CN103048464A - Neutrophile granulocyte gelatinase related lipid transport protein detection kit and preparation method thereof - Google Patents
Neutrophile granulocyte gelatinase related lipid transport protein detection kit and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a neutrophile granulocyte gelatinase related lipid transport protein detection kit and a preparation method thereof. The detection kit is composed of a reagent I and a reagent II independent from each other; the reagent I comprises the components of biobuffer, surfactant, coagulant, preservative, stabilizer, blocker, chelating agent and water; and the reagent II comprises the components of rubber latex particle of enveloping neutrophile granulocyte gelatinase related lipid transport protein antibody, biobuffer, chelating agent, surfactant, preservative, suspending agent, sealing agent, stabilizer and water. According to the invention, the neutrophile granulocyte gelatinase related lipid transport protein antibody of the detection kit can not only be combined with the neutrophile granulocyte gelatinase related lipid transport protein antigen, but also be combined with the MMP-9/NGAL (matrix metallopeptidase-9/ (neutrophil gelatinase associated lipocalin) compound, thereby avoiding the influence of MMP-9/NGAL compound to the detecting result, and having the advantages of good specificity, high sensitivity, wide linear range, favorable stability and the like.
Description
Technical field
The present invention relates to the lipocalin protein reagent box for detecting content, relate in particular to detection kit of neutrophil gelatinase-associated lipocalin (NGAL) content in a kind of human body and preparation method thereof, the invention further relates to the using method of this detection kit NGAL content in human body, belong to the detection field of NGAL content in the human body.
Background technology
Research in recent years is found, a newcomer in the lipocalin protein family---neutrophil gelatinase-associated lipocalin (neutrophil gelatinase-associated lipocalin, NGAL), with acute injury of kidney (acute kidney injury, AKI) and chronic kidney disease (the chronic kidney disease that causes of a variety of causes, CKD) closely related, being the novel markings thing of prediction AKI, also is simultaneously the biological indicator of monitoring CKD progress and kidney function damage.
NGAL is one of member of lipocalin protein family, is found in human neutrophil leucocyte and is separated by Kjeldsen etc. in 1993, and its rat, mouse ortholog are respectively α 2 microballoon associated protein and 24p3 albumen.Molecular weight is arranged is 25kDa, self-polymerization 46kDa homodimer to NGAL or form three kinds of existing waies of 135kDa heterodimer with the MMP-9 polymerization in vivo.NGAL has by 8 β-pleated sheets and interacts and the β autofolding barrel structure that forms, the hydrophobic core of its bottom inside can in conjunction with and transport the lipophilicity part, this design feature has determined the biologically active of NGAL.5 of the β 4-of beta sheet bucket blind end β free sulfydryl (Cys87) is arranged, this has established architecture basics for NGAL in conjunction with proMMP-9.Think that at present NGAL albumen can transport some lipophilic molecules of inducing inflammatory reaction, such as platelet activating factor (PAF), leukotriene B4 (LTB4) and lipopolysaccharides (LPS) etc.; It is active to regulate MMP-9, and may participate in removing inflammatory mediator, regulates immune response, the processes such as the genesis of inhibited apoptosis and tumour, infiltration metastasis.NGAL also by being combined with the siderophore of iron content, forms NGAL-siderophore-iron complexes, participates in generation, development and the repair process of kidney.Under normal physiological status, except neutrophil leucocyte, NGAL has low expression level in the mankind much organize epithelial cell such as bronchus, stomach, small intestine, pancreas, kidney etc.And under pathological state, the epithelial cell of these tissues then has a large amount of NGAL to express, particularly in the proximal renal tubular epithelial cells of damage.
In the research of seeking AKI early diagnosis label, it is found that NGAL is well prediction and a diagnosis index.The scholar is arranged in the AKI of acute ischemia reperfusion and cisplatin induction mouse model, find to occur to detect within a few hours in damage the expression of NGAL and obviously increase.The research at U.S. Cincinnati children center is found, in the 71 routine child patients of accepting the Shunt art, wherein the infant postoperative 2h of 20 routine postoperative acute renal failures urine NGAL level namely obviously raises, the rising of its serum creatinine then occurs in postoperative 1-3d, and prompting urine NGAL is early stage, a sensitive indicator of Ischemic kieney injury.Afterwards many similarly researchs all obtain same conclusion, and namely blood, urine NGAL level all raise during AKI, and urine NGAL level raises more remarkable.And other indexs of the more early stage kidney injury of NGAL rising such as kidney injury molecule (kidney injury molecular1, KIM-1), Cyr61 (cystein richprotein61), B2M etc. occur must be early.Simultaneously, NGAL level and kidney injury degree are proportionate, and blood and urine NGAL can be used as that diagnosis AKI is early stage, responsive, specific early stage biological marker, and can reflect the AKI order of severity and judging prognosis to a certain extent.
The detection method of NGAL has euzymelinked immunosorbent assay (ELISA), radioimmunology, Western-blotting, chemoluminescence method and latex immunoturbidimetry at present.The euzymelinked immunosorbent assay (ELISA) automaticity is not high, and is affected by human factors larger; Radioimmunology exists problem of environmental pollution; The Western-blotting complicated operation, it is low to measure precision; Though and chemoluminescence method sensitivity is high, the mensuration range of linearity is less and testing cost is higher, needs specific chemiluminescence detector, and these reasons cause its range of application less; The latex immunoturbidimetry also is the common method of measuring NGAL concentration in human plasma or the urine, and it is compared with above-mentioned other several methods, has simple to operate, highly sensitive, the outstanding features such as the range of linearity is wide, pollution-free, applied range.Therefore, the latex immunoturbidimetry has great researching value, but latex immunoturbidimetry detection kit in the market exists and can't detect NGAL/MMP-9 compound, poor anti jamming capability, specificity is low, reagent stability is bad or detect the problems such as linear narrow range, haves much room for improvement.
Summary of the invention
One of purpose of the present invention provides a kind of neutrophil gelatinase-associated lipocalin detection kit;
Two of purpose of the present invention provides a kind of method for preparing described neutrophil gelatinase-associated lipocalin detection kit.
Above-mentioned purpose of the present invention is achieved through the following technical solutions:
A kind of neutrophil gelatinase-associated lipocalin detection kit is comprised of reagent I independent of each other and reagent II; The component of described reagent I comprises: biological buffer, surfactant, set accelerator, antiseptic, stabilizing agent, blocking agent, sequestrant and water; The component of described reagent II comprises: latex particle, biological buffer, sequestrant, surfactant, antiseptic, suspending agent, sealer, stabilizing agent and the water of coated neutrophil gelatinase-associated lipocalin (NGAL) antibody; Wherein, described neutrophil gelatinase-associated lipocalin (NGAL) antibody can combine with the MMP-9/NGAL compound again with the combination of neutrophil gelatinase-associated lipocalin antigen.
Preferably, the neutrophil gelatinase-associated lipocalin described in the present invention (NGAL) antibody obtains by the screening of affinity chromatography method; Preferably, described screening technique comprises:
(1) preparation of MMP-9 affinity column mixes MMP-9 albumen with hematocrit CNBr-Sepharose4B, successively through crosslinked, clean, activated group that sealing is residual, again clean, fill post, balance, obtain the MMP-9 affinity column;
(2) preparation of MMP-9/NGAL compound affinity column: albumen NGAL solution is crossed the MMP-9 affinity column; Then use the unnecessary NGAL albumen of phosphate buffer wash-out;
(3) affinity chromatography: the NGAL antibody-solutions by prepared affinity column, is carried out desorb after the removal non-specific adsorption, the Fractional Collections stripping liquid, fully the TB in the stripping liquid is fallen in dialysis, vacuum freeze drying, and get final product.
Wherein, CNBr-Sepharose4B mixes with the ratio of 5-7mg albumen MMP-9 according to every milliliter of hematocrit CNBr-Sepharose4B with 5-7mg albumen MMP-9 in the step (1); 0.1mol/L monoethanolamine-HCl4 ℃ of the quality such as the sealing mode described in the step (1) adds was shaken 1 hour.
Preferably, in the step (2) albumen NGAL is mixed with the solution of 2mg/mL, crosses the MMP-9 affinity column with the NGAL protein solution with the flow velocity of 1mL/min; Then use 0.1mol/L pH8.0 phosphate buffer with the unnecessary NGAL albumen of the flow velocity wash-out of 1mL/min;
Preferably, step is adjusted to 600ug/mL with the NGAL antibody concentration in (3), with 6mL/ hour flow velocity by prepared affinity column, clean to remove non-specific adsorption with 1mol/L NaCl-TB, 0.5%NP-40-TB, TB and distilled water successively again; Then with carrying out desorb in 3mol/L KSCN6mL/ hour; Ultraviolet detects the Fractional Collections stripping liquid.
The preparation method of the latex particle of coated NGAL antibody of the present invention comprises:
(1) preparation carboxylation modified polystyrene latex solution: taking by weighing particle diameter is the polystyrene latex particle drying thing of 105-145nm, adding phosphate buffer disperses, add 1-ethyl-3-(3-dimethylaminopropyl) carbodiimides and sulfo-NHS, stirring reaction under the room temperature; The centrifugal supernatant of abandoning disperses precipitation latex with the phosphate buffer dilution again, obtains carboxylation modified polystyrene latex;
(2) preparation of NGAL antibody-solutions: with screening obtain can with neutrophil gelatinase-associated lipocalin antigen in conjunction with NGAL monoclonal antibody or the polyclonal antibody deionized water dissolving that can combine with the MMP-9/NGAL compound again, obtain the NGAL antibody-solutions;
(3) preparation of the latex particle of coated NGAL antibody: add carboxylation modified polystyrene latex solution after the prepared antibody-solutions of step (2) dilutes with phosphate buffer, stirring reaction at room temperature, after the cessation reaction, the centrifugal supernatant of abandoning, washing precipitation, and get final product.
Described in the present invention latex particle be that particle diameter is the latex particle of the medium particle diameter of 105-145nm, adopt the latex particle in this particle size range both can solve the inadequate problem of small grain size latex detection sensitivity, can also solve large grain size latex and detect the little problem of the range of linearity, the bad problem of product stability that can also avoid large grain size latex in the long storage time process, to sink to causing simultaneously.And the latex particle that adopts a kind of particle diameter prepares detection kit and reduced operation in the kit production run, namely provided cost savings and avoided to cross multiple operation to the impact of product quality.
In order to reach better detection effect, the consumption of each component is in per 1 liter of reagent I: biological buffer 5-100g, surfactant 0.5-20mL, set accelerator 2-50g, antiseptic 0.02-1mL, stabilizing agent 1-60g, blocking agent 1-10mL, sequestrant 0.05-5g, surplus is water;
The consumption of each component is in per 1 liter of reagent II: the latex particle 0.1-10g of coated NGAL antibody, biological buffer 1-100g, sequestrant 0.1-10g, surfactant 0.05-3mL, antiseptic 0.02-1mL, suspending agent 5-50mL, sealer 5-50g, stabilizing agent 50-150g, surplus is water.
Further preferred, the consumption of each component is in per 1 liter of reagent I: biological buffer 7-20g, and surfactant 1-10mL, set accelerator 10-30g, antiseptic 0.1-0.5mL, stabilizing agent 5-15g, blocking agent 3-6mL, sequestrant 0.1-1g, surplus is water;
The consumption of each component is in per 1 liter of reagent II: the latex particle 0.5-5g of coated NGAL antibody, biological buffer 5-30g, sequestrant 0.5-4g, surfactant 0.5-2mL, antiseptic 0.1-0.5mL, suspending agent 10-40mL, sealer 7-15g, stabilizing agent 80-120g, surplus is water.
Most preferred, the consumption of each component is in per 1 liter of reagent I: biological buffer 12g, and surfactant 4mL, set accelerator 15g, antiseptic 0.25mL, stabilizing agent 10g, blocking agent 5mL, sequestrant 0.4g, surplus is water;
The consumption of each component is in per 1 liter of reagent II: the latex particle 1.7g of coated NGAL antibody, and biological buffer 11g, sequestrant 2g, surfactant 1.5mL, antiseptic 0.25mL, suspending agent 20mL, sealer 10g, stabilizing agent 100g, surplus is water.
Biological buffer described in reagent I or the reagent II is to can be used in the various biological buffer of keeping certain pH value, it for example can be trishydroxymethylaminomethane (Tris), Pehanorm base ethyl sulfonic acid (TES), 2-(N-morpholine) ethyl sulfonic acid (MES), N-(2-hydroxyethyl) piperazine-N'-2-ethane sulfonic acid (HEPES), Pehanorm base propane sulfonic acid (TAPS), piperazine-1,4-dihydroxy propane sulfonic acid (POPSO), the various biological buffers commonly used such as 3-(N-morpholine) propane sulfonic acid (MOPS), in order to reach better detection effect, biological buffer described in the reagent I is preferably trishydroxymethylaminomethane (Tris), and the biological buffer described in the reagent II is preferably 3-(N-morpholine) propane sulfonic acid.
Surfactant described in reagent I or the reagent II mainly plays and promotes in the reagent system each component and detect the Uniform Dispersion of each material in the sample and improve the precision that detects, and reaches and reduces the sample turbidity to the purpose of the impact of measurement result; Therefore, existing surfactant with solubilization all can be used as the surfactant described in reagent I or the reagent II, for example, and polysorbas20, polysorbate40, triton x-100, Nonidet P40 etc.Surfactant in reagent I of the present invention and the reagent II is preferably triton x-100.
Antiseptic described in reagent I or the reagent II mainly is in order to prevent that the product that bacteria breed causes from going bad, so any antiseptic that prevents the bacteria breed effect that can play all can be applicable to the present invention; Antiseptic in reagent I of the present invention and the reagent II is preferably Proclin-300, and this product toxicity is little, and using dosage is little, and fungicidal spectrum is wide.
Stabilizing agent described in reagent I or the reagent II, Main Function are the active of protection antibody and aggegation, the sinking that prevents latex particle, can also increase the stability of each component in the kit simultaneously, service time after the shelf life of prolongation product and the uncork.Stabilizing agent commonly used is carbohydrate, alcohols, protide and some amino acid etc., and the reagent I among the present invention and the stabilizing agent in the reagent II are preferably sucrose.
Set accelerator in the reagent I can promote antigen-antibody reaction, is conducive to formation and the increase of latex particle cross-linking agent, improves detection sensitivity and the range of linearity.Set accelerator in the detection kit of the present invention is preferably PEG-6000 or PEG-8000.
Blocking agent in the reagent I can effectively avoid nonspecific reaction to the interference of testing result.Common disturbing factor has following two classes in the detection kit: heterophile antibody disturbs, rheumatoid factor is disturbed.The preferred blocking agent of the present invention is the rheumatoid factor blocking agent.The one class autoantibody that can occur in part rheumatoid arthritis, various collagenic connective tissue disease, hepatopathy and the multiple chronic disease patient body can react with the Fc section antigenic determinant of Autologous IgG molecule, and this antibody-like is called as rheumatoid factor.
Sequestrant in reagent I and the reagent II can complexing detects the metallic ion in the sample, the interference that causes to reduce metallic ion; Therefore, various sequestrants that can complexation of metal ions all can be suitable for, for example: disodium ethylene diamine tetraacetate, aminotriacetic acid, diethylene-triamine pentaacetic acid and salt thereof etc., the preferred disodium ethylene diamine tetraacetate of reagent II of the present invention (EDTA-2Na) is sequestrant.
Suspending agent in the reagent II can help latex particle to keep a good disperse state, prevent that latex particle from sinking to affecting quality and the uncork stability of detection kit, suspending agent commonly used has ethylene glycol, glycerine, lactose and maltose etc., and the preferred ethylene glycol of reagent II of the present invention is as suspending agent.
The effect of the sealer in the reagent II is combined for free-COOH site in latex particle, and harmful effect is caused to testing result in the carboxyl site of avoiding dissociating, and preferred BSA albumen is as sealer in the reagent II of the present invention.
Used every kind of component all can obtain from biological reagent company or pharmaceuticals's purchase by commercial sources in the detection kit of the present invention.
Another object of the present invention provides a kind of method for preparing described detection NGAL kit, comprising:
(1) preparation reagent I: each components dissolved in distilled water or distilled water, is mixed constant volume; (2) preparation reagent II: each components dissolved in distilled water or distilled water, is mixed constant volume; (3) with reagent I and the independent packing of reagent II, sealing, and get final product.
Another purpose of the present invention provides the detection method of NGAL content in the described NGAL detection kit test sample, the method is Two point end assay, may further comprise the steps: (calibration tube is made sample with calibration object to 2.5 μ L sample to be detected, blank take distilled water as sample) the middle abundant mixing of reagent I that adds 120 μ L, in 37 ℃ of constant temperature 5 minutes, in mixed system, add 30 μ L reagent II again, mixing, 37 ℃ of constant temperature are after 1 minute, the blank tube zeroing, wavelength 570nm measures respectively to manage to measure behind the absorbance A Isosorbide-5-Nitrae minute and respectively manages absorbance A 2.Calculate Δ A=A2-A1.According to multiple spot calibration object concentration and corresponding absorbance changing value Δ A, adopt multiple spot gamma correction mode decision working curve, sample absorbance variation corresponding concentration value on working curve is mensuration concentration.
Detection kit of the present invention is improved prior art and prescription on the basis of existing NGAL latex detection kit, and it is poor to have overcome existing latex detection kit anti-interference, has eliminated the impact of NGAL/MMP-9 compound on testing result; In addition, it is the latex particle that carrier prepares coated neutrophil gelatinase-associated lipocalin (NGAL) antibody that detection kit of the present invention adopts the latex particle of medium particle diameter, not only improve detection sensitivity, also efficiently solved linear narrow range and the inadequate problem of product stability of detecting.
Detection kit of the present invention can overcome the impact of interference factor, can detect the compound of NGAL and MMP-9, avoids the NGAL testing result doctor's of causing on the low side erroneous judgement; Simultaneously detection kit of the present invention also has the uncork good stability, detects that the range of linearity is wide, production cost is lower etc. a bit.
Fig. 1 is the standard working curve of detection kit of the present invention.
Fig. 2 is 37 ℃ of heat stabilization test results of detection kit of the present invention.，
Fig. 3 is 37 ℃ of heat stabilization test results of contrast NGAL latex immunoturbidimetry detection kit.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.But these embodiment only are exemplary, scope of the present invention are not consisted of any restriction.It will be understood by those skilled in the art that lower without departing from the spirit and scope of the present invention and can make amendment or replace the details of technical solution of the present invention and form, but these modifications and replacing all fall within the scope of protection of the present invention.
The screening of preparation embodiment 1NGAL antibody
1, the preparation of MMP-9 affinity column: with albumen MMP-9 (available from R﹠amp; D company) with hematocrit CNBr-Sepharose4B (Pharmacia product) mix in the 5-7mg/mL ratio, 4 ℃ were shaken 20 hours, and made it crosslinked.Then clean 3 times with 0.1mol/L pH8.0 carbonate buffer solution, add again 4 ℃ of equivalent 0.1mol/L monoethanolamine-HCl and shook 1 hour, to seal residual activation group.Use at last 0.01mol/L Tris-HCl (TB) to clean, fill post, balance, the volume of pillar is selected 10mL.
2, the preparation of MMP-9/NGAL compound affinity column: with albumen NGAL (available from R﹠amp; D company) be mixed with the solution of 2mg/mL, cross the MMP-9 affinity column with the NGAL protein solution with the flow velocity of 1mL/min, the consumption of protein solution is 60mL; Then use 0.1mol/L pH8.0 phosphate buffer with the unnecessary NGAL albumen of the flow velocity wash-out of 1mL/min, the consumption of eluent is 100mL.
3, affinity chromatography: NGAL antibody (available from Wuhan Sino-American Biotechnology Company), the NGAL antibody concentration is adjusted to 600ug/mL, pass through the affinity column of step 2 preparation, clean with 1mol/L NaCl-TB, 0.5%NP-40-TB, TB and distilled water successively with 6mL/ hour flow velocity, to remove non-specific adsorption.Then with carrying out desorb in 3mol/L KSCN6mL/ hour.Ultraviolet detects the Fractional Collections stripping liquid.With the bag filter TB that fully dialyses, the final vacuum freeze-drying solution both must be through the NGAL antibody of screening.
The preparation of the latex particle of preparation embodiment 2 coated NGAL antibody
The preparation process of the latex particle of coated NGAL antibody is as follows:
1. the preparation of carboxylation modified polystyrene latex solution: taking by weighing the 1.0g particle diameter is the polystyrene latex particle drying thing of 105nm, adding 100mL0.12M phosphate buffer (pH7.5) disperses, add 40mg1-ethyl-3-(3-dimethylaminopropyl) carbodiimides (EDC), the sulfo-NHS that adds again 20mg, at room temperature stirring reaction is 0.5 hour, the centrifugal supernatant of abandoning disperses precipitation latex both to get carboxylation modified polystyrene latex with the dilution of 100mL0.12M phosphate buffer again;
2. the preparation of NGAL antibody-solutions: with the 250 μ g preparation embodiment 1 NGAL antibody that obtains that screens 0.5mL deionized water dissolving, obtain the NGAL antibody-solutions;
3. the preparation of the latex particle of coated NGAL antibody: get 100 μ L and 2. go on foot prepared NGAL antibody-solutions, after the dilution of 5mL0.12M phosphate buffer, add the 5mL step and 1. make latex solution, at room temperature stirring reaction is 2 hours, adds 0.2mL0.1M glycine buffer and 2mL10%BSA solution, stirs 30 minutes, cessation reaction, the centrifugal supernatant of abandoning with 0.12M phosphate buffer washing precipitation 2 times, obtains the latex particle of coated NGAL antibody.
The preparation of the latex particle of preparation embodiment 3 coated NGAL antibody
The preparation process of the latex particle of coated NGAL antibody is as follows:
1. the preparation of carboxylation modified polystyrene latex solution: taking by weighing the 1.0g particle diameter is the polystyrene latex particle drying thing of 130nm, adding 100mL0.12M phosphate buffer (pH7.5) disperses, add 40mg1-ethyl-3-(3-dimethylaminopropyl) carbodiimides (EDC), the sulfo-NHS that adds again 20mg, at room temperature stirring reaction is 0.5 hour, the centrifugal supernatant of abandoning disperses precipitation latex both to get carboxylation modified polystyrene latex with the dilution of 100mL0.12M phosphate buffer again;
2. the preparation of NGAL antibody-solutions: with the 250 μ g preparation embodiment 1 NGAL antibody that obtains that screens 0.5mL deionized water dissolving, obtain the NGAL antibody-solutions;
3. the preparation of the latex particle of coated NGAL antibody: get 100 μ L and 2. go on foot prepared NGAL antibody-solutions, after the dilution of 5mL0.12M phosphate buffer, add the 5mL step and 1. make latex solution, at room temperature stirring reaction is 2 hours, adds 0.2mL0.1M glycine buffer and 2mL10%BSA solution, stirs 30 minutes, cessation reaction, the centrifugal supernatant of abandoning with 0.12M phosphate buffer washing precipitation 2 times, obtains the latex particle of coated NGAL antibody.
The preparation of the latex particle of preparation embodiment 4 coated NGAL antibody
The preparation process of the latex particle of coated NGAL antibody is as follows:
1. the preparation of carboxylation modified polystyrene latex solution: taking by weighing the 1.0g particle diameter is the polystyrene latex particle drying thing of 145nm, adding 100mL0.12M phosphate buffer (pH7.5) disperses, add 40mg1-ethyl-3-(3-dimethylaminopropyl) carbodiimides (EDC), the sulfo-NHS that adds again 20mg, at room temperature stirring reaction is 0.5 hour, the centrifugal supernatant of abandoning disperses precipitation latex both to get carboxylation modified polystyrene latex with the dilution of 100mL0.12M phosphate buffer again;
2. the preparation of NGAL antibody-solutions: with the 250 μ g preparation embodiment 1 NGAL antibody that obtains that screens 0.5mL deionized water dissolving, obtain the NGAL antibody-solutions;
3. the preparation of the latex particle of coated NGAL antibody: get 100 μ L and 2. go on foot prepared NGAL antibody-solutions, after the dilution of 5mL0.12M phosphate buffer, add the 5mL step and 1. make latex solution, at room temperature stirring reaction is 2 hours, adds 0.2mL0.1M glycine buffer and 2mL10%BSA solution, stirs 30 minutes, cessation reaction, the centrifugal supernatant of abandoning with 0.12M phosphate buffer washing precipitation 2 times, obtains the latex particle of coated NGAL antibody.
The preparation of embodiment 1 NGAL latex detection kit:
1, the preparation of reagent I:
Measure each component by described usefulness:
Above-mentioned each components dissolved in distilled water or distilled water, is settled to 1 liter, and sealing is preserved, and is for subsequent use.
2, the preparation of reagent II:
Measure each component by described usefulness:
Above-mentioned each components dissolved in distilled water or distilled water, is settled to 1 liter, and sealing is preserved, and is for subsequent use.
The preparation of embodiment 2 NGAL latex detection kit:
1, the preparation of reagent I:
Measure each component by described usefulness:
Above-mentioned each components dissolved in distilled water or distilled water, is settled to 1 liter, and sealing is preserved, and is for subsequent use.
2, the preparation of reagent II:
Measure each component by described usefulness:
Above-mentioned each components dissolved in distilled water or distilled water, is settled to 1 liter, and sealing is preserved, and is for subsequent use.
The preparation of embodiment 3 NGAL latex detection kit:
1, the preparation of reagent I:
Measure each component by described usefulness:
Above-mentioned each components dissolved in distilled water or distilled water, is settled to 1 liter, and sealing is preserved, and is for subsequent use.
2, the preparation of reagent II:
Measure each component by described usefulness:
Above-mentioned each components dissolved in distilled water or distilled water, is settled to 1 liter, and sealing is preserved, and is for subsequent use.
The preparation of embodiment 4 NGAL latex detection kit:
1, the preparation of reagent I:
Measure each component by described usefulness:
Above-mentioned each components dissolved in distilled water or distilled water, is settled to 1 liter, and sealing is preserved, and is for subsequent use.
2, the preparation of reagent II:
Measure each component by described usefulness:
Above-mentioned each components dissolved in distilled water or distilled water, is settled to 1 liter, and sealing is preserved, and is for subsequent use.
The preparation of embodiment 5 NGAL latex detection kit:
1, the preparation of reagent I:
Measure each component by described usefulness:
Above-mentioned each components dissolved in distilled water or distilled water, is settled to 1 liter, and sealing is preserved, and is for subsequent use.
2, the preparation of reagent II:
Measure each component by described usefulness:
Above-mentioned each components dissolved in distilled water or distilled water, is settled to 1 liter, and sealing is preserved, and is for subsequent use.
The preparation of embodiment 6 NGAL latex detection kit:
1, the preparation of reagent I:
Measure each component by described usefulness:
Above-mentioned each components dissolved in distilled water or distilled water, is settled to 1 liter, and sealing is preserved, and is for subsequent use.
2, the preparation of reagent II:
Measure each component by described usefulness:
Above-mentioned each components dissolved in distilled water or distilled water, is settled to 1 liter, and sealing is preserved, and is for subsequent use.
The preparation of comparative example's 1 NGAL latex detection kit:
One, the preparation of the latex particle of coated NGAL antibody
1. the preparation of carboxylation modified polystyrene latex solution: taking by weighing the 1.0g particle diameter is the polystyrene latex particle drying thing of 100nm, adding 100mL0.12M phosphate buffer (pH7.5) disperses, add 40mg1-ethyl-3-(3-dimethylaminopropyl) carbodiimides (EDC), the sulfo-NHS that adds again 20mg, at room temperature stirring reaction is 0.5 hour, the centrifugal supernatant of abandoning disperses precipitation latex both to get carboxylation modified polystyrene latex with the dilution of 100mL0.12M phosphate buffer again;
2. the preparation of NGAL antibody-solutions: with 250 μ g NGAL antibody 0.5mL deionized water dissolvings, obtain the NGAL antibody-solutions;
3. the preparation of the latex particle of coated NGAL antibody: get 100 μ L and 2. go on foot prepared NGAL antibody-solutions, after the dilution of 5mL0.12M phosphate buffer, add the 5mL step and 1. make latex solution, at room temperature stirring reaction is 2 hours, adds 0.2mL0.1M glycine buffer and 2mL10%BSA solution, stirs 30 minutes, cessation reaction, the centrifugal supernatant of abandoning with 0.12M phosphate buffer washing precipitation 2 times, obtains the latex particle of coated NGAL antibody.
Two, the preparation of kit
1, the preparation of reagent I:
Measure each component by described usefulness:
Above-mentioned each components dissolved in distilled water or distilled water, is settled to 1 liter, and sealing is preserved, and is for subsequent use.
2, the preparation of reagent II:
Measure each component by described usefulness:
Above-mentioned each components dissolved in distilled water or distilled water, is settled to 1 liter, and sealing is preserved, and is for subsequent use.
The preparation of comparative example's 2 NGAL latex detection kit:
One, the preparation of the latex particle of coated NGAL antibody
1. the preparation of carboxylation modified polystyrene latex solution: taking by weighing the 1.0g particle diameter is the polystyrene latex particle drying thing of 50nm, adding 100mL0.12M phosphate buffer (pH7.5) disperses, add 40mg1-ethyl-3-(3-dimethylaminopropyl) carbodiimides (EDC), the sulfo-NHS that adds again 20mg, at room temperature stirring reaction is 0.5 hour, the centrifugal supernatant of abandoning disperses precipitation latex both to get carboxylation modified polystyrene latex with the dilution of 100mL0.12M phosphate buffer again;
2. the preparation of NGAL antibody-solutions: with 250 μ g NGAL antibody 0.5mL deionized water dissolvings, obtain the NGAL antibody-solutions;
3. the preparation of the latex particle of coated NGAL antibody: get 100 μ L and 2. go on foot prepared NGAL antibody-solutions, after the dilution of 5mL0.12M phosphate buffer, add the 5mL step and 1. make latex solution, at room temperature stirring reaction is 2 hours, adds 0.2mL0.1M glycine buffer and 2mL10%BSA solution, stirs 30 minutes, cessation reaction, the centrifugal supernatant of abandoning with 0.12M phosphate buffer washing precipitation 2 times, obtains the latex particle of coated NGAL antibody.
Two, the preparation of kit
1, the preparation of reagent I:
Measure each component by described usefulness:
Above-mentioned each components dissolved in distilled water or distilled water, is settled to 1 liter, and sealing is preserved, and is for subsequent use.
2, the preparation of reagent II:
Measure each component by described usefulness:
Above-mentioned each components dissolved in distilled water or distilled water, is settled to 1 liter, and sealing is preserved, and is for subsequent use.
The preparation of comparative example's 3 NGAL latex detection kit:
One, the preparation of the latex particle of coated NGAL antibody
1. the preparation of carboxylation modified polystyrene latex solution: taking by weighing the 1.0g particle diameter is the polystyrene latex particle drying thing of 150nm, adding 100mL0.12M phosphate buffer (pH7.5) disperses, add 40mg1-ethyl-3-(3-dimethylaminopropyl) carbodiimides (EDC), the sulfo-NHS that adds again 20mg, at room temperature stirring reaction is 0.5 hour, the centrifugal supernatant of abandoning disperses precipitation latex both to get carboxylation modified polystyrene latex with the dilution of 100mL0.12M phosphate buffer again;
2. the preparation of NGAL antibody-solutions: with 250 μ g NGAL antibody 0.5mL deionized water dissolvings, obtain the NGAL antibody-solutions;
3. the preparation of the latex particle of coated NGAL antibody: get 100 μ L and 2. go on foot prepared NGAL antibody-solutions, after the dilution of 5mL0.12M phosphate buffer, add the 5mL step and 1. make latex solution, at room temperature stirring reaction is 2 hours, adds 0.2mL0.1M glycine buffer and 2mL10%BSA solution, stirs 30 minutes, cessation reaction, the centrifugal supernatant of abandoning with 0.12M phosphate buffer washing precipitation 2 times, obtains the latex particle of coated NGAL antibody.
Two, the preparation of kit
1, the preparation of reagent I:
Measure each component by described usefulness:
Above-mentioned each components dissolved in distilled water or distilled water, is settled to 1 liter, and sealing is preserved, and is for subsequent use.
2, the preparation of reagent II:
Measure each component by described usefulness:
Above-mentioned each components dissolved in distilled water or distilled water, is settled to 1 liter, and sealing is preserved, and is for subsequent use.
The preparation of comparative example's 4 NGAL latex detection kit:
One, the preparation of the latex particle of coated NGAL antibody
1. the preparation of carboxylation modified polystyrene latex solution: taking by weighing the 1.0g particle diameter is the polystyrene latex particle drying thing of 500nm, adding 100mL0.12M phosphate buffer (pH7.5) disperses, add 40mg1-ethyl-3-(3-dimethylaminopropyl) carbodiimides (EDC), the sulfo-NHS that adds again 20mg, at room temperature stirring reaction is 0.5 hour, the centrifugal supernatant of abandoning disperses precipitation latex both to get carboxylation modified polystyrene latex with the dilution of 100mL0.12M phosphate buffer again;
2. the preparation of NGAL antibody-solutions: with 250 μ g NGAL antibody 0.5mL deionized water dissolvings, obtain the NGAL antibody-solutions;
3. the preparation of the latex particle of coated NGAL antibody: get 100 μ L and 2. go on foot prepared NGAL antibody-solutions, after the dilution of 5mL0.12M phosphate buffer, add the 5mL step and 1. make latex solution, at room temperature stirring reaction is 2 hours, adds 0.2mL0.1M glycine buffer and 2mL10%BSA solution, stirs 30 minutes, cessation reaction, the centrifugal supernatant of abandoning with 0.12M phosphate buffer washing precipitation 2 times, obtains the latex particle of coated NGAL antibody.
Two, the preparation of kit
1, the preparation of reagent I:
Measure each component by described usefulness:
Above-mentioned each components dissolved in distilled water or distilled water, is settled to 1 liter, and sealing is preserved, and is for subsequent use.
2, the preparation of reagent II:
Measure each component by described usefulness:
Above-mentioned each components dissolved in distilled water or distilled water, is settled to 1 liter, and sealing is preserved, and is for subsequent use.
The standard working curve of test example 1 detection kit of the present invention
Getting concentration is 0ng/mL, 150ng/mL, 600ng/mL, 1500ng/mL, 3000ng/mL, the NGAL standard solution of 5000ng/mL detects it with the embodiment of the invention 1 prepared NGAL detection kit, the NGAL latex immunoturbidimetry detection kit of comparative example's 2 preparations and the NGAL latex detection kit of comparative example's 4 preparations, draw each detection kit standard working curve, the results are shown in Figure 1.
The detection method of NGAL content in each NGAL detection kit test sample, the method is Two point end assay, may further comprise the steps: (calibration tube is made sample with calibration object to 2.5 μ L sample to be detected, blank take distilled water as sample) the middle abundant mixing of reagent I that adds 120 μ L, in 37 ℃ of constant temperature 5 minutes, in mixed system, add 30 μ L reagent II again, mixing, 37 ℃ of constant temperature are after 1 minute, the blank tube zeroing, wavelength 570nm measures respectively to manage to measure behind the absorbance A Isosorbide-5-Nitrae minute and respectively manages absorbance A 2.Calculate Δ A=A2-A1.According to multiple spot calibration object concentration and corresponding absorbance changing value Δ A, adopt multiple spot gamma correction mode decision working curve, sample absorbance variation corresponding concentration value on working curve is mensuration concentration.
By the standard working curve of Fig. 1 as seen, the range of linearity of detection kit of the present invention and sensitivity all are better than comparative example's detection kit.
The test of the high value of test example 2 detection kit of the present invention and the Comparative Examples detection kit range of linearity
Detect with the detection kit of the embodiment of the invention 2 preparation and comparative example's 3 the detection kit high value quality-control product after to serial dilution, the concentration of quality-control product and calculate its deviation from linearity when measuring different extension rate respectively, test findings see Table 1 and table 2:
The high value of table 1 embodiment of the invention 2 detection kit range of linearity result
| Extension rate | Measured value 1 | Measured value 2 | Measure average | Theoretical value | Deviation from linearity (%) |
| 0.1 | 940 | 942 | 941.5 | 956.8 | -1.60 |
| 0.2 | 1904 | 1898 | 1901.0 | 1913.6 | -0.66 |
| 0.3 | 2935 | 2924 | 2929.5 | 2870.4 | 2.06 |
| 0.4 | 3801 | 3807 | 3804.0 | 3827.2 | -0.61 |
| 0.5 | 4753 | 4745 | 4749.0 | 4784 | -0.73 |
| 0.6 | 5780 | 5789 | 5784.5 | 5740.8 | 0.76 |
| 0.7 | 6794 | 6785 | 6789.5 | 6697.6 | 1.37 |
| 0.8 | 7601 | 7611 | 7606.0 | 7654.4 | -0.63 |
| 0.9 | 8656 | 8649 | 8652.5 | 8611.2 | 0.48 |
| 1.0 | 9618 | 9627 | 9622.5 | 9568 | 0.57 |
The high value of table 2 comparative example 3 detection kit range of linearity result
| Extension rate | Measured value 1 | Measured value 2 | Measure average | Theoretical value | Deviation from linearity (%) |
| 0.1 | 569 | 572 | 570.5 | 584.8 | -2.45 |
| 0.2 | 1184 | 1176 | 1180.0 | 1169.6 | 0.89 |
| 0.3 | 1697 | 1684 | 1690.5 | 1754.4 | -3.64 |
| 0.4 | 2298 | 2287 | 2292.5 | 2339.2 | -2.00 |
| 0.5 | 2774 | 2758 | 2766.0 | 2924 | -5.40 |
| 0.6 | 3337 | 3318 | 3327.5 | 3508.8 | -5.17 |
| 0.7 | 3678 | 3664 | 3671.0 | 4093.6 | -10.32 |
| 0.8 | 3787 | 3804 | 3795.5 | 4678.4 | -18.87 |
[0190]?
| 0.9 | 3847 | 3840 | 3843.5 | 5263.2 | -26.97 |
| 1.0 | 3894 | 3912 | 3903.0 | 5848 | -33.26 |
By the result in table 1 and the table 2 as seen, detection kit of the present invention detects the range of linearity will be apparently higher than the control test kit, deviation from linearity is greater than 10% during greater than 4000ng/mL at NGAL content for the Comparative Examples detection kit, and detection kit of the present invention is that 9568 o'clock deviations from linearity still only have 0.57% in NGAL concentration.Illustrate that thus the detection kit range of linearity of the present invention will be much better than the detection kit that conventional method is made.
Test example 3 detection kit of the present invention and the test of the contrast detection kit low value range of linearity
The low value quality-control product of the detection kit that the detection kit of getting the embodiment of the invention 2 preparation and Comparative Examples 3 prepare after to serial dilution detects, the concentration of quality-control product and calculate its deviation from linearity when measuring different extension rate respectively, test findings see Table 3 and table 4:
Table 3 embodiment of the invention 2 detection kit low value range of linearity results
| Extension rate | Measured value 1 | Measured value 2 | Measure average | Theoretical value | Deviation from linearity (%) |
| 0.1 | 62.7 | 64.3 | 63.5 | 63.25 | 0.40 |
| 0.2 | 127.3 | 128.0 | 127.65 | 126.5 | 0.91 |
| 0.3 | 190.4 | 191.1 | 190.75 | 189.75 | 0.53 |
| 0.4 | 254.3 | 254.8 | 254.55 | 253 | 0.61 |
| 0.5 | 318.5 | 318.9 | 318.7 | 316.25 | 0.77 |
| 0.6 | 381.2 | 379.2 | 380.2 | 379.5 | 0.18 |
| 0.7 | 445.2 | 445.6 | 445.4 | 442.75 | 0.60 |
| 0.8 | 505.4 | 505.1 | 505.25 | 506 | -0.15 |
| 0.9 | 570.3 | 569.1 | 569.7 | 569.25 | 0.08 |
| 1.0 | 633.1 | 633.9 | 633.5 | 632.5 | 0.16 |
Table 4 comparative example 3 detection kit low value range of linearity results
| Extension rate | Measured value 1 | Measured value 2 | Measure average | Theoretical value | Deviation from linearity (%) |
| 0.1 | 69.2 | 68.9 | 69.05 | 73.11 | -5.55 |
| 0.2 | 155.3 | 155.1 | 155.2 | 146.22 | 6.14 |
| 0.3 | 231.3 | 230.9 | 231.1 | 219.33 | 5.37 |
[0198]?
| 0.4 | 293.5 | 292.9 | 293.2 | 292.44 | 0.26 |
| 0.5 | 356.8 | 356.7 | 356.75 | 365.55 | -2.41 |
| 0.6 | 426.1 | 425.9 | 426 | 438.66 | -2.89 |
| 0.7 | 523.1 | 523.4 | 523.25 | 511.77 | 2.24 |
| 0.8 | 596.4 | 596.3 | 596.35 | 584.88 | 1.96 |
| 0.9 | 661.7 | 669.2 | 665.45 | 657.99 | 1.13 |
| 1.0 | 741.6 | 743.5 | 742.55 | 731.1 | 1.57 |
By the result in table 3 and the table 4 as seen, the embodiment of the invention 2 detection kit detect the low value range of linearity will be starkly lower than Comparative Examples 3 detection kit, and the deviation from linearity of detected value and theoretical value is less, illustrates that measured value is more accurate.
The comparison when detecting high concentration NGAL/MMP-9 compound sample of test example 4 detection kit of the present invention and Comparative Examples detection kit
The NGAL/MMP-9 compound adopts Human MMP-9/NGAL Complex Immunoassay (available from R﹠amp in the described urine specimen of this test example; D Systems, Inc.) detect, adopt again the detection kit of embodiment of the invention 1-3 preparation and the detection kit of comparative example 1-4 preparation to detect respectively (testing result is averaged) to the sample that MMP-9/NGAL compound content is high in the urine specimen.The difference of concrete testing result sees Table 5:
Table 5 detection kit of the present invention and Comparative Examples detection kit testing result are relatively
The NGAL content that visible detection kit of the present invention detects from table 5 will be higher than the prepared detection kit of comparative example 1-4, illustrate that kit of the present invention can avoid the NGAL/MMP-9 compound on the impact of testing result, avoid the testing result clinical misdiagnosis that causes on the low side and erroneous judgement.
The anti-interference detection test of test example 5 detection kit of the present invention
Final concentration by the rheumatoid factor shown in the table 6 in the 600ng/mL calibration object adds a certain amount of rheumatoid factor, the NGAL latex detection kit of producing with the prepared detection kit of embodiment of the invention 1-3 and Denmark Bioporto Diagnostics company respectively again detects each sample, investigate rheumatoid factor to the impact of each detection kit, the results are shown in following table 6.NGAL detection kit of the present invention is measured the method for NGAL content with test example 1, and the NGAL latex detection kit that Denmark Bioporto Diagnostics company produces detects the method for NGAL content referring to product description.The interference performance of the detection kit resisting rheumatoid disease factor of the present invention will obviously be better than the control test kit as can be known from Table 6.
Table 6NGAL detection kit resisting rheumatoid disease factor jamming performance control experiment result
Test example 6 detection kit heat stabilization tests of the present invention
The detection kit that embodiment of the invention 1-6 is prepared and the NGAL of Beijing Strong Biotechnologies, Inc. latex immunoturbidimetry detection kit, in 37 ℃ of difference thermal treatment 0 day, 3 days, 5 days and 7 days, and after the different processing times, measure respectively the NGAL calibration object, record its Δ A value, and draw its change curve, 37 ℃ of heat stabilization tests of detection kit of the present invention the results are shown in Figure 2, and 37 ℃ of heat stabilization tests of the NGAL of Beijing Strong Biotechnologies, Inc. latex immunoturbidimetry detection kit the results are shown in Figure 3.
From the result of Fig. 2 and Fig. 3 as can be known, 37 ℃ of heat treatment stabilities of detection kit of the present invention are good, and its good stability has determined its widespread use in clinical diagnosis.
Claims (10)
1. a neutrophil gelatinase-associated lipocalin detection kit is comprised of reagent I independent of each other and reagent II; The component of described reagent I comprises: biological buffer, surfactant, set accelerator, antiseptic, stabilizing agent, blocking agent, sequestrant and water; The component of described reagent II comprises: latex particle, biological buffer, sequestrant, surfactant, antiseptic, suspending agent, sealer, stabilizing agent and the water of coated neutrophil gelatinase-associated lipocalin antibody; It is characterized in that: described neutrophil gelatinase-associated lipocalin antibody can combine with the MMP-9/NGAL compound again with the combination of neutrophil gelatinase-associated lipocalin antigen.
2. according to detection kit claimed in claim 1, it is characterized in that described neutrophil gelatinase-associated lipocalin antibody obtains by the screening of affinity chromatography method; Preferably, its screening technique comprises:
(1) preparation MMP-9 affinity column: MMP-9 albumen is mixed with hematocrit CNBr-Sepharose4B, pass through successively crosslinked, clean, seal, again clean, fill post, balance, obtain the MMP-9 affinity column;
(2) preparation MMP-9/NGAL compound affinity column: albumen NGAL solution is crossed the MMP-9 affinity column; The unnecessary NGAL albumen of wash-out again;
(3) affinity chromatography: the NGAL antibody-solutions by the prepared affinity column of step (1), is carried out desorb after the removal non-specific adsorption, the Fractional Collections stripping liquid, dialysis, freeze drying, and get final product.
3. according to detection kit claimed in claim 1, it is characterized in that the preparation method of the latex particle of described coated NGAL antibody comprises:
(1) preparation carboxylation modified polystyrene latex solution: getting particle diameter is the polystyrene latex particle drying thing of 105-145nm, disperse with damping fluid, add 1-ethyl-3-(3-dimethylaminopropyl) carbodiimides and sulfo-NHS, stirring reaction under the room temperature; The centrifugal supernatant of abandoning disperses precipitation latex with the damping fluid dilution again, obtains carboxylation modified polystyrene latex;
(2) preparation of NGAL antibody-solutions: with screening obtain can with neutrophil gelatinase-associated lipocalin antigen in conjunction with NGAL monoclonal antibody or the polyclonal antibody deionized water dissolving that can combine with the MMP-9/NGAL compound again, obtain the NGAL antibody-solutions;
(3) preparation of the latex particle of coated NGAL antibody: the antibody-solutions that step (2) is prepared adds carboxylation modified polystyrene latex solution, at room temperature stirring reaction after diluting with damping fluid, after the cessation reaction, the centrifugal supernatant of abandoning, washing precipitation, and get final product.
4. according to detection kit claimed in claim 1, it is characterized in that: described surfactant is triton x-100; Described antiseptic is Proclin-300; Described stabilizing agent is sucrose; Described set accelerator is PEG-6000 or PEG-8000; Described blocking agent is the rheumatoid factor blocking agent; Described sequestrant is disodium ethylene diamine tetraacetate; Described suspending agent is ethylene glycol; Described sealer is bovine serum albumin(BSA).
5. according to detection kit claimed in claim 1, it is characterized in that: the biological buffer described in the reagent I is trishydroxymethylaminomethane, and the biological buffer described in the reagent II is 3-(N-morpholine) propane sulfonic acid.
6. according to detection kit claimed in claim 1, it is characterized in that: the consumption of each component is in per 1 liter of reagent I: biological buffer 5-100g, surfactant 0.5-20mL, set accelerator 2-50g, antiseptic 0.02-1mL, stabilizing agent 1-60g, blocking agent 1-10mL, sequestrant 0.05-5g, surplus is water;
The consumption of each component is in per 1 liter of reagent II: the latex particle 0.1-10g of coated NGAL antibody, biological buffer 1-100g, sequestrant 0.1-10g, surfactant 0.05-3mL, antiseptic 0.02-1mL, suspending agent 5-50mL, sealer 5-50g, stabilizing agent 50-150g, surplus is water.
7. according to kit claimed in claim 6, it is characterized in that: the consumption of each component is in per 1 liter of reagent I: biological buffer 7-20g, surfactant 1-10mL, set accelerator 10-30g, antiseptic 0.1-0.5mL, stabilizing agent 5-15g, blocking agent 3-6mL, sequestrant 0.1-1g, surplus is water;
The consumption of each component is in per 1 liter of reagent II: the latex particle 0.5-5g of coated NGAL antibody, biological buffer 5-30g, sequestrant 0.5-4g, surfactant 0.5-2mL, antiseptic 0.1-0.5mL, suspending agent 10-40mL, sealer 7-15g, stabilizing agent 80-120g.
8. according to kit claimed in claim 7, it is characterized in that: the consumption of each component is in per 1 liter of reagent I: biological buffer 12g, surfactant 4mL, set accelerator 15g, antiseptic 0.25mL, stabilizing agent 10g, blocking agent 5mL, sequestrant 0.4g, surplus is water;
The consumption of each component is in per 1 liter of reagent II: the latex particle 1.7g of coated NGAL antibody, biological buffer 11g, sequestrant 2g, surfactant 1.5mL, antiseptic 0.25mL, suspending agent 20mL, sealer 10g, stabilizing agent 100g.
9. according to any one detection kit of claim 1-8, it is characterized in that: the scope of the pH value of reagent I is 7.0-8.0, and the scope of the pH value of reagent II is 6.5-7.5.
10. a method for preparing any one described detection kit of claim 1-8 comprises: (1) preparation reagent I: each components dissolved in distilled water or distilled water, is mixed constant volume; (2) preparation reagent II: each components dissolved in distilled water or distilled water, is mixed constant volume; (3) with reagent I and the independent packing of reagent II, sealing, and get final product.
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