CN112485427A - Immune quantitative detection device, detection method and application of tumor marker - Google Patents
Immune quantitative detection device, detection method and application of tumor marker Download PDFInfo
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- 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
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57473—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving carcinoembryonic antigen, i.e. CEA
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- 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
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57476—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving oncofetal proteins
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Abstract
The invention provides an immune quantitative detection device of a tumor marker, which is characterized by comprising the following components: a mobile phase containing device (2), a filter (1), a detection phase containing device (3) and a detector (4); wherein the mobile phase containing device (2) is connected with the filter (1) through a pipeline (8); the filter (1) comprises an inlet (5), a filter element (6) and an outlet (7); the detection phase containing device (3) is connected with the detector (4) through a pipeline (9); the material of the filter core (6) is a substance which can be coupled by a tumor marker specific conjugate. The invention also provides an immune quantitative detection method and an immune quantitative detection amplification method of the tumor marker and related applications. The invention improves the individuation controllable degree of the immune quantitative detection and improves the detection efficiency.
Description
Technical Field
The invention relates to the technical field of immune quantitative detection, in particular to an immune quantitative detection device, an immune quantitative detection method, an immune quantitative amplification detection method and application of a tumor marker.
Background
The immunological detection technology is an experimental means for determining antigens, antibodies, immune cells, chemical components and the like by applying the immunological principle, and is widely applied to samples which are derived from human bodies and animal bodies and can be used for disease diagnosis and health detection and samples for environmental, pharmaceutical, food and industrial analysis. The common immunological detection techniques include immunoturbidimetric technique, solid-phase enzyme immunoassay technique, chemiluminescence detection technique, immunofluorescence labeling technique, flow cytometry, colloidal gold technique, etc.
The immunoturbidimetry technique, also called immunoturbidimetry, refers to the specific binding of soluble antigen and antibody in liquid phase to the substance to be measured, producing a complex of a certain size, forming the refraction or absorption of light, and measuring the transmitted light or scattered light after the refraction or absorption as a calculation unit for quantitative detection. However, this method has low detection sensitivity and is not suitable for detecting trace amounts.
The solid-phase enzyme immunoassay technique is based on immobilization of an antigen or an antibody and enzyme labeling of the antigen or the antibody. The antigen or antibody bound to the surface of the solid phase carrier retains its immunological activity, and the enzyme conjugate of the antigen or antibody retains both its immunological activity and its enzyme activity. When in measurement, the detected sample (the antibody or antigen in the detected sample) and the enzyme-labeled antigen or antibody react with the antigen or antibody on the surface of the solid phase carrier according to different steps, and the method has the remarkable advantages of high sensitivity, wide linear response range, easy realization of automation and the like. However, the long reaction time of the solid-phase enzyme immunoassay technology limits the use of the technology.
The immunochemiluminescence detection technology is a high-sensitivity micro and trace analysis technology, has the obvious advantages of convenient operation, high sensitivity, wide linear response range, easy realization of automation and the like, is widely applied to environment, clinic, pharmaceutical analysis, food and industrial analysis, and is also a solid-phase separation means based on an antigen or an antibody and a luminescent reagent labeling technology based on the antigen or the antibody. However, the immunochemiluminescence detection technology has long detection reaction time and high requirements on detection equipment, and the application of the detection equipment is also influenced.
In addition, immunofluorescence labeling, flow cytometry, and colloidal gold are also commonly used detection techniques, but all have their corresponding disadvantages.
High sensitivity, rapidness, miniaturization, full quantification and automation are development trends of clinical immunoassay technology products at present, but the existing immunoassay technology cannot realize the functions at the same time. Therefore, a novel detection technology which has the characteristics of high sensitivity, full quantification and automation, and has the characteristics of rapid detection and small and portable equipment is developed, so that the detection technology is convenient to use, reduces waste, can obviously improve the working efficiency, and has important practical significance in a plurality of fields of detection, analysis and separation.
Disclosure of Invention
The invention provides an immune quantitative detection device of a tumor marker, which has high sensitivity, full quantification and high detection speed, in particular to an immune quantitative detection device taking a filter as a reaction carrier.
In order to achieve the above object, the present invention adopts a technical solution of an apparatus for quantitative immunoassay of a tumor marker, the apparatus comprising: a mobile phase containing device, a filter, a detection phase containing device and a detector;
wherein,
the mobile phase containing device is connected with the filter through a pipeline;
the filter comprises an inlet, a filter element and an outlet;
the detection phase containing device is connected with the detector through a pipeline;
the material of the filter core is a substance which can be coupled by a tumor marker specific conjugate.
Furthermore, the tumor marker specific binding substance is one of an enzyme, an inhibitor, a ligand, a receptor and a protein. Still further, the protein is selected from an antigen and/or an antibody.
Further, the volume of the filter is less than 1ml, preferably 2 microliters to 1ml, more preferably 3 microliters to 0.3 ml, most preferably 5 microliters to 0.1 ml.
Further, the filtering structure of the filtering core is in a shape with a length larger than a width, wherein the ratio of the length to the width is 2-100, preferably 2-50, more preferably 2-30, and the filtering structure can be in a shape of a cylinder, a cone, a square, a rectangle, a combination thereof and the like; or the filtering structure of the filter core is in a shape with the width larger than the length, wherein the ratio of the width to the length is 1.1-10, preferably 1.1-5, more preferably 1.1-3, and the filtering structure can be in a shape of a cylinder, a cone, a square, a rectangle, a combination thereof and the like.
The filter core is made of water-insoluble solid-phase materials; further, the water-insoluble solid phase material is a granular solid phase material or a mesh-shaped solid phase material; preferably, the granular solid phase substance or the mesh-shaped solid phase substance is a plurality of solid-state substances which can be coupled and combined with a tumor marker specific binding substance such as an antigen or an antibody and do not obviously change the immunological binding characteristics of the tumor marker specific binding substance such as the antigen or the antibody; more preferably, the water-insoluble solid phase material is selected from one or more of gel particles, latex particles and magnetic particles; most preferably, the gel particles are selected from one or more of sephadex particles, sepharose particles, cyanogen bromide activated sepharose particles, NHS activated sepharose particles.
The detector is a mechanical structure capable of quantitatively detecting and recording color or light quantity change; preferably, the detector is selected from one or more of a chemiluminescence detector, a spectrophotometric detector, a fluorescence detector, and an enzyme-linked immunosorbent detector.
Further, the tumor marker is selected from one or more of alpha-fetoprotein, carcinoembryonic antigen, cancer antigen 125, cancer antigen 15-3, carbohydrate antigen 19-9, cancer antigen 50, carbohydrate antigen 242, gastric cancer-associated antigen, ferritin, prostate specific antigen, prostate acid phosphatase, b2 microglobulin, neuron-specific enolase, cytokeratin 19, squamous cell carcinoma antigen, nuclear matrix protein 22, a-L-fucosidase and human epididymis secretory protein 4; preferably, the tumor marker is selected from one of alpha-fetoprotein and carcinoembryonic antigen.
The invention also provides an immune quantitative detection method of the tumor marker, which comprises the following steps:
(1) marking the filter core by a tumor marker specific conjugate to prepare a marked filter core I;
(2) the labeled filter core I is used for capturing tumor markers specifically to prepare a filter core-tumor marker conjugate I;
(3) specifically capturing a detection substance in a detection phase by using the filter core-tumor marker conjugate I to prepare a filter core-tumor marker-detection substance complex, wherein the detection substance is an indication conjugate of the tumor marker specific conjugate and an indicator;
(4) detecting and calculating the content of the tumor marker.
Further, before the step (4), eluting the filter core-tumor marker-detection substance complex with a washing solution to remove the detection phase residue which is not captured and remains in the filter core.
Further, the immune quantitative detection method of the tumor marker comprises the following specific steps:
(1) marking the filter core by a tumor marker specific conjugate to prepare a marked filter core I;
(2) preparing a filter structure of the filter by using a marked filter element I;
(3) filtering the tumor marker by the filter, and specifically capturing the tumor marker by using the marked filter core I to prepare a filter core-tumor marker conjugate I;
(4) eluting the filter core-tumor marker conjugate I by using a cleaning solution so as to remove tumor marker residues which are not captured and remain in the filter core;
(5) filtering a detection phase containing a detection substance by the filter, and specifically capturing the detection substance in the detection phase by using the filter core-tumor marker conjugate I to prepare a filter core-tumor marker-detection substance complex, wherein the detection substance is an indication conjugate of a tumor marker specific conjugate and an indicator;
(6) eluting the filter element-tumor marker-detection substance compound by using a cleaning solution to remove the detection phase residue which is not captured and remains in the filter element;
(7) detecting and calculating the content of the tumor marker.
The step of detecting and calculating the content of the tumor marker is selected from any one of the following methods: 1) directly detecting the filter core-tumor marker-detection substance compound to obtain the amount I of the indicator specifically captured by the filter core, and calculating the content of the tumor marker according to the amount I of the indicator; and/or, 2) directly detecting the eluent which is eluted by the cleaning solution in the step (6) and contains the detection substance to obtain the amount II of the indicator which is not captured and remains in the filter element, and calculating the content of the tumor marker according to the amount II of the indicator; and/or 3) directly detecting the detection phase flowing out after the specific capture in the step (5) to obtain the amount III of the indicator flowing out without being combined, and calculating the content of the tumor marker according to the amount III of the indicator.
The invention also provides an immune quantitative amplification detection method of the tumor marker, which comprises the following steps:
(1) marking the filter core by a tumor marker specific conjugate to prepare a marked filter core II;
(2) the tumor marker is captured by the specificity of the marked filter core II to prepare a filter core-tumor marker conjugate II;
(3) specifically capturing the avidin conjugate in the primary detection phase by using the filter core-tumor marker conjugate II to prepare a compound of the filter core-tumor marker-avidin conjugate;
(4) specifically capturing a biotin detection object in a secondary detection phase by using the filter core-tumor marker-avidin conjugate compound to prepare a filter core-tumor marker-avidin conjugate-biotin detection object compound, wherein the biotin detection object is a conjugate of biotin and an indicator;
(5) detecting and calculating the content of the tumor marker.
Further, before step (5), eluting the filter core-tumor marker-avidin conjugate-biotin detector complex with a washing solution to remove the secondary detection phase residue which is not captured and remains in the filter core.
The avidin is a substance with avidin binding property; preferably, the avidin is selected from one or more of ovalbumin, streptavidin, vitellin and avidin.
Further, the immune quantitative amplification detection method comprises the following specific steps:
(1) marking the filter core by a tumor marker specific conjugate to prepare a marked filter core II;
(2) preparing a filter structure of the filter by using a marked filter element II;
(3) making the tumor marker pass through the filter, and using the marked filter core II to specifically capture the tumor marker to prepare a filter core-tumor marker conjugate II;
(4) eluting the filter core-tumor marker conjugate II by using a cleaning solution to remove tumor marker residues which are not captured and remain in the filter core;
(5) filtering a primary detection phase containing the avidin conjugate through the filter, and specifically capturing the avidin conjugate in the primary detection phase by using the filter core-tumor marker conjugate II to prepare a filter core-tumor marker-avidin conjugate compound;
(6) eluting the filter element-tumor marker-avidin conjugate compound by using a cleaning solution to remove the first-stage detection phase residue which is not captured and remains in the filter element;
(7) filtering a secondary detection phase containing a biotin detection substance through the filter, and specifically capturing the biotin detection substance in the secondary detection phase by using the complex of the filter core-the tumor marker-the avidin conjugate to prepare a filter core-the tumor marker-the avidin conjugate-the biotin detection substance complex, wherein the biotin detection substance is a conjugate of biotin and an indicator;
(8) eluting the filter element-tumor marker-avidin conjugate-biotin detector compound by using a cleaning solution to remove secondary detection phase residues which are not captured and remain in the filter element;
(9) detecting and calculating the content of the tumor marker.
Further, the step of detecting and calculating the content of the tumor marker is selected from any one of the following methods: 1) directly detecting the filter core-tumor marker-avidin conjugate-biotin detection object to obtain the amount IV of the indicator specifically captured by the filter core, and calculating the content of the tumor marker according to the amount IV of the indicator; and/or, 2) directly detecting the eluent which is eluted by the cleaning solution and contains the biotin detection object, so as to obtain the amount V of the indicator which is not captured and remains in the filter element, and calculating the content of the tumor marker according to the amount V of the indicator; and/or 3) directly detecting the secondary detection phase flowing out after the specific capture in the step (3) to obtain the amount VI of the unbound indicator flowing out, and calculating the content of the tumor marker according to the amount VI of the indicator.
Further, the indicator is a substance capable of directly or indirectly producing a change in color or amount of light;
preferably, the indicator is an enzyme substance, a substance capable of directly or indirectly emitting light, a substance capable of generating fluorescence, or a substance with color;
more preferably, the enzyme substance is selected from one or more of horseradish peroxidase (HRP), Alkaline Phosphatase (AP), glucose oxidase, beta-galactosidase, lysozyme and malate dehydrogenase; the substance capable of directly or indirectly emitting light is selected from one or more of luminol and its derivatives, lucigenin, loessine, peroxyoxalate esters, and acridinium esters; the substance capable of generating fluorescence is selected from one or more of Fluorescein Isothiocyanate (FITC) or radamine (RB 200); the self-colored substance is selected from colloidal gold substances.
The invention also provides an immune quantitative detection device, an immune quantitative detection method or an immune quantitative amplification detection method, which are used for immune quantitative detection of a sample containing a tumor marker.
Wherein, the sample containing the tumor marker is a sample which is derived from a human body and an animal body and can be used for disease diagnosis and health detection, or the sample containing the tumor marker is a sample used for environmental analysis, pharmaceutical analysis, food and industrial analysis. The immune quantitative detection device, the immune quantitative detection method or the immune quantitative detection amplification method are applied to the development of various detection technical products, and the detection of samples from human bodies is the main content of clinical detection and is used for diagnosis, auxiliary diagnosis, prediction, disease condition monitoring and the like of various diseases; the sample detection from animal body is also the main content of clinical detection, and is used for diagnosis, auxiliary diagnosis, prediction, disease monitoring, food safety detection and the like of various diseases related to animals.
By adopting the technical scheme, the invention at least has the following advantages:
compared with the prior art, the immune quantitative detection device of the tumor marker with the filter as the reaction carrier has the advantages that the whole operation time is only 13 minutes, but the prior chemiluminescence detection technology basically reaches the balance and needs 30 minutes, the reaction time of the filtration is obviously shortened compared with the prior detection combined reaction time, and the detection speed is improved. Therefore, under the condition that the detection results are basically the same, the detection time of the invention is obviously shorter than that of the prior detection technology, the individuation controllable degree of the tumor marker immune quantitative detection is improved, and the detection efficiency is greatly improved.
In addition, when the detection time is more than 10 minutes, the detection fold is basically unchanged, which shows that the detection sample of the invention has particularly good repeatability of the level of the alpha-fetoprotein and can meet the requirement of detection accuracy.
The invention can complete all reactions at room temperature, avoids a temperature control reaction structure which needs to be arranged in the existing detection equipment, can realize immunoassay by adopting a filter of 3 cubic millimeters, has small volume of the whole detection device, simplifies the design of instruments, and can realize the purposes of miniaturization and portability.
Therefore, the immune quantitative detection device and the detection method of the tumor marker have important significance and good application prospect for improving the existing immune detection technology of the tumor marker.
Drawings
FIG. 1 is a schematic view of an apparatus for quantitative immunoassay of tumor markers according to the present invention.
Detailed Description
To further illustrate the technical means and effects of the present invention adopted to achieve the predetermined purpose, the present invention will be described in detail with reference to the accompanying drawings 1 and the preferred embodiment.
As shown in fig. 1, an apparatus for the quantitative immunoassay of tumor markers comprises: a mobile phase containing device 2, a filter 1, a detection phase containing device 3 and a detector 4; wherein the mobile phase containing device 2 is connected with the filter 1 through a pipeline 8; the filter 1 comprises an inlet 5, a filter element 6 and an outlet 7; the detection phase containing device 3 is connected with the detector 4 through a pipeline 9.
Further, the filter 1 is a reaction vessel for detection reaction, and the main reaction process of the detection reaction is performed on the filter 1, and comprises an inlet 5, a filter core 6 and an outlet 7, wherein the filter core in the filter layer is composed of a labeled water-insoluble solid phase material capable of forming specific binding with a tumor marker and coupled with an antigen or an antibody. The solid phase material may be stacked solid phase particulate matter forming lacunar filter pore sizes between particles, or may be a mesh solid phase matter.
Further, the mobile phase containing apparatus 2 is a container structure containing a sample for disease diagnosis and health examination, including samples for environmental, pharmaceutical, food and industrial analysis, which are derived from human and animal bodies.
Further, the detection phase containing device 3 is a container structure containing a detection solution, and contains a solution of a detection substance capable of specifically binding to a tumor marker and directly or indirectly generating a change in color or light amount.
Further, the detector 4 is a color or light amount detection and analysis device.
Example 1: preparation of the Filter according to the invention
Experimental materials: commercially available microfilters, NHS activated sepharose particles (ACRO BIOSYSTEMS), anti-human fibrinogen polyclonal antibody (genatates Inc.), sodium bicarbonate, hydrochloric acid, ethanolamine.
The experimental method comprises the following steps: taking a commercially available micro filter, and taking out a filter element; dissolving 5mg of anti-human fibrinogen polyclonal antibody in 0.2M sodium bicarbonate solution (pH8.3) and 2 ml; taking 1ml of NHS activated agarose gel particles, and carrying out suction filtration and washing by using 20ml of 1mM hydrochloric acid for three times; mixing the anti-human fibrinogen polyclonal antibody solution with the treated NHS activated agarose gel particles, and carrying out oscillation reaction for 4 hours at 4 ℃; the reacted NHS activated sepharose particles are washed, 10mM ethanolamine and 0.2M sodium carbonate solution are added, the pH value is 8.0, the uncoupled groups are blocked by shaking for 4 hours at room temperature, the solution is washed clean, the prepared NHS activated sepharose particles are used for preparing filter cores, and the filter cores are loaded into a commercial microfilter and blocked for standby.
Example 2: comparison of detection Performance of the present invention with that of the existing chemiluminescence detection technique
Experimental materials: the kit comprises an anti-human fibrinogen polyclonal antibody filter, a horseradish peroxidase labeled anti-human fibrinogen monoclonal antibody, magnetic particles, luminol, p-iodophenol, carbamide peroxide, a chemiluminescence detector and a human fibrinogen solution.
The experimental method comprises the following steps: a human fibrinogen solution of known concentration was diluted with PBS to prepare a 1ug/ml human fibrinogen solution. The experimental method is to observe the influence of different combination reaction time on the luminescence quantity by adopting the invention and the existing chemiluminescence detection technology. Binding reaction time points were observed for 1, 2, 4, 10, 20, 30, 45, 60 minutes.
The current chemiluminescence detection technology group comprises adding 100ul of magnetic particles marked by anti-human fibrinogen polyclonal antibody into each tube, respectively adding 100ul of human fibrinogen solution, performing binding reaction at 37 ℃ for shaking incubation, adsorbing and separating the magnetic particles by a magnetic block, discarding the supernatant, adding 200ul of PBS for washing three times, adsorbing and separating the magnetic particles by a magnetic block, discarding the supernatant, adding 200ul of horse radish peroxidase-marked anti-human fibrinogen monoclonal antibody, performing binding reaction at 37 ℃ for shaking incubation for corresponding reaction time, adsorbing and separating the magnetic particles by a magnetic block, discarding the supernatant, adding 200ul of PBS for washing three times, adsorbing and separating the magnetic particles by a magnetic block, discarding the supernatant, transferring the magnetic particles to a luminescence cup, placing a chemiluminescence detector, adding 100ul of luminol, when the reaction was carried out for 2 minutes in the luminescence substrate working solution prepared from iodophenol, carbamide peroxide, etc., the luminescence amount was recorded for 6 seconds.
The invention group, take the anti-human fibrinogen polyclonal antibody filter made in example 1, dilute 100ul human fibrinogen solution to 1ml with alkaline PBS, filter and wash three times with 1ml alkaline PBS buffer solution, dilute 100ul horseradish peroxidase labeled anti-human fibrinogen monoclonal antibody to 1ml alkaline PBS buffer solution, filter, wash three times with 1ml alkaline PBS buffer solution, filter with 1ml pH4.5 Tris-hydrochloric acid buffer solution, collect filtrate, take 100ul to the luminescence cup, put the chemiluminescence detector, add 100ul luminol, p-iodophenol and urea peroxide and so on the configuration luminescence substrate working solution, when the reaction is carried out for 2 minutes, record the luminescence amount for 6 seconds, multiply by 10 as the above-mentioned existing chemiluminescence detection technology group comparable total count result.
The experimental results are as follows: the luminescence (mV) of the existing chemiluminescence detection technology at 1, 2, 4, 10, 20, 30, 45 and 60 minutes is 3222, 5672, 7968, 9810, 14281, 20339, 19827 and 20513 respectively, the binding reaction reaches an equilibrium basically within 30 minutes, and the operation time of the whole process at 30 minutes is 89 minutes. The detection result of the invention is 21320, and the operation time of the whole process is only 13 minutes.
Example 3: comparison of the detection results of the present invention with the existing chemiluminescence detection technology
Experimental materials: the kit comprises an anti-human fibrinogen polyclonal antibody filter, a horseradish peroxidase labeled anti-human fibrinogen monoclonal antibody, magnetic particles, luminol, p-iodophenol, carbamide peroxide, a chemiluminescence detector, a human fibrinogen solution and healthy human plasma.
The experimental method comprises the following steps: taking a human fibrinogen standard solution with a known concentration, and diluting the human fibrinogen standard solution with a PBS solution to prepare 10, 30, 70, 100, 300 and 700ng/ml human fibrinogen solutions; taking blood plasma of healthy people, and diluting by 10000 times by PBS; the experimental method adopts the invention and the existing chemiluminescence detection technology to detect human fibrinogen solution and draw a standard curve, then the fibrinogen of healthy people is measured, and the fibrinogen concentration is calculated by using the standard curve; taking 42 test tubes, and dividing the test tubes into the invention group and the current chemiluminescence detection technology group; 3 parallel tubes were made for each sample.
The existing chemiluminescence detection technology group comprises the steps of adding 100ul of magnetic particles marked by anti-human fibrinogen polyclonal antibodies into each tube, then respectively adding 100ul of human fibrinogen solution or healthy human plasma, performing binding reaction, shaking and incubating for 30 minutes at 37 ℃, adsorbing and separating the magnetic particles by using a magnetic block, discarding supernatant, adding 200ul of PBS for cleaning for three times, adsorbing and separating the magnetic particles by using the magnetic block, and discarding supernatant; adding 200ul of horseradish peroxidase labeled anti-human fibrinogen monoclonal antibody, performing binding reaction, performing shaking incubation at 37 ℃ for 30 minutes, adsorbing and separating magnetic particles by using a magnetic block, discarding the supernatant, adding 200ul of PBS (phosphate buffer solution) for cleaning for three times, adsorbing and separating the magnetic particles by using the magnetic block, and discarding the supernatant; transferring the magnetic particles to a light-emitting cup, placing the light-emitting cup in a chemiluminescence detector, adding 100ul of luminol, p-iodophenol, carbamide peroxide and other prepared luminescent substrate working solutions, and recording the luminescence amount for 6 seconds when the reaction is carried out for 2 minutes; and drawing a standard curve, and calculating the content of the plasma fibrinogen.
The invention group, take the anti-human fibrinogen polyclonal antibody filter made in example 1, dilute 100ul human fibrinogen solution or healthy human plasma to 1ml each with alkaline PBS, filter; filtering and washing with 1ml of alkaline PBS buffer solution for three times, diluting 100ul of horseradish peroxidase-labeled anti-human fibrinogen monoclonal antibody with alkaline PBS to 1ml, and filtering; filtering and washing the filtrate for three times by using 1ml of alkaline PBS buffer solution, and filtering the filtrate by using 1ml of Tris-hydrochloric acid buffer solution with pH4.5; collecting filtrate, taking 100ul to a luminescence cup, placing the luminescence cup in a chemiluminescence detector, adding 100ul of luminol, p-iodophenol, carbamide peroxide and other prepared luminescence substrate working solutions, and recording the luminescence amount for 6 seconds when the reaction is carried out for 2 minutes; and drawing a standard curve, and calculating the content of the plasma fibrinogen.
The experimental results are as follows: the result of the current chemiluminescence detection technology is 2.51g/L, the result of the invention is 2.58g/L, the results of the two experimental methods are basically consistent, but the experimental completion time of the invention is obviously shorter than that of the current technology, and the comparison experimental result shows that the whole process operation time of the current chemiluminescence detection technology is 89 minutes, and the whole process operation time of the invention is only 13 minutes.
Example 4: influence of the volume of the filter element on the detection result
Experimental materials: commercially available microfilters, NHS activated sepharose particles (ACRO BIOSYSTEMS), anti-human fibrinogen polyclonal antibody (Genagates Inc.), sodium bicarbonate, hydrochloric acid, ethanolamine, human fibrinogen.
The experimental method comprises the following steps: an anti-human fibrinogen polyclonal antibody filter with the volume of 1, 2, 3, 5, 10, 50, 100, 300, 500, 700 and 1000 cubic millimeters is prepared by adopting the method of example 1; diluting with PBS solution to prepare 1ug/ml human fibrinogen solution; diluting 100ul human fibrinogen solution to 1ml with alkaline PBS, filtering and washing with 1ml alkaline PBS buffer solution for three times, diluting 100ul horse radish peroxidase labeled anti-human fibrinogen monoclonal antibody to 1ml with alkaline PBS, and filtering; filtering and washing the filtrate for three times by using 1ml of alkaline PBS buffer solution, and filtering the filtrate by using 1ml of Tris-hydrochloric acid buffer solution with pH4.5; collecting filtrate, taking 100ul to a luminescence cup, placing the luminescence cup in a chemiluminescence detector, adding 100ul of luminol, p-iodophenol, carbamide peroxide and other prepared luminescence substrate working solutions, and recording the luminescence amount for 6 seconds when the reaction is carried out for 2 minutes.
The experimental results are as follows: the luminescence (mV) values recorded for the 1, 2, 3, 5, 10, 50, 100, 300, 500, 700, 1000 cubic millimeter filters were 843, 1597, 1871, 1925, 1967, 1983, 2042, 1956, 1995, 2035, 2068, respectively, and were substantially in equilibrium for a volume of 3 cubic millimeters. Therefore, the invention can realize immunodetection by adopting the filter with 3 cubic millimeters, so that the whole detection device has small volume and is convenient to carry.
Example 5: comparison of the detection results of the invention and the existing enzyme-linked immunoassay technology
Experimental materials: the kit comprises an anti-human fibrinogen polyclonal antibody (Genagates Inc.), an anti-human fibrinogen polyclonal antibody filter, a horseradish peroxidase labeled anti-human fibrinogen monoclonal antibody, o-phenylenediamine, an enzyme linked immunosorbent assay (ELISA) detector, a human fibrinogen solution and healthy human plasma.
The experimental method comprises the following steps: taking a human fibrinogen standard solution with a known concentration, and diluting the human fibrinogen standard solution with a PBS solution to prepare a human fibrinogen solution with the concentration of 30, 70, 100, 300, 700 and 1000 ng/ml; taking blood plasma of healthy people, and diluting by 10000 times by PBS; the experiment adopts the invention and the existing enzyme-linked immunosorbent assay technology to detect the human fibrinogen solution and draw a standard curve, then the fibrinogen of healthy people is determined, and the fibrinogen concentration is calculated by the standard curve; taking 42 test tubes, and dividing the test tubes into the group of the invention and the group of the existing enzyme-linked immunoassay technology; 3 parallel tubes were made for each sample.
The current enzyme-linked immunoassay technology group adopts a 96-hole enzyme-linked plate, 100ul of anti-human fibrinogen polyclonal antibody is added into each tube, the mixture is coated overnight at 4 ℃, the mixture is washed for three times, then 100u of human fibrinogen solution or healthy human plasma is respectively added, the binding reaction is incubated for 120 minutes at 37 ℃, the mixture is washed for three times, 100ul of horseradish peroxidase labeled anti-human fibrinogen monoclonal antibody is added, the binding reaction is incubated for 60 minutes at 37 ℃, the mixture is washed for three times, the supernatant is discarded, 100ul of color development liquid (2.43 ml of 0.1M citric acid, 2.57ml of 0.2M disodium hydrogen phosphate, 5mg of o-phenylenediamine and 5ul of hydrogen peroxide) is added, the mixture is shielded from light for 5 minutes, and 2M sulfuric acid is added to stop the reaction; reading the light absorption value on an enzyme linked immunosorbent assay detector, drawing a standard curve, and calculating the fibrinogen content of the blood plasma.
The invention group, take the anti-human fibrinogen polyclonal antibody filter made in example 1, dilute 100ul human fibrinogen solution or healthy human plasma to 1ml each with alkaline PBS, filter; filtering and washing with 1ml of alkaline PBS buffer solution for three times, diluting 100ul of horseradish peroxidase-labeled anti-human fibrinogen monoclonal antibody with alkaline PBS to 1ml, and filtering; filtering and washing the filtrate for three times by using 1ml of alkaline PBS buffer solution, and filtering the filtrate by using 1ml of Tris-hydrochloric acid buffer solution with pH4.5; collecting filtrate, taking 100ul of the enzyme linked plate with 96 holes, adding 100ul of color development liquid, keeping out of the sun for 5 minutes, and adding 2M sulfuric acid to terminate the reaction; reading the light absorption value on an enzyme linked immunosorbent assay detector, drawing a standard curve, and calculating the fibrinogen content of the blood plasma.
The experimental results are as follows: the determination result of the prior art is 2.56g/L, the determination result of the technique of the invention is 2.50g/L, the results obtained by the two experimental methods are basically consistent, but the experimental completion time of the technique of the invention is obviously shorter than that of the prior art, and the comparison experimental result shows that the whole process operation of the prior enzyme-linked immunoassay technique needs 16 hours, and the whole process operation time of the technique of the invention is only 16 minutes.
Example 6: the invention uses colloidal gold as indicator to compare the detection results
Experimental materials: the system comprises an anti-human fibrinogen polyclonal antibody filter, a colloidal gold labeled anti-human fibrinogen monoclonal antibody, a spectrophotometer and a human fibrinogen solution.
The experimental method comprises the following steps: taking a human fibrinogen standard solution with a known concentration, and diluting the human fibrinogen standard solution with a PBS solution to prepare a human fibrinogen solution with the concentration of 100, 300, 700, 1000 and 3000 ng/ml; another healthy human plasma was diluted 5000-fold with PBS. In the experiment, 100ng, 300ng, 700ng, 1000ng and 3000ng of human fibrinogen solutions are detected by adopting the technology of the invention, standard curves are drawn, then the fibrinogen of healthy people is measured, and the fibrinogen concentration is calculated by using the standard curves. 18 tubes were taken and 3 parallel tubes were made for each sample.
Taking the anti-human fibrinogen polyclonal antibody filter prepared in the embodiment 1, diluting 100ul of human fibrinogen solution or healthy human plasma to 1ml respectively by alkaline PBS, and filtering; filtering and washing with 1ml of alkaline PBS buffer solution for three times, diluting 100ul of colloidal gold labeled anti-human fibrinogen monoclonal antibody with alkaline PBS to 1ml, and filtering; filtering and washing the filtrate for three times by using 1ml of alkaline PBS buffer solution, and filtering the filtrate by using 1ml of Tris-hydrochloric acid buffer solution with pH4.5; collecting filtrate, mixing, reading absorbance at 520nm wavelength with 800ul spectrophotometer, drawing standard curve, and calculating fibrinogen content in plasma.
The experimental results are as follows: the detection result of the technology of the invention is 2.81g/L, which is basically consistent with the detection results of other methods, but the experimental completion time of the invention is obviously shorter than that of the prior art, the whole process operation of the prior colloidal gold detection technology needs 12 hours, and the whole process operation time of the invention is only 13 minutes.
Example 7: the invention adopts experimental research experimental materials for measuring alpha fetoprotein level by HRP chemiluminescence technology: NHS activated sepharose particles (ACRO BIOSYSTEMS), anti-human alpha-fetoprotein monoclonal antibody (genates Inc.), anti-human alpha-fetoprotein polyclonal antibody (genates Inc.), sodium bicarbonate, hydrochloric acid, ethanolamine, human alpha-fetoprotein (genates Inc.), HRP-goat anti-mouse IgG (ping ui organism), pico luminescent reagent (Thermo scientific).
The experimental method comprises the following steps: the method of example 1 is adopted to prepare a micro columnar filter of the anti-human alpha-fetoprotein polyclonal antibody with the volume of 50 cubic millimeters, and 3 groups are selected for repeated measurement; diluting with PBS solution to prepare 500ng/ml human alpha-fetoprotein solution; diluting 100ul human alpha-fetoprotein solution with alkaline PBS to 300ul, and filtering; filtering and washing with 1ml of alkaline PBS buffer solution, diluting 100ul of anti-human alpha-fetoprotein monoclonal antibody with alkaline PBS to 300ul, and filtering; filtering and washing with 1ml of alkaline PBS buffer solution, diluting the horseradish peroxidase-labeled goat anti-mouse polyclonal antibody with alkaline PBS to 300ul, and filtering; filtering and washing with 1ml of alkaline PBS buffer solution, collecting a filter element, adding 200ul of pico luminescence reagent, detecting the luminescence value by using a chemiluminescence apparatus, and recording.
The experimental results are as follows: table 1 shows the experimental results of the alpha fetoprotein level, the human alpha fetoprotein antigen concentration in the experiment is 500ng/ml, the maximum detection times of 3 parallel experiments are all about 40 times, and the results show that the alpha fetoprotein level of the detection sample is particularly good in repeatability.
TABLE 1 Experimental results for alpha-fetoprotein levels
Remarking: the positive and negative detection indexes are luminescence values, which are counts of luminescence values.
Example 8: experimental research for measuring carcinoembryonic antigen level by adopting HRP (horse radish peroxidase) chemiluminescence technology
Experimental materials: NHS activated sepharose particles (ACRO BIOSYSTEMS), anti-human carcinoembryonic antigen monoclonal antibodies (genates Inc.), anti-human carcinoembryonic antigen polyclonal antibodies (genates Inc.), sodium bicarbonate, hydrochloric acid, ethanolamine, human carcinoembryonic antigen (genates Inc.), HRP-goat anti-mouse IgG (ping ui biosome), pico luminescent reagent (Thermo scientific).
The experimental method comprises the following steps: the method of example 1 is adopted to prepare a 50 cubic millimeter anti-human carcino-embryonic antigen polyclonal antibody micro-cylindrical filter, and 3 groups are selected for repeated measurement; diluting with PBS solution to prepare 200ng/ml human carcinoembryonic antigen solution. Diluting 100ul human carcinoembryonic antigen solution to 300ul with alkaline PBS, filtering, and washing with 1ml alkaline PBS buffer solution; diluting 100ul of anti-human carcinoembryonic antigen monoclonal antibody to 300ul with alkaline PBS, and filtering; filtering and washing with 1ml of alkaline PBS buffer solution, diluting the horseradish peroxidase-labeled goat anti-mouse polyclonal antibody with alkaline PBS to 300ul, and filtering; filtering and washing with 1ml of alkaline PBS buffer solution, collecting a filter element, adding 200ul of pico luminescence reagent, detecting the luminescence value by using a chemiluminescence apparatus, and recording.
The experimental results are as follows: table 2 shows the experimental results of the carcinoembryonic antigen level, the human carcinoembryonic antigen concentration in the experiment is 200ng/ml, the maximum detection multiple of 3 parallel experiments is about 20 times, and the results show that the carcinoembryonic antigen level of the detection sample has good repeatability.
TABLE 2 Experimental results for Carcino-embryonic antigen levels
Remarking: the positive and negative detection indexes are luminescence values, which are counts of luminescence values.
Example 9: the invention adopts the avidin-biotin amplification technology to measure the experimental research of the alpha-fetoprotein level
Experimental materials: NHS activated sepharose particles (ACRO BIOSYSTEMS), anti-human alpha-fetoprotein monoclonal antibody (genates Inc.), anti-human alpha-fetoprotein polyclonal antibody (genates Inc.), sodium bicarbonate, hydrochloric acid, ethanolamine, human alpha-fetoprotein (genates Inc.), avidin (sigma), edc (thermo scientific), NHS (thermo scientific), MES (shanghai institute), goat anti-mouse polyclonal antibody (genates Inc.), biotin-labeled AP (shanghai bamann), APLS luminescent reagent (zhengnuo).
The experimental method comprises the following steps:
1. avidin-labeled goat anti-mouse polyclonal antibody: taking 100ul of 10mg/ml avidin, adding 50ug of goat anti-mouse IgG, supplementing MES solution to 150ul, and carrying out ice bath for 10 min; taking 12ul of 0.1mg/ml EDC solution, adding MES to 600ul, mixing uniformly, and slowly adding into the centrifuge tube; taking 18ul of NHS solution of 0.1mg/ml, adding MES to 900ul, mixing uniformly, and slowly adding into the centrifuge tube; after ice-bath for 10min, shaking on a shaker for 3 hours; taking down the centrifuge tube, and adding 600ul 1M Tris to make the final concentration 20 mM; shaking on a shaker for 1 hour; PBS buffer was dialyzed overnight at 4 ℃ and frozen at-20 ℃ for future use.
2. Detection experiment: the method of example 1 is adopted to prepare a micro columnar filter of the anti-human alpha-fetoprotein polyclonal antibody with the volume of 50 cubic millimeters, and 3 groups are selected for repeated measurement; diluting with PBS solution to prepare 500ng/ml human alpha-fetoprotein solution; diluting 100ul human alpha-fetoprotein solution to 300ul with alkaline PBS, filtering, and washing with 1ml alkaline PBS buffer solution; diluting 100ul of anti-human alpha-fetoprotein monoclonal antibody to 300ul by using alkaline PBS, and filtering; filtering and washing with 1ml of alkaline PBS buffer solution, diluting 100ul of avidin-labeled goat anti-mouse polyclonal antibody with alkaline PBS to 300ul, and filtering; filtering and washing with 1ml of alkaline PBS buffer solution, diluting biotin-labeled AP to 300ul with alkaline PBS, and filtering; filtering and cleaning with 1ml of alkaline PBS buffer solution, collecting a filter element, adding 200ul of APLS luminous reagent, detecting the luminous value by using a chemiluminescence apparatus, and recording.
The experimental results are as follows: table 3 shows the results of the level test by the avidin-biotin amplification technology, the antigen concentration of human alpha-fetoprotein in the test is 100ng/ml, the maximum detection times of 3 parallel tests are about 25 times, and the results show that the alpha-fetoprotein level of the test sample has good repeatability and can meet the requirement of accuracy detection.
TABLE 3 results of experiments using avidin-biotin amplification technique to determine alpha-fetoprotein levels
Remarking: the positive and negative detection indexes are luminescence values, which are counts of luminescence values.
While the invention has been described in connection with specific embodiments thereof, it is to be understood that it is intended by the appended drawings and description that the invention may be embodied in other specific forms without departing from the spirit or scope of the invention.
Claims (10)
1. An apparatus for quantitative immunoassay of a tumor marker, comprising: a mobile phase containing device (2), a filter (1), a detection phase containing device (3) and a detector (4); wherein,
the mobile phase containing device (2) is connected with the filter (1) through a pipeline (8);
the filter (1) comprises an inlet (5), a filter element (6) and an outlet (7);
the detection phase containing device (3) is connected with the detector (4) through a pipeline (9);
the material of the filter core (6) is a substance which can be coupled by a tumor marker specific conjugate.
2. The apparatus of claim 1, wherein the tumor marker specific binding substance is one of an enzyme, an inhibitor, a ligand, a receptor, a protein; preferably, the protein is selected from an antigen and/or an antibody.
3. The quantitative immunoassay device of claim 1,
the volume of the filter (1) is less than 1 ml; preferably, the filter element (6) is made of water-insoluble solid-phase material; more preferably, the water-insoluble solid phase material is a particulate solid phase material or a mesh-like solid phase material; most preferably, the water-insoluble solid phase material is selected from one or more of gel particles, latex particles, magnetic particles; further most preferably, the gel particles are selected from one or more of sephadex particles, sepharose particles, cyanogen bromide activated sepharose particles, NHS activated sepharose particles;
and/or the detector (4) is a mechanical structure capable of quantitatively detecting and recording color or light quantity changes; preferably, the detector (4) is selected from one or more of a chemiluminescence detector, a spectrophotometric detector, a fluorescence detector, and an enzyme-linked immunosorbent detector.
4. The quantitative immunoassay device of claim 1, wherein the tumor marker is selected from one or more of alpha-fetoprotein, carcinoembryonic antigen, cancer antigen 125, cancer antigen 15-3, carbohydrate antigen 19-9, cancer antigen 50, carbohydrate antigen 242, gastric cancer-associated antigen, ferritin, prostate specific antigen, prostatic acid phosphatase, b2 microglobulin, neuron-specific enolase, cytokeratin 19, squamous cell carcinoma antigen, nuclear matrix protein 22, a-L-fucosidase, and human epididymis secretory protein 4; preferably, the tumor marker is selected from one of alpha-fetoprotein and carcinoembryonic antigen.
5. An immune quantitative detection method of a tumor marker is characterized by comprising the following steps:
(1) marking the filter core by the tumor marker specific conjugate to prepare a marked filter core I;
(2) the labeled filter core I is used for capturing tumor markers specifically to prepare a filter core-tumor marker conjugate I;
(3) specifically capturing a detection substance in a detection phase by using the filter core-tumor marker conjugate I to prepare a filter core-tumor marker-detection substance complex, wherein the detection substance is an indication conjugate of the tumor marker specific conjugate and an indicator;
(4) detecting and calculating the content of the tumor marker.
6. The method of claim 5, wherein the antibody is immobilized on the surface of the sample,
before the step (4), eluting the filter core-tumor marker-detection substance complex by using a cleaning solution to remove the detection phase residues which are not captured and remain in the filter core;
the step of detecting and calculating the content of the tumor marker in the step (4) is selected from any one of the following methods: 1) directly detecting the filter core-tumor marker-detection substance compound to obtain the amount I of the indicator specifically captured by the filter core, and calculating the content of the tumor marker according to the amount I of the indicator; and/or, 2) directly detecting the eluent which is eluted by the cleaning solution and contains the detection substance to obtain the amount II of the indicator which is not captured and remains in the filter element, and calculating the content of the tumor marker according to the amount II of the indicator; and/or 3) directly detecting the detection phase flowing out after the specific capture in the step (3) to obtain the amount III of the indicator flowing out without being combined, and calculating the content of the tumor marker according to the amount III of the indicator.
7. An immune quantitative amplification detection method of a tumor marker is characterized by comprising the following steps:
(1) marking the filter core by a substance capable of forming specific binding with a tumor marker to prepare a marked filter core II;
(2) the tumor marker is captured by the specificity of the marked filter core II to prepare a filter core-tumor marker conjugate II;
(3) specifically capturing the avidin conjugate in the primary detection phase by using the filter core-tumor marker conjugate II to prepare a compound of the filter core-tumor marker-avidin conjugate;
(4) specifically capturing a biotin detection object in a secondary detection phase by using the filter core-tumor marker-avidin conjugate compound to prepare a filter core-tumor marker-avidin conjugate-biotin detection object compound, wherein the biotin detection object is a conjugate of biotin and an indicator;
(5) detecting and calculating the content of the tumor marker.
8. The method of claim 7, wherein the immunoassay is performed by amplifying the sample,
before the step (5), eluting the filter element-tumor marker-avidin conjugate-biotin detector complex by using a cleaning solution to remove secondary detection phase residues which are not captured and remain in the filter element;
and/or, the avidin is a substance having avidin-binding properties; preferably, the avidin is selected from one or more of ovalbumin, streptavidin, vitellin and avidin;
and/or, the step of detecting and calculating the content of the tumor marker in the step (5) is selected from any one of the following methods: 1) directly detecting the filter core-tumor marker-avidin conjugate-biotin detection object to obtain the amount IV of the indicator specifically captured by the filter core, and calculating the content of the tumor marker according to the amount IV of the indicator; 2) directly detecting eluent which is eluted by a cleaning solution and contains a biotin detection object, so as to obtain the amount V of the indicator which is not captured and remains in the filter element, and calculating the content of the tumor marker according to the amount V of the indicator; 3) and (3) directly detecting the secondary detection phase flowing out after the specific capture in the step (3) to obtain the amount VI of the unbound indicator flowing out, and calculating the content of the tumor marker according to the amount VI of the indicator.
9. The method for quantitative immunoassay according to claim 5 or the method for amplified quantitative immunoassay according to claim 7,
the indicator is a substance capable of directly or indirectly generating a color or light quantity change;
preferably, the indicator is an enzyme substance, a substance capable of directly or indirectly emitting light, a substance capable of generating fluorescence, or a substance with color;
more preferably, the enzyme substance is selected from one or more of horseradish peroxidase (HRP), Alkaline Phosphatase (AP), glucose oxidase, β -galactosidase, lysozyme and malate dehydrogenase; the substance capable of directly or indirectly emitting light is selected from one or more of luminol and derivatives thereof, lucigenin, loving alkali, peroxyoxalate esters and acridine esters; the substance capable of generating fluorescence is selected from one or more of Fluorescein Isothiocyanate (FITC) or radamine (RB 200); the material with the color is selected from colloidal gold materials.
10. Use of the apparatus of any one of claims 1 to 4, the method of any one of claims 5 to 6, or the method of any one of claims 7 to 9 for quantitative immunoassay of tumor markers in the preparation of a sample quantitative immunoassay product.
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