CN111579800A - Hypersensitive procalcitonin detection reagent and preparation method and use method thereof - Google Patents
Hypersensitive procalcitonin detection reagent and preparation method and use method thereof Download PDFInfo
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- 238000001514 detection method Methods 0.000 title claims abstract description 145
- 108010048233 Procalcitonin Proteins 0.000 title claims abstract description 66
- CWCXERYKLSEGEZ-KDKHKZEGSA-N procalcitonin Chemical compound C([C@@H](C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](C(C)C)C(=O)NCC(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)NCC(O)=O)[C@@H](C)O)NC(=O)[C@@H](NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCSC)NC(=O)[C@H]1NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(N)=O)NC(=O)CNC(=O)[C@@H](N)CSSC1)[C@@H](C)O)[C@@H](C)O)[C@@H](C)O)C1=CC=CC=C1 CWCXERYKLSEGEZ-KDKHKZEGSA-N 0.000 title claims abstract description 66
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- 206010020751 Hypersensitivity Diseases 0.000 title abstract description 35
- 238000002360 preparation method Methods 0.000 title abstract description 10
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- 239000007788 liquid Substances 0.000 claims abstract description 89
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- 238000003908 quality control method Methods 0.000 claims abstract description 40
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- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims description 38
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 20
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- JYCQQPHGFMYQCF-UHFFFAOYSA-N 4-tert-Octylphenol monoethoxylate Chemical compound CC(C)(C)CC(C)(C)C1=CC=C(OCCO)C=C1 JYCQQPHGFMYQCF-UHFFFAOYSA-N 0.000 claims description 11
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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
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
-
- 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
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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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
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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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/575—Hormones
- G01N2333/585—Calcitonins
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Abstract
The invention discloses a hypersensitive procalcitonin detection reagent and a preparation method and a use method thereof, wherein the detection reagent comprises the following steps: a reaction buffer and a reagent strip, further comprising a solid first probe and a second probe, wherein: the reagent strip comprises a pad, a nitrocellulose membrane laid on the pad, a sample adding pad, a quality control line, a first detection line, a second detection line and an absorption pad, wherein the sample adding pad, the quality control line, the first detection line, the second detection line and the absorption pad are sequentially arranged on the nitrocellulose membrane at intervals; the preparation method of the hypersensitive procalcitonin detection reagent comprises the following steps: s1, spraying on the nitrocellulose membrane to form a quality control line, a first detection line and a second detection line, and then pasting the sample adding pad and the absorption pad on the nitrocellulose membrane; s2, preparing a reaction buffer solution; s3, preparing a first liquid and a second liquid, and freeze-drying the first liquid and the second liquid; a method for using the hypersensitive procalcitonin detection reagent to detect procalcitonin. The method improves the precision of procalcitonin detection and improves the stability of a detection reagent product.
Description
Technical Field
The invention relates to the field of biotechnology. More specifically, the invention relates to a hypersensitive procalcitonin detection reagent, a preparation method and a use method thereof.
Background
Procalcitonin detection reagents are detection tools for detecting whether a body has systemic inflammatory response syndrome, such as bacterial infection, pancreatitis, burns and the like.
The existing procalcitonin detection reagent comprises a reaction buffer solution and a reagent strip, wherein the reagent strip comprises a pad, a nitrocellulose membrane laid on the pad, a sample adding pad, a binding pad, a quality control line, a first detection line, a second detection line and an absorption pad which are sequentially arranged on the nitrocellulose membrane at intervals, and the binding pad is coated with a fluorescent-labeled PCT antibody complex, a biotin-labeled PCT antibody complex and a fluorescent-labeled Anti-chicken IgY antibody complex. When detection is needed, a sample to be detected and a reaction buffer solution are uniformly mixed to prepare a mixed solution, the mixed solution is added on a sample adding pad of the reagent strip, the mixed solution flows to an absorption pad from the sample adding pad in sequence, and detection equipment is used for detecting a first detection line and a second detection line on the reacted reagent strip, so that the procalcitonin concentration in the sample to be detected can be obtained.
When the sample to be detected flows through the binding pad, the reaction time of the antigen in the sample and the coated fluorescence-labeled PCT antibody complex, the biotin-labeled PCT antibody complex, and the quality control complex is short, and the precision is poor due to incomplete reaction.
Disclosure of Invention
An object of the present invention is to solve at least the above problems and to provide at least the advantages described later.
The invention also aims to provide a hypersensitive procalcitonin detection reagent, a preparation method and a use method thereof, which improve the precision of procalcitonin detection and improve the stability of detection reagent products.
To achieve these objects and other advantages in accordance with the present invention, there is provided a procalcitonin detection reagent comprising a reaction buffer and a reagent strip, further comprising a solid first probe and a solid second probe, wherein:
the first detector contains a biotin-labeled PCT antibody complex and a fluorescence-labeled Anti-chicken IgY antibody complex;
the second detector contains a fluorescent labeled PCT antibody complex;
the reagent strip comprises a pad, a nitrocellulose membrane laid on the pad, and a sample adding pad, a quality control line, a first detection line, a second detection line and an absorption pad which are arranged on the nitrocellulose membrane at intervals in sequence.
Preferably, the reaction buffer has a pH of 7.4 and a composition of: 10mM PBS, 100-30.1-3% Tween20, 0.01-0.5% IGEPAL CA-630, 0.5-3% BSA, wherein the volume of the reaction buffer is not less than 150. mu.L.
Preferably, the first probe is prepared by freeze-drying a first liquid having a pH of 7.4, the first liquid having the composition: 10mM PBS, 0.05-10% BSA, 0.05-5% Sucrose, 0.01-10ug/mL biotin-labeled PCT antibody complex, 0.01-10ug/mL fluorescently-labeled Anti-chicken IgY antibody complex;
the second probe is prepared by freeze-drying a second liquid with the pH of 7.4, and the second liquid comprises the following components: 10mM PBS, 0.05-10% BSA, 0.05-5% Sucrose, 2-25ug/mL fluorescently labeled PCT antibody complex.
Preferably, the quality control line, the first detection line and the second detection line are sequentially spaced by a distance of 2-3 mm.
Preferably, the first and second probes are one of spherical and powdery.
Also provides a preparation method of the hypersensitive procalcitonin detection reagent, which comprises the following steps:
s1, sticking the nitrocellulose membrane on the pad card, spraying the nitrocellulose membrane to form a quality control line, a first detection line and a second detection line, and sticking the sample adding pad and the absorption pad on the nitrocellulose membrane to obtain a reagent strip;
s2, preparing a reaction buffer solution;
s3, preparing the first liquid and the second liquid, and freeze-drying the first liquid and the second liquid to obtain a first detector and a second detector.
Also provides a method for using the hypersensitive procalcitonin detection reagent to detect procalcitonin, which comprises the following steps:
SA, horizontally placing the reagent strip;
SB, mixing 150 mu L of reaction buffer solution with the detector to obtain a mixed reagent;
SC, taking 35 mu L of sample to be detected and uniformly mixing the sample with the mixed reagent to obtain a sample mixed solution;
SD, adding 75 mu L of sample mixed solution to a sample adding pad of the reagent strip, and reacting at room temperature for 12min to obtain a reacted reagent strip;
and SE, detecting the first detection line and the second detection line on the reacted reagent strip by using detection equipment.
The invention at least comprises the following beneficial effects:
the invention makes the fluorescent marked PCT antibody complex, biotin marked PCT antibody complex and fluorescent marked Anti-chicken IgY antibody complex coated on the binding pad of the reagent strip into a detector, and removes the binding pad on the reagent strip. When the concentration of procalcitonin in a sample to be detected is detected, the reaction buffer solution and the detector are fully mixed to obtain a mixed reagent, the sample to be detected is added into the mixed reagent to be fully mixed to obtain a sample mixed solution, and the sample mixed solution is added onto a sample adding pad of the reagent strip.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.
Drawings
FIG. 1 is a schematic diagram of the detection substance and the reaction buffer in the detection reagent according to one embodiment of the present invention;
FIG. 2 is a schematic structural diagram of a test strip according to one embodiment of the present invention;
FIG. 3 is a graph showing the correlation between the serum test value of the experimental system and the serum test value of the control system according to one embodiment of the present invention;
FIG. 4 is a correlation graph of a plasma measurement value and a serum measurement value of an experimental system according to one embodiment of the present invention;
FIG. 5 is a graph showing the correlation between a whole blood measurement value and a serum measurement value in an experimental system according to one embodiment of the present invention.
Reference numerals: the kit comprises a reaction buffer solution 1, a first detector 2, a second detector 3, a pad card 4, a nitrocellulose membrane 5, a sample adding pad 6, a quality control line 7, a first detection line 8, a second detection line 9 and an absorption pad 10.
Detailed Description
The present invention is further described in detail below with reference to examples so that those skilled in the art can practice the invention with reference to the description.
It is to be noted that the experimental methods described in the following embodiments are all conventional methods unless otherwise specified, and the reagents and materials, if not otherwise specified, are commercially available; in the description of the present invention, the indicated orientation or positional relationship is based on the orientation or positional relationship shown in the drawings, and is only for convenience of description and simplification of description, and does not indicate or imply that the indicated device or element must have a particular orientation, be constructed and operated in a particular orientation, and thus should not be construed as limiting the present invention.
As shown in fig. 1-2, the present invention provides a procalcitonin hypersensitivity test reagent, comprising a reaction buffer 1 and a reagent strip, further comprising a solid first probe 2 and a solid second probe 3, wherein:
the first detector 2 contains a biotin-labeled PCT antibody complex and a fluorescence-labeled Anti-chicken IgY antibody complex;
the second detector 3 contains a fluorescent labeled PCT antibody complex;
the reagent strip comprises a pad 4, a nitrocellulose membrane 5 laid on the pad 4, and a sample adding pad 6, a quality control line 7, a first detection line 8, a second detection line 9 and an absorption pad 10 which are arranged on the nitrocellulose membrane 5 at intervals in sequence.
In the technical scheme, the detection reagent for the hypersensitive procalcitonin comprises a reaction buffer solution 1, a reagent strip, a solid first detector 2 and a solid second detector 3, wherein the first detector 2 contains a biotin-labeled PCT antibody complex and a fluorescence-labeled Anti-chicken IgY antibody complex; the second detector 3 contains a fluorescent labeled PCT antibody complex; the reagent strip comprises a pad card 4, a nitrocellulose membrane 5, a sample adding pad 6, a quality control line 7, a first detection line 8, a second detection line 9 and an absorption pad 10, wherein the nitrocellulose membrane 5 is laid on the pad card 4, and the sample adding pad 6, the quality control line 7, the first detection line 8, the second detection line 9 and the absorption pad 10 are sequentially arranged on the nitrocellulose membrane 5 at intervals.
By adopting the technical scheme, the fluorescence-labeled PCT antibody complex, the biotin-labeled PCT antibody complex and the fluorescence-labeled Anti-chicken IgY antibody complex which are coated on the binding pad of the reagent strip are made into probes, and the binding pad on the reagent strip is removed. When the procalcitonin concentration in a sample to be detected is detected, the reaction buffer solution 1 and a detector are fully mixed to obtain a mixed reagent, the sample to be detected is added into the mixed reagent to be fully mixed to obtain a sample mixed solution, and the sample mixed solution is added onto a sample adding pad 6 of a reagent strip.
< example 1>
A reagent for detecting hypersensitive procalcitonin, the pH of reaction buffer solution 1 is 7.4, and the components are as follows: 10mM PBS, 100mM NaCl, 0.01% NaN30.1% Tween20, 0.01% IGEPAL CA-630, 0.5% BSA; the first probe 2 was prepared by freeze-drying a first liquid having a pH of 7.4, the composition of the first liquid being: 10mM PBS, 0.05% BSA, 0.05% Sucrose, 0.01ug/mL biotin-labeled PCT antibody complex, 0.01ug/mL fluorescently-labeled Anti-chicken IgY antibody complex; the second probe 3 was prepared by freeze-drying a second liquid having a pH of 7.4, the composition of the second liquid being: 10mM PBS, 0.05% BSA, 0.05% Sucrose, 2ug/mL fluorescently labeled PCT antibody complex; the reagent strip comprises a pad 4, a nitrocellulose membrane 5 laid on the pad 4, a sample adding pad 6, a quality control line 7, a first detection line 8, a second detection line 9 and an absorption pad 10 which are sequentially arranged on the nitrocellulose membrane 5 at intervals.
A preparation method of a procalcitonin hypersensitivity detection reagent comprises the following steps:
s1, preparing a reagent strip:
s101, screening of antibodies: purchasing streptavidin and chicken IgY and carrying out a pairing experiment on the streptavidin and chicken IgY, screening the streptavidin and the chicken IgY with high specificity and good affinity, and respectively adjusting the streptavidin and the chicken IgY to 1-10mg/mL and 0.01-5mg/mL by using coating diluent;
s102, antibody coating: the nitrocellulose membrane 5 is prepared and correctly placed on a pasting device, by which the nitrocellulose membrane 5 is pasted on the backer 4. And spraying the chicken IgY adjusted by using the coating diluent on the nitrocellulose membrane 5 by using a spraying device to form a control line, and spraying the streptavidin adjusted by using the coating diluent on the nitrocellulose membrane 5 to form a first detection line 8 and a second detection line 9, so as to obtain the coated reagent strip, wherein the spraying amount of the spraying device is 1 mu L/cm, the spraying speed is 9cm/s, and the quality control line 7, the first detection line 8 and the second detection line 9 are sequentially spaced by 2 mm. Then placing the coated reagent strip in an oven, baking for 30-120min at 37 ℃, and taking out;
s103, sticking a film on the reagent strip and cutting; the sample adding pad 6 and the absorption pad 10 are pasted on the cellulose nitrate membrane 5 and are arranged on a quality control line 7, a first detection line 8, a second detection line 9 and two sides of the absorption pad 10, the sample adding pad 6 is pasted on one side face of a pad card 4 with wide width, the length of the side of the cellulose nitrate membrane 5 below the sample adding pad 6, which is parallel to the long side of the pad card 4, is 1-2mm, the absorption pad 10 is pasted on one side face of the pad card 4 with narrow width, and the distance between the end part of the cellulose nitrate membrane 5 below the absorption pad 10 and one end of the pad card 4, which is provided with the absorption pad 10, is less than 1. After the absorption pad 10 and the sample adding pad 6 are adhered, the reagent strip is cut into reagent strips with the width of 3.9mm by using a cutting device, so that the cut reagent strips are obtained, and the cut reagent strips are placed on a pressing shell device and pressed firmly to obtain the reagent strips.
S2, preparing a reaction buffer solution 1; NaCl and NaN were added to 10mM PBS at pH 7.43Tween20, IGEPAL CA-630, BSA to a NaCl concentration of 100mM, NaN3The percentage concentration is 0.01%, the Tween20 percentage concentration is 0.1%, the IGEPAL CA-630 percentage concentration is 0.01%, the BSA percentage concentration is 0.5%, after the components are completely dissolved, the reaction buffer solution 1 is obtained by filtration, and the reaction buffer solution 1 is stored at the temperature of 2-8 ℃;
s3, preparing the first probe 2 and the second probe 3:
s301, configuring a first liquid: adding BSA, Sucrose, a biotin-labeled PCT antibody complex and a fluorescence-labeled Anti-chicken IgY antibody complex into PBS with the pH value of 7.4 and the concentration of 10mM until the percent concentration of BSA is 0.05 percent, the percent concentration of Sucrose is 0.05 percent, the percent concentration of the biotin-labeled PCT antibody complex is 0.01ug/mL, and the concentration of the fluorescence-labeled Anti-chicken IgY antibody complex is 0.01 ug/mL;
s302, configuring a second liquid: adding BSA, Sucrose and a fluorescently-labeled PCT antibody complex into PBS with the pH value of 7.4 and the concentration of 10mM until the percent concentration of BSA is 0.05 percent, the percent concentration of Sucrose is 0.05 percent and the concentration of the fluorescently-labeled PCT antibody complex is 2 ug/mL;
s303, detecting the effectiveness of the first liquid and the second liquid: mixing 0.01-1 μ g of the first liquid and 0.01-3 μ g of the second liquid in the same test tube, adding appropriate amount of reaction buffer solution 1 and quality control product, determining whether the detection result is similar to the expected value, and if so, verifying that the prepared first liquid and second liquid are effective;
s304, subpackaging and freeze-drying the first liquid and the second liquid: installing a 20-80 mu L liquid transferring head on a liquid racking machine, starting to perform racking after the racking times are input on the liquid racking machine, respectively racking the first liquid and the second liquid into corresponding test tubes, and detecting the volumes of the first liquid and the second liquid by calculating the difference between the weight of the test tubes after racking and the weight of empty test tubes before racking. Transferring the obtained 20-80 μ L of the first liquid and 20-80 μ L of the second liquid into corresponding stainless steel containers, freeze-drying in a freeze dryer to obtain powdered first detector 2 and powdered second detector 3, and storing the first detector 2 and the second detector 3 at 18-28 deg.C and humidity of less than 10%.
< example 2>
A reagent for detecting hypersensitive procalcitonin, the pH of reaction buffer solution 1 is 7.4, and the components are as follows: 10mM PBS, 350mM NaCl, 0.25% NaN31.5% Tween20, 0.25% IGEPAL CA-630, 1.75% BSA; the first probe 2 was prepared by freeze-drying a first liquid having a pH of 7.4, the composition of the first liquid being: 10mM PBS, 5% BSA, 2.5% Surcross, 5ug/mL biotin-labeled PCT antibody complex, 5ug/mL fluorescently-labeled Anti-chicken IgY antibody complex; the second probe 3 was prepared by freeze-drying a second liquid having a pH of 7.4, the composition of the second liquid being: 10mM PBS, 5% BSA, 2.5% Sucrose, 13.5ug/mL fluorescently labeled PCT antibody complex; the reagent strip comprises a pad 4, a nitrocellulose membrane 5 laid on the pad 4, a sample adding pad 6, a quality control line 7, a first detection line 8, a second detection line 9 and an absorption pad 10 which are sequentially arranged on the nitrocellulose membrane 5 at intervals.
A preparation method of a procalcitonin hypersensitivity detection reagent comprises the following steps:
s1, preparing a reagent strip:
s101, screening of antibodies: purchasing streptavidin and chicken IgY and carrying out a pairing experiment on the streptavidin and chicken IgY, screening the streptavidin and the chicken IgY with high specificity and good affinity, and respectively adjusting the streptavidin and the chicken IgY to 1-10mg/mL and 0.01-5mg/mL by using coating diluent;
s102, antibody coating: the nitrocellulose membrane 5 is prepared and correctly placed on a pasting device, by which the nitrocellulose membrane 5 is pasted on the backer 4. And spraying the chicken IgY adjusted by using the coating diluent on the nitrocellulose membrane 5 by using a spraying device to form a control line, and spraying the streptavidin adjusted by using the coating diluent on the nitrocellulose membrane 5 to form a first detection line 8 and a second detection line 9, so as to obtain the coated reagent strip, wherein the spraying amount of the spraying device is 1 mu L/cm, the spraying speed is 9cm/s, and the quality control line 7, the first detection line 8 and the second detection line 9 are sequentially spaced by 2.5 mm. Then placing the coated reagent strip in an oven, baking for 30-120min at 37 ℃, and taking out;
s103, sticking a film on the reagent strip and cutting; the sample adding pad 6 and the absorption pad 10 are pasted on the cellulose nitrate membrane 5 and are arranged on a quality control line 7, a first detection line 8, a second detection line 9 and two sides of the absorption pad 10, the sample adding pad 6 is pasted on one side face of a pad card 4 with wide width, the length of the side of the cellulose nitrate membrane 5 below the sample adding pad 6, which is parallel to the long side of the pad card 4, is 1-2mm, the absorption pad 10 is pasted on one side face of the pad card 4 with narrow width, and the distance between the end part of the cellulose nitrate membrane 5 below the absorption pad 10 and one end of the pad card 4, which is provided with the absorption pad 10, is less than 1. After the absorption pad 10 and the sample adding pad 6 are adhered, the reagent strip is cut into reagent strips with the width of 3.9mm by using a cutting device, so that the cut reagent strips are obtained, and the cut reagent strips are placed on a pressing shell device and pressed firmly to obtain the reagent strips.
S2, preparing a reaction buffer solution 1; NaCl and NaN were added to 10mM PBS at pH 7.43Tween20, IGEPAL CA-630, BSA to a NaCl concentration of 350mM, NaN3The percentage concentration is 0.25 percent, and the percentage concentration of Tween201.5 percent, 0.25 percent of IGEPAL CA-630 percent and 1.75 percent of BSA, filtering the mixture after the components are completely dissolved to obtain a reaction buffer solution 1, and storing the reaction buffer solution 1 at the temperature of 2-8 ℃;
s3, preparing the first probe 2 and the second probe 3:
s301, configuring a first liquid: adding BSA, Sucrose, a biotin-labeled PCT antibody complex and a fluorescence-labeled Anti-chicken IgY antibody complex into PBS with the pH value of 7.4 and the concentration of 10mM until the percent concentration of BSA is 5 percent, the percent concentration of Sucrose is 2.5 percent, the concentration of the biotin-labeled PCT antibody complex is 5ug/mL, and the concentration of the fluorescence-labeled Anti-chicken IgY antibody complex is 5 ug/mL;
s302, configuring a second liquid: adding BSA, Sucrose and a fluorescently-labeled PCT antibody complex into PBS with the pH value of 7.4 and the concentration of 10mM until the percent concentration of BSA is 5 percent, the percent concentration of Sucrose is 2.5 percent and the concentration of the fluorescently-labeled PCT antibody complex is 13.5 ug/mL;
s303, detecting the effectiveness of the first liquid and the second liquid: mixing 0.01-1 μ g of the first liquid and 0.1-3 μ g of the second liquid in the same test tube, adding appropriate amount of reaction buffer solution 1 and quality control product, determining whether the detection result is similar to the expected value, and if so, verifying that the prepared first liquid and second liquid are effective;
s304, subpackaging and freeze-drying the first liquid and the second liquid: installing a 20-80 mu L liquid transferring head on a liquid racking machine, starting to perform racking after the racking times are input on the liquid racking machine, respectively racking the first liquid and the second liquid into corresponding test tubes, and detecting the volumes of the first liquid and the second liquid by calculating the difference between the weight of the test tubes after racking and the weight of empty test tubes before racking. Transferring the obtained 20-80 μ L of the first liquid and 20-80 μ L of the second liquid into corresponding stainless steel containers, freeze-drying in a freeze dryer to obtain spherical first detector 2 and second detector 3, and storing the first detector 2 and the second detector 3 at 18-28 deg.C and humidity of less than 10%.
< example 3>
A reagent for detecting hypersensitive procalcitonin, the pH of reaction buffer solution 1 is 7.4, and the components are as follows: 10mM PBS, 600mM NaCl, 0.5% NaN 33% Tween20, 0.5% IGEPAL CA-630, 3% BSA; the first probe 2 was prepared by freeze-drying a first liquid having a pH of 7.4, the composition of the first liquid being: 10mM PBS, 10% BSA, 5% Surcross, 10ug/mL biotin-labeled PCT antibody complex, 10ug/mL fluorescently-labeled Anti-chicken IgY antibody complex; the second probe 3 was prepared by freeze-drying a second liquid having a pH of 7.4, the composition of the second liquid being: 10mM PBS, 10% BSA, 5% Sucrose, 25ug/mL fluorescently labeled PCT antibody complex; the reagent strip comprises a pad 4, a nitrocellulose membrane 5 laid on one side of the pad 4, a sample adding pad 6, a quality control line 7, a first detection line 8, a second detection line 9 and an absorption pad 10 which are sequentially arranged on the nitrocellulose membrane 5 at intervals.
A preparation method of a procalcitonin hypersensitivity detection reagent comprises the following steps:
s1, preparing a reagent strip:
s101, screening of antibodies: purchasing streptavidin and chicken IgY and carrying out a pairing experiment on the streptavidin and chicken IgY, screening the streptavidin and the chicken IgY with high specificity and good affinity, and respectively adjusting the streptavidin and the chicken IgY to 1-10mg/mL and 0.01-5mg/mL by using coating diluent;
s102, antibody coating: the nitrocellulose membrane 5 is prepared and correctly placed on a pasting device, by which the nitrocellulose membrane 5 is pasted on the backer 4. And spraying the chicken IgY adjusted by using the coating diluent on the nitrocellulose membrane 5 by using a spraying device to form a control line, and spraying the streptavidin adjusted by using the coating diluent on the nitrocellulose membrane 5 to form a first detection line 8 and a second detection line 9, so as to obtain the coated reagent strip, wherein the spraying amount of the spraying device is 1 mu L/cm, the spraying speed is 9cm/s, and the quality control line 7, the first detection line 8 and the second detection line 9 are sequentially spaced by 3 mm. Then placing the coated reagent strip in an oven, baking for 30-120min at 37 ℃, and taking out;
s103, sticking a film on the reagent strip and cutting; the sample adding pad 6 and the absorption pad 10 are pasted on the cellulose nitrate membrane 5 and are arranged on a quality control line 7, a first detection line 8, a second detection line 9 and two sides of the absorption pad 10, the sample adding pad 6 is pasted on one side face of a pad card 4 with wide width, the length of the side of the cellulose nitrate membrane 5 below the sample adding pad 6, which is parallel to the long side of the pad card 4, is 1-2mm, the absorption pad 10 is pasted on one side face of the pad card 4 with narrow width, and the distance between the end part of the cellulose nitrate membrane 5 below the absorption pad 10 and one end of the pad card 4, which is provided with the absorption pad 10, is less than 1. After the absorption pad 10 and the sample adding pad 6 are adhered, the reagent strip is cut into reagent strips with the width of 3.9mm by using a cutting device, so that the cut reagent strips are obtained, and the cut reagent strips are placed on a pressing shell device and pressed firmly to obtain the reagent strips.
S2, preparing a reaction buffer solution 1; NaCl and NaN were added to 10mM PBS at pH 7.43Tween20, IGEPAL CA-630, BSA to a NaCl concentration of 600mM, NaN3The percentage concentration is 0.5%, the Tween20 percentage concentration is 3%, the IGEPAL CA-630 percentage concentration is 0.5%, the BSA percentage concentration is 3%, after the components are completely dissolved, the reaction buffer solution 1 is obtained by filtering, and the reaction buffer solution 1 is stored at the temperature of 2-8 ℃;
s3, preparing the first probe 2 and the second probe 3:
s301, configuring a first liquid: adding BSA, Sucrose, a biotin-labeled PCT antibody complex and a fluorescence-labeled Anti-chicken IgY antibody complex into PBS with the pH value of 7.4 and the concentration of 10mM until the BSA percentage concentration is 10 percent, the Sucrose percentage concentration is 5 percent, the biotin-labeled PCT antibody complex concentration is 10ug/mL, and the fluorescence-labeled Anti-chicken IgY antibody complex concentration is 10 ug/mL;
s302, configuring a second liquid: adding BSA, Sucrose and a fluorescently-labeled PCT antibody complex into PBS with the pH value of 7.4 and the concentration of 10mM until the percent concentration of BSA is 10 percent, the percent concentration of Sucrose is 5 percent, and the concentration of the fluorescently-labeled PCT antibody complex is 25 ug/mL;
s303, detecting the effectiveness of the first liquid and the second liquid: mixing 0.01-1 μ g of the first liquid and 0.01-3 μ g of the second liquid in the same test tube, adding appropriate amount of reaction buffer solution 1 and quality control product, determining whether the detection result is similar to the expected value, and if so, verifying that the prepared first liquid and second liquid are effective;
s304, subpackaging and freeze-drying the first liquid and the second liquid: installing a 20-80 mu L liquid transferring head on a liquid racking machine, starting to perform racking after the racking times are input on the liquid racking machine, respectively racking the first liquid and the second liquid into corresponding test tubes, and detecting the volumes of the first liquid and the second liquid by calculating the difference between the weight of the test tubes after racking and the weight of empty test tubes before racking. Transferring the obtained 20-80 μ L of the first liquid and 20-80 μ L of the second liquid into corresponding stainless steel containers, freeze-drying in a freeze dryer to obtain powdered first detector 2 and powdered second detector 3, and storing the first detector 2 and the second detector 3 at 18-28 deg.C and humidity of less than 10%.
< example 4>
A method for using the hypersensitive procalcitonin detection reagent to detect procalcitonin comprises the following steps:
SA, horizontally placing the reagent strip;
SB, mixing 1150 mu L of reaction buffer solution with the detector to obtain a mixed reagent;
SC, taking 35 mu L of sample to be detected and uniformly mixing the sample with the mixed reagent to obtain a sample mixed solution;
SD, adding 75 mu L of sample mixed solution to the sample adding pad 6 of the reagent strip, and reacting at room temperature for 12min to obtain a reacted reagent strip;
and SE, detecting the first detection line 8 and the second detection line 9 on the reacted reagent strip by using detection equipment.
(one) < manufacturing of addition disclosure and ID chip >
And (3) testing the prepared reagent strip, the reaction buffer solution 1 and the PCT calibrator, wherein each calibrator has at least 5 different concentrations, and each concentration is tested at least 5 times. And (3) making a standard curve of the average value and the concentration of the area scanning images of the calibration products with different concentrations to obtain a linear equation, importing the numerical value of the equation into special software, connecting a blank original chip to a computer, importing the numerical value code into the chip through the special software, and pasting a chip label.
(II) < detection of precision of hypersensitive procalcitonin detection reagent >
Detection materials:
the detection reagent for the hypersensitive procalcitonin comprises: using the procalcitonin detection reagent prepared in any one of examples 1 to 3;
fluorescence immunoassay appearance: the type of the fluorescence immunoassay analyzer matched with the hypersensitive procalcitonin detection reagent is A2000 or A5000;
detecting a sample: the PCT quality control product comprises a low-concentration PCT quality control product and a high-concentration PCT quality control product, wherein the low concentration of the PCT quality control product is 1.13ng/mL, and the high concentration of the PCT quality control product is 21.3ng/mL, wherein the manufacturer of the PCT quality control product is Randox company, the product number of the low-concentration PCT quality control product is IAS3117, the batch number is 1854EC, the product number of the high-concentration PCT quality control product is IAS3119, and the batch number is 1856 EC;
the application method of the reagent for detecting the hypersensitive procalcitonin comprises the following steps: the detection was repeated 20 times for each of the low concentration PCT quality control and the high concentration PCT quality control in the same manner as in example 4, and the results are shown in table 1 below:
table 1.
As can be seen from Table 1, the average value of the low concentration of the PCT quality control substance detected by the procalcitonin detection reagent is 1.12ng/mL, the standard deviation is 0.06, the CV is 5.43%, the average value of the high concentration of the PCT quality control substance is 21.12ng/mL, the standard deviation is 1.10, and the CV is 5.23%, wherein the CV values of the low concentration and the high concentration of the PCT quality control substance are both less than 10%, which indicates that the reagent prepared by the invention has good precision and is suitable for clinical detection.
(III) < detection of accuracy of hypersensitive Procalcitonin detection reagent >
Detection materials:
the detection reagent for the hypersensitive procalcitonin comprises: using the procalcitonin detection reagent prepared in any one of examples 1 to 3;
fluorescence immunoassay appearance: the type of the fluorescence immunoassay analyzer matched with the hypersensitive procalcitonin detection reagent is A2000 or A5000;
materials: PCT standard solution prepared from rmpct protein (Boditech med), 100% horse serum (gibco), PCT negative serum sample;
detecting a sample: taking three portions of PCT negative serum with the same volume, adding 0.1mL of 100% horse serum into one portion to prepare a basic sample, and adding 0.1mL of PCT standard solution with the same volume as the other two portions of PCT negative serum, 5ng/mL of PCT standard solution with the same volume as the first portion, 0.1mL of PCT standard solution with the same volume as the second portion, and 30ng/mL of PCT standard solution with the same volume as the first portion to prepare an analysis sample 1 to be recovered and an analysis sample 2 to be recovered;
the application method of the reagent for detecting the hypersensitive procalcitonin comprises the following steps: the same as the use method in the embodiment 4, the basic sample, the sample to be recovered and analyzed 1 and the sample to be recovered and analyzed 2 are respectively subjected to repeated detection for 3 times, the mean value is calculated, the mean value is taken to perform the following calculation, wherein the concentration in the following formula is PCT concentration, the standard solution is PCT standard solution, and n is a non-zero natural number:
1. adding the concentrationnConcentration n × [ volume of standard solution/(sample volume + volume of standard solution) ]]；
2. The recovery rate n ═ n [ mean value of concentration of samples to be recovered-n-mean value of concentration of base samples determined ] × 100% ÷ n concentration added;
3. average recovery rate (recovery rate 1+ recovery rate 2+ … + recovery rate n) × 100% ÷ n;
the results obtained are shown in Table 2 below:
TABLE 2
As can be seen from Table 2, the recovery rates are calculated after the PCT 3 samples are repeatedly measured for 3 times, and the obtained result shows that the average recovery rate of the reagent of the invention is 96.64% when the recovery rate is measured, which indicates that the reagent prepared by the invention has good accuracy, is suitable for clinical detection and meets the differentiation requirements of different clients on different detection occasions.
(IV) < detection of the consistency of the Procalcitonin hypersensitivity detection reagent >
Detection materials:
the detection reagent for the hypersensitive procalcitonin comprises: using the procalcitonin detection reagent prepared in any one of examples 1 to 3;
fluorescence immunoassay appearance: the type of the fluorescence immunoassay analyzer matched with the hypersensitive procalcitonin detection reagent is A2000 or A5000;
detecting a sample: the clinical samples are provided by relevant hospitals, and the total 300 samples with electrochemiluminescence method values comprise 100 blood serums, 100 homologous blood plasmas and 100 homologous whole blood, wherein the PCT content distribution intervals of the three samples are 0.01-50 ng/mL;
the application method of the reagent for detecting the hypersensitive procalcitonin comprises the following steps: the same as the method used in example 4, all the sera, the homologous plasma and the homologous whole blood were tested, each sample was tested 1 time repeatedly, and the correlation between the sample types was analyzed based on the serum test results, as shown in fig. 3-5;
as shown in FIG. 3, a scatter plot was plotted with the serum test value of the control system as the X-axis and the serum test value of the experimental system as the Y-axis, and correlation analysis was performed. Detection of R in 100 clinical serum samples2The gradient is in the range of 0.9-1.1, which indicates that the reagent prepared by the invention has good consistency with the electrochemical luminescence method reagent in the detection of the PCT serum, and meets the requirement of clinical detection of the PCT serum.
As shown in FIG. 4, a scatter plot was plotted with the serum measurement value of the experimental system as the X-axis and the plasma measurement value of the experimental system as the Y-axis, and correlation analysis was performed. 100 clinical plasma samples were tested, R2The gradient is more than or equal to 0.98 and in the range of 0.9-1.1, which shows that the reagent prepared by the invention has good consistency when detecting PCT serum and homologous plasma samples, and meets the requirement of clinical detection of PCT plasma.
As shown in FIG. 5, a scatter diagram was drawn with the serum measurement value of the experimental system as the X-axis and the whole blood measurement value of the experimental system as the Y-axis, and correlation analysis was performed. The test is carried out on 100 clinical whole blood samples,R2the gradient is 0.98 or more and 0.9-1.1, which shows that the reagent prepared by the invention has good consistency when detecting PCT serum and homologous whole blood samples, and meets the requirement of clinical detection of PCT whole blood.
(V) < comparison of the hypersensitive procalcitonin test reagent with the existing reagent >
Detection materials:
the detection reagent for the hypersensitive procalcitonin comprises: the hypersensitive procalcitonin detection reagent prepared in any one of the embodiments 1 to 3 is used, wherein the fluoroimmunoassay analyzer is a fluoroimmunoassay analyzer matched with the hypersensitive procalcitonin detection reagent of the invention, the model is A2000, and the using method is the same as that in the embodiments;
the existing procalcitonin detection reagent comprises the following components: the PCT reagent with the combination pad and the fluorescence immunoassay device have the using method of the conventional procalcitonin detection reagent;
detecting a sample: clinical samples are provided by related hospitals, 20 cases of electrochemiluminescence method-fixed value serum samples are provided, and the distribution interval of the PCT content is 0.01-50 ng/mL;
the test method comprises the following steps: the results of 20 samples respectively detected by the hypersensitive procalcitonin detection reagent and the existing procalcitonin detection reagent are shown in table 3:
table 3.
As shown in Table 3, 20 samples are simultaneously detected by the conventional procalcitonin detection reagent and the procalcitonin detection reagent, wherein the precision CV value of each sample is more than or equal to 10 percent and the relative deviation is more than or equal to 10 percent when the conventional procalcitonin detection reagent is used for testing a PCT serum sample, and the situations that the precision CV is more than or equal to 10 percent and the relative deviation is more than or equal to 10 percent do not occur in the detection range of 0.01-50ng/mL in the conventional procalcitonin detection reagent show that the procalcitonin detection reagent has good performance and meets the requirement of clinical detection of PCT.
While embodiments of the invention have been described above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable in various fields of endeavor to which the invention pertains, and further modifications may readily be made by those skilled in the art, it being understood that the invention is not limited to the details shown and described herein without departing from the general concept defined by the appended claims and their equivalents.
Claims (7)
1. The procalcitonin detection reagent comprises a reaction buffer solution and a reagent strip, and is characterized by further comprising a solid first detector and a solid second detector, wherein:
the first detector contains a biotin-labeled PCT antibody complex and a fluorescence-labeled Anti-chicken IgY antibody complex;
the second detector contains a fluorescent labeled PCT antibody complex;
the reagent strip comprises a pad, a nitrocellulose membrane laid on the pad, and a sample adding pad, a quality control line, a first detection line, a second detection line and an absorption pad which are arranged on the nitrocellulose membrane at intervals in sequence.
2. The procalcitonin detection reagent according to claim 1, wherein the reaction buffer has a pH of 7.4 and comprises the following components: 10mM PBS, 100-30.1-3% Tween20, 0.01-0.5% IGEPAL CA-630, 0.5-3% BSA, wherein the volume of the reaction buffer is not less than 150. mu.L.
3. The procalcitonin detection reagent according to claim 1, wherein the first probe is prepared by freeze-drying a first liquid having a pH of 7.4, the first liquid having the components: 10mM PBS, 0.05-10% BSA, 0.05-5% Sucrose, 0.01-10ug/mL biotin-labeled PCT antibody complex, 0.01-10ug/mL fluorescently-labeled Anti-chicken IgY antibody complex;
the second probe is prepared by freeze-drying a second liquid with the pH of 7.4, and the second liquid comprises the following components: 10mMPBS, 0.05-10% BSA, 0.05-5% Sucrose, 2-25ug/mL fluorescently labeled PCT antibody complex.
4. The procalcitonin detection reagent according to claim 1, wherein the quality control line, the first detection line and the second detection line are sequentially spaced at a distance of 2-3 mm.
5. The procalcitonin detection reagent of claim 1, wherein the first probe and the second probe are one of spherical and powdered.
6. The method for preparing the procalcitonin detection reagent according to any one of claims 1 to 5, comprising the steps of:
s1, sticking the nitrocellulose membrane on the pad card, spraying the nitrocellulose membrane to form a quality control line, a first detection line and a second detection line, and sticking the sample adding pad and the absorption pad on the nitrocellulose membrane to obtain a reagent strip;
s2, preparing a reaction buffer solution;
s3, preparing the first liquid and the second liquid, and freeze-drying the first liquid and the second liquid to obtain a first detector and a second detector.
7. The method of using the procalcitonin detection reagent according to any one of claims 1 to 5 for the detection of procalcitonin, comprising the steps of:
SA, horizontally placing the reagent strip;
SB, mixing 150 mu L of reaction buffer solution with the detector to obtain a mixed reagent;
SC, taking 35 mu L of sample to be detected and uniformly mixing the sample with the mixed reagent to obtain a sample mixed solution;
SD, adding 75 mu L of sample mixed solution to a sample adding pad of the reagent strip, and reacting at room temperature for 12min to obtain a reacted reagent strip;
and SE, detecting the first detection line and the second detection line on the reacted reagent strip by using detection equipment.
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Application publication date: 20200825 |