Immunofluorescence kit for rapidly and quantitatively detecting horse serum amyloid protein A and application of immunofluorescence kit
Through the prepared fluorescent immunochromatography kit, the antibodies and antibodies are labeled with fluorescent microspheres, the rapid, sensitive and accurate quantitative detection of amyloid A in horse serum is achieved, and the problems of low detection sensitivity and narrow range in the prior art are solved. It is suitable for early infection detection of equine disease, reducing the false negative and false positive rates, and has important disease prevention and control significance.
Patent Information
- Application Number
- CN202510564695.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-30
- Publication Date
- 2025-07-18
AI Technical Summary
In the prior art, the detection sensitivity of horse serum amyloid A is not high enough, the detection range is narrow, and it is prone to false negatives and false positives. The detection time is long, so quantitative analysis cannot be carried out quickly on site, which affects the economic benefits of the breeding industry.
The immunofluorescence kit prepared by fluorescent immunochromatography technology includes test strips, sample diluents and calibration information cards. Through the preparation of fluorescent microsphere labeled antibodies and antibodies, combined with the preparation of pads, detection pads and water absorption pads, it is assembled into immunofluorescence test strips to achieve rapid quantitative detection of horse serum amyloid A.
It realizes rapid, sensitive and accurate quantitative detection of horse serum amyloid A, with good specificity, can operate on site, reduce false negative and false positive rates, and reduce economic losses. It has important disease prevention and control significance.
Smart Images
Figure CN120334538A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biological detection technologies, and particularly to an immunofluorescence kit for rapid quantitative detection of equine serum amyloid A and its application. Background Art
[0002] Equine serum amyloid A is an acute-phase protein and an obvious marker of inflammation and tissue damage, with a relative molecular weight of about 12 KDa. Under normal circumstances, the content of SAA in a healthy organism is relatively low. When the organism is stimulated by pathogens such as viruses and bacteria or suffers from tissue damage, the liver will synthesize and secrete a large amount of SAA into the blood, and its production precedes the appearance of clinical symptoms. When the antigens in the organism are cleared, SAA can quickly drop back to normal levels. Therefore, the determination of SAA content can be applied to all stages of diseases. In the early stage of infection, the SAA index can help determine whether it is an inflammatory disease. During the disease infection process, it can be used to evaluate the severity of inflammation, the control situation of the disease, and prognosis monitoring.
[0003] "Early detection, early isolation, early treatment" can effectively control the spread of infectious diseases and is an important measure for group epidemic prevention. The determination of equine SAA content can detect diseases early before clinical symptoms appear in horses, which has very important clinical significance and can judge the health level of horses in the first time, so as to guide the clinical treatment and medication of horses.
[0004] The quantitative detection of SAA mainly uses the Elisa method, but the detection sensitivity is not high enough, the detection range is narrow, there are many influencing factors, it is easy to cause false negatives and false positives, and it takes a long time. The sample needs to be sent to the laboratory for detection. There are SAA detection kits based on different methods, but the order cycle is long and the price is expensive. The kit of the present invention can quantitatively detect the concentration of equine serum SAA by using fluorescence immunochromatography technology, and can be detected and analyzed on-site at the sampling site without the need for large-scale instrument equipment, providing a simple, highly sensitive, highly accurate and cost-effective detection means for the early screening of infectious diseases, and can greatly reduce the economic losses in the breeding industry. Summary of the Invention
[0005] The purpose of the present invention is to provide an immunofluorescence kit for rapid quantitative detection of equine serum amyloid A and its application to solve the problems in the prior art.
[0006] The present application provides an immunofluorescence kit for rapid quantitative detection of equine serum amyloid A, which is prepared by the following method:
[0007] First, the preparation of fluorescent microsphere-labeled antibody, the specific steps are as follows:
[0008] Activated microspheres: Take 100 μl of Eu carboxyl fluorescent microspheres with a solid content of 1%, add 1 ml of 0.05 mol / L boric acid buffer (pH 8.0), mix well, add 100 μl of 10 mg / ml EDC and 100 μl of 10 mg / ml NHS, mix well, and shake at 25 °C and 200 rpm on a horizontal shaker for 30 min;
[0009] Centrifuge the activated fluorescent microspheres at 4 °C and 14000 rpm for 20 min, discard the supernatant, add 1 ml of 0.05 mol / L boric acid buffer (pH 8.0) to resuspend the microspheres and mix well, and perform water bath sonication for 5 min;
[0010] Couple antibodies: Add 50 μg of rabbit anti-horse SAA monoclonal antibody 15B8 to one tube and 20 μg of chicken IgG to the other tube, and couple at 25 °C and 200 rpm on a horizontal shaker for 2 h;
[0011] Block: Add 110 μl of 10% BSA solution (prepared with 0.05 mol / L boric acid buffer), mix well, and block at 25 °C and 200 rpm on a horizontal shaker for 30 min;
[0012] Wash and store: Centrifuge the blocked fluorescent microsphere solution at 4 °C and 14000 rpm for 20 min, discard the supernatant, add 1 ml of 0.05 mol / L boric acid buffer (pH 8.0) to resuspend the microspheres and mix well, repeat the washing twice, and finally add the microsphere storage solution to resuspend the microspheres and mix well;
[0013] Secondly, the preparation of the conjugate pad is as follows:
[0014] Dilute the fluorescent microspheres labeled with two antibodies in equal proportion with 0.01 mol / L PBS solution (pH 7.4) containing 1% BSA, 1% Tween 20, 3% trehalose, and 0.1% PC300, and spray them onto the glass fiber membrane at a rate of 5 μl / cm using a quantitative film spraying instrument, and dry at 37 °C for 4 hours to prepare a microsphere conjugate pad, and store it in a sealed and dry manner;
[0015] Then, the preparation of the test pad is as follows:
[0016] Dilute the horse SAA monoclonal antibody and goat anti-chicken IgG antibody to 2 mg / ml with 0.01 mol / L PBS solution (pH 7.4) containing 0.5% BSA, 3% trehalose, and 0.1% PC300, and spray the two antibodies onto the nitrocellulose membrane at a rate of 1 μl / cm using a quantitative film spraying instrument, respectively as the test line (T line) and the control line (C line), with a 6 mm interval between the two lines, dry at 37 °C for 4 hours, and store it in a sealed and dry manner;
[0017] Then, the preparation of the sample pad is as follows:
[0018] Soak the glass cellulose membrane in a 0.01 mol / L PBS solution (pH 7.4) containing 0.5% BSA, 1% Tween 20, and 0.1% PC300, dry it at 37°C for 4 h, and store it in a sealed and dry condition;
[0019] Then, the preparation of the absorbent pad is as follows:
[0020] Use absorbent filter paper as the absorbent pad;
[0021] Then, the assembly of the immunofluorescence test strip is as follows:
[0022] On the PVC bottom plate, sequentially lap and paste the treated sample pad, the conjugate pad sprayed with fluorescent microsphere-labeled antibody, the nitrocellulose membrane test pad sprayed with the test line T and the control line C, and the absorbent pad; after assembly, cut it into a 3.8 mm test strip and put it into a plastic cartridge;
[0023] Finally, the preparation of the kit. The kit includes 1 test strip prepared above, 1 tube of 500 ml sample diluent, and 1 calibration information card. Put the prepared test strip into an aluminum foil bag, put in a desiccant and then seal it; the composition of the sample diluent is: 0.05 mol / L PBS solution containing 0.1% BSA and 0.1% Tween 20; the calibration information card contains calibration curve information.
[0024] This application also provides the application of the immunofluorescence kit for rapid quantitative detection of equine serum amyloid A in the quantitative detection of equine serum amyloid A.
[0025] Compared with the prior art, the present invention discloses an immunofluorescence kit for rapid quantitative detection of equine serum amyloid A (serum amyloid A, SAA) and its application. The kit includes a test strip, a cartridge, a sample diluent, and a calibration information card. The test strip includes a bottom plate, a sample pad, a conjugate pad, a test pad, and an absorbent pad. Among them, the conjugate pad has a rabbit anti-equine SAA monoclonal antibody and chicken IgG labeled with fluorescent microspheres, and the test pad is coated with a rabbit anti-equine SAA monoclonal antibody and a goat anti-chicken IgG antibody. The present invention can rapidly and quantitatively detect equine SAA, has good specificity, high sensitivity, and a wide detection range. It is an effective detection method and can be used for the early infection detection of some diseases, which is of great significance for the prevention and control of equine diseases. BRIEF DESCRIPTION OF THE DRAWINGS
[0026] Figure 1 It is the standard curve graph of the fluorescence immunoassay of equine SAA provided by the present invention. DETAILED DESCRIPTION OF THE INVENTION
[0027] Embodiments of the present invention will be described in detail below. Examples of the embodiments are shown in the accompanying drawings, where like or similar reference numerals denote like or similar elements or elements having like or similar functions throughout. The embodiments described below by referring to the drawings are exemplary only for explaining the present invention and should not be construed as limiting the present invention.
[0028] In the description of the present invention, it should be understood that the terms "center", "longitudinal", "lateral", "length", "width", "thickness", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", "clockwise", "counterclockwise", "axial", "radial", "circumferential", etc. indicate the orientation or positional relationship based on the orientation or positional relationship shown in the drawings. They are only for facilitating the description of the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and thus should not be construed as limiting the present invention.
[0029] In addition, the terms "first" and "second" are only used for descriptive purposes and should not be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, the features defined with "first" and "second" may explicitly or implicitly include one or more of such features. In the description of the present invention, "a plurality of" means two or more unless otherwise specifically defined.
[0030] In the present invention, unless otherwise clearly specified and defined, the terms such as "mounted", "connected", "coupled", "fixed", etc. should be understood in a broad sense. For example, it may be a fixed connection, a detachable connection, or integrated; it may be a mechanical connection or an electrical connection; it may be directly connected or indirectly connected through an intermediate medium, and it may be the internal communication of two elements or the interaction relationship between two elements. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.
[0031] In the present invention, unless otherwise clearly specified and defined, the first feature being "on" or "under" the second feature may be the direct contact between the first and second features, or the indirect contact between the first and second features through an intermediate medium. Moreover, the first feature being "above", "over" and "on top of" the second feature may be that the first feature is directly above or obliquely above the second feature, or merely indicates that the first feature has a higher horizontal height than the second feature. The first feature being "under", "below" and "beneath" the second feature may be that the first feature is directly below or obliquely below the second feature, or merely indicates that the first feature has a lower horizontal height than the second feature.
[0032] The implementation of the present invention will be described in detail below in conjunction with specific embodiments.
[0033] Example 1. The preparation method of the immunofluorescence test strip for detecting equine serum amyloid A is as follows:
[0034] First, the preparation of the fluorescent microsphere-labeled antibody is carried out according to the following specific steps:
[0035] Activating the microspheres. Take 100 μl of Eu carboxyl fluorescent microspheres with a solid content of 1%, add 1 ml of 0.05 mol / L boric acid buffer (pH 8.0), mix well, add 100 μl of 10 mg / ml EDC and 100 μl of 10 mg / ml NHS, after mixing well, shake at 25 °C and 200 rpm on a horizontal shaker for 30 min.
[0036] Centrifuge the activated fluorescent microspheres at 4 °C and 14000 rpm for 20 min, discard the supernatant, add 1 ml of 0.05 mol / L boric acid buffer (pH 8.0) to resuspend the microspheres and mix well, and perform water bath ultrasonic treatment for 5 min.
[0037] Coupling the antibody. Add 50 μg of rabbit anti-horse SAA monoclonal antibody 15B8 to one tube, and add 20 μg of chicken IgG to the other tube, and couple at 25 °C and 200 rpm on a horizontal shaker for 2 h.
[0038] Blocking. Add 110 μl of 10% BSA solution (prepared with 0.05 mol / L boric acid buffer), mix well, and block at 25 °C and 200 rpm on a horizontal shaker for 30 min.
[0039] Washing and storing. Centrifuge the blocked fluorescent microsphere solution at 4 °C and 14000 rpm for 20 min, discard the supernatant, add 1 ml of 0.05 mol / L boric acid buffer (pH 8.0) to resuspend the microspheres and mix well, repeat the washing twice, and finally add the microsphere storage solution to resuspend the microspheres and mix well.
[0040] Secondly, the preparation of the conjugate pad is carried out according to the following specific steps:
[0041] Dilute the fluorescent microspheres labeled with two kinds of antibodies proportionally with 0.01 mol / L PBS solution (pH 7.4) containing 1% BSA, 1% Tween 20, 3% trehalose, and 0.1% PC300, and spray it on the glass fiber membrane at a rate of 5 μl / cm with a quantitative film spraying instrument, and dry at 37 °C for 4 hours to prepare a microsphere conjugate pad, and store it in a sealed and dry manner.
[0042] Then, the preparation of the test pad is carried out according to the following specific steps:
[0043] Dilute the equine SAA monoclonal antibody and goat anti-chicken IgG antibody to 2 mg / ml with 0.01 mol / L PBS solution (pH 7.4) containing 0.5% BSA, 3% trehalose, and 0.1% PC300. Spray the two antibodies onto the nitrocellulose membrane at a rate of 1 μl / cm using a quantitative spraying film apparatus, serving as the test line (T line) and the quality control line (C line) respectively. The two lines are spaced 6 mm apart and dried at 37°C for 4 hours, then stored in a sealed and dry condition.
[0044] Then, the preparation of the sample pad is as follows:
[0045] Soak the glass fiber membrane with 0.01 mol / L PBS solution (pH 7.4) containing 0.5% BSA, 1% Tween 20, and 0.1% PC300, dry it at 37°C for 4 h, and store it in a sealed and dry condition.
[0046] Then, the preparation of the absorbent pad is as follows:
[0047] Use absorbent filter paper as the absorbent pad.
[0048] Finally, the assembly of the immunofluorescence test strip is as follows:
[0049] On the PVC bottom plate, successively overlap and paste the treated sample pad, the conjugate pad sprayed with fluorescent microsphere-labeled antibody, the nitrocellulose membrane test pad sprayed with the test line T and the quality control line C, and the absorbent pad. After assembly, cut it into test strips with a width of 3.8 mm and put them into plastic cartridges.
[0050] Example 2, Preparation and Detection Method of the Kit
[0051] The main components and quantities of the kit are shown in the following table
[0052] Kit Composition Specification Test Strip 1 piece / bag Sample Diluent 500 μl / tube Calibration Information Card 1 box
[0053] Packaging of the test card: Put the test strip prepared in Example 1 into an aluminum foil bag, put in a desiccant, and then seal it.
[0054] Preparation of the sample diluent: 0.05 mol / L PBS solution containing 0.1% BSA and 0.1% Tween 20.
[0055] The calibration information card contains calibration curve information.
[0056] The detection method is as follows:
[0057] Check the batch numbers of the test card, the sample diluent, and the calibration information card to ensure that the batch numbers of the three are the same;
[0058] Insert the calibration information card into the instrument chip port;
[0059] Pierce the buffer sealing film with a conical cap, take 5 μl of horse blood / serum / plasma, add it to a centrifuge tube containing sample diluent, cover the tube cap, and mix well for 30 s;
[0060] Insert the test strip into the reader, add 3 drops of the above sample to the sample addition hole of the test strip, click the button to start timing and measurement, and the instrument will automatically display the test result after 10 minutes.
[0061] Example 3. Performance analysis of the kit of the present invention
[0062] 1 Draw a standard curve
[0063] Dilute the horse SAA reference product with sample diluent to concentrations of 160 mg / L, 80 mg / L, 40 mg / L, 20 mg / L, 10 mg / L, 5 mg / L, 2.5 mg / L, 1.25 mg / L, 0.625 mg / L, 0 mg / L (i.e., sample diluent) respectively. Detect with the horse SAA fluorescence immunochromatography kit of the present invention, and read the fluorescence signals of the quality control line C and the test line T with a fluorescence analyzer. Each concentration is measured in parallel 10 times. Draw a standard curve with the logarithm of the horse SAA concentration as the abscissa and the logarithm of the mean value of T / C as the ordinate, and obtain the fitting curve equation. The results are as Figure 1 shown.
[0064] It can be seen from the results that the correlation coefficient R2 of this curve equation is greater than 0.99, showing a good linear relationship. The concentration of horse SAA can be quantitatively determined through this standard curve.
[0065] 2 Specificity
[0066] Take hemoglobin (3 g / L), C-reactive protein (0.1 g / L), and horse cholesterol (5 g / L) and mix them with the horse 50 mg / L SAA internal reference product in equal proportions. Detect the concentration of SAA with the kit of the present invention. Each sample is detected 10 times repeatedly, and the average value and relative deviation of the results are calculated. The results are shown in the following table:
[0067] Group Average Measured Value of Equine SAA (mg / L) Relative Deviation (%) Hemoglobin (3 g / L) 26.4 5.68 C-Reactive Protein (0.1 g / L) 23.6 5.48 Cholesterol (5 g / L) 26.8 7.20
[0068] The results show that the relative deviations of the three groups of substances are all less than 10%. It indicates that these three substances in the blood have no influence on the detection of horse SAA. The kit of the present invention has good specificity and strong anti-interference ability.
[0069] 3 Accuracy
[0070] Add equal volumes of horse SAA protein with concentrations of 20 mg / L, 60 mg / L, and 120 mg / L to horse SAA serum (measured value: 0.85 mg / L), detect the concentration of horse SAA protein with the kit of the present invention, and detect each concentration 5 times respectively to calculate its recovery rate.
[0071]
[0072] The results are shown in the following table:
[0073]
[0074] The results show that the recovery rates are all between 90% and 110%, indicating that the accuracy of this kit is good.
[0075] 4 Blank limit
[0076] The SAA internal standard product (i.e., sample diluent) with a concentration of 0 mg / ml was detected using the horse SAA detection kit of the present invention, and the detection was repeated 20 times. The average value of T / C was 0.03. Substituting this value into the calibration curve, the blank limit was obtained as 0.53 mg / L.
[0077] Example 4, Preliminary clinical application of the horse SAA immunofluorescence kit
[0078] 50 horse sera were collected, and the horse SAA concentrations were detected using the kit of the present invention and a commercial kit respectively. The results showed that 45 horses had normal SAA concentrations and 5 had abnormal SAA concentrations when detected with the kit of the present invention, and 45 horses had normal SAA concentrations and 5 had abnormal SAA concentrations when detected with the commercial kit, with a coincidence rate of 100%. For the samples of horses with abnormal SAA concentrations, pathogen detection was carried out. A total of 3 samples were detected positive for equine piroplasm nucleic acid, and no conventional pathogens were detected in the other 2 samples. Therefore, SAA detection can make an early judgment on diseases, detect them early, know the drug use strategies for diseases, thereby reducing the risk of disease transmission and the property losses of farmers.
[0079] In the description of this specification, the description with reference to terms such as "one embodiment", "some embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.
[0080] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.
Claims
1. An immunofluorescence kit for rapid quantitative detection of equine serum amyloid A, characterized in that, It is prepared by the following method: First, the preparation of fluorescent microsphere-labeled antibody, the specific steps are as follows: Activate the microspheres. Take 100 μl of Eu carboxyl fluorescent microspheres with a solid content of 1%, add 1 ml of 0.05 mol / L boric acid buffer (pH 8.0), mix well, add 100 μl of 10 mg / ml EDC and 100 μl of 10 mg / ml NHS, after mixing well, shake at 25 °C and 200 rpm on a horizontal shaker for 30 min; Centrifuge the activated fluorescent microspheres at 4 °C and 14000 rpm for 20 min, discard the supernatant, add 1 ml of 0.05 mol / L boric acid buffer (pH 8.0) to resuspend the microspheres and mix well, and perform water bath ultrasound for 5 min; Couple the antibody. Add 50 μg of rabbit anti-horse SAA monoclonal antibody 15B8 to one tube and 20 μg of chicken IgG to the other tube, and couple at 25 °C and 200 rpm on a horizontal shaker for 2 h; Block. Add 110 μl of 10% BSA solution (prepared with 0.05 mol / L boric acid buffer), mix well, and block at 25 °C and 200 rpm on a horizontal shaker for 30 min; Wash and store. Centrifuge the blocked fluorescent microsphere solution at 4 °C and 14000 rpm for 20 min, discard the supernatant, add 1 ml of 0.05 mol / L boric acid buffer (pH 8.0) to resuspend the microspheres and mix well, repeat the washing twice, and finally add the microsphere storage solution to resuspend the microspheres and mix well; Secondly, the preparation of the conjugate pad, the specific steps are as follows: Dilute the fluorescent microspheres labeled with two kinds of antibodies in equal proportion with 0.01 mol / L PBS solution (pH 7.4) containing 1% BSA, 1% Tween 20, 3% trehalose, and 0.1% PC300, and spray them on the glass fiber membrane at a rate of 5 μl / cm with a quantitative film spraying instrument, and dry at 37 °C for 4 hours to prepare a microsphere conjugate pad, and store it in a sealed and dry manner; Then, the preparation of the test pad, the specific steps are as follows: Dilute the horse SAA monoclonal antibody and goat anti-chicken IgG antibody to 2 mg / ml with 0.01 mol / L PBS solution (pH 7.4) containing 0.5% BSA, 3% trehalose, and 0.1% PC300, and spray the two antibodies on the nitrocellulose membrane at a rate of 1 μl / cm with a quantitative film spraying instrument, respectively as the test line (T line) and the control line (C line), and the two lines are spaced 6 mm apart, and dry at 37 °C for 4 hours, and store it in a sealed and dry manner; Then, the preparation of the sample pad, the specific steps are as follows: Soak the glass fiber membrane with 0.01 mol / L PBS solution (pH 7.4) containing 0.5% BSA, 1% Tween 20, and 0.1% PC300, and dry at 37 °C for 4 h, and store it in a sealed and dry manner; Then, the preparation of the absorbent pad, the specific steps are as follows: Use absorbent filter paper as the absorbent pad; Then, the assembly of the immunofluorescence test strip, the specific steps are as follows: On the PVC bottom plate, the processed sample pad, the conjugate pad sprayed with fluorescent microsphere-labeled antibody, the nitrocellulose membrane test pad sprayed with the test line T and the control line C, and the absorbent pad are successively overlapped and pasted; after assembly, it is cut into a test strip with a width of 3.8 mm and loaded into a plastic cartridge; Finally, the preparation of the kit. The kit includes 1 test strip prepared previously, 1 tube of 500 ml sample diluent, and 1 calibration information card. The prepared test strip is loaded into an aluminum foil bag, and after putting in a desiccant, it is sealed; the composition of the sample diluent is: 0.05 mol / L PBS solution containing 0.1% BSA and 0.1% Tween 20; The calibration information card contains calibration curve information.
2. Application of the immunofluorescence kit for rapid quantitative detection of equine serum amyloid A according to claim 1 in the quantitative detection of equine serum amyloid A.