CN102879562B - Colloidal gold immunofiltration quantitive detection method and reagent kit - Google Patents
Colloidal gold immunofiltration quantitive detection method and reagent kit Download PDFInfo
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Abstract
The invention relates to clinical immunodiagnosis reagents and preparation methods thereof, and discloses a colloidal gold immunofiltration quantitive detection reagent kit and a method for detecting antigens and antibodies by colloidal gold immunofiltration quantitive detection. One or two quality control lines or points which contain known to-be-detected substances are added on the colloidal gold immunofiltration quantitive detection reagent kit. In one reaction, change of color developing degree of detection lines (points) is relevant with change of color developing degree of the quality control lines (points) significantly, deviation of the detection lines (points) can be corrected by the quality control lines (points), and thus, variation caused by deviation of raw materials (nitrocellulose membranes, water absorbent pads), sample viscosity, temperature and sample adding quantity and the like can be overcome effectively.
Description
Technical field
The present invention relates to clinical immunization diagnostic reagent and preparation method thereof, be specifically related to colloidal gold immunity percolation technology and quantivative approach thereof.
Background technology
1971, Faulk and Taylor (Immunochem, 8:1081,1971) first used immune colloid gold to be used for immunoelectronmicroscopy as label, and after this immune colloidal gold technique is widely used as a kind of new immunological method.The colloid gold particle of various different-grain diameter can be prepared easily from gold chloride by reducing process, the most conventional (Nature (Phys.Sci.) 241:20 of the method that Frens introduces, 1973), take trisodium citrate as 10 ~ 70nm collaurum that reductive agent obtains be that particle size accurately can be determined by the addition of trisodium citrate through conventional magnitude range in reagent for clinical diagnosis.Colloid gold particle has very strong adsorptive power to protein, can with the Non-covalent binding such as staphylococcal protein A, immunoglobulin (Ig), toxin, glycoprotein, enzyme, hormone, virus or bacterial antigens, form immune colloid gold conjugate, for immune detection.
At ministry of Health of China higher medical institution programming textbook " immunology and immunological test ", (Tao Yixun edits immune colloidal gold technique, the second edition, Beijing, People's Health Publisher, 1997) chapters and sections have been classified as in, these chapters and sections specifically describe two methods that immune colloid gold the most often uses in medical test: 1) colloidal gold immunity percolation test, and 2) colloidal gold immune chromatography experiment.
Colloid gold immune diagnosis Test paper conventional at present has immune chromatography test paper, can reach quantitative or qualitative detection (as Chinese patent application CN10125640A and US Patent No. 5753517).
Colloidal gold immunity percolation is also a kind of proven technique, commercially there is the diagnostic kit supply of the multiple different testing sample of many manufacturers produce at present, wherein major part is qualitative kit, namely feminine gender and the positive can only be distinguished, quantification kit is at present primarily of the Nycocard of Axis-Shield company production and the U2 of Shanghai Upper Bio-tech Pharma Co., Ltd.'s production and matched reagent box thereof, Qpad and matched reagent box thereof, the detection technique quantitatively realized is single-point reflection method and ccd image disposal route.
But, adopt Micelle growth to carry out detecting also existing problems at present:
Due to raw material (nitrocellulose filter, adsorptive pads), sample viscosity, temperature, the deviation etc. of application of sample amount all can induce reaction error, and general error CV about about 10%, can reach 15% when error is larger.Existing control device is raw material quality control (nitrocellulose filter and adsorptive pads screening meet the lot number of quantitative requirement) and process optimization means pre-service etc. such as () nitrocellulose filter immersion, sprayings.But in existing technology, testing result variation is large, can only be passive utilize raw material screening and increase pretreating process solves these problems, and waste time and energy, efficiency is low, and cost is high, can't deal with problems completely.
Therefore, need to make improvements prior art, a kind of new test paper and detection method are provided, to reduce error when Micelle growth detects.
Summary of the invention
The present invention aims to provide a kind of colloidal gold immunity percolation detection kit and utilizes the method for the quantitative detectable antigens antibody of Micelle growth, can reduce error.
Technical scheme of the present invention is, colloid gold immune detection reaction plate increases by 1 or 2 nature controlling line or Quality Control point, and wherein nature controlling line or Quality Control point are the thing standard model to be detected that content is known.Because in same primary first-order equation, the change of detection line (point) depth that develops the color and the change of nature controlling line (point) depth that develops the color are significant correlativity, utilize nature controlling line (point) to carry out the deviation of correct detection line (point), effectively can overcome the variation that the deviation etc. due to raw material (nitrocellulose filter, adsorptive pads), sample viscosity, temperature, application of sample amount causes.
Being CRP albumen (c reactive protein) as detected thing, just using the CRP albumen sterling (as 1-10mg/L and 50-150mg/L) of concentration known to be coated in nature controlling line position on film.Using special quantitative readout instrument (as detected the instrument of absorbance or CCD numerical value) to read light absorption value or the CCD gray-scale value of detection line and nature controlling line during detection simultaneously, drawing instrument internal value through data processing.Use 7 ~ 11 calibrations to determine curve, according to matched curve through formulae discovery, after instrument internal value being transformed on special readout instrument screen, directly export concentration value.
Technical scheme is: a kind of colloidal gold immunofiltration quantitive detection method, comprises the following steps:
(1) wrap by primary antibodie on the detection line or check point of colloidal gold immunity percolation detection kit; And one or two nature controlling line or Quality Control point are set, nature controlling line or Quality Control point contain identical with testing sample, that content is known standard model; Described primary antibodie is monoclonal antibody, polyclonal antibody or antigen;
Described testing sample comprises CRP, human chorionic gonadotrophin (HCG), albumin (ALB), plasma D-dimer content (DD), mycobacterium tuberculosis antibody (TB), amyloid (SAA), cardiac muscle troponin I (cTnI).
(2) testing sample and gold mark liquid is added in colloidal gold immunity percolation detection kit, containing markd two anti-collaurums in gold mark liquid; Described two resist for monoclonal antibody, polyclonal antibody or antigen; Described collaurum particle diameter is 15 ~ 60nm;
(3), after washing, read absorbance or the CCD of detection line or check point and nature controlling line or Quality Control point on test paper, according to typical curve determination concentration simultaneously;
The approximating method of described typical curve is, is added drop-wise on colloidal gold immunity percolation Test paper with the testing sample standard solution of variable concentrations, then adds gold mark liquid, after washing, reads absorbance or the CCD of detection line or check point and nature controlling line or Quality Control point; With following formulae discovery correction:
R=T/ (C1*C2) or R=T/C
Wherein T is absorbance or the CCD reading of detection line or check point, C1 and C2 is respectively the reading of two nature controlling lines or Quality Control point; The reading that C is Quality Control point or nature controlling line when being one.
Present invention also offers a kind of colloidal gold immunity percolation immue quantitative detection reagent box, comprise detection line or check point, and one or two nature controlling line or Quality Control point; Detection line or check point wrap by primary antibodie; Nature controlling line or Quality Control point bag are by the standard model identical with testing sample.
This method may be used for detecting the antibody antigens such as CRP, human chorionic gonadotrophin (HCG), albumin (ALB), plasma D-dimer content (DD), mycobacterium tuberculosis antibody (TB), amyloid (SAA), cardiac muscle troponin I (cTnI).
Beneficial effect of the present invention and superiority be,
(1) quality detected gets a promotion.In existing technology, the variation between every person-portion is large, average out to CV<15%, adopts CV after after this method to be down to <10%.
(2) productive rate improves: what prior art can only be passive utilizes raw material screening and increase pretreating process to solve these problems, and the effect that wastes time and energy is also bad; Greatly alleviate quality requirements and pretreated technological requirement, the greatly improving production efficiency of raw material after using the present invention, reduce costs, and quality level significantly promotes.
After overcorrect, increased quality, can reduce the requirement that raw material is selected, can greatly simplify the operation of pretreatment of raw material.
Accompanying drawing explanation
Fig. 1 is in embodiment 2, the curve of variable concentrations CRP standard model solution and corresponding quantitative readout instrument and inner reading processing costs thereof.
Embodiment
Embodiment 1 prepares the colloidal gold immunity percolation kit detecting CRP
(1) preparation of reaction plate
A. by adsorptive pads, nitrocellulose filter and plastic casing are assembled into blank reaction plate.There are a circular hole in plastic casing central authorities.
B. quilt is wrapped: the nitrocellulose filter assigned address specking nature controlling line 1 in plastic casing circular hole, detection line, nature controlling line 2.
Detection line wraps by CRP antibody (primary antibodie), concentration is 0.1-2.0mg/ml; Nature controlling line 1 and nature controlling line wrap by CRP standard model, and concentration is 0.1-2.0mg/ml;
C. dry: 37 degree dry 2 ~ 24 hours, load aluminium foil bag after cooling, add one bag of drying agent, seal.
(2) colloid gold label
A. CRP monoclonal antibody (two resist) to be marked is marked on the colloid gold particle that mean grain size is 15 ~ 60nm, centrifugal segregation supernatant, precipitate PEG (molecular weight 15000 ~ 20000) centrifuge washing 1 time with 0.2 ~ 0.5%, be concentrated into 1/10 ~ 1/5,4 degree of original volume with the damping fluid (containing 1%BSA) of PH7.2 ~ 7.6 to save backup.
B. allocate: with the damping fluid (containing 1%BSA) of PH7.2 ~ 7.6, according to CRP serial standards, (concentration is 0mg/L, 1mg/L, 3mg/L, 10mg/L, 20mg/L, 40mg/L, 60mg/L, 80mg/L, 100mg/L, 120mg/L) prepare curve, each concentration does 3 times, determines dilutability, is gold mark liquid after dilution.In general gold mark liquid, antibody content is 0.01-0.2mg/ml.
(3) other component
Cleansing solution: the PB damping fluid of PH7.2 ~ 7.6, surfactant (0.05%-0.5%).
Embodiment 2
Detect with the kit that embodiment 1 obtains.
The CRP standard model solution getting variable concentrations is tested, method is marked by the CRP standard model solution of variable concentrations (5,10,25,50,100,150 and 200mg/L) 3 μ l and 200 μ l gold after liquid fully mixes, get 120 μ l mixed liquors to drip in the circular hole of test paper on nitrocellulose filter, after infiltrating completely, successively add cleansing solution and confining liquid.
Kit is put into quantitative readout instrument, read the CCD gray-scale value of detection line, nature controlling line 1 and nature controlling line 2 simultaneously.Use following formulae discovery:
R=T/ (C1*C2), or R=T/C
Wherein T, C1 and C2 are respectively the instrument internal reading (when only having a nature controlling line, the instrument internal reading of nature controlling line is C) of detection line, nature controlling line 1 and nature controlling line 2.Mean value and the concentration of getting the R=T/ (C1*C2) under each concentration are mapped (when only having a nature controlling line, getting R=T/C numerical value), and result as shown in figure 1 and table 1.In Fig. 1, Y is C protein concentration, and X is the R in formula, i.e. the processing costs of machine intimate reading.
Table 1
Note:
The instrument readings of the detection line (or check point) of T1--------first time experiment,
The instrument readings of the detection line (or check point) of T2--------second time experiment,
The instrument readings (machine intimate value) of C11-------first time experiment nature controlling line 1
The instrument readings (machine intimate value) of C12-------second time experiment nature controlling line 1
The instrument readings (machine intimate value) of C21-------first time experiment nature controlling line 2
The instrument readings (machine intimate value) of C22-------second time experiment nature controlling line 2
Numerical results is as table 2:
Table 2
In table, T1/T2 data are the deviation ratio of the T value do not corrected; Correction 1/ correction 2 data are the deviation ratio after formula correction, have obvious decline.
Embodiment 3 detects the colloidal gold immunity percolation kit of ALB
Prepare the colloidal gold immunity percolation kit detecting ALB by the method identical with embodiment 1, CRP wherein and monoclonal antibody are replaced with ALB albumen and corresponding monoclonal antibody, the quantity of Quality Control point or nature controlling line is one.
The method identical by same embodiment 2 detects, and result is as table 3:
Table 3
| ALB concentration | T1 | T2 | C1 | C2 | T1/C1 | T2/C2 |
| 10mg/L | 58.2 | 70.4 | 100.3 | 130.4 | 0.58 | 0.54 |
| 30mg/L | 169.1 | 138.8 | 104.4 | 103.6 | 1.62 | 1.34 |
| 50mg/L | 330 | 279.6 | 115.4 | 100.9 | 2.86 | 2.77 |
| 100mg/L | 485.8 | 455.8 | 98.1 | 89.5 | 4.95 | 5.09 |
| 200mg/L | 706.8 | 671.2 | 82.4 | 83.2 | 8.58 | 8.07 |
The instrument readings of the detection line (or check point) of T1--------first time experiment,
The instrument readings of the detection line (or check point) of T2--------second time experiment,
The instrument readings (machine intimate value) of C1-------first time experiment nature controlling line 1
The instrument readings (machine intimate value) of C2-------second time experiment nature controlling line 1
CRP(ALB in embodiment 1 ~ 3) and antibody can replace with HCG(or DD, TB, SAA) and corresponding antibodies, prepare kit and colloid gold label liquid; Be added drop-wise in kit after testing sample and gold marks liquid hybrid reaction, or testing sample is added drop-wise to kit adds gold mark liquid again and react.Result shows, revised correction 1/ corrects 2 deviation ratios lower than not calibrated T value deviation ratio.
Claims (1)
1. a colloidal gold immunofiltration quantitive detection method, is characterized in that, comprises the following steps:
(1) wrap by primary antibodie on the detection line or check point of colloidal gold immunity percolation detection kit; And one or two nature controlling line or Quality Control point are set, nature controlling line or Quality Control point contain identical with testing sample, that content is known standard model; Described primary antibodie is monoclonal antibody, polyclonal antibody or antigen;
(2) testing sample and gold mark liquid is added in colloidal gold immunity percolation detection kit, containing markd two anti-collaurums in gold mark liquid; Described two resist for monoclonal antibody, polyclonal antibody or antigen;
(3), after washing, read absorbance or the CCD of detection line or check point and nature controlling line or Quality Control point on test paper, according to typical curve determination concentration simultaneously;
The approximating method of described typical curve is, is added drop-wise on colloidal gold immunity percolation Test paper with the testing sample standard solution of variable concentrations, then adds gold mark liquid, after washing, reads absorbance or the CCD of detection line or check point and nature controlling line or Quality Control point; With following formulae discovery correction:
R=T/ (C1*C2) or R=T/C;
Wherein T is absorbance or the CCD reading of detection line or check point, C1 and C2 is respectively the reading of two nature controlling lines or Quality Control point; The reading that C is Quality Control point or nature controlling line when being one;
Described testing sample is selected from c reactive protein, human chorionic gonadotrophin, albumin, plasma D-dimer content, mycobacterium tuberculosis antibody, amyloid or cardiac muscle troponin I;
Described collaurum particle diameter is 15 ~ 60nm.
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| CN106290902A (en) * | 2016-08-02 | 2017-01-04 | 长春理工大学 | A kind of colloidal-gold detecting-card of serum amyloid A protein 1 and preparation method thereof |
| CN109324046A (en) * | 2017-07-31 | 2019-02-12 | 爱威科技股份有限公司 | Test strips reaction density detection method, device, storage medium and computer equipment |
| WO2021102741A1 (en) * | 2019-11-27 | 2021-06-03 | 深圳加美生物有限公司 | Image analysis method and system for immunochromatographic detection |
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| CN101782577A (en) * | 2010-01-21 | 2010-07-21 | 郑州大学 | Diagnostic reagent kit for trichinosis by employing dot-immunogold filtration assay |
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| CN102680710A (en) * | 2012-05-22 | 2012-09-19 | 成都华神生物技术有限责任公司 | Enzyme-linked immunosorbent assay kit for human oxidized low-density lipoprotein, and using method and application |
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| US20120225422A1 (en) * | 2011-03-03 | 2012-09-06 | RoMonics, LLC | Method and device employing a non-receptor ligand interaction with nanoparticles or other solid phase followed by specific detection |
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| CN1409111A (en) * | 2001-09-28 | 2003-04-09 | 上海数康生物科技有限公司 | Device for simultanously detecting multiple antigen and antibody associated with diseases |
| CN101782577A (en) * | 2010-01-21 | 2010-07-21 | 郑州大学 | Diagnostic reagent kit for trichinosis by employing dot-immunogold filtration assay |
| CN102279263A (en) * | 2011-05-06 | 2011-12-14 | 杭州顿力医疗器械股份有限公司 | CCD-type quantitative analysis system of colloidal gold immunity chromatography diagnosis test papers |
| CN102608321A (en) * | 2012-02-29 | 2012-07-25 | 厦门大学 | Echinococcus multilocularis circulating antigen dot immunogold filtration kit and preparation method |
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