CN101699287A - Homogeneous phase sol particle type cystatin C measuring kit and preparation method thereof - Google Patents
Homogeneous phase sol particle type cystatin C measuring kit and preparation method thereof Download PDFInfo
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Images
Abstract
The invention provides a homogeneous phase sol particle type immunity measuring kit and a preparation method thereof. The homogeneous phase sol particle type immunity measuring kit comprises a reagent R1 and a reagent R2, wherein the reagent R1 is a buffer solution containing polymers, and the reagent R2 is a buffer solution of colloidal gold marked with anti-human cystatin C antibodies, wherein the particle diameter of the colloidal gold is 65-85nm. The kit of the invention can specifically measure the content of cystatin C in blood serum or blood plasma at high sensitivity.
Description
Technical field
The present invention relates to a kind of homogeneous phase sol particle type immunoassay kit and preparation method thereof, more specifically, relate to and be used for homogeneous phase sol particle type cystatin C immunoassay kit of measuring serum or blood plasma bladder chalone C and preparation method thereof.
Background technology
Bladder chalone C (CystatinC) is a kind of low molecular weight protein, can be produced by all karyocytes of body, and generation rate is constant.Bladder chalone C in the circulation only is eliminated through glomerular filtration, it is a kind of ideal endogenous mark that reflects that glomerular filtration rate(GFR changes, and the filtration rate of glomerulus (glomerular filtrationrate, GFR) be an important indicator of monitoring renal function, especially for the patient of kidney transplant, the variation of grasping glomerular filtration rate(GFR fast and accurately is crucial.In addition since the bladder chalone C molecular weight greater than creatinine, and it is positively charged, so early stage variation of its easier reflection glomerular filtration membrane permeability, can when slightly reducing, glomerular filtration rate(GFR raise, more responsive than creatinine, be better than serum creatinine as kidney function test, have important clinical application value.
Bladder chalone C is the only low-molecular-weight secreted protein of 13KD of a kind of molecular weight of being made up of 120 amino acid residues, is one of member of cysteine proteinase mortifier superfamily.Cystatin C genes 5 ' flanking sequence has identical characteristic with the promoter region of " house-keeping gene ", also to belong to " house-keeping gene " be this gene transcribes constantly and expresses in that institute is constant in a organized way, the inorganization specificity, be present in widely in the various body fluid, as cerebrospinal fluid, blood, saliva, seminal fluid etc., body bladder chalone C generation rate is quite constant.And bladder chalone C is a kind of low molecular weight protein, can freely filter through glomerulus, kidney is unique organ of removing bladder chalone C in the circulation, is the endogenous mark that a kind of desirable reflection GFR changes so serum bladder chalone C concentration, this shows bladder chalone C mainly by the GFR decision.
Clinical research low molecular weight protein (B2M, RBP ELISA, bladder chalone C) concentration and GFR (
51Cr-EDTA clearance rate or its goldstandard
99mThe Tc-DTPA clearance rate) correlativity, the susceptibility of serum bladder chalone C, creatine concentration diagnosing abnormal GFR is respectively 78.1% and 57.1%, separately inverse concentration and GFR (
51The Cr-EDTA clearance rate) related coefficient is respectively: 0.81 and 0.50 (referring to non-patent literature 1).Show bladder chalone C be in the low molecular weight protein with the maximally related endogenous mark of GFR, even be better than serum creatinine.Serum B2M concentration is owing to also raising when inflammation, tumour and immunological diseases, so be not desirable GFR endogenous mark.Even and bladder chalone C is under inflammatory conditions, its generation rate also can not change, and serum-concentration is subjected to the influence of change of illness state of age, sex, disease less.The diagnostic accuracy that bladder chalone C is described obviously is better than serum creatinine.
Detection side's science of law of bladder chalone C has experienced the evolution in general 4 stages, and to enzyme immunoassay (EIA), latex finally strengthens method and homogeneous phase sol particle method etc. again from initial unidirectional immunodiffusion, and several method all belongs to immunodetection.The ultimate principle of immunity turbidimetric analysis turbidimetry is: antigen-antibody forms antigen antibody complex fast in special damping fluid, make reactant liquor turbidity occur.When keeping antibody excess in the reactant liquor, the compound of formation increases with the antigen amount, and the turbidity of reactant liquor also increases thereupon, with a series of standard control, can calculate the content of thing to be detected.Form turbidity but the antigen antibody complex that the content low-molecular-weight is little is extremely difficult, unless place the long period; As forming bigger compound, then antigen and antibody consumption are also bigger, obviously do not meet the requirement of traceization.So developed immune latex nephelometry, it is a kind of on-radiation homogeneous phase immunity nephelometry that development is set up on latex agglutination qualitative test basis, its ultimate principle is: antibody is adsorbed on be of moderate size, on the latex particle of uniformity, when running into corresponding antigens, then make latex particle generation aggegation.Single latex particle does not hinder light and sees through within incident light wave, then make through light during two or more latex particle cohesions and reduce, the degree of the degree of this minimizing and latex particle cohesion is proportional, certainly also proportional with the determined antigen amount, can measure the content of determinand in the sample thus.This method can produce immunoprecipitation.The homogeneous phase sol particle immunoassay had appearred again afterwards, its ultimate principle is: antibody is adsorbed on be of moderate size, on the colloid gold particle of uniformity, when running into corresponding antigens, because the interaction of antigen-antibody is condensed the colloid gold particle of absorption antibody.Gold grain cohesion back color is by red stain indigo plant, and absorbance can reduce gradually at wavelength 540nm place.And the degree of the degree of this reduction and colloid gold particle cohesion is proportional, also proportional with the determined antigen amount certainly, can measure the content of determinand in the sample thus.But, present homogeneous phase sol particle immunoassay, its sensitivity for analysis is also unsatisfactory, waits in expectation further to improve the detection sensitivity of this method.
Because bladder chalone C concentration is lower in the serum, so its assay method needs higher sensitivity for analysis and specificity.Adopt simple immunodiffusion method (RID) and enzyme immunoassay (EIA) that bladder chalone C content is measured the earliest, and these two kinds of methods are not only time-consuming, sensitivity is also relatively poor.More simply and the method for sensitiveer radiation, fluorescence and various enzyme immunoassay (EIA) (RIA, FIA and EIA) can improve the reliability of bladder chalone C determining, but minute is still longer, can't conventionally use, and more can't be used for emergency treatment and measure.More than various assay methods all belong to the heterogeneous assays method, be difficult to robotization, therefore limited the extensive clinical practice of bladder chalone C.Adopt above-mentioned latex enhancing immune homogeneous determination method, i.e. coated antibody on latex particle, particle generation aggegation when combining with antigen, the method is easy to robotization.The latex particle agglutination that Kabanda equals the mensuration bladder chalone C of report in 1994 is a kind of grain count immunoassay (referring to a non-patent literature 2).Kyhse Andersen equals to have reported the same year that a kind of particle strengthens transmission immunoturbidimetry (particle-enhanced turbidimetric immunoassay, PETIA), the time of bladder chalone C determining was foreshortened to about 5 minutes, and can on automatic biochemistry analyzer, carry out, make the routine application of bladder chalone C determining become possible (referring to non-patent literature 3).Finney etc. have reported that in 1997 particle strengthens scattering immunoturbidimetry (particle-enhanced nephelometric immunoassay, PENIA), its principle be with anti-human cystatin C labeling of monoclonal antibody on the polystyrene latex particle, the immune scattering reduced turbidity method that adopts latex particle to strengthen is measured (referring to non-patent literature 4).Homogeneous phase sol particle immunoassay (sol particle immunoassays, SPIAs) the earliest by propositions (referring to non-patent literature 5) such as Leuvering, Tanaka in 2004 etc. have done improvement to this method, sensing range 0.15～8mg/L (referring to non-patent literature 6).This method reaction time is short, can be used for automatic biochemical analyzer.
With regard to detection sensitivity, the RID method is the poorest, and EIA, RIA and FIA method all are better than PETIA, PENIA and SPIAs method.Because there is radioactive contamination in the RTA method, the FIA method needs expensive instrument, and RIA, FIA, EIA method minute are long, and can not full-automation, can't be used for the mensuration of clinical sample in enormous quantities.Though PETIA, PENIA and SPIAs method detection sensitivity difference are a little, but still can reach about 0.15mg/L, well below the lower limit of healthy human serum bladder chalone C concentration (less than 1.0mg/L), can meet clinical needs; These three kinds of assay methods are not subjected to the influence of haemoglobin, cholerythrin, rheumatoid factor, triglyceride basically, and the recovery can reach 95%～109%, reach interassay coefficient of variation all less (＜4.5%) in batch; But, be unfavorable for the cleaning of biochemical instruments owing to can produce precipitation after two kinds of method reactions of PETIA and PENIA.And the SPIAs rule can not produce precipitation.So can first-selected SPIAs method in the routine clinical of serum bladder chalone C determining is used.Tanaka has carried out studying (non-patent literature 6) to the SPIAs method, but the sensitivity of the method for its proposition still remains to be improved.
Non-patent literature 1:Newman DJ.Serum cysC measured by automatedimmunoassay:a more sensitive marker of changes in GFR than serum creatinine.Kidney Int.1995,47:312-318.
Non-patent literature 2:Kabamda A.Determinants of the serum con-centrations oflow molecular weight proteins in patients on maintenance hemodialysis.KidneyInt.1994,45 (6): 1689～1696
Non-patent literature 3:Kyhse, Andersen J.Serum Cystatin C, determined by arapid, automated partied-enhanced tirbidimetric method, is better maker thanserum creatinine for glomenular filtration taer.Clin Chem.1994,40 (10): 1921～1926
Non-patent literature 4:Finney H.Initial evaluation of Cystatin C measurement byparticle enhanced immunonephelometry on the Behringnephelometer2systems (BNA, BN II) .Clin Chem.1997,43 (6): 1016～1022
Non-patent literature 5:Leuvering JHW.Sol particle immunoassay (SPIA) .JImmunoassay.1980,1:77-91.
Non-patent literature 6:Tanaka M.A sol particle homogeneous immunoassay formeasuring serum cystatin C, Clinical Biochemistry.2004,37:27-35
Summary of the invention
The problem to be solved in the present invention is the sensitivity for analysis that further improves when measuring bladder chalone C content, and a species specific highly sensitive homogeneous phase sol particle type immunoassay kit is provided, and can be used to measure the bladder chalone C content of 0.06mg/L～8mg/L.The present invention also aims to provide the preparation method of mentioned reagent box.In addition, the present invention also aims to provide the method for using homogeneous phase sol particle type immunoassay kit of the present invention to measure bladder chalone C content.
In order to address the above problem, the present invention adopts homogeneous phase sol particle immunoassay (SPIAs) to detect bladder chalone C content in serum or the blood plasma.Employing is marked on the antibody of the anti-bladder chalone C of collaurum, with the bladder chalone C generation aggregation in the sample to be measured the collaurum color is changed, detect this variation with 540nm, draw the concentration of target detection thing bladder chalone C in the sample with the typical curve of bladder chalone C standard items.
The invention provides following technical scheme.
1. homogeneous phase sol particle type immunoassay kit, it is used to measure the content of bladder chalone C, this kit comprises reagent R1 and reagent R2, in volume ratio, the content ratio of reagent R1 and reagent R2 is 4: 1, randomly contains the bladder chalone C standard items in this kit, and described reagent R1 is the buffer solution that contains polymer, described reagent R2 is the buffer solution that is combined with the collaurum of anti-human cystatin C antibody, and wherein the colloid gold particle diameter is 65～85nm.
2. according to the homogeneous phase sol particle type immunoassay kit of claim 1 record, wherein, described colloid gold particle diameter is 70～80nm.
3. according to the homogeneous phase sol particle type immunoassay kit of claim 1 or 2 records, wherein, in the buffer solution of described collaurum, gold content is 0.1～1.0mg/mL.
4. according to the homogeneous phase sol particle type immunoassay kit of each record in the claim 1～3, wherein, the content of the described polymer among the reagent R1 in w/v g/ml, is 0.1～5.0%.
5. according to the homogeneous phase sol particle type immunoassay kit of each record in the claim 1～4, wherein, described polymer comprise among PEG 20,000 or the Brij-35 at least any.
6. according to the homogeneous phase sol particle type immunoassay kit of each record in the claim 1～5, wherein, the pH value of reagent R1 is 6.5～8.5.
7. the preparation method of the homogeneous phase sol particle type immunoassay kit of each record in the claim 1～6 is characterized in that, may further comprise the steps:
Polymer is dissolved in the damping fluid, makes the buffer solution that contains polymer, be i.e. reagent R1;
Use gold chloride to be the feedstock production colloidal gold solution, the particle diameter of the colloid gold particle in the solution is 65～85nm; After will resisting human cystatin C antibody with the damping fluid dilution, to wherein adding above-mentioned colloidal gold solution, make collaurum and anti-human cystatin C antibodies, make the buffer solution of the collaurum that is combined with anti-human cystatin C antibody, carry out purifying as required, obtain reagent R2.
8. according to the preparation method of the homogeneous phase sol particle type immunoassay kit of claim 7 record, wherein, making buffer concentration used in the step of collaurum and anti-human cystatin C antibodies is 0.004～0.02mol/L, and pH of buffer is 7.5～8.5.
9. the preparation method of homogeneous phase sol particle type immunoassay kit of record according to Claim 8, wherein, described damping fluid is borate buffer solution or HEPES damping fluid.
10. a bladder chalone C content assaying method is characterized in that, uses the homogeneous phase sol particle type immunoassay kit of each record in the claim 1～6.
Use the highly sensitive bladder chalone C that guarantees to detect extremely low-level (0.06mg/L) of method of the present invention, have very high value for clinical application.In addition, kit of the present invention is a liquid double reagent, and it is good, highly sensitive therefore to have specificity, and accuracy reaches advantages such as antijamming capability is strong well, can be used for detecting the concentration of bladder chalone C in serum or the blood plasma, is applicable to clinical automatic clinical chemistry analyzer.
Description of drawings
Fig. 1 shows the typical curve of the bladder chalone C normative reference of 6 kinds of different contents, and each point is represented the bladder chalone C normative reference of different content, and wherein, the x axle is represented the content of bladder chalone C, and Y-axis is represented absorbance.
Fig. 2 is for adopting the reagent and contrast agents (the commercially available bladder chalone C homogeneous phase sol particle immunoassay reagent of preparation example 1 of the present invention respectively, available from Japanese Alfresa company), the AU400 automatic clinical chemistry analyzer that adopts Olympus Corp to make is measured 50 parts of human serums, and measured value is carried out correlation analysis.That wherein X-axis is represented is the patients serum result of the reagent mensuration of preparation example 1 of the present invention; That Y-axis is represented is the relatively patients serum result of product mensuration of bladder chalone C reagent, coefficient R
2=0.9922, regression equation is y=0.9109x+0.05.
Embodiment
The objective of the invention is to measure in high sensitivity specifically bladder chalone C content, among the present invention, when preparation homogeneous phase sol particle type immunoassay reagent, by selecting colloid gold particle with appropriate particle size, with the antibody labeling collaurum time, select suitable flag condition, realized above-mentioned purpose.
Specifically, the invention provides a kind of special highly sensitive homogeneous phase sol particle immunoassay kit that can measure the bladder chalone C content of 0.06mg/L～8mg/L, described kit comprises reagent R1 and reagent R2, and in volume ratio, the content ratio of reagent R1 and reagent R2 is 4: 1.Described reagent R1 is the damping fluid that contains polymer; Described reagent R2 is the collaurum buffer solution that is combined with anti-human cystatin C antibody.The pH value of reagent R1 and reagent R2 can be 6.5～8.5.In the scope that does not influence effect of the present invention, also can contain suitable preservatives, for example NaN among the reagent R1 of the present invention
3Deng, also can contain suitable stabilizing agent among the reagent R2 of the present invention, stabilizing agent commonly used for example has hyclone (BSA) and PEG 20,000, and addition can be respectively hyclone (BSA) final concentration 0.5～2% and PEG 20,000 final concentrations 0.1～1%.
Described colloid gold particle diameter is for being preferably 65～85nm, and more preferably 70～80nm further is preferably 72～78nm, is preferably 73～77nm especially.If the colloid gold particle diameter less than 65nm, can influence sensitivity, if greater than 85nm, can make collaurum easily form precipitation, influence measurement result.
Among the present invention,, use spectrophotometer,, obtain the particle diameter of colloid gold particle according to this maximum absorption band at the maximum absorption band of 37 ℃ of scanning colloidal gold solutions by the wave spectrum scanning method.
Kind, concentration and the pH value of used damping fluid were bigger to mark result influence when collaurum combined (being labelled antibody) with the IgG molecule, at this moment, by selecting suitable damping fluid for use, can realize technique effect of the present invention better.Among the present invention, the concentration of used damping fluid is preferably 0.004～0.02mol/L during labelled antibody, 0.005～0.01mol/L more preferably, preferred especially 0.005～0.008mol/L, the pH value is preferably 7.0～9.0, more preferably 7.5～8.5, this damping fluid is preferably borate buffer solution or HEPES damping fluid, especially preferably uses borate buffer solution.Among the present invention, be that 8.0 borate buffer solution effect is best with 0.005mol/L and pH value.
In homogeneous phase sol particle bladder chalone C determining reagent kit reagent R1 of the present invention, described polymer comprises that molecular weight is 20,000 polyglycol (following brief note is " PEG 20,000 "), polyoxyethylene laurel ether (following brief note is " Brij-35 ") etc.; In reagent R2, be combined with in the colloidal gold solution of anti-human cystatin C antibody, gold content is 0.1～1.0mg/mL, is preferably 0.3～0.7mg/mL, more preferably 0.4～0.6mg/mL most preferably is 0.5mg/mL.Among the reagent R1, described polymer comprises PEG 20,000, Brij-35 etc., and its content is 0.1～5.0% (g/ml); The mark of anti-human cystatin C antibody and collaurum adopts physisorphtion to carry out in the reagent 2.
At homogeneous phase sol particle bladder chalone C determining reagent kit of the present invention, wherein also comprise the bladder chalone C standard items.Bladder chalone C content is 0.0,0.5,1.0,2.0,4.0, six gradient standard items of 8.0mg/L, described standard items are to be added with the human serum of the pure product of recombinant human cystatin C or the liquid of other similar serum matrix, and the pure product of described recombinant human cystatin C can prepare or buy the commercially available prod by known method.Described standard items can adopt suitable gradient and conventional method to prepare as required
In the present invention, in the bladder chalone C determining reagent kit, except the above-mentioned damping fluid of selecting for use especially when being used for mark,, can also use but be not limited to the TRIS damping fluid and other has the damping fluid of similar quality as the damping fluid that can be used for reagent R1 and reagent R2.The pH value of damping fluid is 6.5～9.0, and more preferably the pH value is 7.0～8.5.
The homogeneous phase sol particle immunoassay that adopts among the present invention is that the SPIAs ratio juris is: the collaurum sol particle that is adsorbed with being of moderate size of bladder chalone C antibody, uniformity, when running into corresponding antigens (bladder chalone C), because the interaction of antigen-antibody is condensed the colloid gold particle of absorption antibody.Gold grain cohesion back color is by red stain indigo plant, and absorbance can reduce gradually at wavelength 540nm place.And the degree of the degree of this reduction and colloid gold particle cohesion is proportional, also proportional with the determined antigen amount, measure the change color of reaction system under the specific wavelength, can measure the concentration that purpose detects thing by typical curve (this curve is reference material concentration and colourity relation curve).
Among the present invention, adopt homemade collaurum and bladder chalone C antibody, behind the mark with sample to be measured (serum or blood plasma) in bladder chalone C generation association reaction, form immune complex and make the mark collaurum color take place to assemble by red stain indigo plant, detect this variation with the light intensity in transmission, draw the concentration of target detection thing bladder chalone C in the sample with the typical curve of bladder chalone C standard items.
Embodiment
" w/v " among the embodiment is " g/ml " except that specified otherwise.
Below, prepare detectable of the present invention and bladder chalone C standard items.
＜preparation detectable of the present invention and the employed primary raw material of standard items 〉
1. mouse-anti human cystatin C monoclonal antibody (monoclonal antibody): obtain according to the conventional method manufacturing, this antibody only with the human cystatin C reaction, do not have immunological cross-reaction with other antigens, tire and can satisfy this reagent requirement.
2. gold chloride:, be used for preparing the collaurum that the present invention uses available from sigma company.
3. recombinant human cystatin C albumen: obtain according to the conventional method manufacturing, be used for the preparation standard product.
4. human cystatin C titer: available from Dako company, ProductName: X0974, concentration 7.5mg/L is used for to the standard items assignment.
The preparation of＜bladder chalone C standard items of the present invention 〉
Be dissolved in solution (0.9%NaCl, 0.1%BSA, the 0.1%NaN of similar human serum matrix with recombinant human cystatin C albumen
3, the Tris-Hcl pH of buffer 8.0 of 50mmol/L), the standard items of preparation variable concentrations.With Dako human cystatin C titer is primary standard, adopt collaurum agglutinating reaction method, respectively the standard items of preparation are measured, detect respectively 20 times, obtain average and reach the desired concentration of standard items, promptly 0.0,0.5,1.0,2.0,4.0 and 8.0mg/L.
The preparation of＜detectable of the present invention 〉
Preparation example 1
1. the preparation of reagent R1 of the present invention
In the Tris-HCl of 0.1mol/L damping fluid (pH 7.5) 80ml, add PEG2 ten thousand 2g, Brij-350.5g and NaN
30.1g fully the dissolving back is supplemented to 100ml with distilled water with volume, with the membrane filtration of 0.22 μ m, and generate a reagent R1.Among this reagent R1, the content of PEG2 ten thousand is 2% (w/v), and the content of Brij-35 is 0.5% (w/v), NaN
3Content be 0.1% (w/v).Make reagent R1 of the present invention thus.
Though the polymer in this preparation example is PEG2 ten thousand and Brij-35, the polymer among the reagent R1 of the present invention also can be any among PEG2 ten thousand or the Brij-35.
2. the preparation of reagent R2 of the present invention
I. the preparation of colloidal gold solution
(1) gold chloride (HAuCl of preparation 0.01%
4) aqueous solution, this solution of 1L is heated to boils.
(2) stir the NaOH solution that accurately adds 1ml 1mol/L down, accurately add 1% trisodium citrate (Na after 2 minutes
3C
6H
5O
72H
2O) aqueous solution 7ml.
(3) continue heated and boiled 15 minutes, observe flaxen aqueous solution of chloraurate very fast grizzle after sodium citrate adds, become black then, become peony and stable subsequently gradually.
(4) be cooled to that to return to original volume with distilled water after the room temperature be 1L.
(5) make colloidal gold solution, wherein the particle diameter of colloid gold particle is about 75nm.
II. monoclonal antibody mark collaurum
(1) ratio of collaurum and labelled protein (monoclonal antibody) consumption determines
1) uses 0.2mol/L K
2CO
3Regulate the pH to 8.0 of colloidal gold solution, packing 12 pipes, every pipe 1ml.
2) the mark monoclonal antibody being used 0.005mol/L (pH8.0) borate buffer solution be diluted to concentration respectively is 5 μ g/ml, 10 μ g/ml, 15 μ g/ml, 20 μ g/ml, 25 μ g/ml, 30 μ g/ml, 35 μ g/ml, 40 μ g/ml, 45 μ g/ml, 50 μ g/ml, 55 μ g/ml, 60 μ g/ml, get 0.1ml respectively, add in the above-mentioned colloidal gold solution mixing.Control tube only adds the 0.1ml borate buffer solution.
3) after 5 minutes, in above-mentioned each pipe, add 0.1ml 10%NaCl solution, left standstill behind the mixing 2 hours.
4) result observes: the quantity not sufficient of control tube (not adding monoclonal antibody) and adding monoclonal antibody all can present the coagulation phenomenon by red stain indigo plant with each pipe of stable colloid gold; Meet or exceed each quantitative pipe of minimum steady and can still keep red constant and add protein content.To stablize the minimum amount of the constant minimum protein consumption of 1ml colloidal gold solution redness as this mark monoclonal antibody, according to circumstances, this consumption can suitably increase by 10%～20%.
(2) combine (mark) of collaurum and monoclonal antibody (IgG)
1) with 0.005mol/L (pH8.0) borate buffer solution monoclonal antibody is diluted to 40ug/ml.
2) with 100ml colloidal gold solution and above-mentioned 10ml monoclonal antibody solution respectively with 0.1mol/L K
2CO
3After transferring pH to 8.0, on one side electromagnetic agitation IgG solution, on one side to wherein adding colloidal gold solution, add the back fully and continue to stir 10 minutes.
3) adding an amount of stabilizing agent precipitates to prevent antibody protein and collaurum polymerization.The stabilizing agent that uses in this preparation example is hyclone (BSA) and PEG 20,000.The amount that adds: the BSA final concentration is 1%; PEG 20,000 final concentrations are 0.2%.
III. the purifying of colloidal gold solution behind the mark IgG:
The mark colloidal gold solution is with centrifugal 30 minutes of 15000g, supernatant discarded, sediment with the Tris damping fluid that contains 1%BSA and 0.2%PEG 20,000 (10mmol/L, pH7.5) resuspended, recover centrifugal again behind the original volume.So washing is 3 times.Thoroughly to remove unconjugated protein.
Abandoning supernatant, sediment is resuspended with above-mentioned damping fluid to be 1/10 of original volume, makes reagent R2 of the present invention, this reagent color is an aubergine.
Preparation example 2
1. the preparation of reagent R1 of the present invention
Prepare reagent R1 of the present invention according to the method identical with preparation example 1.
2. the preparation of reagent R2 of the present invention
I. the preparation of colloidal gold solution
(1) gold chloride (HAuCl of preparation 0.01%
4) aqueous solution, this solution of 1L is heated to boils.
(2) stir down accurately adding 1ml 1mol/L K
2CO
3Solution accurately adds 1% trisodium citrate (Na after 1 minute
3C
6H
5O
72H
2O) aqueous solution 8ml.
(3) continue heated and boiled 15 minutes, observe flaxen aqueous solution of chloraurate very fast grizzle after sodium citrate adds, become black then, become peony and stable subsequently gradually.
(4) be cooled to that to return to original volume with distilled water after the room temperature be 1L.
(5) make colloidal gold solution, wherein the particle diameter of colloid gold particle is about 70nm.
II. monoclonal antibody mark collaurum
(1) ratio of collaurum and labelled protein (monoclonal antibody) consumption determines
Except using 0.004mol/L (pH7.5) borate buffer solution dilution mark monoclonal antibody, according to preparation example 1 in identical mode determine the amount ratio of collaurum and labelled protein.
(2) combine (mark) of collaurum and monoclonal antibody (IgG)
1) with 0.004mol/L (pH7.5) borate buffer solution monoclonal antibody is diluted to 40ug/ml.
2) with 100ml colloidal gold solution and above-mentioned 10ml monoclonal antibody solution respectively with 0.1mol/L K
2CO
3After transferring pH to 7.5, on one side electromagnetic agitation IgG solution, on one side to wherein adding colloidal gold solution, add the back fully and continue to stir 10 minutes.
3) adding an amount of stabilizing agent precipitates to prevent antibody protein and collaurum polymerization.The stabilizing agent that uses in this preparation example is hyclone (BSA) and PEG 20,000.The amount that adds: the BSA final concentration is 0.5%; PEG 20,000 final concentrations are 1%.
III. the purifying of colloidal gold solution behind the mark IgG:
According to the method identical with preparation example 1 to mark IgG after colloidal gold solution carry out purifying, make reagent R2 of the present invention, this reagent color is an aubergine.
Preparation example 3
1. the preparation of reagent R1 of the present invention
Prepare reagent R1 of the present invention according to the method identical with preparation example 1.
2. the preparation of reagent R2 of the present invention
I. the preparation of colloidal gold solution
(1) gold chloride (HAuCl of preparation 0.01%
4) aqueous solution, this solution of 1L is heated to boils.
(2) stir down accurately adding 0.5ml 1mol/L NaOH solution, accurately add 1% trisodium citrate (Na after 3 minutes
3C
6H
5O
72H
2O) aqueous solution 6ml.
(3) continue heated and boiled 15 minutes, observe flaxen aqueous solution of chloraurate very fast grizzle after sodium citrate adds, become black then, become peony and stable subsequently gradually.
(4) be cooled to that to return to original volume with distilled water after the room temperature be 1L.
(5) make colloidal gold solution, wherein the particle diameter of colloid gold particle is about 80nm.
II. monoclonal antibody mark collaurum
(1) ratio of collaurum and labelled protein (monoclonal antibody) consumption determines
Except using 0.02mol/L (pH8.5) HEPES damping fluid dilution mark monoclonal antibody, according to preparation example 1 in identical mode determine the amount ratio of collaurum and labelled protein.
(2) combine (mark) of collaurum and monoclonal antibody (IgG)
1) with 0.02mol/L (pH8.5) HEPES damping fluid monoclonal antibody is diluted to 40ug/ml.
2) with 100ml colloidal gold solution and above-mentioned 10ml monoclonal antibody solution respectively with 0.1mol/L K
2CO
3After transferring pH to 8.5, on one side electromagnetic agitation IgG solution, on one side to wherein adding colloidal gold solution, add the back fully and continue to stir 10 minutes.
3) adding an amount of stabilizing agent precipitates to prevent antibody protein and collaurum polymerization.The stabilizing agent that uses in this preparation example is hyclone (BSA) and PEG 20,000.The amount that adds: the BSA final concentration is 2%; PEG 20,000 final concentrations are 0.1%.
III. the purifying of colloidal gold solution behind the mark IgG:
According to the method identical with preparation example 1 to mark IgG after colloidal gold solution carry out purifying, make reagent R2 of the present invention, this reagent color is an aubergine.
Preparation example 4
1. the preparation of reagent R1 of the present invention
Prepare reagent R1 of the present invention according to the method identical with preparation example 1.
2. the preparation of reagent R2 of the present invention
I. the preparation of colloidal gold solution
(1) gold chloride (HAuCl of preparation 0.01%
4) aqueous solution, this solution of 1L is heated to boils.
(2) stir down accurately adding 1.5ml 1mol/L NaOH solution, accurately add 1% trisodium citrate (Na after 4 minutes
3C
6H
5O
72H
2O) aqueous solution 9ml.
(3) continue heated and boiled 15 minutes, observe flaxen aqueous solution of chloraurate very fast grizzle after sodium citrate adds, become black then, become peony and stable subsequently gradually.
(4) be cooled to that to return to original volume with distilled water after the room temperature be 1L.
(5) make colloidal gold solution, wherein the particle diameter of colloid gold particle is about 65nm.
II. monoclonal antibody mark collaurum
(1) ratio of collaurum and labelled protein (monoclonal antibody) consumption determines
Except using 0.01mol/L (pH8.3) HEPES damping fluid dilution mark monoclonal antibody, according to preparation example 1 in identical mode determine the amount ratio of collaurum and labelled protein.
(2) combine (mark) of collaurum and monoclonal antibody (IgG)
1) with 0.01mol/L (pH8.3) HEPES damping fluid monoclonal antibody is diluted to 40ug/ml.
2) with 100ml colloidal gold solution and above-mentioned 10ml monoclonal antibody solution respectively with 0.1mol/L K
2CO
3After transferring pH to 8.2, on one side electromagnetic agitation IgG solution, on one side to wherein adding colloidal gold solution, add the back fully and continue to stir 10 minutes.
3) adding an amount of stabilizing agent precipitates to prevent antibody protein and collaurum polymerization.The stabilizing agent that uses in this preparation example is hyclone (BSA) and PEG 20,000.The amount that adds: the BSA final concentration is 1.4%; PEG 20,000 final concentrations are 0.1%.
III. the purifying of colloidal gold solution behind the mark IgG:
According to the method identical with preparation example 1 to mark IgG after colloidal gold solution carry out purifying, make reagent R2 of the present invention, this reagent color is an aubergine.
Preparation example 5
1. the preparation of reagent R1 of the present invention
Prepare reagent R1 of the present invention according to the method identical with preparation example 1.
2. the preparation of reagent R2 of the present invention
I. the preparation of colloidal gold solution
(1) gold chloride (HAuCl of preparation 0.01%
4) aqueous solution, this solution of 1L is heated to boils.
(2) stir down accurately adding 1% trisodium citrate (Na
3C
6H
5O
72H
2O) aqueous solution 5ml.
(3) continue heated and boiled 15 minutes, observe flaxen aqueous solution of chloraurate very fast grizzle after sodium citrate adds, become black then, become peony and stable subsequently gradually.
(4) be cooled to that to return to original volume with distilled water after the room temperature be 1L.
(5) make colloidal gold solution, wherein the particle diameter of colloid gold particle is about 85nm.
II. monoclonal antibody mark collaurum
(1) ratio of collaurum and labelled protein (monoclonal antibody) consumption determines
Except using 0.008mol/L (pH7.8) borate buffer solution dilution mark monoclonal antibody, according to preparation example 1 in identical mode determine the amount ratio of collaurum and labelled protein.
(2) combine (mark) of collaurum and monoclonal antibody (IgG)
1) with 0.008mol/L (pH7.8) borate buffer solution monoclonal antibody is diluted to 40ug/ml.
2) with 100ml colloidal gold solution and above-mentioned 10ml monoclonal antibody solution respectively with 0.1mol/L K
2CO
3After transferring pH to 7.8, on one side electromagnetic agitation IgG solution, on one side to wherein adding colloidal gold solution, add the back fully and continue to stir 10 minutes.
3) adding an amount of stabilizing agent precipitates to prevent antibody protein and collaurum polymerization.The stabilizing agent that uses in this preparation example is hyclone (BSA) and PEG 20,000.The amount that adds: the BSA final concentration is 1%; PEG 20,000 final concentrations are 0.6%.
III. the purifying of colloidal gold solution behind the mark IgG:
According to the method identical with preparation example 1 to mark IgG after colloidal gold solution carry out purifying, make reagent R2 of the present invention, this reagent color is an aubergine.
Below, by testing the performance that example detects reagent of the present invention.
＜bladder chalone C determining method and experiment parameter 〉
Analytical approach: 2 end-point methods;
The consumption of reagent of the present invention: reagent R1 of the present invention and reagent R2 of the present invention, consumption are respectively 200ul and 50 μ l;
The consumption of sample: 2 μ l;
Detect wavelength: be respectively predominant wavelength 540nm and commplementary wave length 700nm.
Determination step: 200 μ l reagent R1 add 2 μ l samples, add 50 μ l reagent R2 after 5 minutes in 37 ℃, promptly begin to read a little, react and read another point after 5 minutes, obtain the difference of absorbance.
Test example 1: the typical curve of making bladder chalone C standard items of the present invention
Adopt standard items of the present invention (6 kinds of different contents 0.0,0.5,1.0,2.0,4.0 of aforementioned preparation, the bladder chalone C standard items of 8.0mg/L), adopt the AU400 Biochemical Analyzer of automatic Olympus Corp manufacturing, record the typical curve (as shown in Figure 1) of bladder chalone C standard items of the present invention according to the said determination step.Each point among Fig. 1 on the curve is represented the standard items of a content.Wherein X-axis is represented the content (mg/L) of bladder chalone C; What Y-axis was represented is absorbance.
Test example 2 correlation tests
The reagent that uses preparation example 1 of the present invention is (in this test example, be called for short " reagent of the present invention ") and contrast agents (commercially available bladder chalone C homogeneous phase sol particle immunoassay reagent, available from Japanese Alfresa company), the AU400 automatic clinical chemistry analyzer that adopts Olympus Corp to make is measured 50 parts of human serums, and measured value is carried out correlation analysis.According to above-mentioned " bladder chalone C determining method " in identical parameter measure, measurement result is seen Fig. 2, the X among the figure, Y-axis are measured value (the content mg/L of bladder chalone C).
By the result of Fig. 2 as can be known, the related coefficient of reagent of the present invention and contrast agents is R
2=0.9922, regression equation is y=0.9109x+0.05.This result shows that the effect correlativity of bladder chalone C content among this reagent and the contrast agents mensuration patients serum is good, has excellent specificity and accuracy.
In addition, above test is that the AU400 automatic clinical chemistry analyzer that adopts Olympus Corp to make carries out, but reagent of the present invention is not limited to above-mentioned instrument, also be applicable to other full-automatic or semi-automatic biochemical analyzers, and those skilled in the art can according to circumstances do suitable adjustment to location parameter.
Test example 3 sensitivity tests
Sensitivity according to following method of operating detectable:
1. determine that a reagent can detect accurately the low concentration value and come with water relatively blankly, the sample of 0.5mg/L is selected in this test for use.
2. detect water 20 times, record absorbance numerical value, calculating mean value (X
Water) and standard deviation (SD).
3. detect the sample of 20 concentration 0.5mg/L, record absorbance numerical value, calculating mean value (X
0.5) and standard deviation (SD).
4. the absorbance mean value of water adds the absorbance of 3SD as the lowest detectable limit correspondence, because the relation of absorbance and concentration is linear relationship substantially, the concentration that can relatively calculate lowest detectable limit by the absorbance mean value with the 0.5mg/L sample is sensitivity.Formula is 0.5 * (X
Water+ 3SD)/X
0.5
The reagent of making in the preparation example 1 of the present invention is (in this test example, be called for short " reagent of the present invention ") and contrast agents (commercially available bladder chalone C homogeneous phase sol particle immunoassay reagent, available from Japanese Alfresa company, particle diameter is about 50nm) the data result of sensitivity test as shown in table 1.
In the following table, Δ A represents the difference of absorbance.
Table 1
The sensitivity of reagent of the present invention=0.5 * (30.4+3 * 7)/429=0.06mg/L
The sensitivity of contrast agents=0.5 * 27.1+ (3 * 21.8)/339.9=0.14mg/L
The result shows that the sensitivity of reagent of the present invention can reach 0.06mg/L, is better than contrast agents.
By the result of above-mentioned table 1 as can be known, reagent of the present invention highly sensitive in the sensitivity of contrast agents, visible reagent of the present invention has been realized the effect that sensitivity improves.
Test example 4～7 sensitivity tests
Method according to identical with above-mentioned test example 6 detects the sensitivity of the reagent of the present invention of preparation in the preparation example 2～5, and the result is shown in following table 2.
Table 2
| Preparation example 2 | Preparation example 3 | Preparation example 4 | Preparation example 5 | |
| Sensitivity (mg/L) | ??0.11 | ??0.10 | ??0.13 | ??0.09 |
Claims (10)
1. homogeneous phase sol particle type immunoassay kit, it is used to measure the content of bladder chalone C, this kit comprises reagent R1 and reagent R2, in volume ratio, the content ratio of reagent R1 and reagent R2 is 4: 1, randomly contains the bladder chalone C standard items in this kit, and described reagent R1 is the buffer solution that contains polymer, described reagent R2 is the buffer solution that is combined with the collaurum of anti-human cystatin C antibody, and wherein the colloid gold particle diameter is 65～85nm.
2. according to the homogeneous phase sol particle type immunoassay kit of claim 1 record, wherein, described colloid gold particle diameter is 70～80nm.
3. according to the homogeneous phase sol particle type immunoassay kit of claim 1 or 2 records, wherein, in the buffer solution of described collaurum, gold content is 0.1～1.0mg/mL.
4. according to the homogeneous phase sol particle type immunoassay kit of each record in the claim 1～3, wherein, the content of the described polymer among the reagent R1 in w/v g/ml, is 0.1～5.0%.
5. according to the homogeneous phase sol particle type immunoassay kit of each record in the claim 1～4, wherein, described polymer comprise among PEG 20,000 or the Brij-35 at least any.
6. according to the homogeneous phase sol particle type immunoassay kit of each record in the claim 1～5, wherein, the pH value of reagent R1 is 6.5～8.5.
7. the preparation method of the homogeneous phase sol particle type immunoassay kit of each record in the claim 1～6 is characterized in that, may further comprise the steps:
Polymer is dissolved in the damping fluid, makes the buffer solution that contains polymer, be i.e. reagent R1;
Use gold chloride to be the feedstock production colloidal gold solution, the particle diameter of the colloid gold particle in the solution is 65～85nm; After will resisting human cystatin C antibody with the damping fluid dilution, to wherein adding above-mentioned colloidal gold solution, make collaurum and anti-human cystatin C antibodies, make the buffer solution of the collaurum that is combined with anti-human cystatin C antibody, carry out purifying as required, obtain reagent R2.
8. according to the preparation method of the homogeneous phase sol particle type immunoassay kit of claim 7 record, wherein, making buffer concentration used in the step of collaurum and anti-human cystatin C antibodies is 0.004～0.02mol/L, and pH of buffer is 7.5～8.5.
9. the preparation method of homogeneous phase sol particle type immunoassay kit of record according to Claim 8, wherein, described damping fluid is borate buffer solution or HEPES damping fluid.
10. a bladder chalone C content assaying method is characterized in that, uses the homogeneous phase sol particle type immunoassay kit of each record in the claim 1～6.
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2009
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