CN114778831A - Method for manufacturing antigen detection colloidal gold segmented semi-quantitative detection test paper based on double-antibody sandwich method - Google Patents
Method for manufacturing antigen detection colloidal gold segmented semi-quantitative detection test paper based on double-antibody sandwich method Download PDFInfo
- Publication number
- CN114778831A CN114778831A CN202210490629.3A CN202210490629A CN114778831A CN 114778831 A CN114778831 A CN 114778831A CN 202210490629 A CN202210490629 A CN 202210490629A CN 114778831 A CN114778831 A CN 114778831A
- Authority
- CN
- China
- Prior art keywords
- detection
- colloidal gold
- control line
- antibody
- test paper
- Prior art date
- Legal status (The legal status 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 status listed.)
- Pending
Links
Images
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
- G01N33/558—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
- G01N33/53—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
- 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
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56966—Animal cells
-
- 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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
- G01N33/587—Nanoparticles
Abstract
The invention provides a method for manufacturing antigen detection colloidal gold segmented semi-quantitative detection test paper based on a double-antibody sandwich method, and relates to the technical field of colloidal gold detection. The invention develops the colloidal gold immunochromatography into semi-quantitative multi-segment detection which can be directly read by naked eyes, and retains the advantages of the traditional colloidal gold test paper. The purpose of semi-quantitative detection is achieved through the detection means of a plurality of detection lines, and a novel colloidal gold test paper detection tool capable of displaying the concentration range of a detected object in a segmented manner is prepared, is suitable for early disease screening and dynamic assessment, and can be applied to other non-medical scenes, such as pesticide detection, food safety detection and water substance detection. Taking medicine as an example, the sectional color reaction result of the prepared test paper after dripping one drop of blood of a patient can be used for quickly detecting serological indexes, so that the early screening of diseases and the dynamic evaluation of the risk and the severity of certain clinical high-risk special diseases are facilitated, and dangerous people are defined.
Description
Technical Field
The invention belongs to the technical field of colloidal gold detection, and particularly relates to a method for manufacturing antigen detection colloidal gold segmented semi-quantitative detection test paper based on a double-antibody sandwich method.
Background
The Colloidal Gold Immunochromatography (GICA) technology is a rapid detection technology developed on the basis of a Colloidal gold labeling technology, an immunological technology and a chromatography technology, and is widely used for qualitative screening due to convenience and timeliness, but due to the limitation of qualitative screening, the Colloidal gold test paper cannot quantitatively evaluate the severity of critical diseases in a stepped manner, so that the application range of the Colloidal gold test paper is only limited to detect the existence of serological indexes, and the concentration and the amount of biological indexes of diseases cannot be directly and dynamically monitored.
The basic principle of the colloidal gold immunochromatography technology is that a nitrocellulose membrane is used as a carrier, a liquid sample dripped is slowly permeated from one end of a membrane strip to the other end by utilizing the capillary action of a microporous membrane, and the antigen-antibody combination reaction in the test paper is displayed by the aggregation and color development of the colloidal gold. In the traditional colloidal gold method, when the detection line and the quality control line show reddish purple at the same time, the result is effective, and the concentration interval is judged according to the display number of T lines; if the quality control line does not develop color, the test paper result is invalid. The traditional method can only carry out qualitative judgment on the existence of the antigen antibody and can not carry out quantitative judgment on a detection substance.
Disclosure of Invention
In view of the above, the present invention provides a method for manufacturing a gold test paper for antigen detection based on a double-antibody sandwich method, which is suitable for semi-quantitative detection of macromolecular antigens and cells, and the manufactured novel gold test paper detection tool capable of displaying the concentration range of a detected object in a segmented manner is suitable for early disease screening and dynamic assessment, and can also be applied in other non-medical scenes, such as pesticide detection, food safety detection and water substance detection. Taking medicine as an example, the sectional color reaction result of the prepared test paper after dripping one drop of blood of a patient can be used for quickly detecting serological indexes, so that the early screening of diseases and the dynamic evaluation of the risk and the severity of certain clinical high-risk special diseases are facilitated, and dangerous people are defined.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides an antigen detection colloidal gold test strip based on a double-antibody sandwich, which comprises a sample pad, a colloidal gold pad coated with anti-Ab 1, a nitrocellulose membrane coated with anti-Ab 2, a nitrocellulose membrane coated with a secondary antibody and water absorption filter paper, wherein the sample pad, the colloidal gold pad, the nitrocellulose membrane and the water absorption filter paper are sequentially adhered to a PVC (polyvinyl chloride) plate;
the coated nitrocellulose membrane comprises a plurality of detection lines and a quality control line;
each detection line is coated with primary anti-Ab 2 with the same concentration or different concentrations;
and secondary antibodies are coated on the quality control line C.
The secondary antibody is capable of binding to primary anti Ab 1; in which primary anti-Ab 1 and primary anti-Ab 2 are different antibody determinants for the same antigen.
The center of the PVC bottom plate is adhered with the coated nitrocellulose membrane, the lower edge of the nitrocellulose membrane is adhered with absorbent filter paper, the upper edge of the nitrocellulose membrane is adhered with an antibody colloidal gold pad, and the upper edge of the colloidal gold pad is adhered with a sample pad.
The amount of primary anti Ab2 coated on each of the test lines corresponds to the corresponding fractional test concentration.
The antigens include macromolecular antigens and cells, and the following experimental procedure is exemplified by CD4+ T cells (CD4 refers to differentiation determinant antigen 4, and CD4+ T cells refers to T lymphocytes with CD4 molecules on the surface).
3 detection lines are arranged on the nitrocellulose membrane on the colloidal gold test strip; when the antigen is CD4+ T cells, 2.0 muL of anti-CD 4+ T cell antibody Ab2 solution of 1-4 mg/mL is sprayed on the detection line T1, 2.0 muL of anti-CD 4+ T cell antibody Ab2 solution of 1-4 mg/mL is sprayed on the detection line T2, and 4.0 muL of anti-CD 4+ T cell antibody Ab2 solution of 1-4 mg/mL is sprayed on the detection line T3 on 3 detection lines.
When the antigen was CD4+ T cells, the concentration of CD4+ T cells was (0, 12.5X 10) when only the control line C appeared reddish purple6/L]When the control line C and T1 are simultaneously purple, the concentration range of CD4+ T cells is (12.5X 10)6/L，25×106/L](ii) a When the quality control line C, T1 and T2 are simultaneously reddish purple, the concentration interval of CD4+ T cells is (25X 10)6/L，50×106/L](ii) a When the control line C and three T lines are purple, the concentration interval of CD4+ T cells is (50X 10)6L, + ∞); the test results were invalid when the control line C did not develop color.
Has the beneficial effects that: the invention provides a method for manufacturing antigen detection colloidal gold subsection semi-quantitative test paper based on a double-antibody sandwich method, which reserves the advantages of the traditional colloidal gold test paper in multiple scenes such as portability, practicability, rapid detection, suitability for hospitals, disease control, basic health unit detection, family self-test and the like, and suitability for popularization and the like. The prepared novel colloidal gold test paper detection tool capable of displaying the concentration range of the detected object in a segmented manner realizes that the colloidal gold test paper can detect the concentration interval of serological substances for the first time, and the infection state of a patient can be obtained by corresponding to a clinical risk system of related diseases. Is expected to expand the disease species range of early screening by using the colloidal gold test paper, and can assist a clinician to make a treatment scheme in time and improve the prognosis of a patient. Meanwhile, the method can also be applied to other non-medical scenes, such as pesticide detection, food safety detection and water substance detection.
Drawings
FIG. 1 is a structural diagram of the colloidal gold test strip of the present invention;
FIG. 2 is a schematic diagram of the detection result of the colloidal gold test strip of the present invention.
Detailed Description
The invention provides a method for manufacturing antigen detection colloidal gold subsection semi-quantitative detection test paper based on a double-antibody sandwich method, wherein the colloidal gold test paper comprises a sample pad, an anti-Ab 1 coated colloidal gold pad, an anti-Ab 2 coated nitrocellulose membrane, a secondary antibody coated nitrocellulose membrane and absorbent filter paper, which are sequentially adhered to a PVC plate;
the coated nitrocellulose membrane comprises a plurality of detection lines and a quality control line;
each detection line is coated with primary anti-Ab 2 with the same concentration or different concentrations;
coating a secondary antibody on the quality control line; in which primary anti-Ab 1 and primary anti-Ab 2 are different antibody determinants for the same antigen.
The primary anti-Ab 1 and the primary anti-Ab 2 are different monoclonal antibodies generated against the same antigen, and the secondary antibodies and the primary anti-Ab 1 can bind.
The structure of the colloidal gold test strip is preferably as shown in figure 1, a coated nitrocellulose membrane is preferably adhered to the center of the PVC base plate, absorbent filter paper is adhered to the upper edge of the nitrocellulose membrane, an antibody colloidal gold pad is adhered to the upper edge of the nitrocellulose membrane, and a sample pad is adhered to the upper edge of the colloidal gold pad.
In the invention, when a sample is dripped on a colloidal gold test strip, the sample sequentially passes through a colloidal gold binding pad, a detection line T1, a detection line T2 … … detection line Tn and a quality control line C, wherein the detection lines T1-Tn respectively represent biomarkers with different concentration gradients; in the case of CD4+ T cells, when the antigen is a CD4+ T cell, the primary antibody is preferably derived from murine sources: anti-CD 4+ T cell antibody Ab1 and anti-CD 4+ T cell antibody Ab2, the secondary antibodies preferably being derived from rabbit sources: rabbit anti-mouse antibody that binds to murine anti-CD 4+ T cell antibody Ab 1. Because the CD4+ T cell antigen is a cell antigen, according to the detection principle of the colloidal gold test strip, a double-antibody sandwich method is adopted for the CD4+ T cell antigen, and a sample sequentially passes through the gold-labeled pad and the detection line when dropping on the colloidal gold test stripT1, detection line T2, detection line T3, and quality control line C. Neither the detection lines T1-T3 nor the quality control line C developed color before detection. The immune colloidal gold aggregated in reddish purple color and the murine anti-CD 4+ T cell antibody Ab1 conjugate (colloidal gold-anti-Ab 1 complex) were uniformly distributed on the colloidal gold conjugate pad. The detection line T contains a murine anti-CD 4+ T cell antibody Ab1 which has been adsorbed beforehand with an antigen against CD4+ T cells, and can be bound to a colloidal gold-anti-Ab 1 complex. The quality control line C contains rabbit anti-mouse antibody which can be combined with the colloidal gold-anti-Ab 1 compound and combined with the mouse anti-CD 4+ T cell anti-Ab 1. If the sample blood does not contain the CD4+ T cell antigen, the detection line is not colored, and the quality control line is colored. When the antigen is contained in the blood of the sample, the murine anti-CD 4+ T cell antibody binds to CD4+ T cell antigen when the CD4+ T cell antigen in the sample is subjected to the immune colloidal gold. When the immune colloidal gold moves to the detection line along with the blood sample, the immune colloidal gold combined with the antigen in the sample is adsorbed to the detection T line, and the detection line T is purple after further aggregation. The three detection lines T1-T3 are provided with fixed immune colloidal gold combination upper limits among the partitions according to the corresponding disease risks, and under the condition that the blood volume of the sample is fixed, the content range of the antigen in the sample can be obtained through the color combination condition displayed by the three detection lines, so that whether the patient is a high risk group suffering from diseases or not can be judged. The C position of the quality control line adsorbs rabbit anti-mouse antibody combined with murine anti-CD 4+ T cell-anti Ab1, when colloidal gold adsorbing murine anti-CD 4+ T cell antibody is not captured, the C line does not develop color because the rabbit anti-mouse antibody combined with murine anti-CD 4+ T cell antibody Ab1 is colorless protein, but when the colloidal gold adsorbing murine anti-CD 4+ T cell antibody is captured, a red purple band is displayed. Only when the control line C showed a reddish purple color, the CD4+ T cell concentration was (0, 12.5X 10)6/L]When the control lines C and T1 are purple, the concentration range of CD4+ T cells is (12.5X 10)6/L，25×106/L](ii) a When the control line C, T1 and T2 are simultaneously purple, the concentration range of CD4+ T cells is (25X 10)6/L，50×106/L](ii) a When the control line C and three T lines are purple, the concentration interval of CD4+ T cells is (50X 10)6/L，+∞)。
The preparation method of the colloidal gold is not particularly limited, and the colloidal gold can be prepared by a conventional method in the field, a trisodium citrate reduction method or other methods.
The preparation method of the colloidal gold pad preferably comprises the following steps: adjusting the pH value of the colloidal gold solution by using a potassium carbonate solution, mixing the colloidal gold solution with anti-Ab 1, reacting at room temperature for 10-30 min, mixing with a confining liquid, standing for 5-10 min, centrifuging at 4 ℃ and 2000rpm to collect supernatant, centrifuging at 4 ℃ and 12000rpm for 60min, discarding the supernatant, dissolving precipitate in PBS (containing 0.2 mass percent of BSA) to obtain a colloidal gold marker; diluting the colloidal gold marker by PBS (containing BSA with the mass fraction of 0.2%) for 1-2 times, and uniformly spraying the colloidal gold marker on the glass fiber membrane by using a spraying instrument, wherein the spraying amount is 0.1-0.5 mL/cm2And drying the mat for 18 to 24 hours at the temperature of 37 to 40 ℃ under the humidity of 30 percent to obtain the gold label mat. Sealing the gold label pad with a tin foil bag, filling a drying agent in the gold label pad, and storing at normal temperature; opening and use should be below 30% humidity.
The invention is not specially limited for the arrangement of the detection lines and the quality control lines on the nitrocellulose membrane, and takes the arrangement of 3 detection lines and 1 quality control line as an example, primary anti-Ab 2 aiming at the antigen is diluted by PBS to the concentration of 1-4 mg/mL, and is scribed (or sprayed on) on 3 nitrocellulose membranes at equal distance to serve as the detection lines. And (3) drawing a line (or spraying the line on a nitrocellulose membrane) at a position 6mm away from the detection line by using 1-4 mg/mL of rabbit anti-mouse antibody (secondary antibody) correspondingly combined with the murine anti-Ab 1 to serve as a quality control line, and curing the detection strip and the quality control line on the nitrocellulose membrane.
The invention provides a method for manufacturing antigen detection colloidal gold subsection semi-quantitative detection test paper based on a double-antibody sandwich method, which comprises the colloidal gold test paper.
According to the invention, 3 detection lines are preferably arranged on the nitrocellulose membrane on the colloidal gold test strip; when the antigen is CD4+ T cells, 2.0 muL of anti-CD 4+ T cell antibody Ab2 solution of 1-4 mg/mL is sprayed on the detection line T1, 2.0 muL of anti-CD 4+ T cell antibody Ab2 solution of 1-4 mg/mL is sprayed on the detection line T2, and 4.0 muL of anti-CD 4+ T cell antibody Ab2 solution of 1-4 mg/mL is sprayed on the detection line T3 on 3 detection lines; when the antigen is CD4+ T cells, 3.0 muL of anti-CD 4+ T cell antibody Ab2 solution of 1-4 mg/mL is sprayed on the detection line T1, 3.0 muL of anti-CD 4+ T cell antibody Ab2 solution of 1-4 mg/mL is sprayed on the detection line T2, and 6.0 muL of anti-CD 4+ T cell antibody Ab2 solution of 1-4 mg/mL is sprayed on the detection line T3 on 3 detection lines.
In the invention, blood is preferably used as a detection sample, if a quality control line develops color, the test paper is effective, and the concentration range of CD + T cells in blood of a patient can be determined according to the number of detection lines; if the quality control line does not develop color, the test strip is invalid (figure 2); when only the quality control line C shows reddish purple, the result corresponds to the concentration at one stage; when the quality control line C and T1 are simultaneously displayed as magenta, the result corresponds to two-stage concentration; when the quality control line C is purple with T1 and T2, the result corresponds to three-stage concentration; when the quality control line C and the three T lines are simultaneously displayed as purple, the result corresponds to four-stage concentration; more preferably, in the examples, the first-stage concentration of CD4+ T cells is at (0, 12.5X 10)6/L]In the interval, the biphasic concentration of CD4+ T cells was (12.5X 10)6/L，25×106/L]Within the interval; the three-stage concentration of CD4+ T cells was at (25X 10)6/L，50×106/L]Within the interval; the four-stage concentration of CD4+ T cells was (50X 10)6L, + ∞) interval.
The present invention provides a double antibody sandwich-based antigen detection colloidal gold test strip and a segmented semi-quantitative detection system, which are described in detail below with reference to the following examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Preparation of immune colloidal gold semi-quantitative segmental detection test strip by double-antigen sandwich method
1. Preparing colloidal gold:
mixing chloroauric acid (HAuCl)4) Preparing a chloroauric acid solution with the mass fraction of 1%, adding 1.0mL of the chloroauric acid solution into 99mL of deionized water, heating to boil, immediately adding 2.0mL of the sodium citrate solution with the mass fraction of 1%, rapidly stirring uniformly, continuing heating to boil for 10min, cooling to room temperature, and supplementingDeionized water to 100.0mL to obtain a colloidal gold solution.
2. Preparation of colloidal gold marker:
taking 10mL of prepared colloidal gold solution, adjusting the pH value to 5.5-7.0 by using 0.1mol/L potassium carbonate solution, adding murine CD4+ T cell antibody Ab150.0-100.0 mu g, uniformly mixing, reacting at room temperature for 10-30 minutes, then adding BSA to a final concentration (mass fraction) of 0.5-1.0%, uniformly mixing, standing for 5-10 minutes, centrifuging at 4 ℃ and 2000rpm for 20 minutes, and discarding the precipitate. The supernatant was centrifuged at 12000rpm for 60 minutes at 4 ℃ to discard the supernatant, and the precipitate was dissolved in 1.0mL of PBS (containing 0.2% by mass of BSA) to obtain a colloidal gold-labeled substance.
3. Preparation of gold-labeled pad (i.e., conjugate pad with colloidal gold label applied thereto):
diluting the colloidal gold marker by PBS (containing BSA with the mass fraction of 0.2%) for 1-2 times, and uniformly spraying the colloidal gold marker on the glass fiber membrane by using a spraying instrument, wherein the spraying amount is 0.1-0.5 mL/cm2And drying the mat for 18 to 24 hours at the temperature of 37 to 40 ℃ under the humidity of 30 percent to obtain the gold label mat. Sealing the gold label pad with a tin foil bag, filling a drying agent in the gold label pad, and storing at normal temperature. Opening and application should be below 30% humidity.
4. Coating of 3 detection bands and quality control lines
The other antibody Ab2 aiming at the CD4+ T cell antigen is diluted to the concentration of 1-4 mg/mL by PBS, and 3 lines are drawn at equal intervals on a nitrocellulose membrane (or sprayed on the nitrocellulose membrane) to be used as detection bands (T1, T2 and T3). And (3) drawing a line again (or spraying the line on a nitrocellulose membrane) by using 1-4 mg/mL of rabbit anti-mouse antibody (secondary antibody) which is correspondingly combined with the mouse CD4+ T cell antibody Ab1 at a position 6mm away from the farthest detection zone to serve as a quality control line. The 3 test strips and the control line were cured on nitrocellulose membrane.
5. And (3) curing:
after drying the nitrocellulose membrane, blocking was performed by soaking in PBS containing 1% BSA at pH7.2 (4 ℃, 1 h). The nitrocellulose membrane was then washed 3 times with PBS, hung, dried at room temperature for more than 3 hours, and then placed in a 30 ℃ air-drying cabinet for 1 hour of air-drying.
6. Assembling the test strip:
6.1 the test strip mainly comprises a test strip consisting of a sample pad, a gold-labeled pad obtained by the steps, a nitrocellulose membrane (3 detection bands and a quality control line) and a water absorption pad, and a substrate for fixing and supporting the test strip. Wherein, the substrate adopts a PVC plate 1 (test paper strip) or a card shell (test paper card); the water absorption pad can adopt water absorption filter paper to absorb redundant liquid in the detection sample; the sample pad may be a glass fiber membrane that contacts the sample to be tested.
6.2 on the PVC plate, the parts of the test paper are arranged in turn along the chromatographic direction, wherein, the downstream end is a water absorption pad, the upstream end is a sample pad, the middle section takes 3 pieces of nitrocellulose membrane of detection zone (containing another antibody Ab2 aiming at CD4+ T cell antigenic determinant) and quality control line (corresponding to rabbit anti-mouse antibody combined with mouse source CD4+ T cell antibody Ab 1) as the detection reaction and analysis area, and a gold-labeled pad adsorbed with colloidal gold-labeled mouse source CD4+ T cell antibody Ab1 is arranged between the nitrocellulose membrane and the sample pad.
6.3 attaching the test paper to the PVC plate 1, and cutting the test paper into strip-shaped bands with the width of 4 mm; the nitrocellulose membrane is attached to the PVC plate, the water absorption pad and the gold label pad are attached to the PVC plate and partially overlapped with two ends of the nitrocellulose membrane respectively, and the sample pad is attached to the PVC plate and partially overlapped with the gold label pad. And sealing, drying and storing the test strip for later use.
7. And (4) judging a result:
filtering the blood to be detected with a blood filtering membrane to obtain red blood cells, vertically dropping the red blood cells on a gold label pad, timing when the liquid flows, standing for 5min for reaction, and observing the result. Since the rabbit anti-mouse antibody bound to the mouse macromolecular antigen/cellular antibody Ab1 is a colorless protein, the quality control C line does not develop color, but shows a reddish purple band when capturing colloidal gold that adsorbs the mouse macromolecular antigen/cellular antibody. When only the quality control line C shows magenta, the result corresponds to a stage concentration; when the quality control line C and T1 are simultaneously displayed as purple, the result corresponds to two-stage concentration; when the quality control line C shows reddish purple with T1 and T2, the result corresponds to three stages of concentration; when the quality control line C and the three T lines are simultaneously displayed as magenta, the result corresponds to four-stage concentration.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (8)
1. The test paper for the antigen detection of the colloidal gold segmentation semi-quantitative detection based on the double-antibody sandwich method is characterized by comprising a sample pad, a colloidal gold pad coated with anti-Ab 1, a nitrocellulose membrane coated with anti-Ab 2 and water absorption filter paper, which are sequentially adhered to a PVC plate; the coated nitrocellulose membrane comprises a plurality of detection lines and a quality control line; each detection line is coated with primary anti-Ab 2 with the same concentration or different concentrations; coating a secondary antibody on the quality control line; in which primary anti-Ab 1 and primary anti-Ab 2 are different antibody determinants for the same antigen.
2. The colloidal gold test strip of claim 1, wherein the secondary antibody is capable of binding to primary anti-Ab 1.
3. The colloidal gold test strip of claim 1, wherein a coated nitrocellulose membrane is adhered to the center of the PVC base plate, absorbent filter paper is adhered to the upper edge of the nitrocellulose membrane, an antibody colloidal gold pad is adhered to the lower edge of the nitrocellulose membrane, and a sample pad is adhered to the lower edge of the colloidal gold pad.
4. The colloidal gold test strip of claim 1, wherein the amount of primary anti-Ab 2 coated on each test line corresponds to the fractional concentration of the substance to be tested; the antigens include all macromolecules and cells, for example: glutamic-oxaloacetic transaminase, glutamic-pyruvic transaminase, hemoglobin, human serum albumin, neutrophil and eosinophil.
5. The colloidal gold test strip of claim 4, wherein 3 detection lines are disposed on the nitrocellulose membrane on the colloidal gold test strip; when the antigen is CD4+ T cells, 2.0 muL of anti-CD 4+ T cell antibody Ab2 solution of 1-4 mg/mL is sprayed on the detection line T1, 2.0 muL of anti-CD 4+ T cell antibody Ab2 solution of 1-4 mg/mL is sprayed on the detection line T2, and 4.0 muL of anti-CD 4+ T cell antibody Ab2 solution of 1-4 mg/mL is sprayed on the detection line T3 on 3 detection lines.
6. A test paper for antigen detection colloidal gold segmental semi-quantitative detection based on a double antibody sandwich method is characterized by comprising the colloidal gold test paper strip of any one of claims 1 to 5.
7. The system according to claim 6, wherein the concentration of CD4+ T cells is (0, 12.5X 10) when only the control line C shows a reddish purple color6/L]When the control line C and T1 are simultaneously purple, the concentration range of CD4+ T cells is (12.5X 10)6/L，25×106/L](ii) a When the control line C, T1 and T2 are simultaneously purple, the concentration range of CD4+ T cells is (25X 10)6/L，50×106/L](ii) a When the control line C and three T lines show red-purple color, the concentration interval of CD4+ T cells is (50X 10)6/L，+∞)。
8. The system according to claim 7, wherein when only the control line C shows magenta, the result corresponds to a first-stage concentration; when the quality control line C and T1 are simultaneously displayed as magenta, the result corresponds to two-stage concentration; when the quality control line C shows reddish purple with T1 and T2, the result corresponds to three stages of concentration; when the quality control line C and the three T lines are simultaneously displayed as purple, the result corresponds to four-stage concentration; and when the quality control line C does not develop color, the test paper fails.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210490629.3A CN114778831A (en) | 2022-05-07 | 2022-05-07 | Method for manufacturing antigen detection colloidal gold segmented semi-quantitative detection test paper based on double-antibody sandwich method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210490629.3A CN114778831A (en) | 2022-05-07 | 2022-05-07 | Method for manufacturing antigen detection colloidal gold segmented semi-quantitative detection test paper based on double-antibody sandwich method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114778831A true CN114778831A (en) | 2022-07-22 |
Family
ID=82436003
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210490629.3A Pending CN114778831A (en) | 2022-05-07 | 2022-05-07 | Method for manufacturing antigen detection colloidal gold segmented semi-quantitative detection test paper based on double-antibody sandwich method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114778831A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115932269A (en) * | 2022-12-26 | 2023-04-07 | 江阴天泽医学检验实验室有限公司 | Method for quantitative detection of antigen by using immunochromatography colloidal gold reagent |
| CN116990504A (en) * | 2023-09-27 | 2023-11-03 | 苏州旭光科星抗体生物科技有限公司 | Double antibody sandwich ELISA kit for detecting human soluble CD4 |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101158683A (en) * | 2007-05-16 | 2008-04-09 | 山东省农业科学院植物保护研究所 | Colloidal gold chromatography semi-quantitative determination 2,4-D test paper and preparation method thereof |
| US20120034711A1 (en) * | 2010-08-05 | 2012-02-09 | Oil Crops Research Institute, Chinese Academy of Agricultural Science | Digital immunochromatographic test strip for semi-quantitative detection of aflatoxin B1 and preparation method thereof |
| CN103743908A (en) * | 2013-10-31 | 2014-04-23 | 北京陆桥技术有限责任公司 | Neomycin semi-quantitative gold-labeled rapid detection kit, and detection method and application thereof |
| CN104360068A (en) * | 2014-11-16 | 2015-02-18 | 中国海洋大学 | Semi-quantitative rapid detection test paper for white spot syndrome virus (WSSV), and preparation method of test paper |
| WO2021017417A1 (en) * | 2019-07-30 | 2021-02-04 | 南通伊仕生物技术股份有限公司 | Human chorionic gonadotropin semi-quantitative detection test paper and preparation method therefor, human chorionic gonadotropin semi-quantitative detection reagent cup, and applications thereof |
| CN113063938A (en) * | 2021-03-13 | 2021-07-02 | 河南省农业科学院 | High-sensitivity gradient semi-quantitative immunochromatography detection test strip and detection method |
| CN113552361A (en) * | 2021-06-01 | 2021-10-26 | 中国农业科学院农业质量标准与检测技术研究所 | Test strip for semi-quantitatively detecting amantadine and preparation method and application thereof |
| CN114034857A (en) * | 2021-11-20 | 2022-02-11 | 无锡胜为然生物科技有限公司 | Preparation method of colloidal gold immunochromatographic test strip for rapidly and semi-quantitatively detecting serum ferritin by double-antibody sandwich method |
-
2022
- 2022-05-07 CN CN202210490629.3A patent/CN114778831A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101158683A (en) * | 2007-05-16 | 2008-04-09 | 山东省农业科学院植物保护研究所 | Colloidal gold chromatography semi-quantitative determination 2,4-D test paper and preparation method thereof |
| US20120034711A1 (en) * | 2010-08-05 | 2012-02-09 | Oil Crops Research Institute, Chinese Academy of Agricultural Science | Digital immunochromatographic test strip for semi-quantitative detection of aflatoxin B1 and preparation method thereof |
| CN103743908A (en) * | 2013-10-31 | 2014-04-23 | 北京陆桥技术有限责任公司 | Neomycin semi-quantitative gold-labeled rapid detection kit, and detection method and application thereof |
| CN104360068A (en) * | 2014-11-16 | 2015-02-18 | 中国海洋大学 | Semi-quantitative rapid detection test paper for white spot syndrome virus (WSSV), and preparation method of test paper |
| WO2021017417A1 (en) * | 2019-07-30 | 2021-02-04 | 南通伊仕生物技术股份有限公司 | Human chorionic gonadotropin semi-quantitative detection test paper and preparation method therefor, human chorionic gonadotropin semi-quantitative detection reagent cup, and applications thereof |
| CN113063938A (en) * | 2021-03-13 | 2021-07-02 | 河南省农业科学院 | High-sensitivity gradient semi-quantitative immunochromatography detection test strip and detection method |
| CN113552361A (en) * | 2021-06-01 | 2021-10-26 | 中国农业科学院农业质量标准与检测技术研究所 | Test strip for semi-quantitatively detecting amantadine and preparation method and application thereof |
| CN114034857A (en) * | 2021-11-20 | 2022-02-11 | 无锡胜为然生物科技有限公司 | Preparation method of colloidal gold immunochromatographic test strip for rapidly and semi-quantitatively detecting serum ferritin by double-antibody sandwich method |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115932269A (en) * | 2022-12-26 | 2023-04-07 | 江阴天泽医学检验实验室有限公司 | Method for quantitative detection of antigen by using immunochromatography colloidal gold reagent |
| CN115932269B (en) * | 2022-12-26 | 2023-11-07 | 江阴天泽医学检验实验室有限公司 | Method for carrying out antigen quantitative detection by adopting immunochromatography colloidal gold reagent |
| CN116990504A (en) * | 2023-09-27 | 2023-11-03 | 苏州旭光科星抗体生物科技有限公司 | Double antibody sandwich ELISA kit for detecting human soluble CD4 |
| CN116990504B (en) * | 2023-09-27 | 2023-12-01 | 苏州旭光科星抗体生物科技有限公司 | Double antibody sandwich ELISA kit for detecting human soluble CD4 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104730258B (en) | A kind of Portable blood group system detectio test paper and its detection method | |
| CN114778831A (en) | Method for manufacturing antigen detection colloidal gold segmented semi-quantitative detection test paper based on double-antibody sandwich method | |
| AU748387B2 (en) | Neutralization of polycations in a chromatographic device for whole blood use | |
| RU2475758C2 (en) | METHOD OF FAST DETERMINATION OF HUMAN BLOOD TYPE ABO/Rh/MN AND TEST-KIT FOR IT | |
| US5652148A (en) | Method and apparatus for red blood cell separation | |
| KR102322094B1 (en) | Method and device for combined detection of viral and bacterial infections | |
| CN104582571A (en) | Quick test device and method | |
| EP1712913A1 (en) | Convenient detection method, detection apparatus and detection kit | |
| CN101750244B (en) | Method for separating red cells from blood sample and application | |
| CN110520736A (en) | A kind of functionalization biology multilayer hole film immune carrier, preparation method, application | |
| CN202916285U (en) | Whole blood immunochromatography device | |
| US20210389318A1 (en) | Lateral flow assays for differential isotype detection | |
| WO2003083479A1 (en) | Blood processing method, blood processing device, method of measuring hemoglobins and device for measuring hemoglobins | |
| CN104714035B (en) | A kind of high flux blood group system detection means and method | |
| Judd et al. | The evaluation of a positive direct antiglobulin test in pretransfusion testing | |
| CN107389925B (en) | IL-6 and IL-10 bigeminy colloidal gold immunochromatographykit kit for pyemia quick diagnosis | |
| CN202916284U (en) | NGAL (neutrophil gelatinase associated lipocalin) whole blood detecting device | |
| KR20180030207A (en) | Cell surface receptor analysis method of blood cells | |
| JP4426122B2 (en) | Blood antigen detection method and apparatus | |
| WO2021140090A1 (en) | Immunochemical device and lateral-flow immunoassay method for the determination of pharmaceutical residues and contaminants in breast milk | |
| CN211905397U (en) | Human ABO blood type reverse-typing rapid detection test strip | |
| RU2464572C1 (en) | Test system for immunochromatographic assay of cardiac protein binding fatty acids and troponin i in whole blood sample for instant diagnosing of myocardial infarction (versions) | |
| RU169868U1 (en) | Test system for immunochromatographic determination of procalcitonin in samples of whole blood, serum or plasma for the rapid diagnosis of sepsis | |
| CN219935868U (en) | Kit for detecting IgG subtype antibody | |
| JP2017519972A (en) | Device and method for detecting blood group antigens using incomplete antibodies |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |