CN107703307B - IV-type collagen detection kit and use method thereof - Google Patents

IV-type collagen detection kit and use method thereof Download PDF

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CN107703307B
CN107703307B CN201710914665.7A CN201710914665A CN107703307B CN 107703307 B CN107703307 B CN 107703307B CN 201710914665 A CN201710914665 A CN 201710914665A CN 107703307 B CN107703307 B CN 107703307B
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reagent
collagen
type
latex
polyclonal antibody
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CN107703307A (en
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吴铮
丁先骏
吴泽东
庄庆华
张金东
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Anhui Iprocom Biotechnology Co ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
    • 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]

Abstract

The invention discloses a type IV collagen detection kit, which comprises a reagent R1: MOPSO buffer solution, NaCl and NaN3Arabic gum, Tween-80 and PEG-2000, wherein the solvent is purified water; reagent R2: MOPSO buffer solution, NaCl and NaN3BSA, glycerol and a latex-coated type IV collagen polyclonal antibody, wherein the solvent is purified water; the latex-coated type IV collagen polyclonal antibody is prepared from the following raw materials: latex microspheres, type IV collagen polyclonal antibody and polyethylene p-chloromethyl styrene copolymer. The invention also discloses a use method of the IV type collagen detection kit. The invention has the advantages that: (1) the kit has high detection sensitivity and accuracy, and is simple and rapid to operate; (2) the formed antigen-antibody complex has good stability and strong specificity; (3) the cost is low and no pollution is caused; (4) the device is used for a full-automatic biochemical analyzer.

Description

IV-type collagen detection kit and use method thereof
Technical Field
The invention relates to the technical field of medical examination, in particular to a type IV collagen detection kit and a using method thereof.
Background
Collagen is a fibrous glycoprotein, it is α -peptide chain network structure formed by three-strand spirochete, found collagen is more than 10 kinds at present, exist in different tissues, type IV collagen is the important component forming basement membrane, normal intrahepatic basement membrane mainly exists around blood vessel, lymphatic vessel, bile duct, hepatic sinus wall lack, with inflammation development in liver disease, fibrous tissue hyperplasia is active, fibrous tissue formation process have a large amount of collagen deposition, various collagens increase, but wherein the most important is the increase of type IV collagen forming basement membrane.
At present, the method for detecting the type IV collagen is mainly a radioimmunoassay method, but the specific operation of the radioimmunoassay method is relatively complicated, and the problems of certain radioactive pollution, much investment and the like exist.
Therefore, an IV type collagen detection kit which is simple to operate, free of pollution and low in cost is urgently needed at present.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides an IV type collagen detection kit which is simple to operate, free of pollution and low in cost and a using method thereof.
The invention is realized by the following technical scheme: a type IV collagen detection kit comprises independent reagent R1 and reagent R2 double liquid components, and comprises the following components in corresponding content:
reagent R1:
Figure BDA0001425617050000021
the solvent is purified water;
reagent R2:
Figure BDA0001425617050000022
the solvent is purified water;
the latex-coated type IV collagen polyclonal antibody comprises the following raw material components: the kit comprises latex microspheres, a type IV collagen polyclonal antibody and a polyethylene p-chloromethyl styrene copolymer, wherein the content of the polyethylene p-chloromethyl styrene copolymer in the overall reagent R2 is 9-11%.
As one of the preferable modes of the invention, the double-liquid component comprises a reagent R1 and a reagent R2 which are independent from each other, and comprises the following components in corresponding content:
reagent R1:
Figure BDA0001425617050000023
Figure BDA0001425617050000031
the solvent is purified water;
reagent R2:
Figure BDA0001425617050000032
the solvent is purified water;
the latex-coated type IV collagen polyclonal antibody comprises the following raw material components: the reagent comprises latex microspheres, a type IV collagen polyclonal antibody and a polyethylene p-chloromethyl styrene copolymer, wherein the content of the polyethylene p-chloromethyl styrene copolymer in the overall reagent R2 is 10%.
As one of the preferable modes of the invention, the double-liquid component comprises a reagent R1 and a reagent R2 which are independent from each other, and comprises the following components in corresponding content:
reagent R1:
Figure BDA0001425617050000033
Figure BDA0001425617050000041
the solvent is purified water;
reagent R2:
Figure BDA0001425617050000042
the solvent is purified water;
the latex-coated type IV collagen polyclonal antibody comprises the following raw material components: the reagent comprises latex microspheres, a type IV collagen polyclonal antibody and a polyethylene p-chloromethyl styrene copolymer, wherein the content of the polyethylene p-chloromethyl styrene copolymer in the overall reagent R2 is 10.5%.
In a preferred embodiment of the present invention, the reagent R1 has a MOPSO buffer pH of 6.5.
In a preferred embodiment of the present invention, the reagent R2 has a MOPSO buffer pH of 6.5.
A use method of an IV type collagen detection kit comprises the following steps:
(1) mixing a sample to be detected with the reagent R1, and incubating for 5min at 37 ℃;
(2) measuring the absorbance A1 after reaction by using a full-automatic biochemical analyzer;
(3) mixing with a reagent R2, and reacting for 5min at 37 ℃;
(4) measuring the absorbance A2 after reaction by using a full-automatic biochemical analyzer;
(5) and calculating the concentration of the type IV collagen in the sample according to the absorbance change value.
In a preferred embodiment of the present invention, the volume ratio of the reagent R1 in the step (1) to the reagent R2 in the step (3) is 4: 1.
In a preferred embodiment of the present invention, the volume ratio of the sample to be tested in step (1) to the reagent solution comprising the reagents R1 and R2 is 1: 50.
In a preferred embodiment of the present invention, in the step (2) and the step (4), the absorbances A1 and A2 are measured at a wavelength of 660 nm.
In a preferred embodiment of the present invention, in the step (5), the absorbance change value is Δ a, which is a value obtained by subtracting a1 from a 2.
Compared with the prior art, the invention has the advantages that:
(1) the kit has higher detection sensitivity and accuracy, is simple and quick to use and operate, and only needs 10 minutes from detection;
(2) the antigen-antibody complex formed by the invention has good stability, certain absorbance under specific wavelength and strong specificity;
(3) the invention does not need to be equipped with special large-scale instruments and equipment, and has low cost and no pollution;
(4) the device can be used on a full-automatic biochemical analyzer, is suitable for full-automatic testing, and can be developed and popularized on a large scale.
Detailed Description
The following examples are given for the detailed implementation and specific operation of the present invention, but the scope of the present invention is not limited to the following examples.
Example 1
The type IV collagen detection kit comprises two liquid components of a reagent R1 and a reagent R2 which are independent of each other, and comprises the following components in corresponding content:
reagent R1:
Figure BDA0001425617050000061
the solvent is purified water;
reagent R2:
Figure BDA0001425617050000062
the solvent is purified water;
the latex-coated type IV collagen polyclonal antibody comprises the following raw material components: the reagent comprises latex microspheres, a type IV collagen polyclonal antibody and a polyethylene p-chloromethyl styrene copolymer, wherein the content of the polyethylene p-chloromethyl styrene copolymer in the overall reagent R2 is 9%.
Example 2
The type IV collagen detection kit comprises two liquid components of a reagent R1 and a reagent R2 which are independent of each other, and comprises the following components in corresponding content:
reagent R1:
Figure BDA0001425617050000071
the solvent is purified water;
reagent R2:
Figure BDA0001425617050000072
the solvent is purified water;
the latex-coated type IV collagen polyclonal antibody comprises the following raw material components: the reagent comprises latex microspheres, a type IV collagen polyclonal antibody and a polyethylene p-chloromethyl styrene copolymer, wherein the content of the polyethylene p-chloromethyl styrene copolymer in the overall reagent R2 is 11%.
Example 3
The type IV collagen detection kit comprises two liquid components of a reagent R1 and a reagent R2 which are independent of each other, and comprises the following components in corresponding content:
reagent R1:
Figure BDA0001425617050000081
the solvent is purified water;
reagent R2:
Figure BDA0001425617050000082
the solvent is purified water;
the latex-coated type IV collagen polyclonal antibody comprises the following raw material components: the reagent comprises latex microspheres, a type IV collagen polyclonal antibody and a polyethylene p-chloromethyl styrene copolymer, wherein the content of the polyethylene p-chloromethyl styrene copolymer in the overall reagent R2 is 10%.
Example 4
The type IV collagen detection kit comprises two liquid components of a reagent R1 and a reagent R2 which are independent of each other, and comprises the following components in corresponding content:
reagent R1:
Figure BDA0001425617050000083
Figure BDA0001425617050000091
the solvent is purified water;
reagent R2:
Figure BDA0001425617050000092
the solvent is purified water;
the latex-coated type IV collagen polyclonal antibody comprises the following raw material components: the reagent comprises latex microspheres, a type IV collagen polyclonal antibody and a polyethylene p-chloromethyl styrene copolymer, wherein the content of the polyethylene p-chloromethyl styrene copolymer in the overall reagent R2 is 10.5%.
Example 5
The preparation method of the type iv collagen assay kit according to the embodiment of the present invention includes the following steps:
(1) preparing a reagent R1:
① according to the component content of the reagent R1 in the above examples, MOPSO buffer solution is prepared, and the pH is adjusted to 6.5 by dilute hydrochloric acid or sodium hydroxide to be used as R1 buffer solution;
② according to the component content of the reagent R1 in the above examples, NaCl and NaN were added3Dissolving Arabic gum, Tween-80 and PEG-2000 in R1 buffer solution, stirring uniformly, and preparing a reagent R1 after all raw materials are fully dissolved;
(2) preparing a reagent R2:
① according to the component content of the reagent R2 in the above examples, MOPSO buffer solution is prepared, and the pH is adjusted to 6.5 by dilute hydrochloric acid or sodium hydroxide to be used as R2 buffer solution;
② according to the component content of the reagent R2 in the above examples, NaCl and NaN were added3Dissolving BSA and glycerol in R2 buffer solution, and stirring uniformly to obtain R2 dispersion;
③ preparation of latex-coated type iv collagen polyclonal antibodies:
a. taking latex microspheres with the particle sizes of 80nm (20g/L) and 120nm (40g/L) into 100mM MES buffer solution, adding 50g/L EDAC solution, mixing uniformly, incubating and mixing uniformly for 1h at 37 ℃, and centrifuging to remove supernatant; adding 50g/L NHS solution to restore to the original volume, mixing uniformly, incubating and mixing uniformly for 1h at 37 ℃, and centrifuging to remove supernatant to obtain mixed microsphere emulsion;
b. adding the polyethylene p-chloromethyl styrene copolymer with the content of the components into the mixed microsphere emulsion, reacting for 2 hours at the temperature of 37 ℃, and copolymerizing latex with the particle sizes of 80nm and 120 nm; centrifuging and precipitating, dispersing the residual substances in MES buffer solution, repeating for 3 times, recovering to the original volume in MES buffer solution, adding type IV collagen polyclonal antibody, reacting for 3.5h at 37 ℃, centrifuging, dispersing the precipitate in PBS buffer solution of the original volume, repeating for 3 times, dispersing the precipitate in NHS buffer solution of the original volume, and adding 30g/L BSA;
c.4 ℃ for 48h to obtain the finally required latex coated type IV collagen polyclonal antibody;
④ the obtained latex-coated type IV collagen polyclonal antibody was dissolved in the R2 dispersion, and ultrasonically dispersed to finally prepare a reagent R2.
Example 6
The use method of the type iv collagen assay kit according to the present embodiment includes the following steps:
(1) mixing 5uL of a sample to be detected with 200uL of reagent R1, and incubating for 5min at 37 ℃;
(2) measuring the absorbance A1 after the reaction at the wavelength of 660nm by using a full-automatic biochemical analyzer;
(3) mixing with 50uL reagent R2, and reacting at 37 ℃ for 5 min;
(4) measuring the absorbance A2 after the reaction at the wavelength of 660nm by using a full-automatic biochemical analyzer;
(5) and calculating the concentration of the type IV collagen in the sample according to the absorbance change value delta A-A2-A1.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Claims (10)

1. The type IV collagen detection kit is characterized by comprising two independent liquid components of a reagent R1 and a reagent R2, and comprises the following components in corresponding content:
reagent R1:
Figure FDA0002314057740000011
reagent R2:
Figure FDA0002314057740000012
the latex-coated type IV collagen polyclonal antibody comprises the following raw material components: the kit comprises latex microspheres, a type IV collagen polyclonal antibody and a polyethylene p-chloromethyl styrene copolymer, wherein the content of the polyethylene p-chloromethyl styrene copolymer in an integral reagent R2 is 9-11%;
preparation of the R1 reagent:
① according to the component content of the reagent R1, preparing MOPSO buffer solution, and adjusting the pH value to 6.5 by using dilute hydrochloric acid or sodium hydroxide as R1 buffer solution;
② according to the component content of the reagent R1, NaCl and NaN are added3Dissolving Arabic gum, Tween-80 and PEG-2000 in R1 buffer solution, stirring uniformly, and preparing a reagent R1 after all raw materials are fully dissolved;
preparation of the latex-coated type iv collagen polyclonal antibody:
a. taking 20g/L latex microspheres with the particle size of 80nm and 40g/L latex microspheres with the particle size of 120nm into 100mM MES buffer solution, adding 50g/L EDAC solution, mixing uniformly, incubating and mixing uniformly for 1h at 37 ℃, and centrifuging to remove supernatant; adding 50g/L NHS solution to restore to the original volume, mixing uniformly, incubating and mixing uniformly for 1h at 37 ℃, and centrifuging to remove supernatant to obtain mixed microsphere emulsion;
b. adding the polyethylene p-chloromethyl styrene copolymer with the content of the components into the mixed microsphere emulsion, reacting for 2 hours at the temperature of 37 ℃, and copolymerizing latex with the particle sizes of 80nm and 120 nm; centrifuging and precipitating, dispersing the residual substances in MES buffer solution, repeating for 3 times, recovering to the original volume in MES buffer solution, adding type IV collagen polyclonal antibody, reacting for 3.5h at 37 ℃, centrifuging, dispersing the precipitate in PBS buffer solution of the original volume, repeating for 3 times, dispersing the precipitate in NHS buffer solution of the original volume, and adding 30g/L BSA;
and c.4 ℃ sealing and storing for 48h to obtain the final needed latex-coated type IV collagen polyclonal antibody.
2. The type IV collagen assay kit of claim 1, comprising independent reagent R1 and reagent R2 double liquid components, comprising the following components and corresponding contents:
reagent R1:
Figure FDA0002314057740000021
Figure FDA0002314057740000031
reagent R2:
Figure FDA0002314057740000032
the latex-coated type IV collagen polyclonal antibody comprises the following raw material components: the reagent comprises latex microspheres, a type IV collagen polyclonal antibody and a polyethylene p-chloromethyl styrene copolymer, wherein the content of the polyethylene p-chloromethyl styrene copolymer in the overall reagent R2 is 10%.
3. The type IV collagen assay kit of claim 1, comprising independent reagent R1 and reagent R2 double liquid components, comprising the following components and corresponding contents:
reagent R1:
Figure FDA0002314057740000033
reagent R2:
Figure FDA0002314057740000041
the latex-coated type IV collagen polyclonal antibody comprises the following raw material components: the reagent comprises latex microspheres, a type IV collagen polyclonal antibody and a polyethylene p-chloromethyl styrene copolymer, wherein the content of the polyethylene p-chloromethyl styrene copolymer in the overall reagent R2 is 10.5%.
4. The type IV collagen test kit according to any one of claims 1 to 3, wherein the MOPSO buffer solution of the reagent R1 has a pH of 6.5.
5. The type IV collagen test kit according to any one of claims 1 to 3, wherein the MOPSO buffer solution of the reagent R2 has a pH of 6.5.
6. A method of using the collagen type iv assay kit of any one of claims 1 to 3 for non-disease diagnostic purposes comprising the steps of:
(1) mixing a sample to be detected with the reagent R1, and incubating for 5min at 37 ℃;
(2) measuring the absorbance A1 after reaction by using a full-automatic biochemical analyzer;
(3) mixing with a reagent R2, and reacting for 5min at 37 ℃;
(4) measuring the absorbance A2 after reaction by using a full-automatic biochemical analyzer;
(5) and calculating the concentration of the type IV collagen in the sample according to the absorbance change value.
7. The use of the type IV collagen test kit for non-disease diagnostic purposes as claimed in claim 6, wherein the volume ratio of the reagent R1 of step (1) to the reagent R2 of step (3) is 4: 1.
8. The use method of the type IV collagen test kit for non-disease diagnosis according to claim 6, wherein the volume ratio of the sample to be tested in step (1) to the reagent solution comprising the reagent R1 and the reagent R2 is 1: 50.
9. The method of using type IV collagen test kit for non-disease diagnostic purposes as claimed in claim 6 wherein in steps (2) and (4) the absorbance A1 and A2 are measured at 660 nm.
10. The method of using type IV collagen test kit for non-disease diagnostic purposes as claimed in claim 6 wherein in step (5) the absorbance change is Δ A, which is the value obtained by subtracting A1 from A2.
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