CN101603962B - Immune nanometer magnetic bead diagnostic kit - Google Patents
Immune nanometer magnetic bead diagnostic kit Download PDFInfo
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- CN101603962B CN101603962B CN 200810111583 CN200810111583A CN101603962B CN 101603962 B CN101603962 B CN 101603962B CN 200810111583 CN200810111583 CN 200810111583 CN 200810111583 A CN200810111583 A CN 200810111583A CN 101603962 B CN101603962 B CN 101603962B
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Abstract
The invention relates to an immune nanometer magnetic bead diagnostic kit comprising a base, a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad. The sample pad, the combination pad, the nitrocellulose membrane and the water absorption pad are orderly arrayed on the base. The combination pad contains immune nanometer magnetic beads which are formed for the purpose of coupling a specific antibody or antigen of a target protein to nanometer magnetic particles. The nitrocellulose membrane is provided with a testing band and a reference band. The testing band is provided with an antibody or antigen which is fixed on the membrane and corresponds to the target protein. The reference band is provided with an antibody or antigen which is fixed on the membrane. The kit produced according to the technology of the invention has the advantages of simplicity, convenience, fastness, low cost, stable and reliable quality and high flexibility. The kit can test a plurality of test targets simultaneously, can test a plurality of samples such as whole blood, serum, plasma, saliva, and the like, and can be used for disease warning, disease diagnosis, curative effect evaluation and prognosis, and the like.
Description
Technical field
The present invention relates to a kind of immune nanometer magnetic bead diagnostic kit, can be applicable to medical science and detect, belong to biomedical sector.
Background technology
Immune nano magnetic particle technology is the new technology that development in recent years is got up, how for separating of, concentrate and test such as diagnosis in.Magnetic nanoparticle can be combined with covalent manner with protein and not influenced its activity, so be commonly used to catch specific protein or protein isolate.Its principle is as follows: with certain antibody coupled to Nano magnetic particle, put into the solution that contains purpose antigen, after abundant hybrid reaction, form antigen-antibody nanometer magnetic bead potpourri, act on this solution with magnetic, the object that is incorporated on the nanometer magnetic bead is separated with other component, thereby realize the purpose to the easy quick separation of object.Simultaneously it again can be as quantitatively detecting, and can determine what of capture antibody or antigen amount according to magnetic is strong and weak.Therefore, the immune nano magnetic particle is a kind of important carrier and separating tool.Especially in the immunodiagnosis field, the magnetic nanoparticle that this surface is had antigen or antibody just calls immune nanometer magnetic bead in biomedicine.At present, in some flourishing countries of Europe, the United States, this immune nanometer magnetic bead is widely used in the immunodiagnosis field, as: utilize nanometer magnetic bead can carry out the quantitative measurement of micro-bioactivator in the body, immunity separates various nucleic acid and the big molecule of protein biology, eukaryotic magnetic immuno separation etc.
Because the above-mentioned characteristic of immune nanometer magnetic bead, the kit of applying nano magnetic particle coupling specific antigen or antibody is a focus of Recent study.Application number is 01113129.2 Chinese patent, a kind of immune colloid gold and immune nano magnetic particle double-tagging quick diagnosis reagent kit are provided, antibody or antigenic mark one magnetic nanoparticle with destination protein, form a magnetic bead, remove the mark collaurum with this magnetic bead again, make this antibody or antigen have two markers simultaneously.Then with this double-tagging antibody or antigen remove binding purpose antigen or antibody, finally be fixed on corresponding antibodies or antigen capture on the film, form one and detect band.At naked eyes inspection side band is carried out on the basis of preliminary qualitative or half-quantitative detection then, with the magnetic measurement instrument this detection band is quantitatively examined side.
But foregoing invention needs at first with antibody or antigenic mark magnetic particle, the magnetic particle of crossing with collaurum antagonist or antigenic mark mark more again, and the preparation process complexity is difficult for Quality Control, cause product quality to be difficult to stablize, and it is bigger additionally to increase cost.
Therefore, be necessary the mentioned reagent box is further improved.
Summary of the invention
The object of the present invention is to provide a kind of immune nanometer magnetic bead diagnostic kit, this kit preparation process is easy, with low cost, is easy to Quality Control, and constant product quality is highly sensitive, can be used for various purpose antibody or detection of antigens.
Another object of the present invention is to provide a kind of method that immune nanometer magnetic bead carries out testing goal albumen of using.
The immune nanometer magnetic bead diagnostic kit that another purpose of the present invention is to provide above-mentioned is for detection of the purposes of the multiple sample destination protein of whole blood, serum, blood plasma, saliva, urine, tissue fluid and cerebrospinal fluid and the purposes that is used for disease early warning, diagnosis, curative effect assessment and prognosis aspect.
A kind of method that immune nanometer magnetic bead carries out testing goal albumen of using provided by the invention, antibody or antigen that destination protein is special are coupled to magnetic nanoparticle, form immune nanometer magnetic bead, with this coupling the immune nanometer magnetic bead of specific antibody or antigen remove binding purpose antigen or antibody, the immune nanometer magnetic bead of being combined with purpose antigen or antibody finally is fixed on corresponding antibodies or the antigen capture on the film, and forming a detection band is the T band; Estimate testing result then or with the magnetic measurement instrument this detection band is quantitatively detected, to be fixed on the antibody on the film be not i.e. two anti-the catching of primary antibodie or antiantibody to the immune nanometer magnetic bead of being combined with purpose antigen or antibody, and forming a contrast band is the C band.
Wherein, the preparation of described magnetic bead is that to select particle diameter for use be the magnetic nanoparticle of 10-800nm, preferably selecting particle diameter for use is the magnetic nanoparticle of 50-300nm, and the antibody that destination protein is special or antigen covalent coupling form immune nanometer magnetic bead in magnetic nanoparticle.
The immobilization of described immune nanometer magnetic bead is that immune nanometer magnetic bead is sprayed onto on the plain film of glass fibre or the polyester film, becomes a solid phase pad, dried for standby.Dry preferred frozen drying or 37 ℃ of dryings.
This immune nanometer magnetic bead diagnostic kit also comprises the corresponding antibody of testing goal thing or antigen is sprayed onto and forms on the nitrocellulose filter that to detect band be the T band, be the i.e. two anti-another locations that are sprayed onto on the nitrocellulose filter of primary antibodie or antiantibody simultaneously with antibody, forming the contrast band is the C band, with protein liquid sealing, drying.Why the C band can fixedly have primary antibodie or two to resist herein, because of object different, when object is antigen, coupling is the specific antibody of purpose antigen on the immune nanometer magnetic bead, and when immune nanometer magnetic bead was with through T, the purpose antigen corresponding antibodies that is fixed on the film was caught, form double-antibody sandwich, the immune nanometer magnetic bead of not being combined with purpose antigen continues migration, and during through the C band, the antibody that is fixed on the specific antibody on the immune nanometer magnetic bead on the film is antiantibody or two anti-catching.In like manner, when object is antibody, coupling is the specific antigen of purpose antibody on the immune nanometer magnetic bead, when immune nanometer magnetic bead is with through T, the purpose antibody corresponding antigens that is fixed on the film is caught, and forms double antigens sandwich, and the immune nanometer magnetic bead of not being combined with purpose antibody continues migration, during through the C band, the antibody that is fixed on the specific antigen on the immune nanometer magnetic bead on the film is that primary antibodie is caught.
Simultaneously, according to said method, the present invention also provides a kind of immune nanometer magnetic bead diagnostic kit, comprise substrate, sample pad, pad, nitrocellulose filter and adsorptive pads, described sample pad, pad, nitrocellulose filter and adsorptive pads are arranged in order in order in substrate, and described sample pad is used for adding sample; Pad contains immune nanometer magnetic bead, described immune nanometer magnetic bead is that antibody or the antigen that destination protein is special is coupled to magnetic nanoparticle formation, it is that T band and contrast band are the C band that the band of detection is arranged on the nitrocellulose filter, described detection with on have be fixed on the film with the corresponding antibody of destination protein or antigen, described contrast with on the antibody that is fixed on the film is arranged is that primary antibodie or antiantibody are namely two anti-.
Because the magnetic force detector is very sensitive to the magnetism intensity detection of magnetic nanoparticle, so this kit has highly sensitive advantage, especially can detect the lower object of concentration.
Wherein, the preparation of described magnetic bead is that to select particle diameter for use be the magnetic nanoparticle of 10-800nm, preferably selecting particle diameter for use is the magnetic nanoparticle of 50-300nm, and the antibody that destination protein is special or antigen covalent coupling form immune nanometer magnetic bead in magnetic nanoparticle.
The immobilization of described immune nanometer magnetic bead is that immune nanometer magnetic bead is sprayed onto on the plain film of glass fibre or the polyester film, becomes a solid phase pad, dried for standby.Dry preferred frozen drying or 37 ℃ of dryings.
The formation of T band is the corresponding antibody of testing goal thing or antigen are sprayed onto on the nitrocellulose filter and get in this immune nanometer magnetic bead diagnostic kit, the formation of C band is that just antibody is that primary antibodie or antiantibody are the two anti-another locations that are sprayed onto on the nitrocellulose filter, with protein liquid sealing, drying.
Above-mentioned formed detection band is a visible or sightless T band, as seen T band for have in the sample destination protein and dense make detect with on the brown magnetic bead that gathers can be finding of naked eye, invisible T band make for no destination protein in the sample or concentration are very low detection with on the brown magnetic bead that gathers can not be seen by naked eyes.
Wherein, above-mentioned detection band is that the T band can be one or more, can be used for the detection of one or more destination proteins in the sample.
Each group sample pad, pad, nitrocellulose filter and adsorptive pads constitute a detection path in the kit, can contain one or more detection path arranged side by side in the kit, place in the same substrate, detect when being used for one or more destination proteins of one or more samples.
Kit form of the present invention is preferably check-out console or detector bar.
The kit that uses the technology of the present invention to make can use double antibody sandwich method, dual-antigen sandwich method or indirect method.
Use principle below in conjunction with the kit of the present invention of Fig. 2 describes,
(1) will be detected sample and be added on the sample pad, this sample moves forward to the adsorptive pads direction in adsorptive pads effect lower edge sample pad;
(2) sample moves forward to the adsorptive pads direction with immune nanometer magnetic bead reaction and continuation in pad, and the immune nanometer magnetic bead of binding purpose antigen or antibody is forming the positive or weak positive detection band by detecting the antibody or the antigen capture that are fixed on the film when being with;
(3) the continuation reach is fixed on primary antibodie or two anti-the catching on the film, forms contrast and is with;
(4) range estimation testing result or with the magnetic measurement instrument this detections is with and quantitatively detects.
The present invention uses the immune nanometer magnetic bead technology to set up diagnostic kit, can carry out qualitative detection and can do quantitative measurement again simultaneously.The kit that uses the technology of the present invention to set up is compared with similar quantification kit, and preparation process is easy, and is with low cost, is easy to Quality Control, stable and reliable product quality.
The kit that uses the technology of the present invention to produce, less demanding to sensitivity and when only needing qualitative detection, can be according to the colour developing of T band whether, the yin and yang attribute of naked eyes judged result.When detection needs high sensitivity, need do quantitative test with the magnetic measurement instrument.
The kit that uses the technology of the present invention to produce, it is easy to have preparation process, with low cost, is easy to Quality Control, constant product quality, highly sensitive characteristics., also can be prepared into the kit that can detect simultaneously multinomial detection index, several samples.Immune nanometer magnetic bead diagnostic kit of the present invention can be used for surveying multiple sample destination proteins such as whole blood, serum, blood plasma, saliva, urine, tissue fluid and cerebrospinal fluid, and is used for aspects such as disease early warning, diagnosis, curative effect assessment and prognosis.
Description of drawings
Fig. 1 is a kind of reaction principle synoptic diagram of example with hCG (human chorionic gonadotrophin) for the present invention.
Y1 is anti-β-hCG monoclonal antibody
Y2 is anti-α-hCG polyclonal antibody
Y3 is the sheep anti mouse polyclonal antibody
Fig. 2 kit structural representation of the present invention
1, sample pad 2, pad 3, nitrocellulose filter
4, adsorptive pads 5, substrate 6, T band
7, C band
Fig. 3 uses kit testing result of the present invention synoptic diagram when positive
Fig. 4 uses kit testing result of the present invention synoptic diagram when negative
Embodiment
Embodiment 1
The method for making of detection kit of the present invention may further comprise the steps:
1, the preparation of magnetic bead.
2, the immobilization of magnetic bead.
3, antibody or antigen or two anti-bags by to nitrocellulose filter and the sealing.
4, the assembling of kit.
The preparation of magnetic bead is that to select particle diameter for use be the magnetic nanoparticle of 10-800nm, preferably uses particle diameter to be the magnetic nanoparticle of 10-800nm; On purpose antibody or the corresponding antigen of antigen or antibody, become immune nanometer magnetic bead by the carbodlimide method covalency.Running program is:
1, use magnetic separator to wash magnetic nanoparticle with 1mLMES;
When 2, washing for the second time, magnetic nanoparticle is suspended among the 0.25mLMES that contains 10mgEDC or CMC again, under the room temperature in mixer mixing 10min;
3, purpose antibody or the corresponding antigen of antigen or antibody are joined magnetic nanoparticle; Room temperature mixing 2 hours;
4, wash magnetic bead 3 times with 1mLPBS;
5, with the magnetic bead that suspends again in the confining liquid
EDC, CMC are the soluble carbon diimine in the aforesaid operations process, and MES is 2-(N morphine generation) ethane sulfonic acid, and all available from Sigma company, the confining liquid composition is: PBS, and 0.1%BSA, 0.05% Sodium azide, BSA and Sodium azide are also available from Sigma company.
The immobilization of magnetic bead is that the air pressure shower nozzle with hundred morals (Bio-Dot) instrument or Metrix instrument etc. is sprayed onto on the plain film of glass fibre, becomes a solid phase pad, frozen drying or in 37 ℃ of dried for standby.
Antibody or antigen or two anti-bags by to nitrocellulose filter and the sealing, be with corresponding another antibody of testing goal thing or the antigen shower nozzle with hundred morals (Bio-Dot) instrument or Metrix instrument etc., being sprayed onto and forming the detection band on the nitrocellulose filter is the T band, be the i.e. two anti-another locations that are sprayed onto on the nitrocellulose filter of primary antibodie or antiantibody simultaneously with antibody, forming the contrast band is the C band, close frozen drying or in 37 ℃ of dryings with the neutral protein fluid-tight.
The assembling of kit is with each composition (substrate, upper end thieving paper, the film that is sprayed with antigen or antibody, lower end thieving paper) cutting, assembling, packing.
The final kit that forms can be referring to Fig. 2, comprise substrate 5, sample pad 1, pad 2, nitrocellulose filter 3 and adsorptive pads 4, described sample pad, pad, nitrocellulose filter and adsorptive pads are arranged in order in order in substrate, and described sample pad is used for adding sample; Pad contains immune nanometer magnetic bead, described immune nanometer magnetic bead is that antibody or the antigen that destination protein is special is coupled to magnetic nanoparticle formation, have on the nitrocellulose filter detect band be T be with 6 and the contrast band be that C is with 7, described detection with on have be fixed on the film with the corresponding antibody of destination protein or antigen, described contrast with on the antibody that is fixed on the film is arranged is that primary antibodie or antiantibody are namely two anti-.
Present embodiment is with kit of the present invention and uses the result that it detects.Be specifically described below in conjunction with Fig. 1-Fig. 4.
Fig. 1 is a kind of reaction principle synoptic diagram of example with hCG (human chorionic gonadotrophin) for the present invention.
In this embodiment, kit is with Y1 (anti-β-hCG monoclonal antibody) coupling magnetic particle, form immune nanometer magnetic bead, again with this coupling the immune nanometer magnetic bead of Y1 go in conjunction with hCG (being triangle among the figure), the Y2 (anti-α-hCG polyclonal antibody) that finally is fixed on the nitrocellulose filter catches, and forming and detecting band is the T band; With the magnetic measurement instrument this detection band is quantitatively detected then, the Y3 (being the sheep anti mouse polyclonal antibody) that is fixed on the nitrocellulose filter of the immune nanometer magnetic bead of being combined with Y2 does not catch, and forming the contrast band is the C band.
Above-mentioned formed detection band is a visible or sightless T band, using kit of the present invention detects sample, the gained result is referring to Fig. 3 or Fig. 4, the positive result of Fig. 3, T has macroscopic brown magnetic bead as seen from the figure, the negative result of Fig. 4, in the sample no destination protein or concentration very low make detect with on the brown magnetic bead that gathers can not be seen by naked eyes.
Claims (4)
1. immune nanometer magnetic bead diagnostic kit, comprise substrate, sample pad, pad, nitrocellulose filter and adsorptive pads, described sample pad, pad, nitrocellulose filter and adsorptive pads are arranged in order in order in substrate, and described sample pad is used for adding sample; Pad contains immune nanometer magnetic bead, described immune nanometer magnetic bead will be for will resist β-hCG monoclonal antibody to be coupled to magnetic nanoparticle, it is that T band and contrast band are the C band that the band of detection is arranged on the nitrocellulose filter, described detection with on the anti-β-hCG polyclonal antibody that is fixed on the film is arranged, described contrast with on the sheep anti mouse polyclonal antibody that is fixed on the film is arranged;
The preparation of described immune nanometer magnetic bead is that to select particle diameter for use be the magnetic nanoparticle of 300nm, will resist β-hCG monoclonal antibody covalent coupling in magnetic nanoparticle, forms immune nanometer magnetic bead; The immobilization of described immune nanometer magnetic bead is that immune nanometer magnetic bead is sprayed onto on the plain film of glass fibre or the polyester film, becomes a solid phase pad, dried for standby;
Described detection band is that the T band is that anti-β-hCG polyclonal antibody is sprayed onto on the nitrocellulose filter and forms, and the contrast band is that the C band is the sheep anti mouse polyclonal antibody to be sprayed onto on the nitrocellulose filter form, with protein liquid sealing, drying;
One group of sample pad, pad, nitrocellulose filter and adsorptive pads constitute a detection path in the described kit, contain one or more detection path arranged side by side in the described kit, detect when being used for one or more sample destination protein.
2. immune nanometer magnetic bead diagnostic kit according to claim 1 is characterized in that, described kit form is check-out console or detector bar.
3. an immune nanometer magnetic bead diagnostic kit according to claim 1 carries out the method for testing goal albumen, it is characterized in that, to resist β-hCG monoclonal antibody to be coupled to magnetic nanoparticle, form immune nanometer magnetic bead, with this coupling the immune nanometer magnetic bead of anti-β-hCG monoclonal antibody remove binding purpose antigen, anti-β-hCG polyclonal antibody that the immune nanometer magnetic bead of being combined with purpose antigen finally is fixed on the film is caught, and forming a detection band is the T band; Estimate testing result then or with the magnetic measurement instrument this detection band is quantitatively detected, the sheep anti mouse polyclonal antibody that is fixed on the film of the immune nanometer magnetic bead of being combined with purpose antigen is not caught, and forming a contrast band is the C band; Described purpose antigen is β-hCG;
Wherein, the described step that will resist β-hCG monoclonal antibody to be coupled to magnetic nanoparticle is:
(1) use magnetic separator to wash magnetic nanoparticle with 1mLMES;
When (2) washing for the second time, magnetic nanoparticle is suspended among the 0.25mLMES that contains 10mgEDC or CMC again, under the room temperature in mixer mixing 10min;
(3) will resist β-hCG monoclonal antibody to join magnetic nanoparticle; Room temperature mixing 2 hours;
(4) wash magnetic bead 3 times with 1mLPBS;
(5) with the magnetic bead that suspends again in the confining liquid.
4. an application rights requires 1 described immune nanometer magnetic bead diagnostic kit to carry out the method for testing goal albumen, comprising:
(1) will be detected sample and be added on the sample pad, this sample moves forward to the adsorptive pads direction in adsorptive pads effect lower edge sample pad;
(2) with immune nanometer magnetic bead reaction and continue to the reach of adsorptive pads direction, catch and form the positive or weak positive detection band being fixed on anti-β-hCG polyclonal antibody on the film when detecting band by the immune nanometer magnetic bead of binding purpose antigen in pad for sample; Described purpose antigen is β-hCG;
(3) immune nanometer magnetic bead continues the sheep anti mouse polyclonal antibody that reach is fixed on the film and catches, and forms the contrast band;
(4) range estimation testing result or with the magnetic measurement instrument this detections is with and quantitatively detects;
The preparation of described immune nanometer magnetic bead is that to select particle diameter for use be the magnetic nanoparticle of 300nm, will resist β-hCG monoclonal antibody covalent coupling in magnetic nanoparticle, forms immune nanometer magnetic bead; The immobilization of described immune nanometer magnetic bead is that immune nanometer magnetic bead is sprayed onto on the plain film of glass fibre or the polyester film, becomes a solid phase pad, dried for standby;
Described detection band is that the T band is that anti-β-hCG polyclonal antibody is sprayed onto on the nitrocellulose filter and forms, and the contrast band is that the C band is the sheep anti mouse polyclonal antibody to be sprayed onto on the nitrocellulose filter form, with protein liquid sealing, drying.
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| CN101603962B true CN101603962B (en) | 2013-09-11 |
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| CN103308671B (en) * | 2013-05-22 | 2016-02-03 | 北京康彻思坦生物技术有限公司 | A kind of detection film and detection system |
| CN104198699B (en) * | 2014-09-04 | 2016-06-15 | 深圳市领治医学科技有限公司 | A kind of quick diagnosis reagent paper and preparation method thereof |
| CN107015006A (en) * | 2017-03-30 | 2017-08-04 | 北京理工大学 | A kind of cell factor immune chromatography test paper and preparation method thereof |
| CN112067803A (en) * | 2020-08-18 | 2020-12-11 | 上海纳米技术及应用国家工程研究中心有限公司 | Novel coronavirus detection kit prepared by magnetic nanoparticle labeled immunochromatography |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1675547A (en) * | 2002-08-27 | 2005-09-28 | 金伯利-克拉克环球有限公司 | Flow-through assay with an internal calibration system using magnetic particles |
| CN1975423A (en) * | 2006-09-06 | 2007-06-06 | 浙江清华长三角研究院 | Immuno magnetic bead and producing method, and method and test plate for detection |
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| US6046585A (en) * | 1997-11-21 | 2000-04-04 | Quantum Design, Inc. | Method and apparatus for making quantitative measurements of localized accumulations of target particles having magnetic particles bound thereto |
| US7323139B2 (en) * | 2002-07-26 | 2008-01-29 | Quantum Design, Inc. | Accessible assay and method of use |
| EP1798556B1 (en) * | 2005-12-14 | 2009-08-26 | The Jordanian Pharmaceutical Manufacturing Co. | Device for the early and rapid immunochromatographic detection of HIV and uses thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1675547A (en) * | 2002-08-27 | 2005-09-28 | 金伯利-克拉克环球有限公司 | Flow-through assay with an internal calibration system using magnetic particles |
| CN1975423A (en) * | 2006-09-06 | 2007-06-06 | 浙江清华长三角研究院 | Immuno magnetic bead and producing method, and method and test plate for detection |
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