CN117849354A - Kit for rapidly and quantitatively detecting human serum amyloid A - Google Patents
Kit for rapidly and quantitatively detecting human serum amyloid A Download PDFInfo
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- CN117849354A CN117849354A CN202410047466.0A CN202410047466A CN117849354A CN 117849354 A CN117849354 A CN 117849354A CN 202410047466 A CN202410047466 A CN 202410047466A CN 117849354 A CN117849354 A CN 117849354A
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Classifications
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- G—PHYSICS
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- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- 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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
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- 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
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54386—Analytical elements
- G01N33/54387—Immunochromatographic test strips
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- 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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- 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
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/775—Apolipopeptides
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/70—Mechanisms involved in disease identification
- G01N2800/7095—Inflammation
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Abstract
The invention discloses a kit for rapidly and quantitatively detecting human serum amyloid A, which comprises an immunofluorescence test strip and a clamping shell, wherein the immunofluorescence test strip comprises a conjugate gasket, and an SAA monoclonal antibody-biotin-streptavidin-fluorescent dye conjugate is loaded on the conjugate gasket. The invention also discloses a preparation method of the fluorescent dye conjugate, which comprises the following steps: s1, marking a mouse anti-human SAA monoclonal antibody by biotin to form an SAA monoclonal antibody-biotin; labeling the fluorescent dye with streptavidin to form streptavidin-fluorescent dye; s2, mixing the SAA monoclonal antibody-biotin and streptavidin-fluorescent dye into a buffer solution, and incubating. The immunofluorescence kit can rapidly and quantitatively detect the human serum amyloid A, a detection sample does not need to be diluted, and the human serum amyloid A in whole blood, serum or plasma can be rapidly, accurately and highly sensitively detected.
Description
Technical Field
The invention belongs to the technical field of medical instruments, and particularly relates to the technical field of in-vitro diagnosis and detection, in particular to a kit for rapidly and quantitatively detecting human serum amyloid A.
Background
Serum amyloid a (serum amyloid A protein, SAA) is a family of polygene-encoded polymorphic proteins, which are acute phase response proteins, playing an important role in disease diagnosis and treatment. In the acute phase of inflammation or infection, SAA increases rapidly within 48-72 hours and decreases rapidly in the convalescence phase. SAA has high sensitivity, and studies have shown that serum SAA is detected in diseases such as bacterial infection, virus infection, atherosclerosis, coronary heart disease, acute graft rejection, tumor and the like, and SAA has important value for early diagnosis of the diseases.
Current methods for clinical and laboratory detection of SAA include radioimmunoassay, enzyme-linked immunosorbent assay, latex turbidimetry, immunochromatography, and the like. The existence of radiation pollution in radioimmunoassay has gradually exited the clinical examination field; the ELISA method has the advantages of low detection sensitivity, narrow detection range, more influence factors, easy false negative and false positive, and is mainly applied to the items with low sensitivity requirements such as infectious disease screening. The emulsion immunoturbidimetry is simple and rapid to operate, but has low sensitivity and low-value repeatability. Wherein the immunochromatography method comprises a colloidal gold quantitative immunochromatography method, a fluorescent quantitative immunochromatography method and a chemiluminescent quantitative detection method. The principle of the fluorescent quantitative immunochromatography used in clinic at present is a double-antibody sandwich method, and most of the fluorescent markers used are fluorescent microspheres and fluorescent dye direct labeling processes. However, immunofluorescence double-antibody sandwich assay requires dilution of the sample and is cumbersome to perform. Therefore, developing a kit for detecting human serum amyloid A by using more effective fluorescent quantitative immunochromatography and better fluorescent markers has important value. Until now, no other person has developed a human serum amyloid a immunochromatographic assay kit that does not require dilution of the sample.
Disclosure of Invention
In order to solve the problems in the prior art, the invention discloses a kit for quantitatively detecting human serum amyloid A by using a fluorescent immunochromatography technology based on a competition method principle of a novel fluorescent polymer. The specific technical scheme is as follows:
in a first aspect of the invention, a kit for rapid quantitative detection of human serum amyloid A is provided, comprising an immunofluorescence test strip and a cartridge, wherein the immunofluorescence test strip comprises a conjugate gasket, and is characterized in that the conjugate gasket is loaded with a fluorescent dye conjugate, and the fluorescent dye conjugate is SAA monoclonal antibody-biotin-streptavidin-fluorescent dye conjugate.
Further, the fluorescent dye is CF770.
In some specific embodiments, the immunofluorescence test strip comprises a support pad, a detection membrane, a filter pad, a water absorption pad and a sample pad, wherein the support pad is provided with the detection membrane, two ends of the detection membrane are respectively connected with the filter pad and the water absorption pad, and two ends of the coupling compound pad are respectively connected with the filter pad and the sample pad.
Further, the sample pad, the conjugate pad, the filter pad, the detection membrane and the water absorption pad are all arranged on the support pad and are in linear arrangement.
In some embodiments, the sample pad is coated with a red blood cell blocking agent.
Further, the red blood cell blocking agent is a murine anti-human red blood cell monoclonal antibody.
In some embodiments, the detection membrane is provided with a test strip T line coated with human serum amyloid A antigen and a quality control strip C line coated with goat anti-streptavidin polyclonal antibody.
In some embodiments, the sample pad is made of glass fiber; the material of the coupler gasket is glass fiber; the filter gasket is made of glass fiber; the detection membrane is a nitrocellulose membrane; the supporting gasket is made of polyvinyl chloride; the water absorbing pad is made of a mixture of glass fiber and cotton velvet.
The kit adopts an immunofluorescence chromatography technology, and quantitatively detects the concentration of serum amyloid A in a sample based on the principle of a double antigen competition method. Firstly, a sample to be detected is added into a sample adding port of a detection test strip, and along with the flow of liquid, the sample to be detected firstly passes through a sample gasket 1 and then is combined with fluorescent dye conjugates coated on a conjugate gasket 2 to form a reaction complex (antigen 1-antibody reaction). The reaction complex moves forward along the detection membrane 4, the fluorescent conjugate which is not combined with the to-be-detected object is captured by the serum amyloid A antigen fixed on the detection line T of the detection membrane 4 (antigen 2-antibody reaction), the more serum amyloid A in the sample is, the fewer the complex accumulated on the detection line T is, the weaker the fluorescent signal is, the intensity of the fluorescent signal is inversely related to the number of the to-be-detected object in the sample, and the concentration of the serum amyloid A in the to-be-detected sample can be detected through a matched instrument. The fluorescent dye conjugate is captured by the sheep anti-streptavidin polyclonal antibody immobilized on the detection membrane 4 to be used as a quality control line C line.
In a second aspect of the present invention, there is provided a method for preparing a fluorescent dye conjugate, comprising the steps of:
s1, marking a mouse anti-human SAA monoclonal antibody by biotin to form an SAA monoclonal antibody-biotin; labeling the fluorescent dye with streptavidin to form streptavidin-fluorescent dye;
s2, mixing the SAA monoclonal antibody-biotin and streptavidin-fluorescent dye into a buffer solution, and incubating.
In some specific embodiments, in the step S2, SAA monoclonal antibody-biotin and streptavidin-fluorescent dye are mixed in a buffer according to the mass ratio of streptavidin to biotin of 1:3-7, and incubated for 30-60 min at 23-27 ℃.
Further, in the step S2, the SAA monoclonal antibody-biotin and streptavidin-fluorochrome are mixed with biotin 1 according to streptavidin: 4 in a buffer solution, and incubating at 25 ℃ for 30min.
In a third aspect of the invention, there is provided the use of a fluorescent dye conjugate in the preparation of a kit for rapid quantitative detection of human serum amyloid a.
Further, the fluorescent dye conjugate is SAA monoclonal antibody-biotin-streptavidin-fluorescent dye conjugate.
Compared with the prior art, the invention has the following beneficial effects:
the immunofluorescence kit can rapidly and quantitatively detect the human serum amyloid A, a detection sample does not need to be diluted, and the human serum amyloid A in whole blood, serum or plasma can be rapidly, accurately and highly sensitively detected, so that the kit is beneficial to rapidly diagnosing nonspecific inflammation.
The conception, specific structure, and technical effects of the present invention will be further described with reference to the accompanying drawings to fully understand the objects, features, and effects of the present invention.
Drawings
FIG. 1 is a schematic structural diagram of an immunofluorescence test strip of example 1.
FIG. 2 is a graph showing the relationship between the sample concentration and the SAA detection signal of the conjugate-based test strip of example 1 of the present invention.
FIG. 3 is a graph showing the relationship between the concentration of a sample detected by the sandwich method and the signal detected directly.
FIG. 4 is a correlation of concentration and signal for the detection of clinical samples using the conjugate-based dipsticks of example 1.
The figure indicates: 1. a sample pad; 2. a conjugate pad; 3. a filter pad; 4. a detection membrane; 5. a support pad; 6. a water absorbing pad.
Detailed Description
The invention is further described with reference to the following detailed description in order to make the technical means, the inventive features, the achieved objects and the effects of the invention easy to understand. The present invention is not limited to the following examples.
It should be understood that the structures, proportions, sizes, etc. shown in the drawings are for illustration purposes only and should not be construed as limiting the invention to the extent that it can be practiced, since modifications, changes in the proportions, or otherwise, used in the practice of the invention, are not intended to be critical to the essential characteristics of the invention, but are intended to fall within the spirit and scope of the invention.
Besides the already described, various reagents, antibodies and consumables involved in the examples are all commercial products, and commercial products can be adopted.
Example 1
Kit (I)
As shown in figure 1, a kit for rapidly and quantitatively detecting human serum amyloid A, in particular to a kit for quantitatively detecting human serum amyloid A based on the principle of a competition method by adopting a fluorescence immunochromatography technology. Comprises an immunofluorescence test strip, an upper card shell, a lower card shell and a standard curve. The test strip comprises a support gasket 5 and a detection membrane 4 arranged on the support gasket 5, wherein two ends of the detection membrane 4 are respectively connected with a filter gasket 3 and a water absorption gasket 6, the filter gasket 3 is connected with a coupler gasket 2, and the other end of the coupler gasket 2 is connected with a sample gasket 1; the sample pad 1 is coated with an erythrocyte blocker, in particular a mouse anti-human erythrocyte monoclonal antibody, a human serum amyloid A test strip T line and a quality control strip C line are sequentially arranged on the detection membrane 4 from the sample pad 1 to the water absorption pad 6, the two are parallel and perpendicular to the longitudinal axis of the detection membrane 4, the human serum amyloid A test strip T line is coated with a human serum amyloid A antigen, the quality control strip C line is coated with a sheep anti-streptavidin polyclonal antibody, and the conjugate pad 2 is coated with a fluorescent dye conjugate.
The fluorescent dye conjugate is SAA monoclonal antibody-biotin-streptavidin-fluorescent dye conjugate, and the conjugate is applied to an immunochromatography platform for the first time and independently by the company.
In this embodiment, the sample pad 1 is made of glass fiber; the material of the coupler gasket 2 is glass fiber; the filter gasket 3 is made of glass fiber; the detection membrane 4 is a nitrocellulose membrane; the supporting pad 5 is made of polyvinyl chloride; the water absorbing pad 6 is made of a mixture of glass fiber and cotton velvet.
The sample gasket 1, the conjugate gasket 2, the filter gasket 3, the detection membrane 4 and the water absorption gasket 6 are all arranged on the support gasket 5 and are in linear arrangement.
Preparation of (II) conjugate pad
The mouse anti-human SAA monoclonal antibody is marked by biotin and a fluorescent dye CF770 marked by streptavidin is adopted, and then the molar ratio of the streptavidin to the biotin is 1:4 are mixed in phosphate buffer with pH=8.4, incubated for 30min at 25 ℃, and the two are bridged to form SAA monoclonal antibody-biotin-streptavidin-fluorescent dye conjugate. Phosphate buffer solution is used as solvent to prepare coating solution of 0.15 mg/ml. The coating solution is respectively coated on the same glass fiber gasket in a uniform scribing mode, and the coating areas are all in a strip shape and parallel to each other and are perpendicular to the longitudinal axis of the glass fiber gasket.
(III) preparation of detection Membrane
From the sample pad to the water absorbing pad (i.e. according to the direction of sample flow), a human serum amyloid A test strip and a quality control strip are coated on a nitrocellulose membrane in sequence. Serum amyloid A antigen was diluted with phosphate buffer to a final concentration of 1.0mg/ml, and coated on nitrocellulose membrane using a uniform streaking to form a human serum amyloid A test strip. The streptavidin antibody is added into phosphate buffer solution to prepare solution with the final concentration of 0.018mg/ml, and the solution is coated on a nitrocellulose membrane by a uniform scribing mode to form a quality control zone.
Comparative example 1 preparation of immunofluorescence reagent strip for quantitative detection of human serum amyloid A based on fluorescent microspheres
The immunofluorescence reagent strip for quantitatively detecting human serum amyloid A comprises a supporting gasket 5 and a detecting film 4 arranged on the supporting gasket 5, wherein two ends of the detecting film 4 are respectively connected with a filtering gasket 3 and a water absorbing gasket 6, the filtering gasket 3 is connected with a combining gasket 2, the other end of the combining gasket is connected with a sample gasket 1, wherein the sample gasket 1 is coated with a red blood cell blocking agent, a human serum amyloid A testing strip T line and a quality control strip C line are sequentially arranged on the detecting film 4 from the sample gasket 1 to the water absorbing gasket 6, the human serum amyloid A testing strip is coated with human serum amyloid A antigen, the quality control strip C line is coated with goat anti-mouse polyclonal antibody, and the coupling gasket 2 is coated with SAA monoclonal antibody-microsphere markers.
The bonding pad is prepared by the following method: to 0.5ml of MES solution at pH5.5, 5mg of microspheres were activated by adding EDC (the number of moles of EDC is the same as the number of moles of carboxyl groups on the surface of the microspheres), and 0.5mg of murine anti-human SAA monoclonal antibody was added to couple the SAA antibody to the surface of the microspheres. The coating solution is prepared by diluting the microsphere marked with the antibody by taking phosphate buffer solution containing sucrose and BSA as a solvent. The coating solution is respectively coated on the same glass fiber gasket in a uniform scribing mode, and the coating areas are all in a strip shape and parallel to each other and are perpendicular to the longitudinal axis of the glass fiber gasket.
The detection film is prepared by the following method: from the sample pad to the water absorbing pad (i.e. according to the direction of sample flow), a human serum amyloid A test strip and a quality control strip are coated on a nitrocellulose membrane in sequence. Serum amyloid A antigen was diluted with phosphate buffer to a final concentration of 1.0mg/ml, and coated on nitrocellulose membrane using a uniform streaking to form a human serum amyloid A test strip. Adding goat anti-mouse antibody into phosphate buffer solution to prepare solution with final concentration of 0.8mg/ml, and coating on a nitrocellulose membrane by using a uniform streaking mode to form a quality control zone.
Test strips for serum amyloid a detection were laminated according to the structure of fig. 1 in example 1.
Comparative example 2 preparation of immunofluorescence reagent strip for quantitative detection of human serum amyloid A based on fluorescent dye
The immunofluorescence reagent strip for quantitatively detecting human serum amyloid A comprises a supporting gasket 5 and a detecting film 4 arranged on the supporting gasket 5, wherein two ends of the detecting film 4 are respectively connected with a filtering gasket 3 and a water absorbing gasket 6, the filtering gasket 3 is connected with a combining gasket 2, the other end of the combining gasket is connected with a sample gasket 1, wherein the sample gasket 1 is coated with a red blood cell blocking agent, a human serum amyloid A testing strip T line and a quality control strip C line are sequentially arranged on the detecting film 4 from the sample gasket 1 to the water absorbing gasket 6, the human serum amyloid A testing strip is coated with human serum amyloid A antigen, the quality control strip C line is coated with goat anti-mouse polyclonal antibody, and the coupling gasket 2 is coated with SAA monoclonal antibody-dye marker.
The bonding pad is prepared by the following method: the murine anti-human SAA monoclonal antibody was labeled with the fluorescent dye CF770 (molar ratio of the two 1:15) and incubated in phosphate buffer at pH=8.4 for 30min at 25℃to form SAA monoclonal antibody-dye label. Phosphate buffer solution is used as solvent to prepare coating solution of 0.15 mg/ml. The coating solution is respectively coated on the same glass fiber gasket in a uniform scribing mode, and the coating areas are all in a strip shape and parallel to each other and are perpendicular to the longitudinal axis of the glass fiber gasket.
The detection film is prepared by the following method: from the sample pad to the water absorbing pad (i.e. according to the direction of sample flow), a human serum amyloid A test strip and a quality control strip are coated on a nitrocellulose membrane in sequence. Serum amyloid A antigen was diluted with phosphate buffer to a final concentration of 1.0mg/ml, and coated on nitrocellulose membrane using a uniform streaking to form a human serum amyloid A test strip. Adding goat anti-mouse antibody into phosphate buffer solution to prepare solution with final concentration of 0.8mg/ml, and coating on a nitrocellulose membrane by using a uniform streaking mode to form a quality control zone.
Test strips for serum amyloid a detection were laminated according to the structure of fig. 1 in example 1. Example 2 sensitivity and detection Linear Range of immunofluorescence test strip
High concentrations of SAA antigen were formulated at about 400, 200, 100, 50, 25, 5, 1mg/L. Using the 230246008 lot serum amyloid A assay kit (immunofluorescence) marketed by the company (Su Xie, reference 20202400773)
Detecting the prepared sample according to the specification, namely adding 5ul of the sample into 995ul of diluent, uniformly mixing, adding 60ul of diluted sample into a sample adding hole of a kit, incubating for 8min at 18 ℃, then placing the sample into a Rayleigh biological immunofluorescence detector for detection, calculating according to a conventional method to obtain SAA concentration, specifically calculating relative optical density by detecting fluorescence intensity of a test strip and a quality control strip, and calculating by a relative optical density-concentration standard curve to obtain SAA concentration as follows: 400. 152.7, 88.4, 34.2, 18.4, 8.1, 1mg/L.
The kit prepared by the methods of the invention example 1, the comparative example 1 and the comparative example 2 is used for detecting the sample prepared by the antigen, 60ul of the sample is dripped on a sample pad of the test strip, incubated for 8min at 18 ℃, and then placed in a Rayleigh immunofluorescence detector for detection. Regression analysis was performed on two sets of data with the detection concentration as an independent variable x and the detection result signal of the immunofluorescence test strip of the present invention as a dependent variable y, and a linear regression equation (i.e., concentration-signal curve) was found, with the results shown in table 1 and fig. 2 below. In Table 1, the linear range of example 1 is 1-400mg/L, R 2 0.9981; in comparative examples 1 and 2, T bands were "off-line" at concentrations of 34mg/L and 88.4mg/L, respectively, i.e., the signal of the T band was the background signal of the band, and the concentration was upward, and the C/T detected by the band did not show a linear relationship with the concentration.
TABLE 1 sample concentration and SAA detection results for test strips of example 1, comparative example 1, and comparative example 2
As can be seen from table 1 and fig. 2, example 1 has a wider concentration detection range.
At the same time, the company also provides a fluorescent immunity layer close to the wavelength of excitation light and emission light of the experimental fluorescent detectorThe fluorescent dye and fluorescent microsphere of some common markers in the analysis are subjected to comparative experimental study, and NIR770, alexa Fluor 750, CF770, and,800CW, BP Light 800, cy7 and +.>The beads Inforred (715/755), QDOT800, cdTe quantum dots, suzhou Nami 1# and 2# Infrared carboxyl microspheres were compared and studied with reference to the experimental methods of comparative example 2 or comparative example 1. The results of the study showed that these markers were far from the broad range of example 1 in terms of concentration detection. It is apparent that example 1 has a significantly wider concentration detection range.
The sample prepared by the antigen is directly detected (i.e. the sample is not diluted) by adopting the SAA sandwich method kit on the market of the company, 60ul of the sample is dripped on a sample pad of the test strip, incubated for 8min at 18 ℃, and then placed in a Rayleigh immunofluorescence detector for detection. The concentration detected by the sandwich method is taken as an independent variable x, the detection result signal of the immunofluorescence test strip of the invention is taken as a dependent variable y, regression analysis is carried out on two groups of data, a linear regression equation (namely a concentration-signal curve) is obtained, and the results are shown in the following table 2 and figure 3, so that an obvious HOOK effect appears.
TABLE 2 Signal relationship between sample concentration and direct detection for dilution method
Sample concentration (mg/L) | Signal signal |
1 | 4.821 |
8.1 | 3.0819 |
18.4 | 1.7711 |
34.2 | 0.9292 |
88.4 | 0.6424 |
152.7 | 0.4829 |
400 | 0.3478 |
As can be seen from the results of Table 2 and FIG. 3, SAA detection using the conjugate-based dipsticks of the present invention can be directly detected without dilution of the sample, with a linear range of 1mg/l to 400mg/l, and a dose-response curve correlation coefficient (r) of greater than 0.990.
Example 3 test results on clinical samples
6 clinical samples (the sample concentration is obtained by detecting the Siemens kit) are detected by adopting the test strip of the embodiment 1, and each sample is repeatedly detected for 3 times. The specific detection method comprises the following steps: 60ul of clinical patient samples (serum) are taken and added to a sample pad of the test strip, incubated for 8min at 18 ℃, then placed in a Rayleigh biological immunofluorescence detector for detection, the detection results of the same sample are subjected to average and variance analysis by detecting the fluorescence intensity of a test strip and a quality control strip, and the variation coefficient is calculated, and the results are shown in Table 3.
TABLE 3 example 1 conjugate-based test strip for detecting concentration versus Signal relationship and precision of clinical samples
In the colloidal gold immunochromatography detection kit (the pharmaceutical industry standard YYT/1713-2020 of the people's republic of China), the requirement for the repeatability of the quantitative detection kit is not higher than 15%. The above results show that: the variation coefficient of the repeated detection result is less than 10%, which indicates that the reagent has good repeatability of the detection result and high precision.
And detecting a plurality of clinical samples (the sample concentration is detected by a Siemens kit), carrying out regression analysis on two groups of data by taking the existing concentration of the clinical samples as an independent variable x and the signal of the immunofluorescence test strip of the invention as a dependent variable y, and solving a linear regression equation (namely a concentration-signal curve), wherein the results are shown in the following charts 4 and 4.
TABLE 4 example 1 detection of correlation of concentration and Signal in clinical samples
Table 4 and figure 4 the results show: detecting clinical samples, the sample concentration and the detection signal show good correlation (R 2 =0.9926)。
The foregoing describes in detail preferred embodiments of the present invention. It should be understood that numerous modifications and variations can be made in accordance with the concepts of the invention without requiring creative effort by one of ordinary skill in the art. Therefore, all technical solutions which can be obtained by logic analysis, reasoning or limited experiments based on the prior art by the person skilled in the art according to the inventive concept shall be within the scope of protection defined by the claims.
Claims (10)
1. The kit for rapidly and quantitatively detecting the human serum amyloid A comprises an immunofluorescence test strip and a clamping shell, wherein the immunofluorescence test strip comprises a conjugate gasket, and is characterized in that a fluorescent dye conjugate is loaded on the conjugate gasket, and the fluorescent dye conjugate is an SAA monoclonal antibody-biotin-streptavidin-fluorescent dye conjugate.
2. The kit of claim 1, wherein the fluorescent dye is CF770.
3. The kit according to claim 1, wherein the immunofluorescence test strip comprises a support gasket, a detection membrane, a filter gasket, a water absorption gasket and a sample gasket, the detection membrane is arranged on the support gasket, two ends of the detection membrane are respectively connected with the filter gasket and the water absorption gasket, and two ends of the conjugate gasket are respectively connected with the filter gasket and the sample gasket; the sample gasket, the conjugate gasket, the filter gasket, the detection membrane and the water absorption gasket are all arranged on the support gasket and are linearly arranged.
4. A kit according to claim 1 or 3, wherein the sample pad is coated with a red blood cell blocker.
5. The kit of claim 4, wherein the erythrocyte blocker is a murine anti-human erythrocyte monoclonal antibody.
6. The kit according to claim 1 or 3, wherein the detection membrane is provided with a test strip T line coated with human serum amyloid A antigen and a quality control strip C line coated with goat anti-streptavidin polyclonal antibody.
7. A preparation method of a fluorescent dye conjugate is characterized by comprising the following steps:
s1, marking a mouse anti-human SAA monoclonal antibody by biotin to form an SAA monoclonal antibody-biotin; labeling the fluorescent dye with streptavidin to form streptavidin-fluorescent dye;
s2, mixing the SAA monoclonal antibody-biotin and streptavidin-fluorescent dye into a buffer solution, and incubating.
8. The method according to claim 7, wherein in the step S2, the SAA monoclonal antibody-biotin and streptavidin-fluorochrome are mixed with biotin 1: 3-7, and mixing the materials in a buffer solution, and incubating for 30-60 min at the temperature of 23-27 ℃.
9. The method according to claim 8, wherein in the step S2, the SAA monoclonal antibody-biotin and streptavidin-fluorochrome are mixed with biotin 1:4 in a buffer solution, and incubating at 25 ℃ for 30min.
Application of SAA monoclonal antibody-biotin-streptavidin-fluorescent dye conjugate in preparation of kit for rapid quantitative detection of human serum amyloid A.
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