37 of 0.3ml lmol/L to spend 15 minutes, wherein the application of sample amount of TRIS is that 1mg antibody adds 0.15mlTRIS;
8, add 1.5ml PH 7.2 0.1mol/L PB to clean the magnetic bead of mark at every turn, mix 30 seconds, added, remove supernatant, repetitive operation 3 times;
9, preserve liquid with the 100ml magnetic bead magnetic bead is proceeded to the 125ml vial, be 0.05% S-100 magnetic separation agent; It is 0.1%BSA that magnetic bead is preserved formula of liquid, 0.05% Tween-20,0.02%NaN3,20% ethanol, 4 ℃ of preservations.
10, by magnetic bead buffer solution, the ratio according to 1: 1 mixes magnetic separation agent step 9 obtained;
11, post label in 28 ℃ of refrigeration house storages, the magnetic separation agent term of validity is 12 months;
Embodiment 2
One, enzyme reaction thing diluent preparing working specification: formula is in Table 2, and the preparation 1L of take is example:
1, get Tris 6.06g, NaCl 13.0g, Zncl
20.05g, Proclin-300 0.2ml and MgCl
20.05g in flask; Then add the 800ml purified water in flask, fully stir, reagent is dissolved fully;
2, adjust PH with HCl or NaOH, measure and make PH in the 7.35-7.45 scope;
3, taking BSA 3g pours in said vesse;
4, finally be settled to 1000ml, survey pH value, scope meets the requirements between 7.35-7.45, with the 0.2um filter, filters; After having filtered, post label in 2-8 ℃ of refrigeration house storage, the term of validity is 12 months;
Enzyme reaction thing dilution table 2
Two, the coupling of alkaline phosphatase (ALP) and anti-S-100 monoclonal antibody
1, get 10mgALP and add in 5ml physiological saline, join in Centriprep YM10 concentration tube, centrifugal about 20 minutes of 3000RPM, be concentrated into 1 milliliter.
2, obtain to step 1 the 0.1M NaIO that adds 0.2ml in concentrate
4Solution, under room temperature, lucifuge stirs 20 minutes, and in the bag filter of then packing into, with the sodium-acetate buffer dialysis of 1mM PH4.4,4 ℃ are spent the night, and collect and retain liquid;
3, obtain in reservation liquid and add 0.2M PH9.5 carbonate buffer solution to step 2, make PH be elevated to 9.0～9.5, then add immediately the anti-S-100 monoclonal antibody of 2.5mg (day strong bio-pharmaceuticals (Tianjin) company limited, concentration is 2.8mg/ml), the room temperature lucifuge stirs 2 hours gently, then adds the 4mg/ml NaBH of 0.1ml
4(sodium borohydride) solution, mix, then put 4 ℃ lower 2 hours;
4, above-mentioned liquid is packed in bag filter, to 0.15M PH7.4 PBS dialysis, 4 ℃ are spent the night, and collect and retain liquid;
5, dropwise add the equal-volume saturated ammonium sulfate in step 4 to retain liquid, put 4 ℃ 1 hour, then 3000rpm centrifugal half an hour, abandon supernatant, sediment is washed secondary with semi-saturation ammonium sulfate, finally sediment is dissolved in the PBS of a small amount of 0.15M PH7.4;
6, solution step 5 obtained is packed in bag filter, dialyse approximately and within 5 hours, to remove ammonium ion by the PB buffer saline of 0.15M PH7.4, collect and retain after liquid 10,000rpm removes precipitation in centrifugal 30 minutes, collect supernatant, the ratio that is 1: 100 according to volume ratio is added 1M Mgcl
2Solution, obtain the conjugate of alkaline phosphatase (ALP) and S-100 monoclonal antibody, finally by the conjugate of the alkaline phosphatase (ALP) collected and S-100 monoclonal antibody, with above-mentioned enzyme reaction thing dilution, the volume ratio with 1: 1000 mixes, and obtains the enzyme reaction thing.
Embodiment 3
Increased response agent formulation operations rules: formula is shown in (table 3), and the preparation 1L of take is example:
1, take TRIS (trishydroxymethylaminomethane, molecular formula: (HOCH2) 3CNH2) 1.56g and NaCl 4.23g are in the 1L container; With pipettor, Proclin-300 is measured after 0.2ml dissolves fully in the beaker of a small amount of purified water, pour in above-mentioned 1L container;
2, measure the 800ml purified water in the 1L container with graduated cylinder, fully stir, until dissolve fully; Adjust PH with HCL or NaOH, measure its scope between 7.35-7.45;
3, take Mak330.9g in above-mentioned 1L container;
4, last constant volume 1000ml, after dissolving fully, survey pH value, and scope meets the requirements between 7.35-7.45, with the 0.2um filter, filters; After having filtered, post label in 2-8 ℃ of refrigeration house storage, the term of validity is 12 months;
The preparation (table 3) of increased response agent
Embodiment 4
The dilution formula is shown in (table 4), and the preparation 1L of take is example:
1, take NaC19.0g and BSA 60g in the container of 1L;
2, with pipettor, Proclin-300 is measured to 0.5ml and add the dissolving of 10ml purified water, pour in the container of above-mentioned 1L;
3, last constant volume 1000ml, after dissolving fully, filter with the 0.2um filter, posts label in 2-8 ℃ of refrigeration house storage, and the term of validity is 12 months.
The preparation (table 4) of dilution
Embodiment 5
The preparation of calibration object and quality-control product:
1, peak: maximum concentration point is X, and impact point concentration is A, B, and C, D, E, F, while preparing the solution of V volume, need add the volume of raw material for being respectively: table 5
| Concentration | Add calibration object dilution volume | Add the X volume |
| A | V-A*V/X | A*V/X |
| B | V-B*V/X | B*V/X |
| C | V-C*V/X | C*V/X |
| D | V-D*V/X | D*V/X |
| E | V-E*V/X | E*V/X |
| 0F | V F*V/X | F*V/X |
2, to be mixed with concentration point be 0.1,0.25,1.5,5.0,10ng/ml to dilution for people S100 albumen (S-100) quantitative determination reagent kit S-100 calibration object raw material (be purchased from ALD company, concentration is 2.1mg/ml) (concrete formula is shown in embodiment 4); Quality-control product is 0.25,5.0ng/ml by the concentration point of diluent preparing.
3, after dissolving fully, post label in 2-8 ℃ of refrigeration house storage, the term of validity is 12 months.
Embodiment 6:
Cleaning concentrate formulation operations rules: formula is shown in (table 6), and the preparation 1L of take is example:
1, take TRIS 12.54g and NaCl 325.6g in the 1L container;
2, after taking 5g Tween-20 and adding suitable quantity of water in the 100ml container it is dissolved fully, pour in said vesse;
3, with pipettor, Proclin-300 is measured after 0.2ml dissolves fully in the beaker that fills the 10ml purified water, pour in above-mentioned 1L container;
4, measure the 800ml purified water in above-mentioned 1L container with graduated cylinder, fully stir, until dissolve fully;
5, adjust PH with HCL or NaOH, measure its scope between 7.35-7.45;
6, last constant volume 1000ml, survey pH value, and scope meets the requirements between 7.35-7.45, after dissolving fully, with the 0.2um filter, filters; After having filtered, post label in 2-8 ℃ of refrigeration house storage, the term of validity is 12 months;
The preparation (table 6) of cleaning concentrate
Embodiment 7
Substrate solution formulation operations rules: formula is shown in (table 7), and the preparation 1L of take is example:
1, take TRIS 2.35g, NaCl 6.41g, Na
2SO
30.002g with Proclin-300 0.2ml is in the 1L beaker;
2, measure the 600ml purified water in the 1L beaker with graduated cylinder, fully stir, until dissolve fully; Adjust PH with HCl or NaOH, measure its scope between 7.95-8.05;
3, after adding 250ml Lumi-Phos 480, with the 0.2um filter, filter and collect filtrate, by purified water, be settled to 1000ml, after mixing, post label in 2-8 ℃ of refrigeration house storage, the term of validity is 12 months.
The preparation (table 7) of chemical luminous substrate
Method of testing of the present invention is as follows
1, add 45 μ l S-100 calibration objects, quality-control product, sample to be measured to corresponding test tube bottom.
2, add 60 μ l enzyme reaction things to each test tube.
3, add 60 μ l increased response agent to each test tube.
4, add 30 μ l magnetic separation agents to each test tube.
5, cover test tube with plastic sheeting, the multitube vortex mixer after tube shaken frame 30s, is put 37 ℃ of water-baths 30 minutes gently.
6, the test tube frame linking is put to magnetic separator, guarantees that every test tube all contacts with separator surface, precipitates 2 minutes.The separation vessel that reverses is slowly poured out supernatant, and the test tube of reversing is placed on filter paper together with separation vessel, firmly bounces the separation vessel bottom to remove all drops that are bonded on tube wall.
7, the cleaning fluid concentrate is with after 20 times of purified water dilutions, adds cleaning fluid after 200 μ l dilutions to each test tube, puts on the multitube vortex mixer vibration gently and mixes 30s.During application of sample, should avoid the application of sample dynamics excessive and cause magnetic bead to spill.Mix and want thoroughly.
8, repeating step is 6,7,6 one times.
9, add 200 μ l substrate solutions and mix 3 seconds to test tube, detected with ready luminous detector rapidly.
10,, as run into high value HOOK sample, for fear of high value HOOK effect occurring, the suggestion clinician selects suitable extension rate to be diluted sample according to all the other test indexs.
Clinical testing:
1, detect data
Sample picks up from the normal health check-up of 1500 example, blood donor.The sample physical examination result is all without liver, brain, kidney, disease of digestive tract, and in half a year, without blood transfusion and major operation history, the women is not in the gestational period and lactation.Measured value is carried out to statistical analysis, draw normal serum term of reference:<0.105ng/ml.
Notebook data is only for reference, and different regions, Different Individual and employing distinct methods are detected, and measured S-100 level also can be different, advises the range of normal value of each laboratory foundation oneself.The S-100 value that can not only draw with this method is made diagnosis, only as the intermediate data reference role, should, in conjunction with clinical other Data Analysis Results, comprise patient's concrete condition and treatment situation.S-100 concentration value and this kit measurement result in the sample obtained by additive method do not have direct comparability.The sample that exceeds the kit measurement scope, system can't provide definite numerical value.As wish is measured its definite result, after the suggestion dilution, measured again.The testing result of this kit only supplies clinical reference, can not be separately as making a definite diagnosis or the foundation of Excluded cases, for reaching diagnostic purpose, this testing result will be combined with clinical examination, medical history and other inspection.This product can be used for the mensuration of serum sample, for the reliability of other body fluid samples S-100 concentration determination, is fully confirmed.
2, kit performance index of the present invention
Sensitivity for analysis is defined as: to the mensuration of 20 zero calibration objects, get its mean deviation of 2 times, its corresponding concentration on typical curve is sensitivity for analysis;
Sensitivity for analysis: 78pg/mL; Accuracy: variation within batch CV%≤10.0%; Batch variation CV%≤15.0%; Linear coefficient: r >=0.9900; The range of linearity: 78pg/mL-5000pg/mL; Specificity: this kit can detect simultaneously the restructuring or natural people S-100, and with other associated protein no cross reaction.
Claims (1)
1. the quantitative determination reagent kit of a people S100 Protein S-100, is characterized in that, this kit comprises S-100 magnetic separation agent, enzyme reaction thing, increased response agent, dilution, S-100 calibration object, S-100 quality-control product, cleaning fluid concentrate and substrate solution;
In described kit, each component makes according to the method for being prepared as follows:
One, the preparation process of magnetic separation agent
One), magnetic bead buffer solution formulation operations step, the preparation 1L:
1, take TRIS 4.58g and NaCl6.81g in the 1L container, after taking 0.96g TWEEN-20 and adding suitable quantity of water in the 20mL container it is dissolved fully, pour in above-mentioned 1L container;
2, with pipettor, Proclin-300 is measured after 0.2mL dissolves fully in the beaker of 10mL purified water, pour in above-mentioned 1L container, then in above-mentioned 1L container, add the 800mL purified water, fully stir;
3, regulate pH, control pH between 7.95-8.05;
4, taking BSA 3g pours in above-mentioned 1L container;
5, finally be settled to 1000mL, with 0.2 μ m filter, filter and get final product;
Two), the preparation process of magnetic separation agent
1, the 1.0mg disuccinimidyl suberate is dissolved in 50 μ l DMSO, get in the 0.1mol/L PB damping fluid that the anti-S-100 monoclonal antibody of 2mg is dissolved in pH 9.5 to cumulative volume be 1mL;
2, draw in the antibody-solutions that above-mentioned disuccinimidyl suberate joins step 1 with liquid-transfering gun, put room temperature 90min;
3, the solution of step 2 is joined in concentration tube, then put in high speed freezing centrifuge centrifugal 30min under 3000g and be concentrated into 0.5mL;
4, get the 0.5mL magnetic bead, add in the 5mL reaction cup, through magnet adsorption, after 2 minutes, draw supernatant, then add 1.5mL pH9.5 0.1mol/L PB, mix 30 seconds, the antibody-solutions that then adds step 3 to obtain, mix rear room temperature reaction 4 hours;
5, add 37 ℃ of the TRIS solution 15 minutes of 0.3mL 1mol/L, then add 1.5mL pH 7.2 0.1mol/L PB to clean the magnetic bead of mark, mix 30 seconds;
6, preserve liquid with the 100mL magnetic bead magnetic bead is proceeded to vial;
7, solution step 6 obtained and above-mentioned magnetic bead buffer solution mix according to the volume ratio of 1: 1, obtain the magnetic separation agent;
Two, the preparation process of enzyme reaction thing
One), enzyme reaction thing diluent preparing operation steps, the preparation 1L:
1, get Tris 6.06g, NaCl 13.0g, ZnCl
20.05g, Proclin-300 0.2mL and MgCl
20.05g, in flask, then in flask, add the 800mL purified water, fully stir, reagent is dissolved fully;
2, adjust pH, control pH in the 7.35-7.45 scope;
3, taking BSA 3g pours in above-mentioned beaker;
4, finally be settled to 1000mL, with 0.2 μ m filter, filter and get final product;
Two), the coupling of alkaline phosphatase and anti-S-100 monoclonal antibody
1, get 10mgALP and add in 5mL physiological saline, join in concentration tube, centrifugal 20 minutes of 3000rpm, be concentrated into 1 milliliter;
2, obtain to step 1 the 0.1M NaIO that adds 0.2mL in concentrate
4Solution, under room temperature, lucifuge stirs 20 minutes, and in the bag filter of then packing into, with the sodium-acetate buffer dialysis of 1mM pH4.4,4 ℃ are spent the night, and collect and retain liquid;
3, obtain in reservation liquid and add 0.2M pH9.5 carbonate buffer solution to step 2, make pH be elevated to 9.0, then add immediately the anti-S-100 monoclonal antibody of 2.5mg, stir 2 hours, then add the 4mg/mL NaBH of 0.1mL
4Solution, mix, put 4 ℃ lower 2 hours;
4, above-mentioned solution is packed in bag filter, with 0.15M pH7.4 PBS dialysis, 4 ℃ are spent the night, and collect and retain liquid;
5, dropwise add the equal-volume saturated ammonium sulfate in step 4 to retain liquid, put 4 ℃ 1 hour, then 3000rpm centrifugal half an hour, abandon supernatant, sediment is washed secondary with semi-saturation ammonium sulfate, finally sediment is dissolved in the PBS of 0.15M pH7.4 of 20mL;
6, solution step 5 obtained is packed in bag filter, within 5 hours, remove ammonium ion with the dialysis of the PB damping fluid of 0.15M pH7.4, collect and retain after liquid 10,000rpm removes precipitation in centrifugal 30 minutes, collect supernatant, the ratio that is 1: 100 according to volume ratio is added 1M MgCl
2Solution, obtain the conjugate of alkaline phosphatase and anti-S-100 monoclonal antibody; By the alkaline phosphatase and the above-mentioned step 1 of the conjugate of anti-S-100 monoclonal antibody of collecting) the enzyme reaction thing dilution that obtains mixes with the volume ratio of 1: 1000, obtains the enzyme reaction thing;
Three, the preparation process of increased response agent, preparation 1L:
1, get TRIS1.56g and NaCl 4.23g in the 1L container, after getting 0.2mL Proclin-300 and dissolving fully in the beaker of 10mL purified water, pour in above-mentioned 1L container;
2, get the 800mL purified water in above-mentioned 1L container, fully stir until dissolving fully adjusts pH between 7.35-7.45;
3, take Mak33 0.9g in above-mentioned 1L container; Last constant volume 1000mL, after dissolving fully, filter and get final product with 0.2 μ m filter;
Four, the preparation process of dilution, preparation 1L:
1, take NaCl9.0g and BSA 60g in the container of 1L;
2, get 0.5mL Proclin-300 and add the dissolving of 10mL purified water, pour in the container of above-mentioned 1L;
3, last constant volume 1000mL, after dissolving fully, filter and get final product with 0.2 μ m filter;
Five, the preparation of S-100 calibration object and S-100 quality-control product:
In the S-100 calibration object, the concentration of S-100 antigen is respectively 0.1,0.25,1.5,5.0,10ng/mL; In the S-100 quality-control product concentration of S-100 antigen be respectively 0.25,5.0ng/mL;
Six, the preparation process of cleaning fluid concentrate, preparation 1L:
1, get TRIS 12.54g and NaCl 325.6g in the 1L container;
2, after getting 5g Tween-20 and adding 20mL water in the 100mL container it is dissolved fully, pour in above-mentioned 1L container;
3, after getting 0.2mL Proclin-300 and dissolving fully in the beaker that fills the 10mL purified water, pour in above-mentioned 1L container;
4, get the 800mL purified water in above-mentioned 1L container, fully stir, until dissolve fully;
5, adjust pH, control its scope between 7.35-7.45;
6, last constant volume 1000mL, filter and get final product with 0.2 μ m filter after dissolving fully;
Seven, the preparation process of substrate solution, preparation 1L:
1, get TRIS 2.35g, NaCl 6.41g, Na
2SO
30.002g with Proclin-300 0.2mL is in the 1L beaker;
2, get the 600mL purified water in the 1L beaker, fully stir until dissolving fully adjusts pH between 7.95-8.05;
3, add 250mL Lumi-Phos 480, with 0.2 μ m filter, filter to collect filtrate, by purified water, be settled to 1000mL, after mixing and get final product.
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| CN104777315A (en) * | 2015-04-17 | 2015-07-15 | 西安金磁纳米生物技术有限公司 | Chemiluminescence immunoassay method for detecting S100 based on gold magnetic particles |
| CN106645737A (en) * | 2016-06-30 | 2017-05-10 | 深圳市亚辉龙生物科技股份有限公司 | S-100 chemiluminescence immunoassay kit and preparation method thereof |
| CN111487409A (en) * | 2019-01-28 | 2020-08-04 | 艾维可生物科技有限公司 | Chemiluminescence detection kit for S100B protein and use method thereof |
| CN110672849B (en) * | 2019-11-12 | 2022-03-01 | 郑州安图生物工程股份有限公司 | Detection kit for S100 protein |
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| SE9602677D0 (en) * | 1996-07-05 | 1996-07-05 | Sangtec Medical Ab | Methods for determining brain antigens |
| US6821790B2 (en) * | 1999-03-02 | 2004-11-23 | Vijay Mahant | Methods and apparatus for separation of biological fluids |
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| CN101432626A (en) * | 2006-04-04 | 2009-05-13 | 神谷来克斯公司 | Highly sensitive system and methods for analysis of troponin |
| CN201429597Y (en) * | 2009-07-06 | 2010-03-24 | 周坚 | Cardiovascular disease three-channel test card |
| CN101937001A (en) * | 2010-08-30 | 2011-01-05 | 复旦大学附属中山医院 | Kit for diagnosing post-kidney transplant acute rejection reaction |
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| 人S100蛋白的表达及其抗血清的制备和脑脊液中S100含量测定方法的建立;张福萍等;《中华实验和临床病毒学杂志》;20021231(第04期);305－308 * |
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