CN112782409A - Kit for determining IV type collagen in human blood and detection method - Google Patents
Kit for determining IV type collagen in human blood and detection method Download PDFInfo
- Publication number
- CN112782409A CN112782409A CN202011633326.XA CN202011633326A CN112782409A CN 112782409 A CN112782409 A CN 112782409A CN 202011633326 A CN202011633326 A CN 202011633326A CN 112782409 A CN112782409 A CN 112782409A
- Authority
- CN
- China
- Prior art keywords
- type
- collagen
- nano
- reagent
- monoclonal antibody
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 102000008186 Collagen Human genes 0.000 title claims abstract description 149
- 108010035532 Collagen Proteins 0.000 title claims abstract description 149
- 229920001436 collagen Polymers 0.000 title claims abstract description 149
- 210000004369 blood Anatomy 0.000 title claims abstract description 11
- 239000008280 blood Substances 0.000 title claims abstract description 11
- 238000001514 detection method Methods 0.000 title abstract description 22
- 239000002245 particle Substances 0.000 claims abstract description 83
- 239000004005 microsphere Substances 0.000 claims abstract description 73
- 102000004266 Collagen Type IV Human genes 0.000 claims abstract description 48
- 108010042086 Collagen Type IV Proteins 0.000 claims abstract description 48
- 230000008878 coupling Effects 0.000 claims abstract description 30
- 238000010168 coupling process Methods 0.000 claims abstract description 30
- 238000005859 coupling reaction Methods 0.000 claims abstract description 30
- 239000003153 chemical reaction reagent Substances 0.000 claims description 64
- 239000000243 solution Substances 0.000 claims description 63
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 42
- 239000011259 mixed solution Substances 0.000 claims description 42
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 30
- 239000004816 latex Substances 0.000 claims description 29
- 229920000126 latex Polymers 0.000 claims description 29
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 28
- 238000002835 absorbance Methods 0.000 claims description 26
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 26
- 239000002244 precipitate Substances 0.000 claims description 24
- 239000006173 Good's buffer Substances 0.000 claims description 23
- 238000006243 chemical reaction Methods 0.000 claims description 17
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 claims description 15
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 15
- 239000004471 Glycine Substances 0.000 claims description 15
- 239000007995 HEPES buffer Substances 0.000 claims description 15
- 229940098773 bovine serum albumin Drugs 0.000 claims description 15
- 239000000839 emulsion Substances 0.000 claims description 15
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 claims description 14
- 238000003908 quality control method Methods 0.000 claims description 14
- 239000002202 Polyethylene glycol Substances 0.000 claims description 13
- 238000000034 method Methods 0.000 claims description 13
- 238000002156 mixing Methods 0.000 claims description 13
- 229920001223 polyethylene glycol Polymers 0.000 claims description 13
- 239000000047 product Substances 0.000 claims description 13
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 12
- 239000002077 nanosphere Substances 0.000 claims description 12
- 239000008055 phosphate buffer solution Substances 0.000 claims description 12
- 241000486679 Antitype Species 0.000 claims description 11
- 239000003623 enhancer Substances 0.000 claims description 11
- 239000003112 inhibitor Substances 0.000 claims description 11
- 239000011780 sodium chloride Substances 0.000 claims description 11
- 239000004094 surface-active agent Substances 0.000 claims description 11
- 230000010355 oscillation Effects 0.000 claims description 10
- 238000003756 stirring Methods 0.000 claims description 10
- 239000006228 supernatant Substances 0.000 claims description 10
- 239000007853 buffer solution Substances 0.000 claims description 9
- 230000000813 microbial effect Effects 0.000 claims description 8
- 150000001718 carbodiimides Chemical class 0.000 claims description 7
- 238000002360 preparation method Methods 0.000 claims description 7
- QYYMDNHUJFIDDQ-UHFFFAOYSA-N 5-chloro-2-methyl-1,2-thiazol-3-one;2-methyl-1,2-thiazol-3-one Chemical compound CN1SC=CC1=O.CN1SC(Cl)=CC1=O QYYMDNHUJFIDDQ-UHFFFAOYSA-N 0.000 claims description 3
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 claims description 3
- YNLCVAQJIKOXER-UHFFFAOYSA-N N-[tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid Chemical compound OCC(CO)(CO)NCCCS(O)(=O)=O YNLCVAQJIKOXER-UHFFFAOYSA-N 0.000 claims description 3
- 239000004353 Polyethylene glycol 8000 Substances 0.000 claims description 3
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 3
- 239000007983 Tris buffer Substances 0.000 claims description 3
- 239000013504 Triton X-100 Substances 0.000 claims description 3
- 229920004890 Triton X-100 Polymers 0.000 claims description 3
- -1 Tween30 Polymers 0.000 claims description 3
- 244000005700 microbiome Species 0.000 claims description 3
- 229940057838 polyethylene glycol 4000 Drugs 0.000 claims description 3
- 229940085678 polyethylene glycol 8000 Drugs 0.000 claims description 3
- 235000019446 polyethylene glycol 8000 Nutrition 0.000 claims description 3
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 3
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 3
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 claims description 3
- 229920000053 polysorbate 80 Polymers 0.000 claims description 3
- 239000012475 sodium chloride buffer Substances 0.000 claims description 3
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 3
- 230000035945 sensitivity Effects 0.000 abstract description 9
- 230000000052 comparative effect Effects 0.000 description 23
- 208000019425 cirrhosis of liver Diseases 0.000 description 7
- 206010016654 Fibrosis Diseases 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- 230000008859 change Effects 0.000 description 4
- 239000003795 chemical substances by application Substances 0.000 description 4
- 230000007882 cirrhosis Effects 0.000 description 4
- 210000004185 liver Anatomy 0.000 description 4
- 239000008363 phosphate buffer Substances 0.000 description 4
- 210000002469 basement membrane Anatomy 0.000 description 3
- 238000010200 validation analysis Methods 0.000 description 3
- 206010008909 Chronic Hepatitis Diseases 0.000 description 2
- 239000008351 acetate buffer Substances 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 230000002596 correlated effect Effects 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 229920000136 polysorbate Polymers 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 238000009210 therapy by ultrasound Methods 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- 206010003445 Ascites Diseases 0.000 description 1
- 206010019755 Hepatitis chronic active Diseases 0.000 description 1
- 206010019759 Hepatitis chronic persistent Diseases 0.000 description 1
- 206010019799 Hepatitis viral Diseases 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 1
- 206010067125 Liver injury Diseases 0.000 description 1
- 206010046274 Upper gastrointestinal haemorrhage Diseases 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 238000009534 blood test Methods 0.000 description 1
- 238000009535 clinical urine test Methods 0.000 description 1
- 239000007822 coupling agent Substances 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 230000004761 fibrosis Effects 0.000 description 1
- 125000003630 glycyl group Chemical group [H]N([H])C([H])([H])C(*)=O 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 231100000234 hepatic damage Toxicity 0.000 description 1
- 230000002440 hepatic effect Effects 0.000 description 1
- 208000007386 hepatic encephalopathy Diseases 0.000 description 1
- 208000006454 hepatitis Diseases 0.000 description 1
- 210000003494 hepatocyte Anatomy 0.000 description 1
- 206010020718 hyperplasia Diseases 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 201000007270 liver cancer Diseases 0.000 description 1
- 230000008818 liver damage Effects 0.000 description 1
- 238000007449 liver function test Methods 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000003760 magnetic stirring Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000002834 transmittance Methods 0.000 description 1
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 1
- 230000007306 turnover Effects 0.000 description 1
- 201000001862 viral hepatitis Diseases 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Images
Classifications
-
- 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
- G01N33/6887—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from muscle, cartilage or connective tissue
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
-
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- 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
-
- 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
- G01N2333/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/08—Hepato-biliairy disorders other than hepatitis
- G01N2800/085—Liver diseases, e.g. portal hypertension, fibrosis, cirrhosis, bilirubin
Abstract
The invention relates to the technical field of type-IV collagen detection, and discloses a kit and a detection method for determining type-IV collagen in human blood. The kit comprises the following components: the anti-IV type collagen monoclonal antibody with the grain diameter of 50-100nm is coupled with the nano-microsphere, and the anti-IV type collagen monoclonal antibody with the grain diameter of 150-250nm is coupled with the nano-microsphere; the mass concentration ratio of the anti-IV type collagen monoclonal antibody coupling nano-microspheres with the particle size of 150-250nm to the anti-IV type collagen monoclonal antibody coupling nano-microspheres with the particle size of 50-100nm is 1: 2-5. The kit combines the large-particle-size nano microspheres and the small-particle-size nano microspheres for use, so that the detection has higher sensitivity and larger linear range.
Description
Technical Field
The invention relates to the technical field of type IV collagen detection, in particular to a kit and a detection method for determining type IV collagen in human blood.
Background
Cirrhosis is one of the common diseases in gastroenterology. The disease is mainly caused by diffuse liver damage caused by long-term or repeated action of various reasons. The main clinical manifestations of the cirrhosis patients are hepatic cell nodular regeneration, liver fibrous tissue hyperplasia, diffuse necrosis, etc., and serious patients can have complications such as upper gastrointestinal hemorrhage, hepatic ascites, hepatic encephalopathy, liver cancer, etc., thereby endangering the life safety of the patients. Accurate diagnosis and active treatment of the condition of patients with cirrhosis as early as possible are key to improving their prognosis. Currently, the common methods for clinically diagnosing cirrhosis mainly include immunological tests, liver function tests, routine blood tests, routine urine tests, and the like.
Type iv collagen (COL iv) is the main component of the basement membrane network, and is directly involved in the formation of extracellular interstitium after intracellular synthesis. The normal liver lobule gap lacks basement membrane, the type IV collagen content is very little, and the normal liver lobule gap is probably distributed independently; fibrosis of the liver occurs in the early stage, with rapid turnover, and eventually forms an intact basement membrane with the continuously deposited laminin, which is characteristic of "sinohepatic capillaris". Therefore, type IV collagen is a common index for observing liver cirrhosis, and the concentration of the type IV collagen is positively correlated with the liver fibrosis degree, and the increase is seen in liver cirrhosis, chronic active hepatitis, chronic persistent hepatitis and acute viral hepatitis.
Currently, methods for detecting type iv collagen include the Elisa assay and the chemiluminescence assay. The Elisa determination method is generally used for semi-quantitative determination, and has poor determination accuracy, sensitivity and precision, and complicated operation, which is not favorable for wide clinical application. The chemiluminescence measuring method has high sensitivity, but has poor measuring accuracy and reagent stability, needs special chemiluminescence measuring equipment, has low measuring speed and obvious limitation of clinical application.
Chinese patent application No. CN201911384289.0 discloses a detection kit for type IV collagen and a preparation method thereof. The kit comprises a reagent R1 and a reagent R2, wherein the volume ratio of the reagent 1 to the reagent 2 is 3: 1; the reagent R1 comprises the following components: 50mmol/L acetate buffer solution with pH of 6.0-6.5, 0.9g/L bovine serum albumin and 0.01% sodium azide; the reagent R2 comprises the following components: 50mmol/L of acetate buffer solution with the pH value of 6.0-6.5, 20mL/L of IV type collagen antibody latex particles, Triton X-1008 mL/L, 13g/L of glycyl amine hydrochloride, 800025g/L of polyethylene glycol and 0.01% of sodium azide, wherein the IV type collagen antibody latex particles are formed by crosslinking IV type collagen antibodies on carboxyl latex microspheres through a combined coupling agent. This kit adopts type IV collagen antibody latex granule, and it can take place the gathering under type IV collagen's effect, so can survey the content of type IV collagen through the change of absorbance, easy operation, detection speed is fast, but simultaneously, this kit also has the problem that linear range is narrow, and the reason lies in: the large-particle-size nano microspheres can be combined with a small amount of IV type collagen, and cannot be used for detecting high-concentration IV type collagen; after the small-particle-size nano microspheres are combined with a small amount of IV type collagen, obvious turbidity change cannot be shown, so that the small-particle-size nano microspheres cannot be used for detecting low-concentration IV type collagen.
Disclosure of Invention
In order to solve the technical problems, the invention provides a kit and a detection method for determining type IV collagen in human blood. The kit combines the large-particle-size nano microspheres and the small-particle-size nano microspheres for use, so that the detection has higher sensitivity and larger linear range.
The specific technical scheme of the invention is as follows:
a kit for determining type IV collagen in human blood comprises the following components: the anti-IV type collagen monoclonal antibody with the grain diameter of 50-100nm is coupled with the nano-microsphere, and the anti-IV type collagen monoclonal antibody with the grain diameter of 150-250nm is coupled with the nano-microsphere.
The invention utilizes the anti-IV type collagen monoclonal antibody to couple with the nano-microsphere for IV type collagen detection, when the IV type collagen is combined with the antibody on the nano-microsphere, the nano-microsphere can be rapidly gathered, the light transmittance of the system is changed, and the turbidity (absorbance) is positively correlated with the content of the IV type collagen.
After the large-particle-size nano microspheres are combined with the IV type collagen, turbidity is easy to appear, the detection sensitivity is high, but the amount of the combined IV type collagen is small, so that the large-particle-size nano microspheres cannot be used for detecting the high-concentration IV type collagen; the small-particle-size nano microspheres have larger specific surface area, can combine more IV type collagen, and have advantages in the detection of samples with higher IV type collagen concentration, but when the IV type collagen concentration is lower, the small-particle-size nano microspheres cannot show obvious turbidity change after being combined with the IV type collagen, so that the small-particle-size nano microspheres cannot be used for detecting low-concentration IV type collagen. Therefore, the invention uses the large-particle-size nano microspheres and the small-particle-size nano microspheres in a matching way, and when in detection, the IV-type collagen enables the nano microspheres with the two particle sizes to be crosslinked and aggregated, so that the detection has higher sensitivity and larger linear range.
Preferably, the collagen quality control agent comprises an agent A, an agent B, IV collagen calibrator and an IV collagen quality control product; the reagent A comprises the following components: microbial inhibitors, EDTA, glycerol, bovine serum albumin, surfactants, reaction enhancers, sodium chloride and Good's buffer solution; the reagent B comprises the following components: microorganism inhibitor, EDTA, glycerol, bovine serum albumin, surfactant, reaction enhancer, sodium chloride, anti-IV type collagen monoclonal antibody coupling nano-microsphere with the particle size of 50-100nm, anti-IV type collagen monoclonal antibody coupling nano-microsphere with the particle size of 150-250nm and Good's buffer solution.
Preferably, in the reagent B, the mass concentration of the anti-IV type collagen monoclonal antibody coupling nano-microsphere with the particle size of 50-100nm is 0.1-0.5g/L, and the mass concentration of the anti-IV type collagen monoclonal antibody coupling nano-microsphere with the particle size of 150-250nm is 0.1-0.5 g/L.
Preferably, the mass concentration ratio of the anti-IV type collagen monoclonal antibody coupling nano-microsphere with the particle size of 150-250nm to the anti-IV type collagen monoclonal antibody coupling nano-microsphere with the particle size of 50-100nm is 1: 2-5.
Preferably, in the reagent A and the reagent B, the mass concentration of the microbial inhibitor is 0.001-1.1g/L, the mass concentration of EDTA is 0.05-5.0g/L, the mass concentration of glycerol is 0.1-10.0g/L, the mass concentration of bovine serum albumin is 0.1-5.0g/L, the mass concentration of the surfactant is 0.01-3.0g/L, the mass concentration of the reaction enhancer is 0.5-5.0g/L, and the mass concentration of sodium chloride is 0.1-8.0 g/L; in the reagent A and the reagent B, the concentrations of Good's buffer solutions are both 25-100mmol/L, pH and are both 6-8; the mass ratio of the reagent A to the reagent B is 1-5: 1.
Preferably, the type IV collagen calibrator is a solution containing type IV collagen, and consists of 25-100mmol/L, pH 6-8 Good's buffer and 400-600ng/mL type IV collagen.
Preferably, the IV type collagen quality control product is a solution containing IV type collagen, and consists of 25-100mmol/L, pH 6-8 Good's buffer solution and 40-60ng/mL IV type collagen.
Preferably, the preparation method of the anti-type IV collagen monoclonal antibody coupled nano-microsphere comprises the following steps:
(i) adding 10-12 w/v% of carboxyl nano latex particle emulsion, phosphate buffer solution, two anti-IV type collagen monoclonal antibodies for identifying different sites on the IV type collagen and 0.1-0.15g/mL of carbodiimide into a reaction container according to the proportion of 200-;
in the step (i), carbodiimide is used as a water loss agent to activate carboxyl on the nano latex particles, so that the carboxyl is condensed with amino in the antibody, and the antibody is coupled to the nano latex particles;
(ii) (ii) resuspending the precipitate subjected to ultrasonic oscillation treatment in the step (i) by using a HEPES buffer solution, adding 0.1-0.2mol/L glycine solution, wherein the volume ratio of the glycine solution to the carboxyl nano latex particle emulsion in the step (i) is 2-2.2:5-6, stirring for 30-40min, centrifuging at 3-5 ℃, and discarding the supernatant to obtain a precipitate;
in step (ii), the amino group in glycine binds to the carboxyl group of the unconjugated antibody on the latex nanoparticle, blocking the free carboxyl group, and preventing it from non-specifically binding to other proteins than type iv collagen when detected. The purpose of resuspension before glycine addition was: the centrifuged latex has overlarge density, and can fully react with glycine after being dispersed by heavy suspension;
(iii) and (3) resuspending the precipitate obtained in the step (ii) by using a HEPES buffer solution to obtain the anti-type IV collagen monoclonal antibody coupled nano-microsphere.
Preferably, in the step (ii), the volume ratio of the precipitates treated by ultrasonic oscillation in the step (i) to the HEPES buffer solution is 1: 1-2.
Preferably, in step (iii), the volume ratio of the precipitate obtained in step (ii) to the HEPES buffer is 1: 1-2.
Preferably, the carboxyl nano latex particles obtained in the step (i) have a particle size of 50-100nm, and the anti-type IV collagen monoclonal antibody coupled nano microspheres obtained in the step (iii) have a particle size of 50-100 nm; or, the particle size of the carboxyl nano latex particle in the step (i) is 150-.
Preferably, in the step (i) and the step (ii), the rotation speed of the centrifugation is 12000-15000r/min, and the time is 1-2 h.
Preferably, in step (i), the specific process of the ultrasonic oscillation is as follows: and (4) carrying out ultrasonic treatment for 1s at intervals of 3-4 s.
The ultrasonic oscillation is carried out in ice bath and adopts an intermittent mode, so that the damage of the activity of the antibody caused by overhigh temperature can be prevented.
Preferably, in step (i), the concentration of the phosphate buffer is 0.01 to 0.02mol/L, and the pH is 7.2 to 7.6.
Preferably, in the steps (ii) and (iii), the concentration of the HEPES buffer solution is 0.1-0.15mol/L, and the pH is 7.2-7.6.
Preferably, the microbial inhibitor is at least one of sodium azide, ProClin 200 and ProClin 300.
Preferably, the surfactant is at least one of Tween20, Tween30, Tween80 and Triton X-100.
Preferably, the reaction enhancer is at least one of polyethylene glycol 2000, polyethylene glycol 4000, polyethylene glycol 8000 and polyethylene glycol 20000.
Preferably, the Good's buffer is at least one of a phosphate buffer, a glycine buffer, a borate buffer, a tris buffer, and an N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid buffer.
A method for detecting type IV collagen by using the kit comprises the following steps:
(1) mixing a sample to be detected with the reagent A, and incubating for 5-7 min at 35-37 ℃ to obtain a mixed solution 1; measuring the absorbance of the mixed solution 1 and recording as A1;
(2) mixing the mixed solution 1 with the reagent B, and incubating for 5-7 min at 35-37 ℃ to obtain a mixed solution 2; measuring the absorbance of the mixed solution 2 and recording as A2;
(3) changing the sample to be detected into a calibrator, repeating the steps (1) to (2), and measuring the absorbance A3 of the calibrator mixed solution 1 and the absorbance A4 of the calibrator mixed solution 2;
(4) calculating the type IV collagen concentration of the sample to be tested: the concentration of the IV type collagen of the sample to be tested is (A2-A1)/(A4-A3). times.the concentration of the IV type collagen of the calibrator.
Compared with the prior art, the invention has the following advantages: the combination use of the large-particle-size nano microspheres and the small-particle-size nano microspheres can ensure that the detection has higher sensitivity and larger linear range.
Drawings
FIG. 1 is a graph showing the linear correlation between the kit of example 1 and a commercially available kit.
Detailed Description
The present invention will be further described with reference to the following examples.
General examples
A kit for measuring type IV collagen in human blood comprises a reagent A, a reagent B, IV type collagen calibrator and a type IV collagen quality control product. The mass ratio of the reagent A to the reagent B is 1-5: 1.
The reagent A comprises the following components: 0.001-1.1g/L of microbial inhibitor, 0.05-5.0g/L of EDTA, 0.1-10.0g/L of glycerol, 0.1-5.0g/L of bovine serum albumin, 0.01-3.0g/L of surfactant, 0.5-5.0g/L of reaction enhancer, 0.1-8.0g/L of sodium chloride and 6-8 of Good's buffer solution with the concentration of 25-100mmol/L, pH.
The reagent B comprises the following components: 0.001-1.1g/L of microbial inhibitor, 0.05-5.0g/L of EDTA, 0.1-10.0g/L of glycerol, 0.1-5.0g/L of bovine serum albumin, 0.01-3.0g/L of surfactant, 0.5-5.0g/L of reaction enhancer, 0.1-8.0g/L of sodium chloride, Good's buffer solution with the concentration of 25-100mmol/L, pH of 6-8, 0.1-0.5g/L of anti-IV type collagen monoclonal antibody coupling nano microsphere with the particle size of 50-100nm, and 0.2-0.5g/L of anti-IV type collagen monoclonal antibody coupling nano microsphere with the particle size of 150-250 nm. The mass concentration ratio of the anti-IV type collagen monoclonal antibody coupling nano-microspheres with the particle size of 150-250nm to the anti-IV type collagen monoclonal antibody coupling nano-microspheres with the particle size of 50-100nm is 1: 2-5.
The IV type collagen calibrator is a solution containing IV type collagen, wherein the calibrator buffer solution is a Good's buffer solution with the concentration of 25-100mmol/L, pH 6-8, and the content of the IV type collagen is 400-600 ng/mL;
the quality control product of the IV type collagen is a solution containing the IV type collagen, wherein the buffer solution of the quality control product is Good's buffer solution of 25-100mmol/L, pH 6-8, and the content of the IV type collagen is 40-60 ng/mL.
The microorganism inhibitor is at least one of sodium azide, ProClin 200 and ProClin 300.
The surfactant is at least one of Tween20, Tween30, Tween80 and Triton X-100.
The reaction enhancer is at least one of polyethylene glycol 2000, polyethylene glycol 4000, polyethylene glycol 8000 and polyethylene glycol 20000.
The Good's buffer solution is at least one of phosphate buffer solution, glycine buffer solution, borate buffer solution, tris buffer solution, and N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid buffer solution.
The preparation method of the anti-IV type collagen monoclonal antibody coupled nano-microsphere comprises the following steps:
(i) 10-12 w/v% of carboxyl nano latex particle emulsion, phosphate buffer solution (0.01-0.02mol/L, pH 7.2-7.6), two anti-IV type collagen monoclonal antibodies for identifying different sites on IV type collagen and 0.1-0.15g/mL carbodiimide are added into a reaction vessel according to the proportion of 200 plus 220 muL: 1500 plus 1800 muL: 1-2mg:1-2mg:500 plus 600 muL, after magnetic stirring for 4-6h, centrifugation is carried out for 1-2h at 3-5 ℃ and 00 plus 15000 r/120min, supernatant is discarded, precipitate is placed into an ice bath, ultrasonic vibration is carried out for 1s, the interval is 3-4s, and the duration is 3-4 min;
(ii) (ii) resuspending the precipitate subjected to ultrasonic oscillation treatment in the step (i) by using 1-2 times of HEPES buffer solution (0.1-0.15mol/L, pH 7.2-7.6), adding 0.1-0.2mol/L glycine solution, wherein the volume ratio of the glycine solution to the carboxyl nano latex particle emulsion in the step (i) is 2-2.2:5-6, stirring for 30-40min, centrifuging for 1-2h at 3-5 ℃ and 12000-;
(iii) and (3) resuspending the precipitate obtained in the step (ii) with 1-2 times volume of HEPES buffer solution (0.1-0.15mol/L, pH 7.2-7.6) to obtain the anti-type IV collagen monoclonal antibody coupled nano-microspheres.
Adopting carboxyl nano latex particles with the particle size of 50-100nm to prepare the anti-IV type collagen monoclonal antibody coupled nano microspheres with the particle size of 50-100nm according to the steps; the carboxyl nano latex particles with the particle size of 150-250nm can be used for preparing the anti-IV type collagen monoclonal antibody coupled nano microspheres with the particle size of 150-250nm according to the steps.
A method for detecting type IV collagen by using the kit comprises the following steps:
(1) mixing a sample to be detected with the reagent A, and incubating for 5-7 min at 35-37 ℃ to obtain a mixed solution 1; measuring the absorbance of the mixed solution 1 and recording as A1;
(2) mixing the mixed solution 1 with the reagent B, and incubating for 5-7 min at 35-37 ℃ to obtain a mixed solution 2; measuring the absorbance of the mixed solution 2 and recording as A2;
(3) changing the sample to be detected into a calibrator, repeating the steps (1) to (2), and measuring the absorbance A3 of the calibrator mixed solution 1 and the absorbance A4 of the calibrator mixed solution 2;
(4) calculating the type IV collagen concentration of the sample to be tested: the concentration of the IV type collagen of the sample to be tested is (A2-A1)/(A4-A3). times.the concentration of the IV type collagen of the calibrator.
Example 1
A kit for measuring type IV collagen in human blood comprises a reagent A, a reagent B, IV type collagen calibrator and a type IV collagen quality control product. The mass ratio of the reagent A to the reagent B is 3: 1.
The reagent A comprises the following components: 0.001g/L of sodium azide, 5.0g/L of EDTA, 0.1g/L of glycerol, 5.0g/L of bovine serum albumin, 203.0 g/L of Twen, 20000.5 g/L of polyethylene glycol, 8.0g/L of sodium chloride and phosphate buffer solution with the concentration of 100mmol/L, pH being 6.
The reagent B comprises the following components: 0.001g/L of sodium azide, 5.0g/L of EDTA, 0.1g/L of glycerol, 5.0g/L of bovine serum albumin, 203.0 g/L of Twenn, 20000.5 g/L of polyethylene glycol, 8.0g/L of sodium chloride, phosphate buffer solution with the concentration of 100mmol/L, pH of 6, 0.1g/L of anti-IV type collagen monoclonal antibody coupling nano-microspheres with the average particle size of about 50nm, and 0.5g/L of anti-IV type collagen monoclonal antibody coupling nano-microspheres with the average particle size of about 150 nm.
The IV type collagen calibrator is a solution containing IV type collagen, and consists of 100mmol/L, pH 6 Good's buffer solution and 500ng/mL IV type collagen.
The quality control product of the IV type collagen is a solution containing the IV type collagen, and consists of 100mmol/L, pH 6 Good's buffer solution and 50ng/mL IV type collagen.
The preparation method of the anti-IV type collagen monoclonal antibody coupled nano-microsphere comprises the following steps:
(i) adding 200 μ L of carboxyl nano latex particle emulsion (Bangs Laboratories, Inc. 10 w/v%, average particle size 50nm), 1500 μ L of phosphate buffer solution (0.01mol/L, pH 7.4), two anti-IV type collagen monoclonal antibodies YKCO-1A7 and YKCO-4D9 (Shanghai friend science and technology Co., Ltd.) with mass of 1mg and capable of identifying different sites on IV type collagen, and 500 μ L of 0.1g/mL carbodiimide into a reaction vessel, magnetically stirring for 4h, centrifuging for 1h at 4 ℃ and 15000r/min, discarding supernatant, placing precipitate in an ice bath, performing ultrasonic vibration for 1s, spacing for 3s, and continuing for 3 min;
(ii) (ii) resuspending the precipitate subjected to ultrasonic oscillation treatment in the step (i) with an isovolumetric HEPES buffer solution (0.1mol/L, pH 7.4), adding 500. mu.L of 0.1mol/L glycine solution, stirring for 30min, centrifuging for 1h at 4 ℃ and 15000r/min, and discarding the supernatant to obtain a precipitate;
(iii) and (3) resuspending the precipitate obtained in the step (ii) with an equal volume of HEPES buffer solution (0.1mol/L, pH 7.4) to obtain the anti-IV type collagen monoclonal antibody coupled nanospheres with the average particle size of about 50 nm.
And (3) replacing the carboxyl nano latex particle emulsion with the average particle size of 50nm with the carboxyl nano latex particle emulsion with the average particle size of 150nm, and preparing the anti-IV type collagen monoclonal antibody coupled nano microsphere with the average particle size of about 150nm according to the steps (i) to (ii).
A method for detecting type IV collagen by using the kit comprises the following steps:
(1) mixing a sample to be detected with the reagent A, and incubating for 6min at 37 ℃ to obtain a mixed solution 1; measuring the absorbance of the mixed solution 1 and recording as A1;
(2) mixing the mixed solution 1 with the reagent B, and incubating for 6min at 37 ℃ to obtain a mixed solution 2; measuring the absorbance of the mixed solution 2 and recording as A2;
(3) changing the sample to be detected into a calibrator, repeating the steps (1) to (2), and measuring the absorbance A3 of the calibrator mixed solution 1 and the absorbance A4 of the calibrator mixed solution 2;
(4) calculating the type IV collagen concentration of the sample to be tested: the concentration of the IV type collagen of the sample to be tested is (A2-A1)/(A4-A3). times.the concentration of the IV type collagen of the calibrator.
Example 2
A kit for measuring type IV collagen in human blood comprises a reagent A, a reagent B, IV type collagen calibrator and a type IV collagen quality control product. The mass ratio of the reagent A to the reagent B is 4: 1.
The reagent A comprises the following components: ProClin 2000.5 g/L, EDTA 2.0g/L, glycerol 5g/L, bovine serum albumin 2.0g/L, Twen 301.0 g/L, polyethylene glycol 40002 g/L, sodium chloride 3.0g/L, 50mmol/L, pH is 7 phosphate buffer.
The reagent B comprises the following components: 2000.5 g/L ProClin, 2.0g/L EDTA, 5g/L glycerol, 2.0g/L bovine serum albumin, 301.0 g/L tween, 40002 g/L polyethylene glycol, 3.0g/L sodium chloride, 50mmol/L, pH phosphate buffer solution of 7, 0.2g/L anti-IV type collagen monoclonal antibody coupling nano microsphere with average particle size of about 50nm, and 0.4g/L anti-IV type collagen monoclonal antibody coupling nano microsphere with average particle size of about 150 nm.
The IV type collagen calibrator is a solution containing IV type collagen, and consists of 100mmol/L, pH 6 Good's buffer solution and 500ng/mL IV type collagen.
The quality control product of the IV type collagen is a solution containing the IV type collagen, and consists of 100mmol/L, pH 6 Good's buffer solution and 50ng/mL IV type collagen.
The preparation method of the anti-IV type collagen monoclonal antibody coupled nano-microsphere comprises the following steps:
(i) adding 200 μ L of carboxyl nano latex particle emulsion (Bangs Laboratories, Inc. 10 w/v%, average particle size 80nm), 1600 μ L of phosphate buffer solution (0.01mol/L, pH 7.4), two anti-IV type collagen monoclonal antibodies YKCO-1A7 and YKCO-4D9 (Shanghai friend science and technology Co., Ltd.) with mass of 1mg and capable of identifying different sites on IV type collagen, and 500 μ L of 0.1g/mL carbodiimide into a reaction vessel, magnetically stirring for 4h, centrifuging for 1h at 4 ℃ and 15000r/min, discarding supernatant, placing precipitate in an ice bath, performing ultrasonic vibration for 1s, spacing for 3s, and continuing for 3 min;
(ii) (ii) resuspending the precipitate subjected to ultrasonic oscillation treatment in the step (i) with an isovolumetric HEPES buffer solution (0.1mol/L, pH 7.4), adding 600. mu.L of 0.1mol/L glycine solution, stirring for 30min, centrifuging for 1h at 4 ℃ and 15000r/min, and discarding the supernatant to obtain a precipitate;
(iii) and (3) resuspending the precipitate obtained in the step (ii) with an equal volume of HEPES buffer solution (0.1mol/L, pH 7.4) to obtain the anti-IV type collagen monoclonal antibody coupled nanospheres with the average particle size of about 80 nm.
And (3) replacing the carboxyl nano latex particle emulsion with the average particle size of 80nm with the carboxyl nano latex particle emulsion with the average particle size of 200nm, and preparing the anti-IV type collagen monoclonal antibody coupled nano microsphere with the average particle size of about 200nm according to the steps (i) to (ii).
A method for detecting type IV collagen by using the kit comprises the following steps:
(1) mixing a sample to be detected with the reagent A, and incubating for 6min at 37 ℃ to obtain a mixed solution 1; measuring the absorbance of the mixed solution 1 and recording as A1;
(2) mixing the mixed solution 1 with the reagent B, and incubating for 6min at 37 ℃ to obtain a mixed solution 2; measuring the absorbance of the mixed solution 2 and recording as A2;
(3) changing the sample to be detected into a calibrator, repeating the steps (1) to (2), and measuring the absorbance A3 of the calibrator mixed solution 1 and the absorbance A4 of the calibrator mixed solution 2;
(4) calculating the type IV collagen concentration of the sample to be tested: the concentration of the IV type collagen of the sample to be tested is (A2-A1)/(A4-A3). times.the concentration of the IV type collagen of the calibrator.
Example 3
A kit for measuring type IV collagen in human blood comprises a reagent A, a reagent B, IV type collagen calibrator and a type IV collagen quality control product. The mass ratio of the reagent A to the reagent B is 1: 1.
The reagent A comprises the following components: ProClin 3001.1 g/L, EDTA0.05g/L, glycerol 10g/L, bovine serum albumin 1.0g/L, Tween 800.01g/L, polyethylene glycol 80005.0 g/L, sodium chloride 0.1g/L, 100mmol/L, pH is 8 phosphate buffer.
The reagent B comprises the following components: ProClin 3001.1 g/L, EDTA0.05g/L, glycerol 10g/L, bovine serum albumin 1.0g/L, Twenn 800.01g/L, polyethylene glycol 80005.0 g/L, sodium chloride 0.1g/L, phosphate buffer solution with concentration of 100mmol/L, pH of 8, anti-IV type collagen monoclonal antibody coupling nano-microsphere with average particle size of about 100nm of 0.1g/L, anti-IV type collagen monoclonal antibody coupling nano-microsphere with average particle size of about 250nm of 0.3 g/L.
The IV type collagen calibrator is a solution containing IV type collagen, and consists of 100mmol/L, pH 6 Good's buffer solution and 500ng/mL IV type collagen.
The quality control product of the IV type collagen is a solution containing the IV type collagen, and consists of 100mmol/L, pH 6 Good's buffer solution and 50ng/mL IV type collagen.
The preparation method of the anti-IV type collagen monoclonal antibody coupled nano-microsphere comprises the following steps:
(i) adding 220 mu L of carboxyl nano latex particle emulsion (Bangs Laboratories, Inc. in the United states; 10 w/v%, average particle size is 100nm), 1800 mu L of phosphate buffer solution (0.01mol/L, pH 7.4), two anti-IV type collagen monoclonal antibodies YKCO-1A7 and YKCO-4D9 (Shanghai friend science and technology Co., Ltd.) with the mass of 1mg and used for identifying different sites on IV type collagen, and 600 mu L of 0.1g/mL carbodiimide into a reaction vessel, magnetically stirring for 6h, centrifuging for 2h at 4 ℃ and 12000r/min, discarding supernatant, placing precipitate in an ice bath, performing ultrasonic oscillation for 1s, performing ultrasonic treatment at intervals of 4s, and continuing for 4 min;
(ii) (ii) resuspending the precipitate subjected to ultrasonic oscillation treatment in the step (i) by using an equal volume of HEPES buffer solution (0.1mol/L, pH 7.4), adding 600 mu L of 0.1mol/L glycine solution, stirring for 40min, centrifuging for 2h at 4 ℃ and 12000r/min, and removing the supernatant to obtain a precipitate;
(iii) and (3) resuspending the precipitate obtained in the step (ii) with an equal volume of HEPES buffer solution (0.1mol/L, pH 7.4) to obtain the anti-IV type collagen monoclonal antibody coupled nanospheres with the average particle size of about 100 nm.
And (3) replacing the carboxyl nano latex particle emulsion with the average particle size of 100nm with the carboxyl nano latex particle emulsion with the average particle size of 250nm, and preparing the anti-IV type collagen monoclonal antibody coupled nano microsphere with the average particle size of about 250nm according to the steps (i) to (ii).
A method for detecting type IV collagen by using the kit comprises the following steps:
(1) mixing a sample to be detected with the reagent A, and incubating for 6min at 37 ℃ to obtain a mixed solution 1; measuring the absorbance of the mixed solution 1 and recording as A1;
(2) mixing the mixed solution 1 with the reagent B, and incubating for 6min at 37 ℃ to obtain a mixed solution 2; measuring the absorbance of the mixed solution 2 and recording as A2;
(3) changing the sample to be detected into a calibrator, repeating the steps (1) to (2), and measuring the absorbance A3 of the calibrator mixed solution 1 and the absorbance A4 of the calibrator mixed solution 2;
(4) calculating the type IV collagen concentration of the sample to be tested: the concentration of the IV type collagen of the sample to be tested is (A2-A1)/(A4-A3). times.the concentration of the IV type collagen of the calibrator.
Comparative example 1
The difference between the comparative example and example 1 is that "0.1 g/L of the type IV collagen monoclonal antibody coupling nanosphere with an average particle size of about 50nm and 0.5g/L of the type IV collagen monoclonal antibody coupling nanosphere with an average particle size of about 150 nm" was replaced with "0.6 g/L of the type IV collagen monoclonal antibody coupling nanosphere with an average particle size of about 50 nm".
Comparative example 2
The difference between the comparative example and example 1 is that "0.1 g/L of the type IV collagen monoclonal antibody coupling nanosphere with an average particle size of about 50nm and 0.5g/L of the type IV collagen monoclonal antibody coupling nanosphere with an average particle size of about 150 nm" was replaced with "0.6 g/L of the type IV collagen monoclonal antibody coupling nanosphere with an average particle size of about 150 nm".
Test example 1
The kit of example 1 was used as an experimental group, and an IV type collagen kit (shenzhen, a new product biomedical engineering corporation, njn) with excellent accuracy, which was approved in the market, was used as a control group to perform a comparative experiment, and 40 samples were tested, and the results are shown in table 1.
TABLE 1
The results of the test group are shown in FIG. 1, and the results of the test group are plotted on the x-axis and the control group on the y-axis. As can be seen from fig. 1, the linear equation is y-0.9903 x +2.7656, and the linear correlation coefficient R-0.9913 > 0.99, so the correlation is good, which indicates that the kit of the present invention has high consistency with the commercially recognized IV collagen kit with excellent accuracy.
Test example 2
The linear range of example 1, comparative example 1 and comparative example 2 was determined by the following method:
serum samples with concentrations of 0ng/mL (L) and 400ng/mL (H) were taken for linear range verification by mixing L and H in the following ratios, 8/0, 7/1, 6/2, 4/4, 0/8, and the samples of the above ratios were examined 3 times using example 1, comparative example 1, and comparative example 2, respectively, for linear range verification, and the results are shown in Table 2, Table 3, and Table 4.
Table 2 example 1 linear range validation results
Table 3 comparative example 1 linear range validation results
Table 4 comparative example 2 linear range validation results
The results show that the correlation coefficients r of example 1, comparative example 1 and comparative example 2 are 0.9982, 0.9656 and 0.9676, respectively, and the correlation coefficient of example 1 is greater than that of comparative example 1 and comparative example 2, so that the linearity of example 1 is better than that of comparative example 1 and comparative example 2, and the low concentration band deviation of comparative example 1 is greater than that of comparative example 2.
And (4) conclusion: the linear range of the kit of the example 1 is [30,400], the linear correlation coefficient r is more than 0.99 and is more than that of the comparative example 1 and the comparative example 2, and the detection linearity of the low-concentration IV type collagen of the comparative example 1 is poor; comparative example 2 the high concentration of type IV collagen was detected with poor linearity. The reason is presumed to be: after the large-particle-size nano microspheres are combined with the IV type collagen, turbidity is easy to appear, the detection sensitivity is high, but the amount of the combined IV type collagen is small, so that the large-particle-size nano microspheres cannot be used for detecting the high-concentration IV type collagen; the small-particle-size nano microspheres have larger specific surface area, can combine more IV type collagen, and have advantages in the detection of samples with higher IV type collagen concentration, but when the IV type collagen concentration is lower, the small-particle-size nano microspheres cannot show obvious turbidity change after being combined with the IV type collagen, so that the small-particle-size nano microspheres cannot be used for detecting low-concentration IV type collagen. Therefore, the invention uses the large-particle-size nano microspheres and the small-particle-size nano microspheres in a matching way, and when in detection, the IV-type collagen enables the nano microspheres with the two particle sizes to be crosslinked and aggregated, so that the detection has higher sensitivity and larger linear range.
The raw materials and equipment used in the invention are common raw materials and equipment in the field if not specified; the methods used in the present invention are conventional in the art unless otherwise specified.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and all simple modifications, alterations and equivalents of the above embodiments according to the technical spirit of the present invention are still within the protection scope of the technical solution of the present invention.
Claims (10)
1. A kit for determining type IV collagen in human blood is characterized by comprising the following components: the anti-IV type collagen monoclonal antibody with the grain diameter of 50-100nm is coupled with the nano-microsphere, and the anti-IV type collagen monoclonal antibody with the grain diameter of 150-250nm is coupled with the nano-microsphere.
2. The kit of claim 1 comprising reagent a, reagent B, IV type collagen calibrator and type IV collagen quality control; the reagent A comprises the following components: microbial inhibitors, EDTA, glycerol, bovine serum albumin, surfactants, reaction enhancers, sodium chloride and Good's buffer solution; the reagent B comprises the following components: microorganism inhibitor, EDTA, glycerol, bovine serum albumin, surfactant, reaction enhancer, sodium chloride, anti-IV type collagen monoclonal antibody coupling nano-microsphere with the particle size of 50-100nm, anti-IV type collagen monoclonal antibody coupling nano-microsphere with the particle size of 150-250nm and Good's buffer solution.
3. The kit as claimed in claim 2, wherein in the reagent B, the mass concentration of the anti-type IV collagen monoclonal antibody coupling nanospheres with the particle size of 50-100nm is 0.1-0.5g/L, and the mass concentration of the anti-type IV collagen monoclonal antibody coupling nanospheres with the particle size of 150-250nm is 0.2-0.5 g/L.
4. The kit as claimed in claim 3, wherein in the reagent B, the ratio of the mass concentration of the anti-type IV collagen monoclonal antibody coupled nano-microsphere with the particle size of 150-250nm to the mass concentration of the anti-type IV collagen monoclonal antibody coupled nano-microsphere with the particle size of 50-100nm is 1: 2-5.
5. The kit according to claim 2, wherein in the reagent A and the reagent B, the microbial inhibitor has a mass concentration of 0.001 to 1.1g/L, the EDTA has a mass concentration of 0.05 to 5.0g/L, the glycerol has a mass concentration of 0.1 to 10.0g/L, the bovine serum albumin has a mass concentration of 0.1 to 5.0g/L, the surfactant has a mass concentration of 0.01 to 3.0g/L, the reaction enhancer has a mass concentration of 0.5 to 5.0g/L, and the sodium chloride has a mass concentration of 0.1 to 8.0 g/L; in the reagent A and the reagent B, the concentrations of Good's buffer solutions are both 25-100mmol/L, pH and are both 6-8; the mass ratio of the reagent A to the reagent B is 1-5: 1.
6. The kit of claim 2, wherein:
the IV type collagen calibrator is a solution containing IV type collagen, and consists of 25-100mmol/L, pH 6-8 Good's buffer solution and 400-600ng/mL IV type collagen;
the quality control product of the IV type collagen is a solution containing the IV type collagen, and consists of 25-100mmol/L, pH 6-8 Good's buffer solution and 40-60ng/mL IV type collagen.
7. The kit of any one of claims 1 to 4, wherein the preparation method of the anti-type IV collagen monoclonal antibody coupled nanospheres is as follows:
(i) adding 10-12 w/v% of carboxyl nano latex particle emulsion, phosphate buffer solution, two anti-IV type collagen monoclonal antibodies for identifying different sites on IV type collagen and 0.1-0.15g/mL of carbodiimide into a reaction container according to the proportion of 200 plus 220 muL to 1500 plus 1800 muL to 1-2mg to 500 plus 600 muL, magnetically stirring for 4-6h, centrifuging at 3-5 ℃, discarding supernatant, placing the precipitate in an ice bath, and ultrasonically shaking for 3-4 min;
(ii) (ii) resuspending the precipitate subjected to ultrasonic oscillation treatment in the step (i) by using a HEPES buffer solution, adding 0.1-0.2mol/L glycine solution, wherein the volume ratio of the glycine solution to the carboxyl nano latex particle emulsion in the step (i) is 2-2.2:5-6, stirring for 30-40min, centrifuging at 3-5 ℃, and discarding the supernatant to obtain a precipitate;
(iii) and (3) resuspending the precipitate obtained in the step (ii) by using a HEPES buffer solution to obtain the anti-type IV collagen monoclonal antibody coupled nano-microsphere.
8. The kit of claim 7, wherein:
(iv) the particle size of the carboxyl nano latex particles in the step (i) is 50-100nm, and the particle size of the anti-type IV collagen monoclonal antibody coupling nano microspheres obtained in the step (iii) is 50-100 nm; or
The particle size of the carboxyl nano latex particles in the step (i) is 150-250nm, and the particle size of the anti-IV type collagen monoclonal antibody coupling nano microspheres obtained in the step (iii) is 150-250 nm.
9. The kit of claim 2, 5 or 6, wherein:
the microbial inhibitor is at least one of sodium azide, ProClin 200 and ProClin 300; and/or
The surfactant is at least one of Tween20, Tween30, Tween80 and Triton X-100; and/or
The reaction enhancer is at least one of polyethylene glycol 2000, polyethylene glycol 4000, polyethylene glycol 8000 and polyethylene glycol 20000; and/or
The Good's buffer solution is at least one of phosphate buffer solution, glycine buffer solution, borate buffer solution, tris buffer solution and N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid buffer solution.
10. A method for detecting type IV collagen using the kit of any one of claims 2 to 9, comprising the steps of:
(1) mixing a sample to be detected with the reagent A, and incubating for 5-7 min at 35-37 ℃ to obtain a mixed solution 1; measuring the absorbance of the mixed solution 1 and recording as A1;
(2) mixing the mixed solution 1 with the reagent B, and incubating for 5-7 min at 35-37 ℃ to obtain a mixed solution 2; measuring the absorbance of the mixed solution 2 and recording as A2;
(3) changing the sample to be detected into a calibrator, repeating the steps (1) to (2), and measuring the absorbance A3 of the calibrator mixed solution 1 and the absorbance A4 of the calibrator mixed solution 2;
(4) calculating the type IV collagen concentration of the sample to be tested: the concentration of the IV type collagen of the sample to be tested = (A2-A1)/(A4-A3). times.the concentration of the calibrator IV type collagen.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011633326.XA CN112782409A (en) | 2020-12-31 | 2020-12-31 | Kit for determining IV type collagen in human blood and detection method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011633326.XA CN112782409A (en) | 2020-12-31 | 2020-12-31 | Kit for determining IV type collagen in human blood and detection method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112782409A true CN112782409A (en) | 2021-05-11 |
Family
ID=75753366
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011633326.XA Pending CN112782409A (en) | 2020-12-31 | 2020-12-31 | Kit for determining IV type collagen in human blood and detection method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112782409A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113552366A (en) * | 2021-07-07 | 2021-10-26 | 浙江亚培生物技术有限公司 | IV-type collagen determination kit and preparation method thereof |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100184049A1 (en) * | 2007-04-27 | 2010-07-22 | Steve Goodison | Glycoprotein Profiling of Bladder Cancer |
| CN102955033A (en) * | 2012-10-22 | 2013-03-06 | 金华市强盛生物科技有限公司 | Kit for determining glycocholic acid in human blood |
| CN105823893A (en) * | 2016-05-17 | 2016-08-03 | 北京豪迈生物工程有限公司 | IV type collagen test kit and test method thereof |
| CN105911296A (en) * | 2016-06-30 | 2016-08-31 | 深圳市亚辉龙生物科技股份有限公司 | IV-type collagen chemiluminescence immunoassay kit and preparation method thereof |
| CN111004325A (en) * | 2020-01-02 | 2020-04-14 | 安徽合创健康生物技术有限公司 | III type procollagen N-terminal peptide nano antibody, and coding sequence and application thereof |
| CN111157740A (en) * | 2019-12-28 | 2020-05-15 | 王贤俊 | Detection kit for IV type collagen and preparation method thereof |
| CN111157739A (en) * | 2019-12-28 | 2020-05-15 | 王贤俊 | IV type collagen antibody latex particle, preparation method and special combined coupling agent thereof |
-
2020
- 2020-12-31 CN CN202011633326.XA patent/CN112782409A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100184049A1 (en) * | 2007-04-27 | 2010-07-22 | Steve Goodison | Glycoprotein Profiling of Bladder Cancer |
| CN102955033A (en) * | 2012-10-22 | 2013-03-06 | 金华市强盛生物科技有限公司 | Kit for determining glycocholic acid in human blood |
| CN105823893A (en) * | 2016-05-17 | 2016-08-03 | 北京豪迈生物工程有限公司 | IV type collagen test kit and test method thereof |
| CN105911296A (en) * | 2016-06-30 | 2016-08-31 | 深圳市亚辉龙生物科技股份有限公司 | IV-type collagen chemiluminescence immunoassay kit and preparation method thereof |
| CN111157740A (en) * | 2019-12-28 | 2020-05-15 | 王贤俊 | Detection kit for IV type collagen and preparation method thereof |
| CN111157739A (en) * | 2019-12-28 | 2020-05-15 | 王贤俊 | IV type collagen antibody latex particle, preparation method and special combined coupling agent thereof |
| CN111004325A (en) * | 2020-01-02 | 2020-04-14 | 安徽合创健康生物技术有限公司 | III type procollagen N-terminal peptide nano antibody, and coding sequence and application thereof |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113552366A (en) * | 2021-07-07 | 2021-10-26 | 浙江亚培生物技术有限公司 | IV-type collagen determination kit and preparation method thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP5031208B2 (en) | Assay | |
| JP4668768B2 (en) | Immunoassay method and non-specific reaction suppression method | |
| US7759074B2 (en) | Immunological latex turbidimetry method and reagent therefor | |
| US7955792B2 (en) | Diluent for norovirus or sapovirus specimen and method for detecting virus | |
| CA1146852A (en) | Reagent for latex agglutination | |
| JP2007516422A (en) | Method for assessing samples containing cell targets and soluble analytes substantially simultaneously | |
| EP0064274A1 (en) | Method for assaying antigen-antibody reactions and reagent therefor | |
| CN111551730B (en) | Fluorescent microsphere sealing liquid and kit using same | |
| CN112782409A (en) | Kit for determining IV type collagen in human blood and detection method | |
| CN102879569A (en) | Immunoturbidimetry kit for detecting cysteine proteinase inhibitor C and preparation method thereof | |
| JPWO2002048711A1 (en) | Immunological analysis reagent and analysis method | |
| JP2000258420A (en) | Stabilizing method of human-hemoglobin in solution and stabilizing solution | |
| JP2001502419A (en) | Assay for the detection of antibodies to p53 | |
| CN111320696A (en) | MMP-3 antibody compound based on streptavidin latex and kit thereof | |
| JP5191291B2 (en) | Method and reagent kit for measuring hemoglobin in stool specimen | |
| JPH1123573A (en) | Immunological measuring method | |
| JP2019028050A (en) | Means for preventing deterioration of immunoassay reagent containing insoluble carrier particles | |
| EP1298438A1 (en) | Insoluble carrier particle nephelometric immunoassay reagent | |
| JP2004012434A (en) | Method for measuring pepsinogen and measuring kit | |
| CN108414775A (en) | Neutrophil gelatinase-associated lipocalin detection kit | |
| CN114200129B (en) | Hematuria simultaneous detection kit of tissue metalloproteinase inhibitor-2 latex immunoturbidimetry | |
| JP2001091516A (en) | METHOD FOR DETERMINING QUANTITY OF HUMAN alpha-FETOPROTEIN AND MEASUREMENT KIT | |
| JP4185589B2 (en) | Method and reagent for measuring rheumatoid factor | |
| JPS61296270A (en) | Reagent for immunological reaction | |
| CN116256517A (en) | anti-NGAL monoclonal antibody composite fluorescent microsphere compound, preparation method thereof and NGAL detection kit |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |