WO2015184847A1 - Immunochromatographic test method and test paper - Google Patents

Immunochromatographic test method and test paper Download PDF

Info

Publication number
WO2015184847A1
WO2015184847A1 PCT/CN2015/072273 CN2015072273W WO2015184847A1 WO 2015184847 A1 WO2015184847 A1 WO 2015184847A1 CN 2015072273 W CN2015072273 W CN 2015072273W WO 2015184847 A1 WO2015184847 A1 WO 2015184847A1
Authority
WO
WIPO (PCT)
Prior art keywords
sample
detection
test paper
tested
quality control
Prior art date
Application number
PCT/CN2015/072273
Other languages
French (fr)
Chinese (zh)
Inventor
陈岩松
Original Assignee
陈岩松
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 陈岩松 filed Critical 陈岩松
Publication of WO2015184847A1 publication Critical patent/WO2015184847A1/en

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G01N33/76—Human chorionic gonadotropin including luteinising hormone, follicle stimulating hormone, thyroid stimulating hormone or their receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54306—Solid-phase reaction mechanisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/575—Hormones
    • G01N2333/59—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g. HCG; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]

Definitions

  • the present invention belongs to the field of in vitro diagnostic reagents, and particularly relates to an immunochromatographic detection method and a test strip and a test strip used.
  • Immunochromatography is a rapid diagnostic technique based on immunocolloidal gold technology that was developed in the 1990s.
  • the principle is to immobilize specific antibodies on the reaction membrane 2 first, and the sample pad 4 of the test paper is immersed for inspection. After the sample, due to capillary action, the sample to be tested will move forward along the membrane. When moving to the region where the antibody is immobilized, the corresponding antigen in the sample to be tested will specifically bind to the antibody, if immunogold is used. This area can be displayed in a certain color to achieve a specific immunodiagnosis.
  • the immunochromatographic test paper technology mainly represented by colloidal gold test paper has been widely used in various aspects of immunoassay, and the technique mainly fixes specific antibodies or antigens on the reaction membrane 2 in a strip shape.
  • the color development result of one strip-shaped detection line 8 in the detection area T is qualitatively analyzed, and the color development result of the plurality of strip-shaped detection lines 8 is semi-quantitatively detected.
  • each detection line 8 on the test strip provides only one type of detection information, and increasing the number of detection lines 8 in a certain area may cause confusion or even misinterpretation of the identification. .
  • the invention patent No. 201210200364.5 discloses a colloidal gold immunochromatographic quantitative test strip and a detection method thereof.
  • the method has at least three detection lines 8, and the characteristics include 3, 4, 5, and 6 detection lines 8 . Cannot be applied to test strips with more than 7 test strips.
  • the detection area T adopts a combination of a dot shape, a polygonal shape, another shape of a figure, a different shape of a figure, or a combination of a shape of a strip chart and a shape of another figure as the detection point 6, and the setting of the detection point 6 It may be the same antibody or antigen at the same concentration or different concentrations, or it may be a plurality of antibodies or antigens of the same concentration or different concentrations.
  • Qualitative, semi-quantitative detection by visual inspection of the coloration of multiple detection points 6 and QC 7 or the use of a signal detection function to collect the characteristic signals of detection points 6 and QC 7 on the test strip for quantitative detection Chromatographic detection methods have never been reported.
  • Immunochromatographic techniques include, but are not limited to, double antibody sandwich methods, indirect methods, competitive inhibition methods.
  • the double antibody sandwich method which is a non-competitive binding assay, is suitable for detecting multivalent antigens having at least two antigenic determinants in a molecule.
  • the basic working principle is as follows: The antibody and the labeled antibody attached to the reaction membrane 2 are respectively combined with two antigenic determinants on the detected antigen molecule in the sample to be tested to form a solid phase antibody-antigen-labeled antibody immune complex.
  • the indirect method is a non-competitive binding assay.
  • the principle is that the antigen is solidified in the reaction membrane 2, and the antibody to be tested is first bound to the labeling molecule to form a complex, and then combined with the antigen on the reaction membrane 2 to form a labeling molecule - the antibody-antigen complex to be detected.
  • competition methods are available for antigen and antibody assays.
  • the basic principle is that the test antigen in the sample to be tested and the coated antigen of the solid phase compete for binding with the labeled antibody.
  • the labeled antibody is It does not bind to the solid phase coating antigen; if there is no antigen in the sample to be tested, the labeled antibody binds directly to the solid phase coating antigen.
  • the measurement method of the immunochromatographic detection method includes a qualitative method of visually observing the color of the detection line 8 of the detection area T, a semi-quantitative method of visually observing the coloration of the plurality of detection lines 8 of the detection area T, and a dedicated method.
  • the apparatus measures a quantitative method for determining the color intensity of the detection line 8 of the detection zone T.
  • Reactive film 2 coating determining the type of coated antibody or antigen based on the target to be detected in the sample to be examined
  • the test paper is composed of a bottom plate 1, and a sample pad 4, a conjugate release pad 5, a reaction film 2, and an absorbent pad 3 which are sequentially disposed on the bottom plate 1;
  • testing the sample to be inspected adding a sample to be inspected in the test paper sample pad 4, and visually or semi-quantitatively detecting the sample to be inspected by visually observing the color development of the plurality of detection points 6 and QC 7 , or
  • the signal with the signal detection function can be used to collect the characteristic signals of the detection point 6 and the quality control point 7 on the test paper for quantitative detection. Or firstly, according to the color development condition of the plurality of detection points 6, the object to be inspected in the sample to be inspected is determined, and then the coloration of the plurality of detection points 6 and the quality control point 7 is observed by the naked eye, and different samples to be inspected can be inspected.
  • Qualitative or semi-quantitative detection of the target, or the use of an instrument with signal detection function to collect the characteristic signals of the detection point 6 and the quality control point 7 on the test paper can be quantitatively detected.
  • An immunochromatographic test strip for qualitative, semi-quantitative or quantitative detection of a sample to be inspected; a test strip consisting of a bottom plate 1, and a sample pad 4, a conjugate release pad 5, which are sequentially disposed on the bottom plate 1, reacting The film 2 and the absorbent pad 3 are formed.
  • the reaction film 2 is divided into two regions, one is a detection zone T adjacent to the bond release pad 5, and the other is a quality control zone adjacent to the absorption pad 3 (:.
  • the detection area ⁇ is provided with 2 to 30 detection points 6, and the detection point 6 does not include only the shape of the strip chart, and the detection point 6 is a combination of a dot shape, a polygonal shape, another shape of a figure or a shape of a different figure. It can also be a combination of a strip chart shape and other graph shapes.
  • the detection point 6 can be set to the same concentration or different concentration of the same antibody or antigen. By visually observing the coloration of multiple detection points 6 and QC 7, the sample to be tested can be qualitatively or semi-quantitatively detected, or used.
  • the signal with the signal detection function can detect the characteristic signals of the detection point 6 and the quality control point 7 on the test paper for quantitative detection.
  • the detection point 6 is coated with different antibodies or antigens of the same concentration or different concentrations.
  • the object to be inspected in the sample to be inspected is determined according to the color development of the plurality of detection points 6, and then the plurality of detection points are observed by the naked eye. 6 and the color control of the quality control point 7 can be qualitatively or semi-quantitatively detected in different objects to be inspected in the sample to be inspected, or the characteristic signal of the detection point 6 and the quality control point 7 on the test paper is collected by an instrument having a signal detection function. Quantitative testing is possible.
  • the quality control area C is provided with 1 to 30 quality control points 7, and the quality control points 7 are a combination of a dot shape, a polygonal shape, a strip chart shape, other graphic shapes or different graphic shapes;
  • the 7 pack is coated with an antibody or antigen that binds to the conjugate on the conjugate release pad 5.
  • the conjugate release pad 5 is labeled with a specific antibody or antigen associated with a single target to be detected, or a plurality of specific antibodies or antigens associated with each target to be detected.
  • the immunochromatographic detection method includes, but is not limited to, quantum dot labeling, colloidal gold labeling, colloidal selenium labeling, colored or fluorescent latex labeling, magnetic particle labeling, inter-turnpoint resolution layer Analytical method, chemiluminescence method.
  • the sample to be tested is from clinical or non-clinical blood, body fluid, urine, saliva, reproductive tract secretion or other liquid sample or viscous sample.
  • the material of the bottom plate 1 includes, but is not limited to, a PVC (polyvinyl chloride) plate
  • the material of the reaction film 2 includes, but is not limited to, a nitrocellulose film and a cellulose acetate film
  • materials of the absorbent pad 3 include, but are not limited to, filter paper, samples.
  • Pad 4 materials include, but are not limited to, glass fibers and nonwovens
  • conjugate release pads 5 include, but are not limited to, glass fibers or nonwovens.
  • An immunochromatographic assay method and test strip comprising the immunochromatographic test strip of any one of claims 1-8.
  • the present invention is an immunochromatographic detection method and a test paper.
  • the test paper used in the method is simple, rapid, and convenient to use, and does not require operations by professionals in the field, and can meet the needs of different levels of personnel, and is easy to popularize. Grassroots community hospitals and ordinary families.
  • the detection area T adopts a combination of a dot shape, a polygonal shape, another shape of the figure, a different shape of the figure, or a combination of the shape of the strip chart and the shape of the other figure as the detection point 6, and the plurality of detection points 6 and the quality control are observed by the naked eye.
  • Qualitative, semi-quantitative or quantitative detection of the sample to be inspected can be achieved by the color development of point 7 or by using the signal detection function to collect the characteristic signals of detection point 6 and quality control point 7 on the test paper.
  • the operation is simple and rapid, and is widely used in the field of in vitro diagnostic reagents.
  • An immunochromatographic detection method and test paper of the present invention differ from the existing methods and test strips in that:
  • the existing immunochromatographic detection method and the test strip detection area T are only one or more strip-shaped detection lines 8, each of which provides only one information, and increases the number of detection lines 8 in a certain area. It will cause confusion and even misinterpretation of the identification.
  • An immunochromatographic detection method of the present invention and a detection zone T of the test paper thereof are provided with 2 to 30 detection points 6, and the detection point 6 does not include only the shape of the strip chart, and the detection point 6 is a dot shape, A combination of a polygonal shape, other graphic shapes, or different graphic shapes may also be a combination of a striped shape and other graphic shapes.
  • the detection point 6 can be set to the same concentration or to different concentrations of the same antibody or antigen.
  • the detection point 6 can also be set as a plurality of antibodies or antigens, and the detection points 6 of different antibodies or antigens can be represented by different graph shapes.
  • An immunochromatographic detection method and a test strip thereof according to the present invention can visually or semi-quantitatively detect a sample to be inspected by visually observing the color development of a plurality of detection points 6 and QC points, or use a signal
  • the characteristic signals of the detection point 6 and the quality control point 7 on the instrument collection test paper of the detection function can be quantitatively detected.
  • the object to be inspected in the sample to be inspected is determined, and then the coloration of the plurality of detection points 6 and the quality control point 7 is observed by the naked eye, and different samples to be inspected can be inspected.
  • Qualitative or semi-quantitative detection of the target, or the use of an instrument with signal detection function to collect the characteristic signals of the detection point 6 and the quality control point 7 on the test paper can be quantitatively detected.
  • the present invention provides more different detection points 6 and better identification of different patterns.
  • FIG. 1 is a schematic view of an immunochromatographic detection method and a test paper of the present invention.
  • FIG. 2 is a schematic view of a conventional immunochromatographic detection method and test paper.
  • Figure 3 is a schematic diagram of a human chorionic gonadotropin test strip (colloidal gold immunochromatography).
  • FIG. 4 is a schematic diagram of a combined test strip of urine microalbumin, urine 2 microglobulin, and cystatin C (colloidal gold immunochromatography).
  • Reactive film 2 coating determining the type of coated antibody or antigen based on the target to be detected in the sample to be examined
  • the test paper is composed of a bottom plate 1, and a sample pad 4, a conjugate release pad 5, a reaction film 2, and an absorbent pad 3 which are sequentially disposed on the bottom plate 1;
  • testing the sample to be inspected adding a sample to be inspected in the test paper sample pad 4, and visually or semi-quantitatively detecting the sample to be inspected by visually observing the color development of the plurality of detection points 6 and QC 7 , or use the instrument with signal detection function to collect the characteristic signals of the detection point 6 and the quality control point 7 on the test paper for quantitative detection. Or firstly, according to the color development condition of the plurality of detection points 6, the object to be inspected in the sample to be inspected is determined, and then the coloration of the plurality of detection points 6 and the quality control point 7 is observed by the naked eye, and different samples to be inspected can be inspected. Targets are qualitatively or semi-quantitatively tested, or acquired using instruments with signal detection capabilities The characteristic signals of the detection point 6 and the quality control point 7 on the test paper can be quantitatively detected.
  • the reaction film 2 is divided into two regions, one is a detection zone T adjacent to the bond release pad 5, and the other is a quality control zone adjacent to the absorption pad 3 ⁇
  • the detection area ⁇ is provided with 2 to 30 detection points 6, and the detection point 6 does not include only the shape of the strip chart, and the detection point 6 is a combination of a dot shape, a polygonal shape, another shape of a figure or a shape of a different figure. It can also be a combination of the shape of the strip chart and the shape of the other graphs.
  • the setting of the detecting point 6 can be the same antibody or antigen of the same concentration or different concentrations, and the plurality of detecting points 6 and the control points 7 are observed by the naked eye.
  • the color condition can be qualitatively or semi-quantitatively detected by the sample to be inspected, or the characteristic signal of the detection point 6 and the quality control point 7 on the test paper can be quantitatively detected by using an instrument with a signal detection function.
  • the detection point 6 is coated with different antibodies or antigens of the same concentration or different concentrations.
  • the color control of the quality control point 7 can be qualitatively or semi-quantitatively detected in different objects to be inspected in the sample to be inspected, or the characteristic signal of the detection point 6 and the quality control point 7 on the test paper is collected by an instrument having a signal detection function. Quantitative testing is possible.
  • the quality control area C is provided with 1 to 30 quality control points 7, and the quality control points 7 are a combination of a dot shape, a polygonal shape, a strip chart shape, other graphic shapes or different graphic shapes;
  • the 7 pack is coated with an antibody or antigen that binds to the conjugate on the conjugate release pad 5.
  • the conjugate release pad 5 is labeled with a specific antibody or antigen associated with a single target to be detected, or a plurality of specific antibodies or antigens associated with each target to be detected.
  • the labeling reagent is generally used as a carrier for immobilizing an antibody or antigen in immunochromatographic detection.
  • colloidal gold particles colloidal selenium particles, colored or fluorescent latex particles and magnetic particles, colloidal gold particles are particularly preferred.
  • the support layer of the bottom plate 1 is a non-absorbent material, and the support material is prevented from absorbing the sample to be inspected to affect the movement in the horizontal direction.
  • the material of the bottom plate 1 includes PVC (polyvinyl chloride) board and other plastic materials, preferably PVC (polyvinyl chloride) board.
  • the material of the reaction film 2 includes, but is not limited to, a nitrocellulose membrane, a nylon membrane or a cellulose acetate membrane.
  • a nitrocellulose membrane is preferred.
  • the absorbent pad 3 refers to a liquid absorbing portion that absorbs a sample to be inspected through the reaction film 2 to control diffusion of the sample.
  • the material of the absorbent pad 3 may be, but not limited to, a filter paper.
  • the absorbent pad 3 can be used in one layer or Use multiple layers.
  • the sample pad 4 is a portion that receives the sample to be inspected, and contains any material and form as long as the liquid and the object to be inspected are allowed to pass.
  • Specific materials suitable for the sample pad 4 include, but are not limited to, glass fibers, acrylic fibers, hydrophilic polyethylene materials, dried paper, nonwoven fabrics, and fiber fabrics. Glass fibers and nonwoven fabrics are preferably used.
  • the sample pad 4 can be used in one layer or in multiple layers.
  • materials suitable for the conjugate release pad 5 include, but are not limited to, paper, cellulose mixture, nitrocellulose, polyester fiber, acrylonitrile copolymer, glass fiber, and nonwoven fabric.
  • a nonwoven fabric is used.
  • a plastic card in addition to an immunochromatographic detection method and test paper, a plastic card is included, and the test paper can be installed in a plastic card.
  • the plastic card conforms to the size of the test paper, the manner and location of the sample addition, and the position at which the antibody or antigen is cured on the reaction membrane 2.
  • Bioactive raw materials ot-HCG monoclonal antibodies, ⁇ -HCG monoclonal antibodies, and IgG polyclonal antibodies were all commercially available.
  • Bovine serum albumin was purchased from the market.
  • the nitrocellulose membrane is commercially available.
  • test paper preparation procedure is as follows:
  • the coated antibody species is determined according to the target to be detected in the sample to be tested: ot-HCG monoclonal antibody, IgG polyclonal antibody. [0064] setting the concentration and quantity of the detection point 10 and the quality control point 11 according to the object to be inspected in the sample to be inspected: the detection point 10 is set to 6 points, 4 different concentrations; the quality control point 11 is set to 2 points, 1 concentration.
  • the centrifuged concentrate was diluted and spread on a 90 cm x 30 cm nonwoven fabric, the amount of liquid added was 10-11 ml/sheet, dried for 24 hours, the temperature was 37 ° C, and the humidity was ⁇ 40 ⁇ 3 ⁇ 4 RH.
  • the glass fiber and the nonwoven fabric which are cut into 83.5 cm ⁇ 30 cm are used to contain 0.5 ⁇ 3 ⁇ 4 NaCl (potassium chloride), 0.5% sucrose, 0.1% BSA (bovine serum albumin) and Tris-HCl.
  • the bottom plate 1 is placed flat on the operation table, and double-sided tape is attached.
  • the coated reaction film 2 is attached to the substrate 1.
  • the absorbent pad 3 was pressed 2 mm of the reaction film 2, and the other end was aligned with the bottom plate 1.
  • a layer of the nonwoven fabric pressure reaction film 2 was attached to 2 mm.
  • the conjugate release pad 5 is pressed against the nonwoven fabric.
  • the glass fibers are pressed against the conjugate release pad 5 - half position, and the other end is aligned with the bottom plate 1.
  • One end of a nonwoven fabric is aligned with the upper end of the bond release pad 5, and the other end is aligned with the bottom plate 1.
  • the preparation example 2 of the immunochromatographic test strip of the present invention prepares and detects three combined test strips of urine microalbumin, urine 2 microglobulin and cystatin C (colloidal gold immunochromatography).
  • Bioactive raw materials human albumin monoclonal antibody, mouse anti-human albumin monoclonal antibody, Cys-C (cytosine C) monoclonal antibody, mouse anti-human Cys-C (cytosine C) antibody, 2 microglobulin monoclonal antibodies, murine anti-human 2 microglobulin antibodies, and IgG polyclonal antibodies were purchased from the market.
  • Bovine serum albumin was purchased from the market.
  • the nitrocellulose membrane is commercially available.
  • test paper preparation procedure is as follows:
  • the coated antibody species is determined according to the target to be detected in the sample to be tested: human albumin monoclonal antibody, Cys-C (cytosine C) monoclonal antibody, 2 microglobulin monoclonal antibody, IgG polyclonal antibody.
  • the detection point 12 is set with 3 points, each point is a detection antibody; the quality control point 13 is set 2 points, 1 concentration.
  • Microglobulin monoclonal antibody, 2.42 mg/ml Cys-C (cystatin C) monoclonal antibody, 8.02 mg/ml IgG polyclonal antibody were coated on a nitrocellulose membrane according to the pattern of Fig. 4, dried 24h, temperature is 37 ° C, humidity ⁇ 40 ⁇ 3 ⁇ 4 RH.
  • the centrifuged concentrate was diluted and spread on a 90 cm x 30 cm nonwoven fabric, the amount of liquid added was 10-11 ml/sheet, dried for 24 hours, the temperature was 37 ° C, and the humidity was ⁇ 40 ⁇ 3 ⁇ 4 RH.
  • the microglobulin antibody was prepared as a marker and placed at room temperature for 5 min, and a BSA (bovine serum albumin) stabilizer was added in a ratio of 0.8%.
  • BSA bovine serum albumin
  • the centrifuged concentrate is diluted to a predetermined concentration, and spread on a 90 cm x 30 cm nonwoven fabric, and the amount of liquid added is 10
  • BSA bovine serum albumin
  • the centrifuged concentrate is diluted according to the specified concentration, and spread on a 90 cm x 30 cm nonwoven fabric, and the amount of liquid added is 10
  • the glass fiber and non-woven fabric which are cut into 83.5 cm ⁇ 30 cm are used to contain 0.5 ⁇ 3 ⁇ 4 NaCl (potassium chloride), 0.5% sucrose, 0.1% BSA (bovine serum albumin) and Tris-HCl buffer (trimethylol). Aminomethane-hydrochloric acid) (pH 7.4)
  • the bottom plate 1 is placed on the operation table, and double-sided tape is attached.
  • the coated reaction film 2 is attached to the substrate 1.
  • the absorbent pad 3 was pressed 2 mm of the reaction film 2, and the other end was aligned with the bottom plate 1.
  • a layer of the nonwoven fabric pressure reaction film 2 was attached to 2 mm.
  • the urinary microalbumin conjugate release pad, the urinary 2 microglobulin conjugate release pad, and the cystatin C conjugate release pad are sequentially pressed onto the nonwoven fabric.
  • the glass fiber is pressed against the conjugate release pad 5 - half position, and the other end is aligned with the bottom plate 1.
  • One end of a nonwoven fabric is aligned with the upper end of the bond release pad 5, and the other end is aligned with the bottom plate 1.

Abstract

An immunochromatographic test method and test paper. One or more specific antibodies or antigens are fixed on a reaction film in the manner of either a combination of two or more dot shapes, a polygonal shape, other shapes or different shapes, or a combination of a strip shape and other shapes; labeled specific antibodies or antigens are adsorbed onto a conjugate release pad; after a sample to be tested is dropwise added onto a sample pad, the sample to be tested moves forwards under the effect of a capillary tube and dissolves the labeled substances on the conjugate release pad, followed by mutual reaction to generate conjugates; and when the sample to be tested then moves to a test area T and a quality control area C, the conjugates are trapped by being conjugated with the antibodies or antigens on test points or quality control points, and gather on the test points and the quality control points. Qualitative, semiquantitative or quantitative tests on the sample to be tested can be realized by either observing, with naked eyes, or scanning, by instruments, different developing results on the test points and the quality control points, and the method can be applied in the field of in-vitro diagnostic reagents.

Description

一种免疫层析检测方法及试纸 技术领域  Immunochromatographic detection method and test paper
[0001] 本发明属于体外诊断试剂领域， 具体涉及一种免疫层析检测方法以及所使用的 试纸和试纸盒。  [0001] The present invention belongs to the field of in vitro diagnostic reagents, and particularly relates to an immunochromatographic detection method and a test strip and a test strip used.
背景技术  Background technique
[0002] 免疫层析法是九十年代兴起的一种基于免疫胶体金技术的快速诊断技术， 其原 理是将特异性的抗体先固定于反应膜 2上， 当试纸的样本垫 4浸入待检样本后， 由于毛细管作用， 待检样本将沿着该膜向前移动， 当移动至固定有抗体的区域 吋， 待检样本中相应的抗原即与该抗体发生特异性结合， 若用免疫胶体金可使 该区域显示一定的颜色， 从而实现特异性的免疫诊断。  [0002] Immunochromatography is a rapid diagnostic technique based on immunocolloidal gold technology that was developed in the 1990s. The principle is to immobilize specific antibodies on the reaction membrane 2 first, and the sample pad 4 of the test paper is immersed for inspection. After the sample, due to capillary action, the sample to be tested will move forward along the membrane. When moving to the region where the antibody is immobilized, the corresponding antigen in the sample to be tested will specifically bind to the antibody, if immunogold is used. This area can be displayed in a certain color to achieve a specific immunodiagnosis.
[0003] 以胶体金试纸为主要代表的免疫层析试纸技术现已广泛用于免疫检测的各个方 面， 该技术主要是将特异性的抗体或抗原以条带状固定在反应膜 2上， 通过检测 区 T一条条带状检测线 8的显色结果进行定性分析， 多条条带状检测线 8的显色结 果进行半定量检测。  [0003] The immunochromatographic test paper technology mainly represented by colloidal gold test paper has been widely used in various aspects of immunoassay, and the technique mainly fixes specific antibodies or antigens on the reaction membrane 2 in a strip shape. The color development result of one strip-shaped detection line 8 in the detection area T is qualitatively analyzed, and the color development result of the plurality of strip-shaped detection lines 8 is semi-quantitatively detected.
[0004] 现有的免疫层析检测方法及试纸， 试纸上的每条检测线 8只提供一种检测信息 ， 在一定面积中增加较多的检测线 8数量会给识别造成信息混乱甚至判读错误。  [0004] Existing immunochromatographic detection methods and test strips, each detection line 8 on the test strip provides only one type of detection information, and increasing the number of detection lines 8 in a certain area may cause confusion or even misinterpretation of the identification. .
[0005] 申请号为 201210200364.5的发明专利公幵了一种胶体金免疫层析定量检测试纸 及其检测方法， 该方法检测线 8至少有三条， 特征包括 3、 4、 5、 6条检测线 8。 无法适用于检测线 8超过 7条的试纸。  [0005] The invention patent No. 201210200364.5 discloses a colloidal gold immunochromatographic quantitative test strip and a detection method thereof. The method has at least three detection lines 8, and the characteristics include 3, 4, 5, and 6 detection lines 8 . Cannot be applied to test strips with more than 7 test strips.
[0006] 目前， 检测区 T采用圆点状、 多边形状、 其它图形状、 不同图形状的组合， 或 者条带图形状与其它图形状组合的图形作为检测点 6， 且检测点 6的设定可以是 同浓度或者不同浓度的同一种抗体或抗原， 也可以是同浓度或者不同浓度的多 种抗体或抗原。 通过肉眼观察多个检测点 6和质控点 7的显色情况进行定性、 半 定量检测或采用具有信号检测功能的仪器采集试纸上检测点 6和质控点 7的特征 信号进行定量检测的免疫层析检测方法从未有报道过。  [0006] At present, the detection area T adopts a combination of a dot shape, a polygonal shape, another shape of a figure, a different shape of a figure, or a combination of a shape of a strip chart and a shape of another figure as the detection point 6, and the setting of the detection point 6 It may be the same antibody or antigen at the same concentration or different concentrations, or it may be a plurality of antibodies or antigens of the same concentration or different concentrations. Qualitative, semi-quantitative detection by visual inspection of the coloration of multiple detection points 6 and QC 7 or the use of a signal detection function to collect the characteristic signals of detection points 6 and QC 7 on the test strip for quantitative detection Chromatographic detection methods have never been reported.
[0007] 免疫层析技术包括但不限于双抗体夹心法、 间接法、 竞争抑制法。 [0008] 双抗体夹心法， 属于非竞争结合测定， 适用于检测分子中具有至少两个抗原决 定簇的多价抗原。 其基本工作原理是： 利用连接于反应膜 2上的抗体和标记抗体 分别与待检样本中被检测抗原分子上两个抗原决定簇结合， 形成固相抗体-抗原- 标记抗体免疫复合物。 [0007] Immunochromatographic techniques include, but are not limited to, double antibody sandwich methods, indirect methods, competitive inhibition methods. [0008] The double antibody sandwich method, which is a non-competitive binding assay, is suitable for detecting multivalent antigens having at least two antigenic determinants in a molecule. The basic working principle is as follows: The antibody and the labeled antibody attached to the reaction membrane 2 are respectively combined with two antigenic determinants on the detected antigen molecule in the sample to be tested to form a solid phase antibody-antigen-labeled antibody immune complex.
[0009] 间接法， 测定抗体最常用的方法， 属非竞争结合测定。 其原理是将抗原固化于 反应膜 2， 待检样本中待测抗体首先与标记分子结合， 形成复合物， 再与反应膜 2上的抗原结合， 形成标记分子 -待检抗体-抗原复合物。  [0009] The indirect method, the most commonly used method for determining antibodies, is a non-competitive binding assay. The principle is that the antigen is solidified in the reaction membrane 2, and the antibody to be tested is first bound to the labeling molecule to form a complex, and then combined with the antigen on the reaction membrane 2 to form a labeling molecule - the antibody-antigen complex to be detected.
[0010] 竞争法可用于抗原和抗体测定。 以测定抗原为例， 其基本原理是待检样本中的 受检抗原和固相的包被抗原竞争与标记抗体结合， 当待检样本中的受检抗原的 量大于试剂的阈值， 标记抗体则不会与固相的包被抗原结合； 若待检样本中无 受检抗原， 标记抗体直接与固相的包被抗原结合。  [0010] Competition methods are available for antigen and antibody assays. Taking the antigen as an example, the basic principle is that the test antigen in the sample to be tested and the coated antigen of the solid phase compete for binding with the labeled antibody. When the amount of the test antigen in the sample to be tested is greater than the threshold of the reagent, the labeled antibody is It does not bind to the solid phase coating antigen; if there is no antigen in the sample to be tested, the labeled antibody binds directly to the solid phase coating antigen.
[0011] 免疫层析检测方法的测定方法包括用肉眼观察确定检测区 T的检测线 8颜色的定 性方法、 用肉眼观察检测区 T的多个检测线 8显色情况的半定量方法和通过专用 仪器设备测定检测区 T的检测线 8颜色强度的定量方法。  [0011] The measurement method of the immunochromatographic detection method includes a qualitative method of visually observing the color of the detection line 8 of the detection area T, a semi-quantitative method of visually observing the coloration of the plurality of detection lines 8 of the detection area T, and a dedicated method. The apparatus measures a quantitative method for determining the color intensity of the detection line 8 of the detection zone T.
技术问题  technical problem
[0012] 本发明的目的在于， 针对现有技术， 提供一种免疫层析检测方法及试纸， 可实 现对待检样本的定性、 半定量或定量检测。  [0012] It is an object of the present invention to provide an immunochromatographic detection method and a test paper for the prior art, which can realize qualitative, semi-quantitative or quantitative detection of a sample to be inspected.
问题的解决方案  Problem solution
技术解决方案  Technical solution
[0013] 一种免疫层析检测方法， 采用如下步骤： [0013] An immunochromatographic detection method, using the following steps:
[0014] (1) 反应膜 2包被： 根据待检样本中的待检目标物确定包被抗体或抗原的种类 (1) Reactive film 2 coating: determining the type of coated antibody or antigen based on the target to be detected in the sample to be examined
， 设定检测点 6及质控点 7的浓度和数量； , setting the concentration and quantity of the detection point 6 and the quality control point 7;
[0015] (2) 根据待检样本中的待检目标物制备结合物释放垫 5; [0015] (2) preparing a conjugate release pad 5 according to the object to be inspected in the sample to be inspected;
[0016] (3) 样本垫 4制备； [0016] (3) preparation of sample pad 4 ;
[0017] (4) 组装试纸： 试纸由底板 1， 及依次设置在底板 1上的样本垫 4、 结合物释放 垫 5、 反应膜 2、 吸收垫 3构成；  [0017] (4) Assembling the test paper: the test paper is composed of a bottom plate 1, and a sample pad 4, a conjugate release pad 5, a reaction film 2, and an absorbent pad 3 which are sequentially disposed on the bottom plate 1;
[0018] (5) 对待检样本进行检测： 在试纸样本垫 4中加入待检样本， 通过肉眼观察多 个检测点 6和质控点 7的显色情况可以对待检样本进行定性或者半定量检测， 或 采用具有信号检测功能的仪器采集试纸上检测点 6和质控点 7的特征信号可以进 行定量检测。 或者首先根据多个检测点 6的显色情况确定待检样本中的待检目标 物， 再通过肉眼观察多个检测点 6和质控点 7的显色情况可以对待检样本中不同 的待检目标物进行定性或者半定量检测， 或采用具有信号检测功能的仪器采集 试纸上检测点 6和质控点 7的特征信号可以进行定量检测。 [0018] (5) Testing the sample to be inspected: adding a sample to be inspected in the test paper sample pad 4, and visually or semi-quantitatively detecting the sample to be inspected by visually observing the color development of the plurality of detection points 6 and QC 7 , or The signal with the signal detection function can be used to collect the characteristic signals of the detection point 6 and the quality control point 7 on the test paper for quantitative detection. Or firstly, according to the color development condition of the plurality of detection points 6, the object to be inspected in the sample to be inspected is determined, and then the coloration of the plurality of detection points 6 and the quality control point 7 is observed by the naked eye, and different samples to be inspected can be inspected. Qualitative or semi-quantitative detection of the target, or the use of an instrument with signal detection function to collect the characteristic signals of the detection point 6 and the quality control point 7 on the test paper can be quantitatively detected.
[0019] 一种免疫层析检测试纸， 用于实现对待检样本的定性、 半定量或定量检测； 试 纸由底板 1， 及依次设置在底板 1上的样本垫 4、 结合物释放垫 5、 反应膜 2、 吸收 垫 3构成。 [0019] An immunochromatographic test strip for qualitative, semi-quantitative or quantitative detection of a sample to be inspected; a test strip consisting of a bottom plate 1, and a sample pad 4, a conjugate release pad 5, which are sequentially disposed on the bottom plate 1, reacting The film 2 and the absorbent pad 3 are formed.
[0020] 所述反应膜 2分成两个区域， 一个是与结合物释放垫 5相邻接的检测区 T， 一个 是与吸收垫 3相邻接的质控区 (：。 [0020] The reaction film 2 is divided into two regions, one is a detection zone T adjacent to the bond release pad 5, and the other is a quality control zone adjacent to the absorption pad 3 (:.
[0021] 所述检测区 Τ设有 2至 30个检测点 6， 检测点 6不包括仅有条带图形状， 检测点 6 为圆点状、 多边形状、 其它图形状或不同图形状的组合， 亦可为条带图形状与 其它图形状的组合。 检测点 6的设定可以是同浓度或者不同浓度的同一种抗体或 抗原， 通过肉眼观察多个检测点 6和质控点 7的显色情况可以对待检样本进行定 性或者半定量检测， 或采用具有信号检测功能的仪器采集试纸上检测点 6和质控 点 7的特征信号可以进行定量检测。 或者是检测点 6包被有同浓度或者不同浓度 的不同的抗体或抗原， 首先根据多个检测点 6的显色情况确定待检样本中的待检 目标物， 再通过肉眼观察多个检测点 6和质控点 7的显色情况可以对待检样本中 不同的待检目标物进行定性或者半定量检测， 或采用具有信号检测功能的仪器 采集试纸上检测点 6和质控点 7的特征信号可以进行定量检测。  [0021] The detection area Τ is provided with 2 to 30 detection points 6, and the detection point 6 does not include only the shape of the strip chart, and the detection point 6 is a combination of a dot shape, a polygonal shape, another shape of a figure or a shape of a different figure. It can also be a combination of a strip chart shape and other graph shapes. The detection point 6 can be set to the same concentration or different concentration of the same antibody or antigen. By visually observing the coloration of multiple detection points 6 and QC 7, the sample to be tested can be qualitatively or semi-quantitatively detected, or used. The signal with the signal detection function can detect the characteristic signals of the detection point 6 and the quality control point 7 on the test paper for quantitative detection. Alternatively, the detection point 6 is coated with different antibodies or antigens of the same concentration or different concentrations. First, the object to be inspected in the sample to be inspected is determined according to the color development of the plurality of detection points 6, and then the plurality of detection points are observed by the naked eye. 6 and the color control of the quality control point 7 can be qualitatively or semi-quantitatively detected in different objects to be inspected in the sample to be inspected, or the characteristic signal of the detection point 6 and the quality control point 7 on the test paper is collected by an instrument having a signal detection function. Quantitative testing is possible.
[0022] 所述质控区 C设有 1至 30个质控点 7， 质控点 7为圆点状、 多边形状、 条带图形状 、 其它图形状或不同图形状的组合； 质控点 7包被有可与结合物释放垫 5上结合 物结合的抗体或抗原。  [0022] The quality control area C is provided with 1 to 30 quality control points 7, and the quality control points 7 are a combination of a dot shape, a polygonal shape, a strip chart shape, other graphic shapes or different graphic shapes; The 7 pack is coated with an antibody or antigen that binds to the conjugate on the conjugate release pad 5.
[0023] 所述结合物释放垫 5上标记有单一待检目标物相关的特异性抗体或抗原， 或标 记有各待检目标物相关的多个特异性抗体或抗原。  [0023] The conjugate release pad 5 is labeled with a specific antibody or antigen associated with a single target to be detected, or a plurality of specific antibodies or antigens associated with each target to be detected.
[0024] 所述的免疫层析检测方法， 其标记方法包括但不限于量子点标记法、 胶体金标 记法、 胶体硒标记法、 有色或荧光乳胶标记法、 磁性颗粒标记法、 吋间分辨层 析法、 化学发光法。 [0025] 所述的待检样本， 是来自临床或非临床的血液、 体液、 尿液、 唾液、 生殖道分 泌液或其他液态样品或粘稠状样品。 [0024] The immunochromatographic detection method, the labeling method includes, but is not limited to, quantum dot labeling, colloidal gold labeling, colloidal selenium labeling, colored or fluorescent latex labeling, magnetic particle labeling, inter-turnpoint resolution layer Analytical method, chemiluminescence method. [0025] The sample to be tested is from clinical or non-clinical blood, body fluid, urine, saliva, reproductive tract secretion or other liquid sample or viscous sample.
[0026] 所述底板 1材料包括但不限于 PVC(聚氯乙烯)板， 反应膜 2材料包括但不限于硝 酸纤维素膜和醋酸纤维素膜， 吸收垫 3的材料包括但不限于滤纸， 样本垫 4材料 包括但不限于玻璃纤维和无纺布， 结合物释放垫 5包括但不限于玻璃纤维或无纺 布。 The material of the bottom plate 1 includes, but is not limited to, a PVC (polyvinyl chloride) plate, and the material of the reaction film 2 includes, but is not limited to, a nitrocellulose film and a cellulose acetate film, and materials of the absorbent pad 3 include, but are not limited to, filter paper, samples. Pad 4 materials include, but are not limited to, glass fibers and nonwovens, and conjugate release pads 5 include, but are not limited to, glass fibers or nonwovens.
[0027] 一种免疫层析检测方法及试纸， 包括权利要求 1-8任一项的免疫层析试纸。  An immunochromatographic assay method and test strip comprising the immunochromatographic test strip of any one of claims 1-8.
发明的有益效果  Advantageous effects of the invention
有益效果  Beneficial effect
[0028] 本发明是一种免疫层析检测方法及试纸， 该方法使用的试纸操作简单， 快速， 使用方便， 不需要本领域的专业人员进行操作， 能够满足不同层次人员的需要 ， 容易普及到基层社区医院及普通家庭。 且检测区 T采用圆点状、 多边形状、 其 它图形状、 不同图形状的组合， 或者条带图形状与其它图形状组合的图形作为 检测点 6， 通过肉眼观察多个检测点 6和质控点 7的显色情况或采用具有信号检测 功能的仪器采集试纸上检测点 6和质控点 7的特征信号， 可实现对待检样本的定 性、 半定量或定量检测。 操作简单、 快速， 广泛应用于体外诊断试剂领域。  [0028] The present invention is an immunochromatographic detection method and a test paper. The test paper used in the method is simple, rapid, and convenient to use, and does not require operations by professionals in the field, and can meet the needs of different levels of personnel, and is easy to popularize. Grassroots community hospitals and ordinary families. And the detection area T adopts a combination of a dot shape, a polygonal shape, another shape of the figure, a different shape of the figure, or a combination of the shape of the strip chart and the shape of the other figure as the detection point 6, and the plurality of detection points 6 and the quality control are observed by the naked eye. Qualitative, semi-quantitative or quantitative detection of the sample to be inspected can be achieved by the color development of point 7 or by using the signal detection function to collect the characteristic signals of detection point 6 and quality control point 7 on the test paper. The operation is simple and rapid, and is widely used in the field of in vitro diagnostic reagents.
[0029] 本发明的一种免疫层析检测方法及试纸不同于现有的方法和试纸之处在于： [0029] An immunochromatographic detection method and test paper of the present invention differ from the existing methods and test strips in that:
[0030] 现有的免疫层析检测方法及试纸检测区 T只是一条或多条条带状检测线 8， 每一 条检测线 8只提供一个信息， 在一定面积中增加较多的检测线 8数量会给识别造 成信息混乱甚至判读错误。 [0030] The existing immunochromatographic detection method and the test strip detection area T are only one or more strip-shaped detection lines 8, each of which provides only one information, and increases the number of detection lines 8 in a certain area. It will cause confusion and even misinterpretation of the identification.
[0031] 本发明的一种免疫层析检测方法及其试纸的检测区 T设有 2至 30个检测点 6， 检 测点 6不包括仅有条带图形状， 检测点 6为圆点状、 多边形状、 其它图形状或不 同图形状的组合， 亦可为条带图形状与其它图形状的组合。 检测点 6的设定可以 是相同浓度， 亦可为不同浓度的同一种抗体或抗原。 检测点 6还可以设定为多种 抗体或抗原， 且不同种抗体或抗原的检测点 6可以用不同的图形状表示。  [0031] An immunochromatographic detection method of the present invention and a detection zone T of the test paper thereof are provided with 2 to 30 detection points 6, and the detection point 6 does not include only the shape of the strip chart, and the detection point 6 is a dot shape, A combination of a polygonal shape, other graphic shapes, or different graphic shapes may also be a combination of a striped shape and other graphic shapes. The detection point 6 can be set to the same concentration or to different concentrations of the same antibody or antigen. The detection point 6 can also be set as a plurality of antibodies or antigens, and the detection points 6 of different antibodies or antigens can be represented by different graph shapes.
[0032] 本发明的一种免疫层析检测方法及其试纸， 可以通过肉眼观察多个检测点 6和 质控点 7的显色情况可以对待检样本进行定性或者半定量检测， 或采用具有信号 检测功能的仪器采集试纸上检测点 6和质控点 7的特征信号可以进行定量检测。 或者首先根据多个检测点 6的显色情况确定待检样本中的待检目标物， 再通过肉 眼观察多个检测点 6和质控点 7的显色情况可以对待检样本中不同的待检目标物 进行定性或者半定量检测， 或采用具有信号检测功能的仪器采集试纸上检测点 6 和质控点 7的特征信号可以进行定量检测。 [0032] An immunochromatographic detection method and a test strip thereof according to the present invention can visually or semi-quantitatively detect a sample to be inspected by visually observing the color development of a plurality of detection points 6 and QC points, or use a signal The characteristic signals of the detection point 6 and the quality control point 7 on the instrument collection test paper of the detection function can be quantitatively detected. Or firstly, according to the color development condition of the plurality of detection points 6, the object to be inspected in the sample to be inspected is determined, and then the coloration of the plurality of detection points 6 and the quality control point 7 is observed by the naked eye, and different samples to be inspected can be inspected. Qualitative or semi-quantitative detection of the target, or the use of an instrument with signal detection function to collect the characteristic signals of the detection point 6 and the quality control point 7 on the test paper can be quantitatively detected.
[0033] 相比较现有的方法本发明提供了更多的不同的检测点 6和更好识别的不同图形 f π息。 [0033] Compared to existing methods, the present invention provides more different detection points 6 and better identification of different patterns.
对附图的简要说明  Brief description of the drawing
附图说明  DRAWINGS
[0034] 附图 1是本发明的一种免疫层析检测方法及试纸的示意图。  1 is a schematic view of an immunochromatographic detection method and a test paper of the present invention.
[0035] 附图 2是现有的免疫层析检测方法及试纸的示意图。  2 is a schematic view of a conventional immunochromatographic detection method and test paper.
[0036] 附图 3人绒毛膜促性腺激素检测试纸 （胶体金免疫层析法） 示意图。  Figure 3 is a schematic diagram of a human chorionic gonadotropin test strip (colloidal gold immunochromatography).
[0037] 附图 4尿微量白蛋白、 尿 2微球蛋白、 抑胱素 C三项联合检测试纸 （胶体金免疫 层析法） 示意图。 [0037] FIG. 4 is a schematic diagram of a combined test strip of urine microalbumin, urine 2 microglobulin, and cystatin C (colloidal gold immunochromatography).
实施该发明的最佳实施例  BEST MODE FOR CARRYING OUT THE INVENTION
本发明的最佳实施方式  BEST MODE FOR CARRYING OUT THE INVENTION
[0038] 实施步骤： [0038] Implementation steps:
[0039] ( 1) 反应膜 2包被： 根据待检样本中的待检目标物确定包被抗体或抗原的种类 (1) Reactive film 2 coating: determining the type of coated antibody or antigen based on the target to be detected in the sample to be examined
， 设定检测点 6及质控点 7的浓度和数量； , setting the concentration and quantity of the detection point 6 and the quality control point 7;
[0040] (2) 根据待检样本中的待检目标物制备结合物释放垫 5 ; [0040] (2) preparing a conjugate release pad 5 according to the target to be inspected in the sample to be inspected;
[0041] (3) 样本垫 4制备； [0041] (3) sample pad 4 preparation;
[0042] (4) 组装试纸： 试纸由底板 1， 及依次设置在底板 1上的样本垫 4、 结合物释放 垫 5、 反应膜 2、 吸收垫 3构成；  [0042] (4) Assembling the test paper: the test paper is composed of a bottom plate 1, and a sample pad 4, a conjugate release pad 5, a reaction film 2, and an absorbent pad 3 which are sequentially disposed on the bottom plate 1;
[0043] (5) 对待检样本进行检测： 在试纸样本垫 4中加入待检样本， 通过肉眼观察多 个检测点 6和质控点 7的显色情况可以对待检样本进行定性或者半定量检测， 或 采用具有信号检测功能的仪器采集试纸上检测点 6和质控点 7的特征信号可以进 行定量检测。 或者首先根据多个检测点 6的显色情况确定待检样本中的待检目标 物， 再通过肉眼观察多个检测点 6和质控点 7的显色情况可以对待检样本中不同 的待检目标物进行定性或者半定量检测， 或采用具有信号检测功能的仪器采集 试纸上检测点 6和质控点 7的特征信号可以进行定量检测。 [0043] (5) Testing the sample to be inspected: adding a sample to be inspected in the test paper sample pad 4, and visually or semi-quantitatively detecting the sample to be inspected by visually observing the color development of the plurality of detection points 6 and QC 7 , or use the instrument with signal detection function to collect the characteristic signals of the detection point 6 and the quality control point 7 on the test paper for quantitative detection. Or firstly, according to the color development condition of the plurality of detection points 6, the object to be inspected in the sample to be inspected is determined, and then the coloration of the plurality of detection points 6 and the quality control point 7 is observed by the naked eye, and different samples to be inspected can be inspected. Targets are qualitatively or semi-quantitatively tested, or acquired using instruments with signal detection capabilities The characteristic signals of the detection point 6 and the quality control point 7 on the test paper can be quantitatively detected.
[0044] 反应膜 2分成两个区域， 一个是与结合物释放垫 5相邻接的检测区 T， 一个是与 吸收垫 3相邻接的质控区^  The reaction film 2 is divided into two regions, one is a detection zone T adjacent to the bond release pad 5, and the other is a quality control zone adjacent to the absorption pad 3^
[0045] 所述检测区 Τ设有 2至 30个检测点 6， 检测点 6不包括仅有条带图形状， 检测点 6 为圆点状、 多边形状、 其它图形状或不同图形状的组合， 亦可为条带图形状与 其它图形状的组合， 检测点 6的设定可以是同浓度或者不同浓度的同一种抗体或 抗原， 通过肉眼观察多个检测点 6和质控点 7的显色情况可以对待检样本进行定 性或者半定量检测， 或采用具有信号检测功能的仪器采集试纸上检测点 6和质控 点 7的特征信号可以进行定量检测。 或者是检测点 6包被有同浓度或者不同浓度 的不同的抗体或抗原， 首先根据多个检测点 6的显色情况确定待检样本中的待检 目标物， 再通过肉眼观察多个检测点 6和质控点 7的显色情况可以对待检样本中 不同的待检目标物进行定性或者半定量检测， 或采用具有信号检测功能的仪器 采集试纸上检测点 6和质控点 7的特征信号可以进行定量检测。  [0045] The detection area Τ is provided with 2 to 30 detection points 6, and the detection point 6 does not include only the shape of the strip chart, and the detection point 6 is a combination of a dot shape, a polygonal shape, another shape of a figure or a shape of a different figure. It can also be a combination of the shape of the strip chart and the shape of the other graphs. The setting of the detecting point 6 can be the same antibody or antigen of the same concentration or different concentrations, and the plurality of detecting points 6 and the control points 7 are observed by the naked eye. The color condition can be qualitatively or semi-quantitatively detected by the sample to be inspected, or the characteristic signal of the detection point 6 and the quality control point 7 on the test paper can be quantitatively detected by using an instrument with a signal detection function. Alternatively, the detection point 6 is coated with different antibodies or antigens of the same concentration or different concentrations. First, the object to be inspected in the sample to be inspected is determined according to the color development of the plurality of detection points 6, and then the plurality of detection points are observed by the naked eye. 6 and the color control of the quality control point 7 can be qualitatively or semi-quantitatively detected in different objects to be inspected in the sample to be inspected, or the characteristic signal of the detection point 6 and the quality control point 7 on the test paper is collected by an instrument having a signal detection function. Quantitative testing is possible.
[0046] 所述质控区 C设有 1至 30个质控点 7， 质控点 7为圆点状、 多边形状、 条带图形状 、 其它图形状或不同图形状的组合； 质控点 7包被有可与结合物释放垫 5上结合 物结合的抗体或抗原。  [0046] The quality control area C is provided with 1 to 30 quality control points 7, and the quality control points 7 are a combination of a dot shape, a polygonal shape, a strip chart shape, other graphic shapes or different graphic shapes; The 7 pack is coated with an antibody or antigen that binds to the conjugate on the conjugate release pad 5.
[0047] 所述结合物释放垫 5上标记有单一待检目标物相关的特异性抗体或抗原， 或标 记有各待检目标物相关的多个特异性抗体或抗原。  [0047] The conjugate release pad 5 is labeled with a specific antibody or antigen associated with a single target to be detected, or a plurality of specific antibodies or antigens associated with each target to be detected.
[0048] 在本发明中， 标记试剂通常是在免疫层析检测中可用作固定抗体或抗原的载体In the present invention, the labeling reagent is generally used as a carrier for immobilizing an antibody or antigen in immunochromatographic detection.
。 例如， 胶体金颗粒， 胶体硒颗粒， 有色或荧光乳胶颗粒和磁性颗粒， 特别优 选胶体金颗粒。 . For example, colloidal gold particles, colloidal selenium particles, colored or fluorescent latex particles and magnetic particles, colloidal gold particles are particularly preferred.
[0049] 在本发明中， 底板 1支撑层为不吸水的材料， 避免支撑材料吸收待检样本从而 影响水平方向的移动。 底板 1的材料包括 PVC (聚氯乙烯)板及其他塑料材料， 优 选 PVC(聚氯乙烯)板。  In the present invention, the support layer of the bottom plate 1 is a non-absorbent material, and the support material is prevented from absorbing the sample to be inspected to affect the movement in the horizontal direction. The material of the bottom plate 1 includes PVC (polyvinyl chloride) board and other plastic materials, preferably PVC (polyvinyl chloride) board.
[0050] 在本发明中， 反应膜 2的材料包括但不限于硝酸纤维素膜、 尼龙膜或醋酸纤维 素膜。 优选硝酸纤维素膜。  In the present invention, the material of the reaction film 2 includes, but is not limited to, a nitrocellulose membrane, a nylon membrane or a cellulose acetate membrane. A nitrocellulose membrane is preferred.
[0051] 在本发明中， 吸收垫 3指吸收通过反应膜 2的待检样本以控制样品扩散的液体吸 收部分。 吸收垫 3的材料可以是但不限于滤纸。 吸收垫 3可以使用一层也可以使 用多层。 In the present invention, the absorbent pad 3 refers to a liquid absorbing portion that absorbs a sample to be inspected through the reaction film 2 to control diffusion of the sample. The material of the absorbent pad 3 may be, but not limited to, a filter paper. The absorbent pad 3 can be used in one layer or Use multiple layers.
[0052] 在本发明中， 样本垫 4是接收待检样本的部分， 包含任何材料和形式， 只要能 让液体和待检目标物通过。 适合于样本垫 4的材料的具体包括但不限于： 玻璃纤 维、 丙烯酸纤维、 亲水聚乙烯材料、 干燥的纸、 无纺布和纤维织物。 优选使用 玻璃纤维和无纺布。 样本垫 4可以使用一层也可以使用多层。  In the present invention, the sample pad 4 is a portion that receives the sample to be inspected, and contains any material and form as long as the liquid and the object to be inspected are allowed to pass. Specific materials suitable for the sample pad 4 include, but are not limited to, glass fibers, acrylic fibers, hydrophilic polyethylene materials, dried paper, nonwoven fabrics, and fiber fabrics. Glass fibers and nonwoven fabrics are preferably used. The sample pad 4 can be used in one layer or in multiple layers.
[0053] 在本发明中， 适合于结合物释放垫 5的材料包括但不限于纸、 纤维素混合物、 硝酸纤维素、 聚酯纤维、 丙烯腈共聚物、 玻璃纤维、 和无纺布。 优选使用无纺 布。  In the present invention, materials suitable for the conjugate release pad 5 include, but are not limited to, paper, cellulose mixture, nitrocellulose, polyester fiber, acrylonitrile copolymer, glass fiber, and nonwoven fabric. Preferably, a nonwoven fabric is used.
[0054] 在本发明中， 除了一种免疫层析法检测方法及试纸， 还包括塑料卡， 可以将试 纸安装在塑料卡之内使用。 所述的塑料卡与试纸的尺寸、 样本加入的方式和位 置、 抗体或抗原在反应膜 2上固化的位置相吻合。  In the present invention, in addition to an immunochromatographic detection method and test paper, a plastic card is included, and the test paper can be installed in a plastic card. The plastic card conforms to the size of the test paper, the manner and location of the sample addition, and the position at which the antibody or antigen is cured on the reaction membrane 2.
本发明的实施方式 Embodiments of the invention
[0055] [0008]提供下述实施例是为了更好地进一步理解本发明， 并不局限于所述最佳 实施方式， 不对本发明的内容和保护范围构成限制， 任何人在本发明的启示下 或是将本发明的其他现有技术的特征进行组合而得出的任何与本发明相同或相 近似的产品， 均属于本发明的保护范围之内。  The following examples are provided to better understand the present invention, and are not to be construed as limited to the preferred embodiments. Any product that is identical or similar to the present invention, which is obtained by combining other prior art features of the present invention, is within the scope of the present invention.
[0056] 实施例 1  Embodiment 1
[0057] 本发明的免疫层析检测试纸的制备实施例 1， 制备及检测人绒毛膜促性腺激素  [0057] Preparation of immunochromatographic test strip of the present invention Example 1, preparation and detection of human chorionic gonadotropin
(HCG) 检测试纸 （胶体金免疫层析法） 。  (HCG) test strip (colloidal gold immunochromatography).
[0058] 生物活性原料： ot-HCG单克隆抗体、 β-HCG单克隆抗体、 IgG多克隆抗体均来 自市场购买。 [0058] Bioactive raw materials: ot-HCG monoclonal antibodies, β-HCG monoclonal antibodies, and IgG polyclonal antibodies were all commercially available.
[0059] 试剂： 牛血清白蛋白来自市场购买。 Reagents: Bovine serum albumin was purchased from the market.
[0060] 硝酸纤维素膜来自市场购买。 [0060] The nitrocellulose membrane is commercially available.
[0061] 试纸制备程序如下： [0061] The test paper preparation procedure is as follows:
[0062] 步骤 1.反应膜 2包被 Step 1. Reaction film 2 coating
[0063] 根据待检样本中的待检目标物确定包被抗体种类： ot-HCG单克隆抗体、 IgG多 克隆抗体。 [0064] 根据待检样本中的待检目标物设定检测点 10及质控点 11的浓度和数量： 检测点 10设置 6个点， 4个不同浓度； 质控点 11设置 2个点， 1个浓度。 [0063] The coated antibody species is determined according to the target to be detected in the sample to be tested: ot-HCG monoclonal antibody, IgG polyclonal antibody. [0064] setting the concentration and quantity of the detection point 10 and the quality control point 11 according to the object to be inspected in the sample to be inspected: the detection point 10 is set to 6 points, 4 different concentrations; the quality control point 11 is set to 2 points, 1 concentration.
[0065] 将 15.42mg/ml的 α-HCG单克隆抗体、 8.02mg/ml的 IgG多克隆抗体用按照附图 3 的图形包被于硝酸纤维素膜上， 干燥 24h， 温度为 37°C， 湿度≤40%RH。  [0065] 15.42 mg/ml of α-HCG monoclonal antibody, 8.02 mg/ml of IgG polyclonal antibody were coated on a nitrocellulose membrane according to the pattern of FIG. 3, dried for 24 h, and the temperature was 37 ° C. Humidity ≤ 40% RH.
[0066] 步骤 2.结合物释放垫 5制备  Step 2. Preparation of the conjugate release pad 5
[0067] 取 40nm的胶体金溶液， 按 1.2%比例加入 0.2M的 K 2CO 3 (碳酸钾） 调节 PH值， 按 15 g/ml比例加入 β-HCG单克隆抗体， 制成标记物， 放置室温 5min， 按 0.8%比 例加入 BSA (牛血清白蛋白） 稳定剂。 [0067] Take 40nm colloidal gold solution, add 0.2M K 2 CO 3 (potassium carbonate) in a ratio of 1.2% to adjust the pH value, add β-HCG monoclonal antibody in a ratio of 15 g / ml, make a marker, place BSA (bovine serum albumin) stabilizer was added at a rate of 0.8% at room temperature for 5 min.
[0068] 4500r离心 30min， 取浓缩沉淀物； 将上清液继续 6500r离心 45min， 取浓缩沉淀 物； 将上清液继续 9000r离心 60min， 取浓缩沉淀物； 将三次离心浓缩沉淀物混合 均匀。 [0068] After centrifugation at 4500r for 30 min, the concentrated precipitate was taken; the supernatant was centrifuged at 6500 rpm for 45 min, and the concentrated precipitate was taken; the supernatant was centrifuged at 9000 rpm for 60 min to obtain a concentrated precipitate; and the precipitate was concentrated by centrifugation three times.
[0069] 将离心好的浓缩液稀释好， 铺于 90cmx30cm的无纺布上， 加液量 10- 11ml/张， 干燥 24h， 温度 37°C， 湿度≤40<¾RH。  [0069] The centrifuged concentrate was diluted and spread on a 90 cm x 30 cm nonwoven fabric, the amount of liquid added was 10-11 ml/sheet, dried for 24 hours, the temperature was 37 ° C, and the humidity was ≤ 40 < 3⁄4 RH.
[0070] 步骤 3.样本垫 4的制备 [0070] Step 3. Preparation of Sample Pad 4
[0071] 将切割成 83.5cmx30cm的玻璃纤维和无纺布用包含 0.5<¾NaCl (氯化钾） 、 0.5% 蔗糖、 0.1%BSA (牛血清白蛋白） 和 Tris-HCl  [0071] The glass fiber and the nonwoven fabric which are cut into 83.5 cm×30 cm are used to contain 0.5<3⁄4 NaCl (potassium chloride), 0.5% sucrose, 0.1% BSA (bovine serum albumin) and Tris-HCl.
缓冲液 (三羟甲基氨基甲烷-盐酸) (pH7.4)工作液浸泡。 在 37°C将所述垫干燥 24h， 获得样本垫 4。  Soak the buffer (trimethylolamine-hydrochloride) (pH 7.4) working solution. The pad was dried at 37 ° C for 24 h to obtain a sample pad 4.
[0072] 步骤 4.组装试纸 [0072] Step 4. Assembling the test paper
[0073] 将底板 1平放在操作台上， 贴上双面胶。  [0073] The bottom plate 1 is placed flat on the operation table, and double-sided tape is attached.
[0074] 将包被好的反应膜 2贴在底板 1上。 [0074] The coated reaction film 2 is attached to the substrate 1.
[0075] 将吸收垫 3压反应膜 2的 2mm贴上， 另一端与底板 1对齐。  [0075] The absorbent pad 3 was pressed 2 mm of the reaction film 2, and the other end was aligned with the bottom plate 1.
[0076] 将一层无纺布压反应膜 2的 2mm处贴上。 [0076] A layer of the nonwoven fabric pressure reaction film 2 was attached to 2 mm.
[0077] 将结合物释放垫 5齐压于无纺布上面。 [0077] The conjugate release pad 5 is pressed against the nonwoven fabric.
[0078] 将玻璃纤维压于结合物释放垫 5—半位置， 另一端与底板 1对齐。  [0078] The glass fibers are pressed against the conjugate release pad 5 - half position, and the other end is aligned with the bottom plate 1.
[0079] 再将一层无纺布一端与结合物释放垫 5上端对齐， 另一端与底板 1对齐。 [0079] One end of a nonwoven fabric is aligned with the upper end of the bond release pad 5, and the other end is aligned with the bottom plate 1.
[0080] 步骤 5.产品检测 [0080] Step 5. Product Inspection
[0081] 取含有 HCG的待检样本尿液滴加到样本垫 4上， 5min吋判读结果， 显色， 其 它 T点无显色， 表示待检样本尿液中含 HCG浓度为 25-125mIU/ml; T T 2显色 ， 其它 Τ无显色， 表示待检样本尿液中含 HCG浓度为 125-500mIU/ml; Τ Τ 2 、 Τ 3显色， 1\无显色， 表示待检样本尿液中含 HCG浓度为 500-2500mIU/ml; T 全部显色， 则表示待检样本尿液中含 HCG浓度为高于 2500mIU/ml。 [0081] taking urine droplets of the sample to be inspected containing HCG, and adding it to the sample pad 4, and reading the result for 5 minutes, color development, It has no coloration at point T, indicating that the concentration of HCG in the urine of the sample to be tested is 25-125 mIU/ml; TT 2 develops color, and other sputum has no coloration, indicating that the concentration of HCG in the urine of the sample to be tested is 125-500 mIU/ Ml; Τ Τ 2 , Τ 3 color, 1\ no color, indicating that the concentration of HCG in the urine of the sample to be tested is 500-2500 mIU/ml; T all color development, indicating the concentration of HCG in the urine of the sample to be tested It is above 2500 mIU/ml.
[0082] 实施例 2 Example 2
[0083] 本发明的免疫层析检测试纸的制备实施例 2， 制备及检测尿微量白蛋白、 尿 2 微球蛋白、 抑胱素 C三项联合检测试纸 （胶体金免疫层析法） 。 [0083] The preparation example 2 of the immunochromatographic test strip of the present invention prepares and detects three combined test strips of urine microalbumin, urine 2 microglobulin and cystatin C (colloidal gold immunochromatography).
[0084] 生物活性原料： 人白蛋白单克隆抗体、 鼠抗人白蛋白单克隆抗体、 Cys-C (抑 胱素 C) 单克隆抗体、 鼠抗人 Cys-C (抑胱素 C) 抗体、 2微球蛋白单克隆抗体、 鼠抗人 2微球蛋白抗体、 IgG多克隆抗体均来自市场购买。 Bioactive raw materials: human albumin monoclonal antibody, mouse anti-human albumin monoclonal antibody, Cys-C (cytosine C) monoclonal antibody, mouse anti-human Cys-C (cytosine C) antibody, 2 microglobulin monoclonal antibodies, murine anti-human 2 microglobulin antibodies, and IgG polyclonal antibodies were purchased from the market.
[0085] 试剂： 牛血清白蛋白来自市场购买。  Reagents: Bovine serum albumin was purchased from the market.
[0086] 硝酸纤维素膜来自市场购买。  [0086] The nitrocellulose membrane is commercially available.
[0087] 试纸制备程序如下：  [0087] The test paper preparation procedure is as follows:
[0088] 步骤 1.反应膜 2包被  Step 1. Reaction film 2 coating
[0089] 根据待检样本中的待检目标物确定包被抗体种类： 人白蛋白单克隆抗体、 Cys- C (抑胱素 C) 单克隆抗体、 2微球蛋白单克隆抗体、 IgG多克隆抗体。 [0089] The coated antibody species is determined according to the target to be detected in the sample to be tested: human albumin monoclonal antibody, Cys-C (cytosine C) monoclonal antibody, 2 microglobulin monoclonal antibody, IgG polyclonal antibody.
[0090] 根据待检样本中的待检目标物设定检测点 12及质控点 13的浓度和数量： 检测点 12设置 3个点， 每个点为一种检测抗体； 质控点 13设置 2个点， 1个浓度。  [0090] setting the concentration and quantity of the detection point 12 and the quality control point 13 according to the object to be inspected in the sample to be inspected: the detection point 12 is set with 3 points, each point is a detection antibody; the quality control point 13 is set 2 points, 1 concentration.
[0091] 将 2.5mg/ml的人白蛋白单克隆抗体、 2.26mg/m 2 2.5 mg/ml human albumin monoclonal antibody, 2.26 mg/m 2
微球蛋白单克隆抗体、 2.42mg/ml的 Cys-C (抑胱素 C) 单克隆抗体、 8.02mg/ml的 IgG多克隆抗体根据附图 4的图形包被于硝酸纤维素膜上， 干燥 24h， 温度为 37°C ， 湿度≤40<¾RH。  Microglobulin monoclonal antibody, 2.42 mg/ml Cys-C (cystatin C) monoclonal antibody, 8.02 mg/ml IgG polyclonal antibody were coated on a nitrocellulose membrane according to the pattern of Fig. 4, dried 24h, temperature is 37 ° C, humidity ≤ 40 < 3⁄4 RH.
[0092] 步骤 2.结合物释放垫 5制备  Step 2. Preparation of the conjugate release pad 5
[0093] 尿微量白蛋白金标结合物释放垫制备  Preparation of urine microalbumin gold standard conjugate release pad
[0094] 取 40nm的胶体金溶液， 按 1.2%比例加入 0.2M的 K 2CO 3 (碳酸钾） 调节 PH值， 按 12 g/ml比例加入鼠抗人白蛋白单克隆抗体， 制成标记物， 放置室温 5min， 按 0 .8%比例加入 BSA (牛血清白蛋白） 稳定剂。 [0094] Take a 40 nm colloidal gold solution, add 0.2 M K 2 CO 3 (potassium carbonate) in a ratio of 1.2% to adjust the pH value, and add a mouse anti-human albumin monoclonal antibody in a ratio of 12 g/ml to prepare a label. , place at room temperature for 5 min, add BSA (bovine serum albumin) stabilizer at a ratio of 0.8%.
[0095] 4500r离心 30min， 取浓缩沉淀物； 将上清液继续 6500r离心 45min， 取浓缩沉淀 物； 将上清液继续 9000r离心 60min， 取浓缩沉淀物； 将三次离心浓缩沉淀物混合 均匀。 [0095] Centrifuge at 4500r for 30 min, and concentrate the precipitate; the supernatant was centrifuged at 6500 rpm for 45 min to obtain a concentrated precipitate. The supernatant was centrifuged at 9000 rpm for 60 min to obtain a concentrated precipitate; the precipitate was concentrated by centrifugation three times.
[0096] 将离心好的浓缩液稀释好， 铺于 90cmx30cm的无纺布上， 加液量 10- 11ml/张， 干燥 24h， 温度 37°C， 湿度≤40<¾RH。  [0096] The centrifuged concentrate was diluted and spread on a 90 cm x 30 cm nonwoven fabric, the amount of liquid added was 10-11 ml/sheet, dried for 24 hours, the temperature was 37 ° C, and the humidity was ≤ 40 < 3⁄4 RH.
[0097] 尿 β 2微球蛋白金标结合物释放垫制备 Preparation of urinary β 2 microglobulin gold standard conjugate release pad
[0098] 取 40nm的胶体金溶液， 按 1.2%比例加入 0.2M的 K 2CO 3 (碳酸钾） 调节 PH值， 按 l(Vg/ml比例加入鼠抗人 β 2 [0098] Take a 40 nm colloidal gold solution, add 0.2 M K 2 CO 3 (potassium carbonate) in a ratio of 1.2% to adjust the pH value, and add 1 (Vg/ml ratio to the mouse anti-human β 2
微球蛋白抗体， 制成标记物， 放置室温 5min， 按 0.8%比例加入 BSA (牛血清白 蛋白） 稳定剂。  The microglobulin antibody was prepared as a marker and placed at room temperature for 5 min, and a BSA (bovine serum albumin) stabilizer was added in a ratio of 0.8%.
[0099] 4500r离心 30min， 取浓缩沉淀物； 将上清液继续 6500r离心 45min， 取浓缩沉淀 物； 将上清液继续 9000r离心 60min， 取浓缩沉淀物； 将三次离心浓缩沉淀物混合 均匀。  [0099] After centrifugation at 4500r for 30 min, the concentrated precipitate was taken; the supernatant was centrifuged at 6500 rpm for 45 min to obtain a concentrated precipitate; the supernatant was further centrifuged at 9000 rpm for 60 min to obtain a concentrated precipitate; and the precipitate was concentrated by centrifugation three times.
[0100] 将离心好的浓缩液按规定浓度稀释好， 铺于 90cmx30cm的无纺布上， 加液量 10 [0100] The centrifuged concentrate is diluted to a predetermined concentration, and spread on a 90 cm x 30 cm nonwoven fabric, and the amount of liquid added is 10
-11ml/张， 干燥 24h， 温度 37°C， 湿度≤40<¾RH。 -11ml/sheet, dry for 24h, temperature 37°C, humidity ≤40<3⁄4RH.
[0101] 抑胱素 C金标结合物释放垫制备 Preparation of Cystatin C Gold Label Conjugate Release Pad
[0102] 取 40nm的胶体金溶液， 按 1.2%比例加入 0.2M的 K 2CO 3 (碳酸钾） 调节 PH值， 按 l l g/ml比例加入鼠抗人 Cys-C (抑胱素 C) 抗体， 制成标记物， 放置室温 5min[0102] Take a 40 nm colloidal gold solution, add 0.2 M K 2 CO 3 (potassium carbonate) in a ratio of 1.2% to adjust the pH value, and add a mouse anti-human Cys-C (cytosine C) antibody in a ratio of llg/ml. Made into a marker, placed at room temperature for 5 min
， 按 0.8%比例加入 BSA (牛血清白蛋白） 稳定剂。 , BSA (bovine serum albumin) stabilizer was added in a ratio of 0.8%.
[0103] 4500r离心 30min， 取浓缩沉淀物； 将上清液继续 6500r离心 45min， 取浓缩沉淀 物； 将上清液继续 9000r离心 60min， 取浓缩沉淀物； 将三次离心浓缩沉淀物混合 均匀。 [0103] After centrifugation at 4500r for 30 min, the concentrated precipitate was taken; the supernatant was centrifuged at 6500 rpm for 45 min to obtain a concentrated precipitate; the supernatant was further centrifuged at 9000 rpm for 60 min to obtain a concentrated precipitate; and the precipitate was concentrated by centrifugation three times.
[0104] 将离心好的浓缩液按规定浓度稀释好， 铺于 90cmx30cm的无纺布上， 加液量 10 [0104] The centrifuged concentrate is diluted according to the specified concentration, and spread on a 90 cm x 30 cm nonwoven fabric, and the amount of liquid added is 10
-11ml/张， 干燥 24h， 温度 37°C， 湿度≤40<¾RH。 -11ml/sheet, dry for 24h, temperature 37°C, humidity ≤40<3⁄4RH.
[0105] 步骤 3.样本垫 4的制备 [0105] Step 3. Preparation of sample pad 4
[0106] 将切割成 83.5cmx30cm的玻璃纤维和无纺布用包含 0.5<¾NaCl (氯化钾） 、 0.5% 蔗糖、 0.1%BSA (牛血清白蛋白） 和 Tris- HC1缓冲液 (三羟甲基氨基甲烷-盐酸) (pH7.4)  [0106] The glass fiber and non-woven fabric which are cut into 83.5 cm×30 cm are used to contain 0.5<3⁄4 NaCl (potassium chloride), 0.5% sucrose, 0.1% BSA (bovine serum albumin) and Tris-HCl buffer (trimethylol). Aminomethane-hydrochloric acid) (pH 7.4)
工作液浸泡。 在 37°C将所述垫干燥 24h， 获得样本垫 4。 [0107] 步骤 4.组装试纸 Soak the working solution. The pad was dried at 37 ° C for 24 h to obtain a sample pad 4. [0107] Step 4. Assembling the test paper
[0108] 将底板 1放在操作台上， 贴上双面胶。  [0108] The bottom plate 1 is placed on the operation table, and double-sided tape is attached.
[0109] 将包被好的反应膜 2贴在底板 1上。 [0109] The coated reaction film 2 is attached to the substrate 1.
[0110] 将吸收垫 3压反应膜 2的 2mm贴上， 另一端与底板 1对齐。  [0110] The absorbent pad 3 was pressed 2 mm of the reaction film 2, and the other end was aligned with the bottom plate 1.
[0111] 将一层无纺布压反应膜 2的 2mm处贴上。 [0111] A layer of the nonwoven fabric pressure reaction film 2 was attached to 2 mm.
[0112] 将尿微量白蛋白结合物释放垫、 尿 2微球蛋白结合物释放垫、 抑胱素 C结合物 释放垫依次齐压于无纺布上面。 [0112] The urinary microalbumin conjugate release pad, the urinary 2 microglobulin conjugate release pad, and the cystatin C conjugate release pad are sequentially pressed onto the nonwoven fabric.
[0113] 将玻璃纤维压于结合物释放垫 5—半位置， 另一端与底板 1对齐。 [0113] The glass fiber is pressed against the conjugate release pad 5 - half position, and the other end is aligned with the bottom plate 1.
[0114] 再将一层无纺布一端与结合物释放垫 5上端对齐， 另一端与底板 1对齐。 [0114] One end of a nonwoven fabric is aligned with the upper end of the bond release pad 5, and the other end is aligned with the bottom plate 1.
[0115] 步骤 5.产品检测 [0115] Step 5. Product Inspection
[0116] 取待检样本尿液样本垫 4上， 8min吋， 判读结果， 显色， 表示待检样本尿液 中尿微量白蛋白为阳性； T 2显色， 表示待检样本尿液中含尿 2微球蛋白为阳性 ； τ 3显色， 表示待检样本尿液中含抑胱素 C为阳性。 [0116] taking the urine sample pad 4 of the sample to be inspected, 8 min 吋, the interpretation result, color development, indicating that the urine microalbumin in the urine of the sample to be tested is positive; T 2 coloring, indicating that the sample to be tested contains urine Urinary 2 microglobulin is positive; τ 3 is colored, indicating that the urine of the sample to be tested contains positive for cystatin C.
[0117] 以上所述仅为本发明的实施例， 并不限制本发明， 凡在本发明基础上， 所作的 任何修改、 改进等， 均应包含在发明的保护范围之内。  The above description is only the embodiment of the present invention, and the present invention is not limited thereto, and any modifications, improvements, etc. made on the basis of the present invention should be included in the scope of the invention.

Claims

权利要求书 一种免疫层析检测方法， 用于实现对待检样本的定性、 半定量或 定量检测； 其特征在于： 采用如下步骤： Claims an immunochromatographic detection method, used to achieve qualitative, semi-quantitative or quantitative detection of samples to be tested; It is characterized by: adopting the following steps:
(1)反应膜包被： 根据待检样本中的待检目标物确定包被抗体或抗 原的种类， 设定检测点及质控点的浓度和数量； (1) Reactive membrane coating: Determine the type of coating antibody or antigen based on the target substance to be tested in the sample to be tested, and set the concentration and quantity of the detection points and quality control points;
(2)根据待检样本中的待检目标物制备结合物释放垫； (2) Prepare the conjugate release pad according to the target substance to be detected in the sample to be detected;
(3)制备样本垫； (3) Prepare sample pad;
(4)组装试纸： 试纸由底板， 及依次设置在底板上的样本垫、 结合 物释放垫、 反应膜、 吸收垫构成； (4) Assemble the test paper: The test paper consists of a base plate, and a sample pad, a conjugate release pad, a reaction membrane, and an absorption pad that are arranged on the base plate in sequence;
(5)对待检样本进行检测： 在试纸样本垫中加入待检样本， 通过肉 眼观察多个检测点和质控点的显色情况可以对待检样本进行定性 或者半定量检测， 或采用具有信号检测功能的仪器采集试纸上检 测点和质控点的特征信号可以进行定量检测； 或者首先根据多个 检测点的显色情况确定待检样本中的待检目标物， 再通过肉眼观 察多个检测点和质控点的显色情况可以对待检样本中不同的待检 目标物进行定性或者半定量检测， 或采用具有信号检测功能的仪 器采集试纸上检测点和质控点的特征信号可以进行定量检测。 根据权利要求 1所述的免疫层析检测方法， 其特征在于： 反应膜分 成两个区域， 一个是与结合物释放垫相邻接的检测区， 一个是与 吸收垫相邻接的质控区； 检测区设有 2至 30个检测点， 检测点不包 括仅有条带图形状， 检测点为圆点状、 多边形状、 其它图形状或 不同图形状的组合， 亦可为条带图形状与其它图形状的组合； 检 测点的设定可以是同浓度或者不同浓度的同一种抗体或抗原， 也 可以是同浓度或者不同浓度的多种抗体或抗原； 所述质控区设有 1 至 30个质控点， 质控点为圆点状、 多边形状、 条带图形状、 其它 图形状或不同图形状的组合； 质控点包被有可与结合物释放垫上 结合物结合的抗体或抗原。 (5) Test the sample to be tested: Add the sample to be tested into the test paper sample pad, and visually observe the color development of multiple testing points and quality control points to conduct qualitative or semi-quantitative testing of the sample to be tested, or use signal detection The functional instrument collects the characteristic signals of the detection points and quality control points on the test paper for quantitative detection; or first determines the target object to be detected in the sample to be tested based on the color development of multiple detection points, and then observes the multiple detection points with the naked eye. The color development of the test paper and the quality control point can be used for qualitative or semi-quantitative detection of different targets in the sample to be tested, or an instrument with a signal detection function can be used to collect the characteristic signals of the test point and quality control point on the test paper for quantitative detection. . The immunochromatographic detection method according to claim 1, characterized in that: the reaction membrane is divided into two areas, one is the detection area adjacent to the conjugate release pad, and the other is the quality control area adjacent to the absorption pad. ; The detection area is equipped with 2 to 30 detection points. The detection points do not include only strip chart shapes. The detection points are dot-shaped, polygonal, other graphic shapes or a combination of different graphic shapes. They can also be strip chart shapes. Combination with other graph shapes; The setting of the detection point can be the same antibody or antigen at the same concentration or different concentrations, or multiple antibodies or antigens at the same concentration or different concentrations; The quality control area is provided with 1 to 30 quality control points, the quality control points are in the shape of dots, polygons, strip diagrams, other diagram shapes or a combination of different diagram shapes; the quality control points are coated with antibodies that can bind to the conjugate on the conjugate release pad or antigen.
根据权利要求 1所述的免疫层析检测方法， 其特征在于： 所述结合 物释放垫上标记有单一待检目标物相关的特异性抗体或抗原， 或 标记有各待检目标物相关的多个特异性抗体或抗原。 The immunochromatographic detection method according to claim 1, characterized in that: the combination The substance release pad is marked with a specific antibody or antigen related to a single target object to be detected, or multiple specific antibodies or antigens related to each target object to be detected.
[权利要求 4] 根据权利要求 1所述的免疫层析检测方法， 其特征在于： 所述标记 方法包括量子点标记法、 胶体金标记法、 胶体硒标记法、 有色或 荧光乳胶标记法、 磁性颗粒标记法、 吋间分辨层析法、 化学发光 法。 [Claim 4] The immunochromatographic detection method according to claim 1, characterized in that: the labeling method includes quantum dot labeling, colloidal gold labeling, colloidal selenium labeling, colored or fluorescent latex labeling, magnetic Particle labeling method, time resolution chromatography method, chemiluminescence method.
[权利要求 5] 根据权利要求 1所述的免疫层析检测方法， 其特征在于： 所述方法 包括双抗体夹心法、 间接法、 竞争抑制法。 [Claim 5] The immunochromatographic detection method according to claim 1, characterized in that: the method includes a double-antibody sandwich method, an indirect method, and a competitive inhibition method.
[权利要求 6] 根据权利要求 1所述的免疫层析检测方法， 其特征在于： 所述待检 样本是来自临床或非临床的血液、 体液、 尿液、 唾液、 生殖道分 泌液或其他液态样品或粘稠状样品。 [Claim 6] The immunochromatographic detection method according to claim 1, characterized in that: the sample to be tested is from clinical or non-clinical blood, body fluids, urine, saliva, reproductive tract secretions or other liquids sample or viscous sample.
[权利要求 7] 根据权利要求 1所述的免疫层析检测试纸， 其特征在于： 用于实现 对待检样本的定性、 半定量或定量检测； 试纸由底板， 及依次设 置在底板上的样本垫、 结合物释放垫、 反应膜、 吸收垫构成。 [Claim 7] The immunochromatographic detection test paper according to claim 1, characterized in that: used to achieve qualitative, semi-quantitative or quantitative detection of the sample to be tested; the test paper consists of a bottom plate and a sample pad sequentially arranged on the bottom plate , conjugate release pad, reaction membrane, and absorption pad.
[权利要求 8] 根据权利要求 1所述的免疫层析检测试纸， 其特征在于： 所述底板 材料是 PVC(聚氯乙烯)板， 反应膜材料是硝酸纤维素膜或醋酸纤 维素膜， 吸收垫的材料是滤纸， 样品垫材料为玻璃纤维和无纺布[Claim 8] The immunochromatographic detection test paper according to claim 1, characterized in that: the bottom plate material is a PVC (polyvinyl chloride) plate, the reaction membrane material is a nitrocellulose membrane or a cellulose acetate membrane, and the absorption The pad material is filter paper, and the sample pad material is glass fiber and non-woven fabric.
， 结合物释放垫材料是玻璃纤维或无纺布。 , the conjugate release pad material is fiberglass or non-woven fabric.
[权利要求 9] 根据权利要求 1-8所述的免疫层析检测试纸， 其特征在于： 试纸还 可装配在与试纸的尺寸、 样本加入的方式和位置、 抗体或抗原在 反应膜上固化的位置相吻合的塑料卡内成为试纸盒。 [Claim 9] The immunochromatographic detection test paper according to claims 1-8, characterized in that: the test paper can also be assembled with the size of the test paper, the way and position of adding the sample, and the solidification of the antibody or antigen on the reaction membrane. The plastic card with matching position becomes the test strip box.
[权利要求 10] —种免疫层析检测方法的成套试纸盒， 其特征在于： 包括待检样 本稀释液和上述权利要求 1-9所述试纸和试纸盒。 [Claim 10] A complete set of test paper boxes for an immunochromatographic detection method, characterized by: including a diluent of the sample to be tested and the test paper and test paper box described in claims 1-9.
PCT/CN2015/072273 2014-05-14 2015-02-05 Immunochromatographic test method and test paper WO2015184847A1 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
CN201410202928 2014-05-14
CN201410244152.6A CN104764877A (en) 2014-05-14 2014-06-04 Immunity chromatography test method and test paper
CN201410244152.6 2014-06-04

Publications (1)

Publication Number Publication Date
WO2015184847A1 true WO2015184847A1 (en) 2015-12-10

Family

ID=53646838

Family Applications (3)

Application Number Title Priority Date Filing Date
PCT/CN2015/072273 WO2015184847A1 (en) 2014-05-14 2015-02-05 Immunochromatographic test method and test paper
PCT/CN2015/078601 WO2015172689A1 (en) 2014-05-14 2015-05-08 Immuno-chromatographic test strip and testing method therefor
PCT/CN2015/078600 WO2015172688A1 (en) 2014-05-14 2015-05-08 Human chorionic gonadotropin immuno-chromatographic testing strip and preparation method therefor

Family Applications After (2)

Application Number Title Priority Date Filing Date
PCT/CN2015/078601 WO2015172689A1 (en) 2014-05-14 2015-05-08 Immuno-chromatographic test strip and testing method therefor
PCT/CN2015/078600 WO2015172688A1 (en) 2014-05-14 2015-05-08 Human chorionic gonadotropin immuno-chromatographic testing strip and preparation method therefor

Country Status (2)

Country Link
CN (3) CN104764877A (en)
WO (3) WO2015184847A1 (en)

Families Citing this family (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104749358A (en) * 2014-05-14 2015-07-01 陈岩松 Semiquantitative immunochromatographic test method and test paper
CN106290925A (en) * 2016-07-31 2017-01-04 江翠珍 Immunochromatography detection method and immunochromatographytest test kit
CN106290924A (en) * 2016-07-31 2017-01-04 江翠珍 A kind of immunochromatographiassays assays test kit
CN106443012B (en) * 2016-09-12 2018-11-06 三诺生物传感股份有限公司 A kind of test strips and preparation method thereof and the application in microdose urine protein and β2-microglobulin joint-detection
CN106896231A (en) * 2017-04-13 2017-06-27 余凌泰 A kind of HCG Test papers that HCG concentration is detected using two response lines
CN107402302A (en) * 2017-08-06 2017-11-28 潘荣兰 A kind of environmental monitoring prokaryotes quantity rapid evaluation chip
CN107290547A (en) * 2017-08-07 2017-10-24 广州市微米生物科技有限公司 A kind of infertile quick detection kit and preparation method thereof
CN107367605A (en) * 2017-08-17 2017-11-21 武汉璟泓万方堂医药科技股份有限公司 Collaurum/fluorescent test paper chip
CN110785116B (en) * 2017-09-07 2023-07-18 普默特株式会社 Chromatographic bands with multiple test lines, diagnostic kit comprising same and qualitative, semi-quantitative and quantitative analysis method comprising multiple competition reaction determination steps
CN109444413B (en) * 2018-09-26 2019-08-27 深圳市疾病预防控制中心（深圳市卫生检验中心、深圳市预防医学研究所） A kind of detection device
CN111521769A (en) * 2019-02-01 2020-08-11 湖南达道生物工程有限公司 Biological sample detection method and device
CN112730855A (en) * 2020-12-28 2021-04-30 杭州新脉生物科技有限公司 Colloidal gold chromatography test strip based on anti-human beta-HCG bispecific antibody
CN117186205A (en) * 2022-05-18 2023-12-08 江苏尤里卡生物科技有限公司 Preparation method of human chorionic gonadotrophin

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
CN102707061A (en) * 2012-06-18 2012-10-03 深圳市博卡生物技术有限公司 Gold immunochromatographic assay quantitative detection test paper and detection method
CN102879583A (en) * 2012-09-21 2013-01-16 田伏洲 Test card for rapidly diagnosing pancreatic trauma and acute pancreatitis and preparation method thereof
CN103620408A (en) * 2011-01-18 2014-03-05 Symbolics有限责任公司 Lateral flow assays using two dimensional features
CN204065099U (en) * 2014-06-11 2014-12-31 陈岩松 A kind of immunity chromatography detection test paper and complete paper box

Family Cites Families (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU3942993A (en) * 1992-03-31 1993-11-08 Abbott Laboratories Method of multiplex ligase chain reaction
US5968839A (en) * 1996-05-13 1999-10-19 Metrika, Inc. Method and device producing a predetermined distribution of detectable change in assays
CN1141580C (en) * 2001-02-27 2004-03-10 周思亮 Rapid joint assay method for viral antibodies of AIDS and hepatitis C
US7939342B2 (en) * 2005-03-30 2011-05-10 Kimberly-Clark Worldwide, Inc. Diagnostic test kits employing an internal calibration system
CN201035010Y (en) * 2007-04-20 2008-03-12 北京万泰生物药业有限公司 Vertical type immune chromatography test paper strip
CN201298042Y (en) * 2008-08-28 2009-08-26 河南省农业科学院 Test strip for detecting porcine parvovirus and Japanese encephalitis antiviral antibody
CN101825640A (en) * 2010-03-25 2010-09-08 沈鹤柏 Qualitative and quantitative detection dual-purpose HCG test paper for colloidal gold immunochromatography assay
CN101900728A (en) * 2010-08-05 2010-12-01 中国农业科学院油料作物研究所 Multi-test-line immunochromatographic test strip for semi-quantitatively detecting aflatoxin B1 and preparation method thereof
CN102539751A (en) * 2010-12-09 2012-07-04 苏州生物医学工程技术研究所 Immunofluorescence test paper strip and quantitative detection method thereof
CN102174109A (en) * 2011-01-21 2011-09-07 中华人民共和国吉林出入境检验检疫局 Acquired immune deficiency syndrome virus and treponema pallidum fusion protein as well as preparation method and application thereof
CN102253222B (en) * 2011-04-21 2014-07-16 中国农业科学院农业质量标准与检测技术研究所 Method and special test paper for estrogen detection
CN104749358A (en) * 2014-05-14 2015-07-01 陈岩松 Semiquantitative immunochromatographic test method and test paper

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
CN103620408A (en) * 2011-01-18 2014-03-05 Symbolics有限责任公司 Lateral flow assays using two dimensional features
CN102707061A (en) * 2012-06-18 2012-10-03 深圳市博卡生物技术有限公司 Gold immunochromatographic assay quantitative detection test paper and detection method
CN102879583A (en) * 2012-09-21 2013-01-16 田伏洲 Test card for rapidly diagnosing pancreatic trauma and acute pancreatitis and preparation method thereof
CN204065099U (en) * 2014-06-11 2014-12-31 陈岩松 A kind of immunity chromatography detection test paper and complete paper box

Also Published As

Publication number Publication date
CN104764877A (en) 2015-07-08
WO2015172689A1 (en) 2015-11-19
CN104820104A (en) 2015-08-05
CN104820094A (en) 2015-08-05
WO2015172688A1 (en) 2015-11-19

Similar Documents

Publication Publication Date Title
WO2015184847A1 (en) Immunochromatographic test method and test paper
WO2015188633A1 (en) Immunochromatography detection method and test paper
JP4846573B2 (en) Lateral flow assay device and method with natural analyte as reference
KR102322094B1 (en) Method and device for combined detection of viral and bacterial infections
JP2000321278A (en) Improved immunochromatographic analysis
WO2015080286A1 (en) Immunochromatography-assisted detection method
JP7267211B2 (en) Sandwich-type assays using the decreasing signal portion of the dose-response curve to measure analytes, such as high-concentration analytes
JP4286157B2 (en) Membrane assay
WO2007061098A1 (en) Analyzer and analysis method
WO2005062052A1 (en) Simple membrane assay method and kit
US20090203151A1 (en) Immunoassay method
JP7451403B2 (en) Lateral flow assays and methods for detecting high concentrations of analytes
JP3848599B2 (en) Simple membrane assay and kit
CN106526166A (en) Rapid detection of lean meat powder in pork
CN104749358A (en) Semiquantitative immunochromatographic test method and test paper
JP4365329B2 (en) Simple assay device and method for detecting a plurality of objects to be detected
CN204065099U (en) A kind of immunity chromatography detection test paper and complete paper box
CN104749359A (en) Immunochromatographic test method and test paper for multi-item joint tests
JP6405339B2 (en) Simple membrane assay and kit
JP2002122599A (en) Immunoassay
JP5911404B2 (en) Simple membrane assay and kit
JP2009002961A (en) Easy membrane assay method and kit
CN204462151U (en) A kind of immunity chromatography detection test paper
JP6405269B2 (en) Simple membrane assay and kit
JP2024057619A (en) A multiplex lateral flow assay for distinguishing viral and bacterial infections

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 15802376

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 15802376

Country of ref document: EP

Kind code of ref document: A1