CN112485449A - Spot immunogold filtration kit for detecting cat SAA and semi-quantitative detection method - Google Patents
Spot immunogold filtration kit for detecting cat SAA and semi-quantitative detection method Download PDFInfo
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- CN112485449A CN112485449A CN202011320032.1A CN202011320032A CN112485449A CN 112485449 A CN112485449 A CN 112485449A CN 202011320032 A CN202011320032 A CN 202011320032A CN 112485449 A CN112485449 A CN 112485449A
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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
- G01N33/68—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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54346—Nanoparticles
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
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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
Abstract
The invention relates to a dot immunogold filtration kit for detecting cat SAA, which comprises a filtration device, a cat SAA antibody marked by colloidal gold, confining liquid and washing liquid, wherein the filtration device sequentially comprises a filtrate layer, a microporous filter membrane, a water absorption cushion layer and a bottom plate from top to bottom, the filtrate layer is provided with through holes corresponding to the microporous filter membrane, and the microporous filter membrane is coated with the cat SAA antibody. The kit and the semi-quantitative detection method can be used for quickly and accurately detecting the SAA content in the cat body, have the advantages of high specificity, low cost, simple and portable instrument, high sensitivity and the like, and can be used for field inspection.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a dot immunogold filtration kit for detecting cat SAA and a semi-quantitative detection method.
Background
Serum Amyloid A (SAA) is a sensitive acute phase reaction protein secreted into serum by hepatocytes, is a precursor substance of Amyloid A (AA), has higher sensitivity and specificity than C-reactive protein (CRP), has become a new research hotspot in the field of modern medical hygiene, can better reflect the degree of inflammation, and when an organism is infected or injured, the protein can be sharply increased in a short time and rapidly reduced to a normal level after the infection of the organism is eliminated. Feline serum amyloid A (i.e., feline SAA) is the most sensitive agent in the indicators of systemic inflammatory response in cats and must be diseased when the SAA value increases. The presence of inflammation in cats can also be determined by detecting the presence of SAA in the cat when clinical symptoms are not clear. When the sick cat is rechecked after operation or treatment, the operation effect or the postoperative recovery condition, whether postoperative infection exists or not and the like can be judged by detecting whether the cat body contains the SAA or not.
The clinical laboratory generally uses methods such as an immunoturbidimetry method, chemiluminescence immunoassay, an enzyme-linked immunosorbent assay and the like for detecting the cat SAA, wherein the immunoturbidimetry method has relatively high detection cost and needs to be used by a matched instrument; chemiluminescence immunoassay has poor selectivity, can react to a series of compounds, is not specific to a single compound, has emission intensity depending on various environmental factors, and has large difference between curves of the emission intensity and time in different environmental systems, so that various external factors must be strictly controlled; the enzyme-linked immunosorbent assay combines an antibody and an enzyme compound, and then detects through color development, has the advantages of high detection speed, low cost, simple and portable instrument, high sensitivity, strong selectivity and the like, can be used for field detection, but the specificity and the repeatability of the enzyme-linked immunosorbent assay need to be improved.
In 1971, Faulk and Taylor (Immunochem, 8: 1081, 1971) first used immunocolloidal gold as a marker for immunoelectron microscopy, and thereafter immunocolloidal gold technology was widely used as a new immunological method. Common gold labeling immune methods include dot immuno gold filtration assay (dot immuno gold filtration assay) and lateral immuno gold chromatography (bipolar immuno gold chromatography), and the lateral immuno gold chromatography has strict requirements on the environment and can be influenced by air humidity after long-time detection, so the technology is clinically restricted. Colloidal immunogold filtration is also a well established technology, and diagnostic kits for a variety of different analytes are currently available on the market from a number of manufacturers, but there is no dot immunogold filtration kit for the detection of feline SAA.
Disclosure of Invention
The invention aims to quickly and accurately detect cat SAA, and provides a dot immunogold filtration kit and a detection method for detecting cat SAA.
In order to realize above-mentioned purpose, the application provides a detect cat SAA's spot immune gold filtration kit, including filtration device, colloidal gold mark's cat SAA antibody, confining liquid, washing liquid, the filtration device down includes filtrate layer, millipore filtration membrane, water absorption bedding course and bottom plate from last in proper order, be equipped with the through-hole corresponding with millipore filtration membrane on the filtrate layer, the millipore filtration membrane upper cladding has cat SAA antibody.
As a further improvement of the application, the concentration of the cat SAA antibody coated on the microporous filter membrane is 0.3 mg/ml-1.5 mg/ml, the pH value of the confining liquid is 6.5-7.0, and the pH value of the washing liquid is 6.5-7.5.
As a further improvement of the present application, the blocking solution comprises 0.05% Triton-100 and 0.2% BSA, and the washing solution is 50mol/L PBS solution with pH 7.2.
As a further refinement of the present application, the concentration of the feline SAA antibody coated on the microfiltration membrane is 0.8 mg/ml.
As a further improvement of the present application, the colloidal gold-labeled feline SAA antibody is as followsThe preparation method comprises the following steps: s1, preparing a colloidal gold solution: boiling 100ml of 0.01% chloroauric acid solution, slowly adding 1-2 ml of 1% trisodium citrate under heating and stirring, continuously boiling for 15min until the color is stable to red, cooling, and recovering the original volume with double distilled water to obtain colloidal gold solution; with 0.1mol/L of K2CO3Adjusting the pH value of the colloidal gold solution to 6.5-7.0 by using the solution, centrifuging the colloidal gold solution at the rotating speed of 2000rpm, and removing the precipitate to obtain the colloidal gold solution; s2, preparation of colloidal gold labeled cat SAA antibody: diluting the cat SAA antibody with PBS (phosphate buffer solution) with the pH value of 7.2, dropwise adding the diluted cat SAA antibody into the colloidal gold solution obtained in the step S1, stirring the solution uniformly, continuously dropwise adding 2.5ml, 5% BSA and 0.1ml, 1% sodium azide, mixing the solution uniformly, standing the mixture at 4 ℃ for 15-22 hours to obtain a colloidal gold-labeled cat SAA antibody; s3, purification of colloidal gold labeled feline SAA antibody: firstly, centrifuging the colloidal gold labeled cat SAA antibody at the rotating speed of 2000rpm, discarding a first precipitate, reserving a first supernatant, then centrifuging the first supernatant at the rotating speed of 10000rpm, discarding a second supernatant, resuspending the second precipitate by using 1ml of 1mol/L PBS buffer solution with the pH value of 7.2 to obtain the colloidal gold labeled cat SAA antibody, and storing at 4 ℃ for later use.
As a further improvement of the present application, in step S2, the concentration of the cat SAA antibody diluted is 0.2mg/ml to 0.3 mg/ml.
As a further development of the present application, the microfiltration membrane coated with feline SAA antibody is prepared by the following steps: diluting the cat SAA antibody by PBS buffer solution, spotting the cat SAA antibody on a microporous filter membrane, naturally airing, immersing the microporous filter membrane in 1% BSA solution, sealing, naturally drying at room temperature, sealing, drying and storing.
As a further improvement of the application, the sample application device is a micro sample applicator, and the sample application amount is 1 ul-2 ul.
In order to achieve the above object, the present application also provides a semi-quantitative detection method using the dot immunogold filtration kit for detecting cat SAA, comprising the steps of: s1, dropwise adding a confining liquid to the microporous filter membrane through the through hole in the percolation device until the confining liquid completely permeates the microporous filter membrane; s2, dropwise adding a sample to be detected on the microporous filter membrane in the step S1 through the through hole in the percolation device, so that the sample to be detected fully permeates the microporous filter membrane; s3, dropwise adding a washing solution to the microporous filter membrane in the step S2 through the through hole in the percolation device, so that the washing solution completely permeates the microporous filter membrane; s4, dropwise adding the cat SAA antibody marked by the colloidal gold to the microporous filter membrane in the step S3 through the through hole on the percolation device until the cat SAA antibody marked by the colloidal gold completely permeates the microporous filter membrane; s5, dropwise adding the washing solution to the microporous filter membrane in the step S4 through the through hole in the percolation device, so that the washing solution completely permeates the microporous filter membrane; s6, performing numerical analysis on the color development result by using a gold-labeled reading instrument; s7, a set of standard sample to be tested containing cat SAA antigen is set, the concentration of cat SAA antigen in the standard sample to be tested is respectively set to 0ug/ml, 0.5ug/ml, 1.0ug/ml, 2.0ug/ml, 4.0ug/ml, 8.0ug/ml, 10.0ug/ml, 14.0ug/ml, 18.0ug/ml, the standard sample to be tested is respectively tested according to the steps of S1-S6, a standard curve is made by taking the test result of the standard sample to be tested as the reference, in the standard curve: taking the concentration of the cat SAA antigen as an abscissa and the numerical value of the gold-labeled reading instrument as an ordinate; and detecting an unknown sample to be detected with unknown cat SAA concentration according to the steps of S1-S6, and judging a result according to the standard curve.
As a further improvement of the present application, in step S1, the amount of the blocking solution is 50 ul; in step S2, the amount of the sample to be measured is 25 ul; in step S3, the amount of the washing solution is 50 ul; in step S4, the amount of the colloidal gold-labeled feline SAA antibody is 25 ul; in step S5, the amount of the washing solution was 50 ul.
The beneficial effects of this application lie in, this application provides a detect cat SAA spot immune gold filtration kit, including filtration device, colloidal gold mark's cat SAA antibody, confining liquid, washing liquid, the filtration device is from last down including filtrating layer, millipore filtration membrane, the bedding course and the bottom plate that absorb water in proper order, be equipped with the through-hole corresponding with millipore filtration membrane on the filtrating layer, the millipore filtration membrane upper cladding has cat SAA antibody, and this application still provides an application and detects the half quantitative detection method of cat SAA spot immune gold filtration kit. The kit and the semi-quantitative detection method can be used for quickly and accurately detecting the SAA in the cat, and have the advantages of high sensitivity and specificity, simple marker, no need of radioactive isotopes, enzyme chromogenic substrates with potential carcinogens, no need of a fluorescence microscope, high detection speed, low cost, simple and portable instruments, high sensitivity and the like, and can be used for field inspection.
Drawings
FIG. 1 is a schematic diagram of the construction of the diafiltration unit in a dot immunogold filtration kit for detecting cat SAA;
in the figure: 1. a filtrate layer; 2. a microporous filtration membrane; 3. a water-absorbing cushion layer; 4. a base plate.
Detailed Description
In order to make the objects, technical solutions and advantages of the present application more apparent, the technical solutions of the present application will be described in detail and completely with reference to the following specific embodiments of the present application and the accompanying drawings. It should be understood that the described embodiments are only a few embodiments of the present application, not all embodiments, and are not intended to limit the scope of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.
In order to improve cat SAA's detection speed and precision, reduce and detect the cost, the application provides a detect cat SAA's spot immune gold infiltration kit, including infiltration device, colloidal gold mark's cat SAA antibody, confining liquid, washing liquid, the infiltration device down includes filtrate layer 1, microfiltration membrane 2, water absorption bedding course 3 and bottom plate 4 from last in proper order, be equipped with the through-hole corresponding with microfiltration membrane 2 on the filtrate layer 1, the 2 coats of microfiltration membrane has cat SAA antibody. In the present application, the microfiltration membrane 2 is further preferably a nitrocellulose membrane; as a preferable embodiment, the concentration of the cat SAA antibody coated on the microfiltration membrane 2 is 0.3mg/ml to 1.5mg/ml, the pH value of the confining liquid is 6.5 to 7.0, and the pH value of the washing liquid is 6.5 to 7.5. Further preferably, the concentration of the feline SAA antibody coated on the microfiltration membrane 2 is 0.8 mg/ml.
In the present application, the microfiltration membrane 2 coated with the feline SAA antibody is prepared by the following steps: diluting the cat SAA antibody by PBS buffer solution, spotting the cat SAA antibody on a microporous filter membrane 2, naturally drying, immersing the microporous filter membrane 2 in 1% BSA solution, sealing, naturally drying at room temperature, sealing, drying and storing. Further preferably, the sample application device is a micro sample applicator, and the sample application amount is 1 ul-2 ul. In this application, as a preferred embodiment, the infiltration device is a plastic capsule with a size of 3cm x 0.6cm, and is divided into an upper cover layer, a lower cover layer and a bottom layer, the cover layer is the infiltration layer, the center of the cover layer is provided with a small hole with a diameter of 0.5cm, the bottom layer is filled with a padding material with strong water absorption, the padding material is equivalent to the water absorption pad layer 3, the bottom layer is equivalent to the bottom plate 4, a microporous filter membrane 2 with a diameter of 1.2cm is placed under the small hole of the cover layer in a manner of being tightly attached to the padding material, the microporous filter membrane 2 is further preferably an NC membrane, and the cover layer and the bottom layer are tightly closed, namely the infiltration. In the further percolation device, 1 ul-2 ul of cat SAA antibody diluted by PBS and having the concentration of 0.8mg/ml is spotted on the microporous filter membrane 2 by using a microspotter, and the cat SAA antibody is naturally dried, immersed in 1% BSA for 1min for sealing, naturally dried at room temperature and stored in a sealed plastic bag with a drying agent. As a preferred example, the blocking solution comprises 0.05% Triton-100 and 0.2% BSA, and the washing solution is 50mol/L PBS solution with pH 7.2.
In this application, as another preferred embodiment, the infiltration device down includes filtrating layer 1, microfiltration membrane 2, water absorption bed course 3 and bottom plate 4 from last in proper order, be equipped with the through-hole corresponding with microfiltration membrane 2 on the filtrating layer 1, water absorption bed course 3 is located on the bottom plate 4, be equipped with the through-hole relative with microfiltration membrane 2 on the filtrating layer 1, the 2 coats of microfiltration membrane has cat SAA antibody. The filtrate layer 1 is prepared from a non-water-absorbing material, the diameter of the through hole formed in the center of the filtrate layer 1 is preferably 0.5cm, and the non-water-absorbing material is preferably release paper; the microporous filter membrane 2 is a microporous membrane with affinity with protein, and is further preferably an NC membrane, and a cat SAA antibody is coated on the microporous filter membrane; the water absorption cushion layer 3 is made of water absorption materials. As the preferred embodiment, the water absorption cushion layer 3 is provided with a groove in the center, the center of the groove is provided with a bulge matched with the shape and size of the coated probe, the coated region of the probe is tightly connected with the bulge part of the water absorption cushion, and the rest part of the microporous filter membrane 2 is not contacted with the water absorption cushion layer 3.
In the present application, as a preferred embodiment, the colloidal gold-labeled feline SAA antibody is prepared by the following steps: s1, preparing a colloidal gold solution: boiling 100ml of 0.01% chloroauric acid solution, slowly adding 1-2 ml of 1% trisodium citrate under heating and stirring, continuously boiling for 15min, rapidly changing the solution color from light yellow to gray, then to black, finally stabilizing to red until the color is stabilized to red, cooling, and recovering to the original volume by using double distilled water after cooling to obtain a colloidal gold solution; with 0.1mol/L of K2CO3Adjusting the pH value of the colloidal gold solution to 6.5-7.0 by using the solution, centrifuging the colloidal gold solution at the rotating speed of 2000rpm, precipitating precipitated colloidal gold particles after adjusting the pH value, and removing the precipitate to obtain the colloidal gold solution; s2, preparation of cat SAA antibody labeled with colloidal gold: diluting the cat SAA antibody with PBS (phosphate buffer solution) with the pH value of 7.2, dropwise adding the diluted cat SAA antibody into the colloidal gold solution obtained in the step S1, stirring the solution uniformly, mixing the solution uniformly, continuously and dropwise adding 2.5ml, 5% BSA and 0.1ml, 1% sodium azide, mixing the solution uniformly, standing the solution at 4 ℃ for 15-22 h, preferably 17h, and obtaining the cat SAA antibody marked by the colloidal gold; s3, purification of colloidal gold labeled feline SAA antibody: centrifuging the colloidal gold-labeled cat SAA antibody at the rotating speed of 2000rpm, discarding a first precipitate, reserving a first supernatant, centrifuging the first supernatant at the rotating speed of 10000rpm, discarding a second supernatant, and carefully sucking the second supernatant to obtain a second precipitate, and re-suspending 1ml of 1mol/L PBS buffer solution with the pH value of 7.2 to obtain the colloidal gold-labeled cat SAAntibody A, stored at 4 ℃ for further use. Further, in the step S2, both the 5% BSA and the 1% sodium azide are filtered through a microporous filter membrane 2; in the step S2, the dilution concentration of the cat SAA antibody is 0.2 mg/ml-0.3 mg/ml; in the step S3, the time for centrifuging the first supernatant at 10000rpm is 30 min.
In the present application, as a preferred embodiment, the present application provides an operating method of a dot immunogold filtration kit for detecting cat SAA, wherein the filtration device is a plastic capsule with a size of 3cm × 0.6cm, and is divided into an upper cover layer, a lower cover layer and a bottom layer, the microfiltration membrane 2 in the filtration device is an NC membrane, 1) the filtration device is taken out, and 2 drops (about 50ul) of a blocking solution are dropped on the NC membrane of the through hole; 2) adding 1 drop (about 25ul) of the sample to be detected on the NC membrane after the sample permeates into the cover layer; 3) when the cleaning solution permeates into the cover layer, 2 drops (about 50ul) of cleaning solution are dripped on the NC membrane; 4) adding 1 drop (about 25ul) of colloidal gold-labeled cat SAA antibody into the NC membrane after the cat SAA antibody permeates into the cover layer, and reacting the cat SAA antibody with the sample adsorbed on the NC membrane; 5) when the cleaning solution permeates into the cover layer, 2 drops (about 50ul) of cleaning solution are dripped on the NC membrane; 6) until the cleaning solution completely permeates into the cover layer. And (4) observing results: when a red reaction appears on the through-hole NC film, the film is positive, and the film is negative and not colored.
In order to improve the detection speed and accuracy of cat SAA and reduce the detection cost, the application also provides a semi-quantitative detection method using the cat SAA spot immunogold filtration detection kit, which comprises the following steps: s1, dropwise adding a confining liquid to the microporous filter membrane 2 through the through hole on the percolation device until the confining liquid completely permeates into the microporous filter membrane 2; s2, dropwise adding a sample to be detected on the microporous filter membrane 2 in the step S1 through the through hole in the percolation device, so that the sample to be detected fully permeates the microporous filter membrane 2; s3, dropwise adding a washing solution to the microfiltration membrane 2 in the step S2 through the through hole in the percolation device, so that the washing solution completely permeates into the microfiltration membrane 2; s4, dropwise adding the cat SAA antibody marked by the colloidal gold to the microfiltration membrane 2 in the step S3 through the through hole on the percolation device until the cat SAA antibody marked by the colloidal gold completely permeates the microfiltration membrane 2; s5, dropwise adding the washing solution to the microfiltration membrane 2 in the step S4 through the through hole in the percolation device, so that the washing solution completely permeates into the microfiltration membrane 2; s6, performing numerical analysis on the color development result by using a gold-labeled reading instrument; s7, a set of standard sample to be tested containing cat SAA antigen is set, the concentration of cat SAA antigen in the standard sample to be tested is respectively set to 0ug/ml, 0.5ug/ml, 1.0ug/ml, 2.0ug/ml, 4.0ug/ml, 8.0ug/ml, 10.0ug/ml, 14.0ug/ml, 18.0ug/ml, the standard sample to be tested is respectively tested according to the steps of S1-S6, a standard curve is made by taking the test result of the standard sample to be tested as the reference, in the standard curve: taking the concentration of the cat SAA antigen as an abscissa and the numerical value of the gold-labeled reading instrument as an ordinate; and detecting an unknown sample to be detected with unknown cat SAA antigen concentration according to the steps of S1-S6, and judging a result according to the standard curve. Further preferably, the standard samples to be tested are tested for at least 5 times, and an average measurement value is selected.
In the present application, as a preferred embodiment: in step S1, the amount of the confining liquid is 50 ul; in step S2, the amount of the sample to be measured is 25 ul; in step S3, the amount of the washing solution is 50 ul; in step S4, the amount of the colloidal gold-labeled feline SAA antibody is 25 ul; in step S5, the amount of the washing solution was 50 ul.
In the present application, as a preferred embodiment, the present application further provides a semi-quantitative detection method using the feline SAA dot immunogold filtration assay kit, wherein 0.5ug/ml, 1.0ug/ml, 2.0ug/ml, 4.0ug/ml, 8.0ug/ml, 10.0ug/ml, 14.0ug/ml and 18.0ug/ml of the standard sample to be detected containing the feline SAA antigen are detected by using the kit, and each concentration is measured for 5 times, and 0ug/ml is used as a blank control. And (5) carrying out interpretation quantitative analysis on the color development result by using a gold-labeled reading instrument. And (3) taking the concentration of the cat SAA antigen as an abscissa and a gold-labeled reading instrument (the detection result of each sample is the average value of 5 samples under the concentration) as an ordinate to obtain a standard curve. On the standard curve, the measured signal value is increased along with the increase of the concentration of the standard sample to be detected, the highest peak is reached when the concentration is 10.0ug/ml, and then the curve is gradually gentle. According to the calculation of the standard curve, the kit can partially detect samples with the concentrations of 0.5ug/ml and 1.0ug/ml and completely detect samples with the concentration of 2.0ug/ml, the lowest detection limit is less than or equal to 2.0ug/ml, and the shade of the reaction color is in direct proportion to the concentration of the cat SAA antigen. Interpretation of the results: when the concentration is less than or equal to 2.0ug/ml, no red reaction appears on the small-hole NC membrane, and the result is negative; when the concentration is greater than 2.0ug/ml, a red reaction appears on the small-pore NC membrane, the result is positive, and the darker the color, the higher the concentration. In this application, the theory of operation of gold mark reading appearance detects the colour that absorbs on the detection card through soaking the sample with the detection card, and the gold mark count appearance is the reflection photometer. The colloidal gold has selectivity for light absorption (reflection), and the reflectance for green light (wavelength around 530 nm) decreases with the increase of the precipitation amount of the colloidal gold, so that the concentration of the analyte can be calculated. Basically, the gold marker can perform rapid detection only by using a detection card on the market.
The basic principle of the invention is as follows: the antigen-antibody reaction and washing are rapidly completed by liquid infiltration through the membrane on a special infiltration device by using the microfiltration membrane 2 as a carrier and utilizing the filterability of the microfiltration membrane 2. The dot immunodiafiltration assay was originally developed on the basis of a dot ELISA, using a conjugate that is enzyme-labeled, and is called a dot enzyme immunodiafiltration assay. In the early 90 s, dot immuno-filtration assay (DIGFA), also called gold drop immunoassay (abbreviated as gold drop method), was developed using colloidal gold as a marker. The reaction of enzyme to substrate is not needed in the gold dropping method, the result can be directly observed after the operation of the method is finished, the detection speed is high, the result is clear at a glance, and the defects of complicated ELISA operation and long time consumption are overcome.
To validate the performance of the feline SAA dot immunogold filtration kit, the following performance tests were performed:
and (3) stability testing: and (4) respectively taking the cat SAA dot immunogold filtration kits of the same batch (all stored at 37 ℃) for stability experiments at 3d, 7d, 14d and 28d, testing by using a standard substance with the concentration of 2.0ug/ml, and carrying out interpretation quantitative analysis on the color development result by using a gold standard reading instrument, wherein the results are consistent, which indicates that the cat SAA dot immunogold filtration kit for detection has good stability.
And (3) sensitivity test: randomly selecting 100 clinical cat serum samples, and detecting by using the kit and an immunofluorescence chromatography detection card method, wherein the quantity of the obtained SAA content range samples is shown in a table I:
table one: comparison of detection results of dot immunogold filtration kit and immunofluorescence chromatography detection card
As can be seen from the above table, the dot immunogold filtration kit for detecting cat SAA has good stability.
Intra-batch difference: the same batch of cat SAA dot immunogold filtration kits are tested by using a standard substance of 2.0ug/ml, and the color development depth of the obtained results is consistent, which indicates that the cat SAA dot immunogold filtration kits have good batch-to-batch difference.
The difference between batches: the cat SAA dot immunogold filtration kit of different batches is tested by using a standard substance of 2.0ug/ml, and the color development depth of the obtained result is consistent, which indicates that the cat SAA dot immunogold filtration kit has good batch-to-batch difference.
To sum up, the application provides a detect cat SAA's spot immune gold filtration kit, including filtration device, colloidal gold mark's cat SAA antibody, confining liquid, washing liquid, the filtration device down includes filtrate layer, millipore filtration membrane, water absorption bedding course and bottom plate from last in proper order, be equipped with the through-hole corresponding with millipore filtration membrane on the filtrate layer, the millipore filtration membrane upper cladding has cat SAA antibody, and the application still provides an application and detects cat SAA's spot immune gold filtration kit's semi-quantitative detection method. The kit and the semi-quantitative detection method can be used for quickly and accurately detecting the SAA in the cat, and have the advantages of high sensitivity and specificity, simple marker, no need of radioactive isotopes, enzyme chromogenic substrates with potential carcinogens, no need of a fluorescence microscope, high detection speed, low cost, simple and portable instruments, high sensitivity and the like, and can be used for field inspection.
Although the description is given in terms of embodiments, not every embodiment includes only a single embodiment, and such description is for clarity only, and those skilled in the art will recognize that the embodiments described herein may be combined as a whole to form other embodiments as would be understood by those skilled in the art.
The above-listed detailed description is only a specific description of a possible embodiment of the present invention, and they are not intended to limit the scope of the present invention, and equivalent embodiments or modifications made without departing from the technical spirit of the present invention should be included in the scope of the present invention.
Claims (10)
1. The utility model provides a detect cat SAA's spot immune gold filtration kit, its characterized in that, includes filtration device, colloidal gold mark's cat SAA antibody, confining liquid, washing liquid, the filtration device includes filtrate layer (1), millipore filtration membrane (2), water absorption bedding course (3) and bottom plate (4) from last down in proper order, be equipped with the through-hole corresponding with millipore filtration membrane (2) on filtrate layer (1), millipore filtration membrane (2) is upcoated with cat SAA antibody.
2. The dot immunogold filtration kit according to claim 1, wherein the concentration of the cat SAA antibody coated on the microfiltration membrane (2) is 0.3mg/ml to 1.5mg/ml, the pH of the blocking solution is 6.5 to 7.0, and the pH of the washing solution is 6.5 to 7.5.
3. The dot immunogold filtration kit of claim 2 for detecting cat SAA wherein the blocking solution comprises 0.05% Triton-100 and 0.2% BSA and the wash solution is 50mol/L in PBS ph 7.2.
4. The dot immunogold filtration kit of claim 2 for detecting feline SAA wherein the concentration of said feline SAA antibody coated on said microfiltration membrane (2) is 0.8 mg/ml.
5. The dot immunogold filtration kit of any one of claims 1 to 4 for detecting cat SAA wherein the colloidal gold labeled cat SAA antibody is prepared by the steps of:
s1, preparing a colloidal gold solution: boiling 100ml of 0.01% chloroauric acid solution, slowly adding 1-2 ml of 1% trisodium citrate under heating and stirring, continuously boiling for 15min until the color is stable to red, cooling, and recovering the original volume with double distilled water to obtain colloidal gold solution; with 0.1mol/L of K2CO3Adjusting the pH value of the colloidal gold solution to 6.5-7.0 by using the solution, centrifuging the colloidal gold solution at the rotating speed of 2000rpm, and removing the precipitate to obtain the colloidal gold solution;
s2, preparation of colloidal gold labeled cat SAA antibody: diluting the cat SAA antibody with PBS (phosphate buffer solution) with the pH value of 7.2, dropwise adding the diluted cat SAA antibody into the colloidal gold solution obtained in the step S1, stirring the solution uniformly, continuously dropwise adding 2.5ml, 5% BSA and 0.1ml, 1% sodium azide, mixing the solution uniformly, standing the mixture at 4 ℃ for 15-22 hours to obtain a colloidal gold-labeled cat SAA antibody;
s3, purification of colloidal gold labeled feline SAA antibody: firstly, centrifuging the colloidal gold labeled cat SAA antibody at the rotating speed of 2000rpm, discarding a first precipitate, reserving a first supernatant, then centrifuging the first supernatant at the rotating speed of 10000rpm, discarding a second supernatant, resuspending the second precipitate by using 1ml of 1mol/L PBS buffer solution with the pH value of 7.2 to obtain the colloidal gold labeled cat SAA antibody, and storing at 4 ℃ for later use.
6. The dot immunogold filtration kit of claim 5 wherein in step S2 the concentration of feline SAA antibody diluted is from 0.2mg/ml to 0.3 mg/ml.
7. The dot immunogold filtration kit according to any one of claims 1 to 4 for detecting feline SAA wherein said microfiltration membrane (2) coated with feline SAA antibody is prepared by the steps of: diluting the cat SAA antibody by PBS buffer solution, spotting the cat SAA antibody on a microporous filter membrane (2), naturally drying, immersing the microporous filter membrane (2) in 1% BSA solution, sealing, naturally drying at room temperature, sealing, drying and storing.
8. The dot immunogold filtration kit of claim 7 for detecting cat SAA wherein the spotting device is a micropipette and the amount of spotting is between 1ul and 2 ul.
9. A semi-quantitative detection method of the dot immunogold filtration kit of claim 1 for detecting cat SAA comprising the steps of:
s1, dropwise adding a confining liquid to the microporous filter membrane (2) through the through hole on the percolation device until the confining liquid completely permeates into the microporous filter membrane (2);
s2, dropwise adding a sample to be detected on the microporous filter membrane (2) in the step S1 through the through hole in the percolation device, so that the sample to be detected fully permeates the microporous filter membrane (2);
s3, dropwise adding a washing solution to the microfiltration membrane (2) in the step S2 through the through hole on the percolation device, so that the washing solution completely permeates into the microfiltration membrane (2);
s4, dropwise adding the cat SAA antibody marked by the colloidal gold to the microfiltration membrane (2) in the step S3 through the through hole on the percolation device until the cat SAA antibody marked by the colloidal gold completely permeates the microfiltration membrane (2);
s5, dropwise adding the washing solution to the microfiltration membrane (2) in the step S4 through the through hole on the percolation device, so that the washing solution completely permeates into the microfiltration membrane (2);
s6, performing numerical analysis on the color development result by using a gold-labeled reading instrument;
s7, a set of standard sample to be tested containing cat SAA antigen is set, the concentration of cat SAA antigen in the standard sample to be tested is respectively set to 0ug/ml, 0.5ug/ml, 1.0ug/ml, 2.0ug/ml, 4.0ug/ml, 8.0ug/ml, 10.0ug/ml, 14.0ug/ml, 18.0ug/ml, the standard sample to be tested is respectively tested according to the steps of S1-S6, a standard curve is made by taking the test result of the standard sample to be tested as the reference, in the standard curve: taking the concentration of the cat SAA antigen as an abscissa and the numerical value of the gold-labeled reading instrument as an ordinate; and detecting an unknown sample to be detected with unknown cat SAA concentration according to the steps of S1-S6, and judging a result according to the standard curve.
10. The semi-quantitative detection method of dot immunogold filtration kit for detecting cat SAA according to claim 9 wherein in step S1, the amount of blocking solution is 50 ul; in step S2, the amount of the sample to be measured is 25 ul; in step S3, the amount of the washing solution is 50 ul; in step S4, the amount of the colloidal gold-labeled feline SAA antibody is 25 ul; in step S5, the amount of the washing solution was 50 ul.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113970535A (en) * | 2021-10-18 | 2022-01-25 | 广州市微米生物科技有限公司 | Detector and using method thereof |
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| CN102608321A (en) * | 2012-02-29 | 2012-07-25 | 厦门大学 | Echinococcus multilocularis circulating antigen dot immunogold filtration kit and preparation method |
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