CN114544940B - Colloidal gold test strip for rapidly detecting hyaluronic acid - Google Patents
Colloidal gold test strip for rapidly detecting hyaluronic acid Download PDFInfo
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- CN114544940B CN114544940B CN202210155773.1A CN202210155773A CN114544940B CN 114544940 B CN114544940 B CN 114544940B CN 202210155773 A CN202210155773 A CN 202210155773A CN 114544940 B CN114544940 B CN 114544940B
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- hyaluronic acid
- test strip
- colloidal gold
- pad
- detection
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- 239000008363 phosphate buffer Substances 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 229910000027 potassium carbonate Inorganic materials 0.000 description 1
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- 230000008569 process Effects 0.000 description 1
- 108010070643 prolylglutamic acid Proteins 0.000 description 1
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- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 229940010747 sodium hyaluronate Drugs 0.000 description 1
- YWIVKILSMZOHHF-QJZPQSOGSA-N sodium;(2s,3s,4s,5r,6r)-6-[(2s,3r,4r,5s,6r)-3-acetamido-2-[(2s,3s,4r,5r,6r)-6-[(2r,3r,4r,5s,6r)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2- Chemical compound [Na+].CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 YWIVKILSMZOHHF-QJZPQSOGSA-N 0.000 description 1
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Classifications
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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
- G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
-
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Peptides Or Proteins (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
The invention relates to the technical field of immunochromatography detection, in particular to a colloidal gold test strip for rapidly detecting hyaluronic acid. The sequence represented by SEQ ID NO:1, specifically comprising a PVC glue plate, a sample pad, a gold-labeled pad, a nitrocellulose membrane and a water absorbing pad sequentially connected to the PVC glue plate, wherein the nitrocellulose membrane is provided with a detection line and a quality control line, the gold-labeled pad contains a colloidal gold particle labeled hyaluronic acid specific binding protein with a GST label, the detection line is anchored with hyaluronic acid, the quality control line is anchored with a GST monoclonal antibody, and the protein with a GST label can be specifically bound. The colloidal gold test strip provided by the invention is convenient and quick to use, can complete chromatographic reaction within 10 minutes, and realizes quick detection of hyaluronic acid in a sample.
Description
Technical Field
The invention relates to the technical field of immunochromatography detection, in particular to a colloidal gold test strip for rapidly detecting hyaluronic acid.
Background
Hyaluronic Acid (HA) is produced by fermentation of streptococcus zooepidemicus (streptococcus zooepidemicus) and is a glycosaminoglycan composed of D-glucuronic acid and N-acetyl-D-glucosamine disaccharide units, and is a linear macromolecular polysaccharide, with glucose, yeast powder, peptone and the like as culture mediums. Because the hyaluronic acid molecule is in a rigid spiral column shape in a space structure, the hydroxyl groups on the inner side of the column have high hydrophilicity, and simultaneously, the hydroxyl groups are continuously and directionally arranged to form a highly hydrophobic region on a hyaluronic acid molecular chain, and even when the concentration is very low, the hyaluronic acid can also form a three-dimensional network structure, so that the hyaluronic acid has very strong hygroscopicity, water retention and viscoelasticity, is a good natural moisturizing factor and is commonly used as a cosmetic ingredient. The national health committee promulgates, 2021, 1 and 7, approves sodium hyaluronate as a "new food raw material" and can be applied to common food additives.
Binding proteins are proteins with autonomous folding and specific recognition of complex carbohydrate arrangements, mainly present in proteins that recognize polysaccharides. It exists as single or multiple domains at the C-or N-terminus of the catalytic domain of a protein, having only binding capacity and no catalytic activity. Polysaccharides are often poor immunogens and specific antibodies to polysaccharides are difficult to obtain compared to conventional antibody preparation methods. The problem of HA antibody deficiency can be effectively solved by adopting the binding protein to bind with HA.
The conventional detection methods of hyaluronic acid at present include an HPLC method, a colorimetric method, a CTAB turbidimetry method and a carbazole chromogenic method. The method has the advantages of complex operation, long detection period, reliance on professional equipment and detection personnel with good experimental skills, and difficulty in realizing the method for on-site rapid detection.
Immunological detection has been a rapidly developing detection means in recent years due to its strong specificity and high sensitivity. The colloidal gold test strip is widely applied to the field of on-site rapid detection due to the advantages of short detection period, easy storage, simple and clear result, and HAs no product for rapid detection of HA in the market at present, so that it is necessary to develop a colloidal gold test strip suitable for HA in cosmetics or foods to make up for the blank, thereby better guiding enterprises to control production quality and monitor law enforcement.
Disclosure of Invention
The invention aims to solve the technical problems that the conventional detection methods of hyaluronic acid at present comprise an HPLC method, a colorimetric method, a CTAB turbidimetry method and a carbazole chromogenic method. The method has the advantages of complex operation, long detection period, reliance on professional equipment and detection personnel with good experimental skills, and difficulty in realizing the method for on-site rapid detection with large sample size.
In order to solve the problems, the invention provides a colloidal gold immunochromatography test strip for detecting hyaluronic acid, which uses hyaluronic acid specific binding protein, so that the colloidal gold immunochromatography test strip has higher specificity, repeatability and accuracy when being applied to hyaluronic acid detection.
In order to achieve the above purpose, the invention is realized by the following technical scheme: a colloidal gold immunochromatographic test strip for rapidly detecting hyaluronic acid, which comprises a nucleotide sequence represented by SEQ ID NO:1 is a detection antigen.
SEQ ID NO.1
EETTTNLVKNGDFTTTTKTSGAWSGQAAKDWETWVPGNVKKENGKVTVDNGRLHISSTDTYRVAVHQTVDVDPSKKYLLGYDVETKDLKGSGVRVRLHGLTAEGKDLEPKEFIHTPFKNGSKKERVEQVLTVSPQTRKLKVELFFENSIGQAWLDNVSLVEHIEKVPETPEQKPEPAQPES
Further, the nucleotide sequence of the gene encoding the hyaluronic acid binding protein is SEQ ID NO.2 or all the genes of SEQ ID NO.1 can be translated.
SEQ ID NO.2
GAAGAGACTACGACTAATTTGGTAAAAAATGGGGATTTTACAACCACAACAAAGACTTCAGGGGCCTGGTCAGGACAGGCAGCTAAAGACTGGGAAACTTGGGTGCCGGGTAATGTGAAAAAGGAAAATGGAAAGGTAACCGTTGATAACGGGCGACTCCATATCTCTTCAACCGATACATACCGAGTAGCGGTTCACCAAACTGTGGATGTTGATCCTAGTAAAAAATATCTCTTAGGCTATGATGTGGAAACTAAGGATCTGAAAGGTAGCGGAGTGCGCGTTCGGTTACACGGTTTGACAGCAGAAGGGAAGGATTTGGAGCCGAAAGAATTTATCCATACCCCTTTTAAAAATGGCAGCAAGAAAGAGCGTGTAGAGCAAGTCTTAACTGTCTCACCACAAACTCGCAAACTGAAGGTCGAACTATTTTTTGAAAATAGTATCGGTCAGGCTTGGCTTGACAACGTTTCGTTGGTTGAACATATTGAAAAAGTGCCAGAAACACCGGAACAAAAACCAGAACCTGCTCAGCCAGAATCG
The recombinant expression method of the hyaluronic acid binding protein comprises the following steps:
the hyaluronic acid binding protein gene is inserted between EcoRI and XhoI restriction enzyme sites of the vector pET28a-GST, other sequences of the pET28a-GST are kept unchanged, and the obtained recombinant expression vector is transformed into the escherichia coli expression engineering bacterium BL21 so as to be inserted into the escherichia coli genome to stably express the target protein.
The specificity of the expressed hyaluronic acid binding protein is tested by utilizing a polysaccharide microarray, and cross tests are performed on the polysaccharide such as hyaluronic acid, chondroitin sulfate, heparin, pectin, algin and the like. The results showed that only the hyaluronic acid sample reacted positively, indicating that the hyaluronic acid binding protein was highly specific for detection.
Based on the test results, the invention provides application of the hyaluronic acid binding protein in preparing immunochromatography products for detecting hyaluronic acid. Preferably, the immunochromatography product is a colloidal gold immunochromatography test strip.
Further, the test strip comprises a back lining, a sample pad, a gold label pad, a nitrocellulose membrane and a water absorption pad which are sequentially stuck on the back lining, wherein the nitrocellulose membrane is marked with a detection line coated with hyaluronic acid and a quality control line coated with GST mouse monoclonal antibody.
Further, the spraying amount of the hyaluronic acid on the detection line is 0.5-2 mu L/cm, and the spraying amount of the GST mouse monoclonal antibody on the quality control line is 0.5-2 mu L/cm.
Further, the gold-labeled pad is sprayed with hyaluronic acid binding protein compound of labeled colloidal gold particles, and the spraying amount of the compound on the gold-labeled pad is 5-8 mu L/cm. In the specific embodiment of the invention, the particle size of the colloidal gold is 20nm, and the labeling ratio content of the colloidal gold to the hyaluronic acid binding protein in the labeling process is 1mL of colloidal gold: 10 μg of hyaluronic acid binding protein.
Further, the back lining is a polyethylene back lining, the sample pad is a VL98 polyester film, the gold-labeled pad is an RB45 glass fiber film, the nitrocellulose film is CN140, and the water absorbing pad is CH27.
Further, the formula of the chromatographic liquid comprises the following components: 20mmol/L pH7.5 PBS, 0.5-2% Tween-20, 1-3% BSA, 10% sucrose, 0.9% NaCl.
The invention has the beneficial effects that:
(1) The novel hyaluronic acid binding protein provided by the invention has good specificity, repeatability and accuracy when being used as hyaluronic acid detection protein, can be applied to related immunochromatography detection products, has visual and macroscopic judgment results, can rapidly and specifically detect hyaluronic acid, is convenient for product quality control and supervision, and has higher practical value.
(2) The hyaluronic acid immunochromatography test strip adopts the principle of immune competition, the hyaluronic acid in the sample and the detection line compete with the hyaluronic acid binding protein on the gold-labeled pad together, and when the content of the hyaluronic acid in the sample is gradually increased, the hyaluronic acid can be combined with more binding proteins, so that the amount of the binding proteins which can be combined with the hyaluronic acid on the T line is reduced, the color of the T line is weakened, the C line is a quality control line, and the color is not influenced by the change of the content of the hyaluronic acid.
(3) The colloidal gold test strip has the advantages of being convenient to carry, high in analysis speed, simple in operation steps and the like, breaks through the limitation of the traditional hyaluronic acid detection method, can complete chromatographic reaction within 10 minutes, realizes rapid detection of hyaluronic acid in samples, provides favorable technical support for rapid on-site detection of large sample volumes, and brings great convenience to production quality control of enterprises.
Drawings
FIG. 1 is a schematic diagram of a two-dimensional structure of a hyaluronic acid colloidal gold rapid detection test strip according to the present invention; wherein, 1, the bottom plate; 2. a sample pad; 3. a gold mark pad; 4. a nitrocellulose membrane; 5. a detection line; 6. a quality control line; 7. a water absorbing pad;
FIG. 2 is a graph showing the particle size distribution of colloidal gold of a rapid test strip for detecting colloidal gold of hyaluronic acid according to the present invention;
FIG. 3 is a graph showing the detection limit of a sample of hyaluronic acid detected by the rapid detection test strip of colloidal gold for hyaluronic acid according to the present invention; wherein 1, 0.2 mug/mL hyaluronic acid; 2. 2 μg/mL hyaluronic acid; 3. 20 μg/mL hyaluronic acid; 4. 200 μg/mL hyaluronic acid;
FIG. 4 is a graph showing the cross-reaction results of the rapid detection of other 8 polysaccharides by the hyaluronic acid colloidal gold test strip according to the present invention; wherein, 1, hyaluronic acid; 2. pectin; 3. chondroitin sulfate a; 4. chondroitin sulfate B; 5. chondroitin sulfate C; 6. heparin; 7. algin; the polysaccharide solutions were all 20. Mu.g/mL in concentration.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. It will be apparent that the described embodiments are some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1:
Referring to fig. 1, a colloidal gold test strip for rapidly detecting hyaluronic acid in food includes: a backing 1, a sample pad 2 arranged on the backing, a gold mark pad 3 and a water absorption pad 7 which are arranged adjacent to the sample pad 2;
A nitrocellulose membrane 4 is arranged between the gold mark pad 3 and the back lining 1;
and a detection line 5 and a quality control line 6 are arranged on the nitrocellulose membrane 4.
The detection line 5 and the quality control line 6 are arranged on the nitrocellulose membrane 4, the detection line 5 is close to the sample pad 2, and the quality control line 6 is close to the water absorption pad 7.
The detection line is sprayed with 2mg/mL of hyaluronic acid, the quality control line is sprayed with GST mouse monoclonal antibody, and the gold label pad is sprayed with hyaluronic acid binding protein marked by colloidal gold particles.
The colloid Jin Lijing is 20nm.
The hyaluronic acid colloidal gold test strip adopts the principle of immune competition: when the sample is dropped onto the sample pad 2, the sample is chromatographed along the direction of the sample pad 2, the gold-labeled pad 3, the nitrocellulose membrane 4, and the absorbent pad 7 with the chromatography. If the sample does not contain hyaluronic acid, when the sample flows through the gold-labeled pad 3, the colloidal gold-hyaluronic acid binding protein complex is chromatographically combined with the hyaluronic acid on the detection line 5, and the unbound complex continues to move to the quality control line 6, so that the hyaluronic acid binding protein is captured by GST mouse monoclonal antibody due to the GST label, and at the moment, both the detection line and the quality control line show macroscopic red. If the sample contains hyaluronic acid, the sample and the hyaluronic acid in the detection line compete with the colloidal gold-hyaluronic acid binding protein complex on the gold-labeled pad 3 together, so that the amount of the complex which can be bound with the hyaluronic acid on the detection line 5 is reduced, the color of the detection line 5 is weakened until the complex disappears, the complex which is not bound with the hyaluronic acid on the detection line 5 continues to move to the quality control 6 line to be captured by GST mouse monoclonal antibody, and at the moment, the detection line 5 does not develop color and the quality control line 6 shows macroscopic red. The result is interpreted as: the detection line 5 and the quality control line 6 are both red, and the detection sample is negative; the quality control line 6 shows red, the detection line 5 shows light red or does not show color, and the detection sample is positive; if the quality control line 6 does not develop, the test strip is invalid.
In this embodiment, the preparation method of the colloidal gold test strip for rapidly detecting hyaluronic acid in food comprises the following steps:
1. Acquisition and purification of the hyaluronic acid binding protein HBP-SR
E.coli harboring the HBP-SR gene of interest was subcultured in LB liquid medium (kanamycin resistance) and incubated at 37℃at 160r/min until OD600 became about 0.4, and IPTG was added to a final concentration of 0.5mmol/L and induced at 17℃for 12 hours. The cells were resuspended in 20mM/L PBS (pH 7.5) and sonicated to obtain HBP-SR crude protein. The crude protein was purified by affinity chromatography using HISTRAPTM HP chromatography column, gradient eluted with 0-0.5mol/L imidazole in a mobile phase of 20mM PBS (pH 7.5) containing 0.3mol/LNaCl, and fractions with UV absorption signal were collected. Desalting the collected components with HiPrepTM/10 DESALTING chromatographic column with water as mobile phase to obtain purified HBP-SR.
2. Process for preparing colloidal gold
1) Preparation of 1% chloroauric acid: 1g of chloroauric acid powder is taken, 100mL of sterilized ultrapure water is added to prepare a 1% chloroauric acid solution, and the solution is placed at 4 ℃ for standby.
2) 1% Trisodium citrate: 0.5679g of trisodium citrate dihydrate was dissolved in 50mL of ultrapure water to prepare a 1% trisodium citrate solution, which was ready for use.
3) The colloidal gold particles were prepared by a citrate reduction method, 99mL of ultrapure water was added to a beaker, then 1mL of 1% chloroauric acid solution was added, and 100mL of chloroauric acid solution with a mass concentration of 0.01% was obtained after stirring uniformly. 100mL of this 0.01% chloroauric acid solution was heated to boiling in a water bath with stirring at 500 rpm. 3mL of 1% sodium citrate solution is accurately measured and rapidly added into chloroauric acid solution, the rotation speed of 500rpm is kept unchanged, heating is continued for 12min, and heating is stopped when the solution color is transparent mauve and kept unchanged. Cooling at room temperature, and storing at 4deg.C in dark place.
3. Preparation of colloidal gold-hyaluronic acid binding protein complexes
Taking 1mL of 20nm colloidal gold solution, adding 5 mu L of 0.1mol/L potassium carbonate to regulate the solution to the optimal pH value, adding 10 mu g of hyaluronic acid binding protein, quickly mixing uniformly, mixing uniformly on a shaking table at room temperature for 30min, then adding BSA to a final concentration of 1%, continuing mixing uniformly for 30min, centrifuging the gold standard solution at 12000r/min at 4 ℃ for 10min, carefully sucking the supernatant, re-suspending the precipitate by using 1% BSA solution in an amount of 1/10 volume of the original gold solution, and preserving at 4 ℃ for later use.
4. Nitrocellulose membrane detection line, quality control line spraying and gold mark pad preparation
The 0.2mg/mL hyaluronic acid solution was uniformly scribed at the T line position of the nitrocellulose membrane at a speed of 1. Mu.L/cm using an XYZ three-dimensional metal-sprayed streaker. GST mouse monoclonal antibody is diluted to 1mg/ml and evenly marked on the C line position of a nitrocellulose membrane at the speed of 1 mu L/cm, and is dried for 2 hours at 37 ℃ for standby. The colloidal gold-hyaluronic acid binding protein complex is evenly sprayed on a gold-labeled pad at 8 mu L/cm, and is dried for 2 hours at 37 ℃ for standby.
5. Assembly of test strips
The sample pad, gold-labeled pad, nitrocellulose membrane, and water absorbing pad were sequentially adhered to the polyethylene backing. The assembly structure is shown in fig. 1, a nitrocellulose membrane 4 is arranged on a polyethylene back lining 1, a sample pad 2 is flatly attached to the left side of the nitrocellulose membrane 4, a water absorbing pad 7 is flatly attached to the right side of the nitrocellulose membrane 4, and a gold mark pad 3 is flatly attached between the sample pad 2 and the nitrocellulose membrane 4, so that one end of the gold mark pad is pressed below the sample pad 2, and the other end of the gold mark pad is covered on the nitrocellulose membrane 4. The overlapping length is about 1 mm. After the bonding is finished, a cutting machine is used for cutting the test paper into test paper strips with the width of 3 mm. The assembled test strip is placed in a plastic card for vacuum packaging and preservation at normal temperature.
6. Application of colloidal gold test strip for rapidly detecting hyaluronic acid
1 Mu L of sample to be tested and 99 mu L of chromatographic liquid are taken and are fully mixed, then the mixture is dripped on the test paper sample pad of the invention, the result is observed after 10min, and meanwhile, known positive and negative samples are respectively taken as positive and negative controls. And (3) result judgment: if the detection line and the quality control line are both red, a negative result is obtained; if the detection line does not develop color and the quality control line develops red, the result is positive; if the quality control line does not develop color, the test paper is invalid. The chromatographic solution is 0.02M phosphate buffer solution containing 1% BSA, 1% Tween 20, 10% sucrose and 0.9% NaCl, and can be used for fully releasing the colloidal gold-hyaluronic acid binding protein complex in the gold-labeled pad.
7. Test strip sensitivity test
Chemically pure hyaluronic acid was weighed in 20mM phosphate buffer pH8, standard solutions of hyaluronic acid of different concentrations were prepared, the procedure described in 6 above was repeated for testing, and three replicates were made. The results are shown in FIG. 2.
FIG. 2 shows the test result of the hyaluronic acid colloidal gold test strip for detecting limit of hyaluronic acid in the example of the invention, and as can be seen from FIG. 2, after the hyaluronic acid solution with the final concentration of 200, 20 and 2 mug/mL is dripped, the T line of the test strip does not develop color, after the hyaluronic acid solution with the final concentration of 0.2 mug/mL is dripped, the T line develops color, which shows that the detection limit of the test strip can reach 2 mug/mL, the three repeated results are the same, and the stability of the test strip is good.
8. Test strip specificity experiment
2Mg/mL of hyaluronic acid, pectin, chondroitin sulfate A, B, C, heparin and algin solution are selected for cross experiments. The procedure described in 6 above was repeated for the test, i.e., the final concentrations were 20. Mu.g/mL, and three replicates were made. The results are shown in FIG. 3.
FIG. 3 shows the cross-reaction results of the hyaluronic acid colloidal gold test strip for detecting different polysaccharides in the examples of the present invention. As can be seen from FIG. 3, after 20. Mu.g/mL of 6 other uronic acid-containing polysaccharides was added to the sample pad, the detection lines were developed, and a negative result was shown. The results show that the test strip has no cross reaction with other uronic acid-containing polysaccharide.
The foregoing description of the embodiments has been provided for the purpose of illustrating the general principles of the invention, and is not meant to limit the scope of the invention, but to limit the invention to the particular embodiments, and any modifications, equivalents, improvements, etc. that fall within the spirit and principles of the invention are intended to be included within the scope of the invention.
Sequence listing
<110> University of ocean in China
<120> Colloidal gold test strip for rapid detection of hyaluronic acid
<130> University of ocean in China
<140> 1
<141> 2022-02-08
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 181
<212> PRT
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 1
Glu Glu Thr Thr Thr Asn Leu Val Lys Asn Gly Asp Phe Thr Thr Thr
1 5 10 15
Thr Lys Thr Ser Gly Ala Trp Ser Gly Gln Ala Ala Lys Asp Trp Glu
20 25 30
Thr Trp Val Pro Gly Asn Val Lys Lys Glu Asn Gly Lys Val Thr Val
35 40 45
Asp Asn Gly Arg Leu His Ile Ser Ser Thr Asp Thr Tyr Arg Val Ala
50 55 60
Val His Gln Thr Val Asp Val Asp Pro Ser Lys Lys Tyr Leu Leu Gly
65 70 75 80
Tyr Asp Val Glu Thr Lys Asp Leu Lys Gly Ser Gly Val Arg Val Arg
85 90 95
Leu His Gly Leu Thr Ala Glu Gly Lys Asp Leu Glu Pro Lys Glu Phe
100 105 110
Ile His Thr Pro Phe Lys Asn Gly Ser Lys Lys Glu Arg Val Glu Gln
115 120 125
Val Leu Thr Val Ser Pro Gln Thr Arg Lys Leu Lys Val Glu Leu Phe
130 135 140
Phe Glu Asn Ser Ile Gly Gln Ala Trp Leu Asp Asn Val Ser Leu Val
145 150 155 160
Glu His Ile Glu Lys Val Pro Glu Thr Pro Glu Gln Lys Pro Glu Pro
165 170 175
Ala Gln Pro Glu Ser
180
<210> 2
<211> 543
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 2
gaagagacta cgactaattt ggtaaaaaat ggggatttta caaccacaac aaagacttca 60
ggggcctggt caggacaggc agctaaagac tgggaaactt gggtgccggg taatgtgaaa 120
aaggaaaatg gaaaggtaac cgttgataac gggcgactcc atatctcttc aaccgataca 180
taccgagtag cggttcacca aactgtggat gttgatccta gtaaaaaata tctcttaggc 240
tatgatgtgg aaactaagga tctgaaaggt agcggagtgc gcgttcggtt acacggtttg 300
acagcagaag ggaaggattt ggagccgaaa gaatttatcc ataccccttt taaaaatggc 360
agcaagaaag agcgtgtaga gcaagtctta actgtctcac cacaaactcg caaactgaag 420
gtcgaactat tttttgaaaa tagtatcggt caggcttggc ttgacaacgt ttcgttggtt 480
gaacatattg aaaaagtgcc agaaacaccg gaacaaaaac cagaacctgc tcagccagaa 540
tcg 543
Claims (9)
1. A colloidal gold immunochromatographic test strip for rapidly detecting hyaluronic acid takes a protein with a sequence shown as SEQ ID NO.1 as a detection antigen.
2. The colloidal gold immunochromatographic test strip for rapid detection of hyaluronic acid according to claim 1, wherein: the nucleotide sequence of the gene for encoding the hyaluronic acid binding protein is shown as SEQ ID NO. 2.
3. The colloidal gold immunochromatographic test strip for rapid detection of hyaluronic acid according to claim 1, wherein: the test strip comprises a backing, and a sample pad, a gold mark pad, a nitrocellulose membrane and a water absorption pad which are sequentially stuck on the backing.
4. The colloidal gold immunochromatographic test strip for rapid detection of hyaluronic acid according to claim 3, wherein: and a detection line coated with hyaluronic acid and a quality control line coated with GST mouse monoclonal antibody are marked on the nitrocellulose membrane.
5. The colloidal gold immunochromatographic test strip for rapid detection of hyaluronic acid according to claim 4, wherein: the spraying amount of the hyaluronic acid on the detection line is 0.5-2 mu L/cm, and the spraying amount of the GST mouse monoclonal antibody on the quality control line is 0.5-2 mu L/cm.
6. The colloidal gold immunochromatographic test strip for rapid detection of hyaluronic acid according to claim 3, wherein: the gold-labeled pad is sprayed with a hyaluronic acid binding protein compound for labeling colloidal gold particles.
7. The colloidal gold immunochromatographic test strip for rapid detection of hyaluronic acid according to claim 6, wherein: the spraying amount of the compound on the gold mark pad is 5-8 mu L/cm.
8. The colloidal gold immunochromatographic test strip for rapid detection of hyaluronic acid according to claim 3, wherein: the back lining is a polyethylene back lining, the sample pad is a VL98 polyester film, the gold-labeled pad is an RB45 glass fiber film, the nitrocellulose film is CN140, and the water absorption pad is CH27.
9. The colloidal gold immunochromatographic test strip for rapid detection of hyaluronic acid according to claim 3, wherein: the formula of the chromatographic liquid is as follows: 20mmol/L pH7.5 PBS, 0.5-2% Tween-20, 1-3% BSA, 10% sucrose, 0.9% NaCl.
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| KR101202622B1 (en) * | 2005-07-30 | 2012-11-19 | 태건식 | Diagnosis strip for inhalation of toluene and diagnosis kit comprising the same |
| US8470321B2 (en) * | 2007-11-16 | 2013-06-25 | The Rockefeller University | Antibodies specific for the protofibril form of beta-amyloid protein |
| CN102645538B (en) * | 2012-03-28 | 2014-05-07 | 广西壮族自治区兽医研究所 | HA epitope colloidal gold rapid detection test strip of H5 subtype avian influenza virus antibody |
| US20140134601A1 (en) * | 2012-11-09 | 2014-05-15 | Korea University Research And Business Foundation | Use of protein nanoparticle based hydrogel |
| CN103076450A (en) * | 2012-12-15 | 2013-05-01 | 中国农业科学院兰州兽医研究所 | Haemophilus parasuis disease antibody detecting test strip and preparation method thereof |
| CN106496328A (en) * | 2016-11-22 | 2017-03-15 | 浙江达美生物技术有限公司 | Preparation method of hyaluronan-binding protein polyclonal antibody |
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