CN115656521A - Chemiluminescence kit for diagnosing depressive disorder by detecting CXCL4 protein - Google Patents
Chemiluminescence kit for diagnosing depressive disorder by detecting CXCL4 protein Download PDFInfo
- Publication number
- CN115656521A CN115656521A CN202211612897.4A CN202211612897A CN115656521A CN 115656521 A CN115656521 A CN 115656521A CN 202211612897 A CN202211612897 A CN 202211612897A CN 115656521 A CN115656521 A CN 115656521A
- Authority
- CN
- China
- Prior art keywords
- antibody
- reagent
- conjugate
- fsa0014
- fsa0013
- 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
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to the field of biotechnology and medical detection and diagnosis, in particular to a chemiluminescence kit for diagnosing depressive disorder by detecting CXCL4 protein and application of a combination of a pair of antibodies specifically binding the CXCL4 protein in preparing the chemiluminescence kit for diagnosing depressive disorder. The chemiluminescence kit comprises the following components: the kit comprises an anti-reagent A, an anti-reagent B, an anti-reagent C, a magnetic particle reagent, a calibrator and a quality control product, wherein the anti-reagent A comprises a conjugate of fluorescein isothiocyanate and an antibody FSA0013, the anti-reagent B comprises a conjugate of biotin and an antibody FSA0014, the anti-reagent C comprises a conjugate of alkaline phosphatase and streptavidin, the magnetic particle reagent comprises a conjugate of goat anti-fluorescein isothiocyanate antibody and magnetic carboxyl beads, and the calibrator and the quality control product are respectively buffers with CXCL4 antigens in different concentrations. The chemiluminescence kit can realize the rapid, accurate and economic diagnosis of depressive disorder on a molecular level.
Description
Technical Field
The invention relates to the field of biotechnology and medical detection and diagnosis, in particular to a chemiluminescence kit for diagnosing depressive disorder by detecting CXCL4 protein, and application of a pair of antibodies specifically binding to the CXCL4 protein in preparing the chemiluminescence kit for diagnosing depressive disorder.
Background
Mental disorders (mental disorders), also known as mental disorders, are a general term for all pathological mental activities that manifest as a syndrome characterized by a clinically significant disturbance of an individual's cognition, affective conditioning, or behavior. Common mental diseases comprise schizophrenia, mood disorders, anxiety disorders, depressive disorders and other various symptoms.
Currently, the classification standards of mental diseases include DSM system based on american society for mental medicine, ICD system based on world health organization, CCMD system based on china, etc. However, these systems are based on clinical symptom assessment, and lack objective biological diagnostic indicators, i.e., subjects with a similar group of symptoms, which may be caused by completely different biological/pathological factors, are classified as the same disease category. In contrast, subjects with different symptoms, although classified as different diseases according to clinical symptom assessment, may be caused by the same biological/pathological factors. This presents difficulties for the accurate treatment of mental disorders.
Therefore, a biomarker is searched and established, an antibody capable of specifically binding the biomarker is developed, and a detection kit capable of being quickly, accurately and economically applied to clinical application is developed, so that objective diagnosis of depressive disorder is realized, and the method has important significance for clinically diagnosing depressive disorder.
Disclosure of Invention
Technical problem to be solved
Aiming at the problems in the prior art, the invention aims to develop a pair of high-specificity antibody pairs and further provide a detection kit capable of clinically performing molecular level diagnosis on depressive disorder based on the antibody pairs, so that rapid, accurate and economical diagnosis on depressive disorder is realized, and the risk of misdiagnosis caused by simple symptom evaluation at present is reduced.
Technical scheme
In order to solve the above problems, the present invention adopts the following technical solutions.
In one aspect, the present invention provides a chemiluminescent kit for diagnosing depressive disorders by specifically detecting CXCL4 protein, wherein the chemiluminescent kit comprises the following components: an anti-reagent A, an anti-reagent B, an anti-reagent C, a magnetic particle reagent, a calibrator and a quality control material, wherein,
-said anti-agent a comprises a conjugate of fluorescein isothiocyanate and antibody FSA0013, said antibody FSA0013 being expressed by transient co-transfection of recombinant plasmids comprising the heavy chain of antibody FSA0013 and the light chain of antibody FSA0013, respectively, into the chinese hamster ovary cell expihcho-s expression system, followed by purification using Protein a affinity chromatography to obtain said antibody FSA0013;
the recombinant plasmid comprising the heavy chain coding sequence of antibody FSA0013 is contained in Escherichia coli FSV0003-H, which Escherichia coli FSV0003-H is deposited at the China center for type culture Collection, the address of university of Wuhan, china, the date of deposition: 2022, 11 months and 10 days, with a preservation number of CCTCC M20221754; and the recombinant plasmid comprising the light chain coding sequence of antibody FSA0013 is contained in Escherichia coli FSV0003-L, which Escherichia coli FSV0003-L is deposited at the China center for type culture Collection, the address of university of Wuhan, china, the date of deposition: 2022, 11 months and 10 days, and the preservation number is CCTCC M20221755;
-said anti-agent B comprises a conjugate of biotin and antibody FSA0014, said antibody FSA0014 being expressed by transient co-transfection of recombinant plasmids comprising the heavy chain of antibody FSA0014 and the light chain of antibody FSA0014, respectively, into the chinese hamster ovary cell ExpiCHO-s expression system, followed by purification using Protein a affinity chromatography to obtain said antibody FSA0014;
the recombinant plasmid containing the heavy chain coding sequence of the antibody FSA0014 is contained in Escherichia coli FSV0004-H, wherein the Escherichia coli FSV0004-H is preserved in China center for type culture Collection, the address of university of Wuhan, china, the preservation date is as follows: 2022, 11 months and 10 days, and the preservation number is CCTCC M20221752; and the recombinant plasmid comprising the light chain coding sequence of antibody FSA0014 is contained in Escherichia coli FSV0004-L, which Escherichia coli FSV0004-L is deposited at the China center for type culture Collection, the address of university of Wuhan, china, the date of deposition: in 2022, 11 months and 10 days, the preservation number is CCTCC M20221753;
-said anti-agent C comprises a conjugate of alkaline phosphatase and streptavidin;
-the magnetic particle reagent comprises a conjugate of goat anti-fluorescein isothiocyanate antibody and a carboxylic magnetic bead; and is
-the calibrator and the quality control are buffers of CXCL4 antigen at different concentrations, respectively.
In one embodiment, the anti-agent a is prepared by the following method:
(1) Coupling of fluorescein isothiocyanate to antibody FSA 0013:
the preparation concentration of fluorescein isothiocyanate in Tris buffer solution is 1.0-5.0 mg/mL, and the volume ratio is 1:100 to 1:10000, diluting fluorescein isothiocyanate to obtain a diluent, adding the FSA0013 into the diluent according to the molar ratio of 1 to 2 to 1;
(2) Purification of conjugate of fluorescein isothiocyanate and antibody FSA 0013:
eluting the conjugate of fluorescein isothiocyanate and the antibody FSA0013 by using a bicarbonate buffer solution with the pH = 8-9, collecting an eluent, and adding the obtained conjugate of fluorescein isothiocyanate and the antibody FSA0013 into an anti-reagent buffer solution to prepare the anti-reagent A.
In one embodiment, the anti-agent B is prepared by the following method:
(1) Coupling of biotin to antibody FSA 0014:
preparing biotin into a carbonate buffer solution with the pH of 8-9, wherein the concentration of the biotin is 1.0 to 5.0mg/mL, adding the antibody FSA0014 into the biotin according to the molar ratio of 1 to 5 to 1, and uniformly mixing for 1 to 2 hours at the temperature of 20 +/-5 ℃ to obtain a conjugate of the biotin and the antibody FSA0014;
(2) Purification of conjugate of biotin and antibody FSA 0014:
and (3) carrying out ultrafiltration and centrifugation on the conjugate of the biotin and the antibody FSA0014 at the rotating speed of 3000 rpm for 10min to obtain mother liquor containing the conjugate of the biotin and the antibody FSA0014, and adding an anti-reagent buffer solution to prepare an anti-reagent B.
In one embodiment, the anti-agent C is prepared by the following method:
(1) Activation of alkaline phosphatase:
diluting 25wt.% glutaraldehyde to 5wt.% with a carbonate buffer solution with pH9-10, dissolving alkaline phosphatase in 0.5ml 5wt.% glutaraldehyde, combining with a water bath at 37 ℃ for 2-3 h, washing with absolute ethanol, discarding the supernatant, adding 0.5ml of a carbonate buffer solution with pH9-10 to dissolve the precipitate, and obtaining activated alkaline phosphatase;
(2) Coupling of alkaline phosphatase to streptavidin:
streptavidin was added to the mixture at a molar ratio of 1:2 to 1:10, adding the alkaline phosphatase solution activated in the step (1), and uniformly mixing for 4 to 5 hours at the temperature of 20 +/-5 ℃ to obtain a conjugate of the alkaline phosphatase and the streptavidin;
(3) Purification of alkaline phosphatase and streptavidin conjugate:
and (3) eluting with bicarbonate buffer solution with the pH of =8 to 9, collecting eluates in parts, and adding the conjugate of the alkaline phosphatase and the streptavidin into the anti-reagent buffer solution to prepare the anti-reagent C.
In one embodiment, the magnetic microparticle reagent is prepared by the following method:
(1) Fully and uniformly mixing the concentrated solution of the carboxyl magnetic beads, taking out the carboxyl magnetic beads, placing the carboxyl magnetic beads in a magnetic field with the magnetic field strength of 3800mT for 15min, sucking the supernatant after the carboxyl magnetic beads are completely settled, adding a phosphoric acid buffer solution with the volume 5 times that of the carboxyl magnetic beads, uniformly mixing for 20 to 30min, placing the mixture in a magnetic field with the magnetic field strength of 3800mT, sucking the supernatant after the magnetic beads are completely settled for 15min, and repeating the cleaning step for 3 times to fully clean the carboxyl magnetic beads;
(2) And (3) fixing the volume of the carboxyl magnetic bead solution to 10-50mg/mL, and according to the volume of the carboxyl magnetic beads: the mass ratio of the goat anti-fluorescein isothiocyanate antibody is 100:1, adding an goat-anti-fluorescein isothiocyanate antibody, and reacting for 18 hours at 2 to 8 ℃ in a uniformly mixed state to obtain a conjugate of the goat-anti-fluorescein isothiocyanate antibody and a carboxyl magnetic bead;
(3) Adding a mixed sealing solution of 0.5 to 2M Tris solution, 5 to 12wt.% of bovine serum albumin, 7 to 15wt.% of casein solution and 1 to 10g of fish skin gel, sealing, uniformly mixing for 3 to 4 hours at 20 +/-5 ℃, washing for 3 times by using a phosphate buffer solution, and performing constant volume by using a magnetic particle buffer solution to obtain 10 to 20mg/mL of concentrated magnetic particle reagent containing the conjugate of the goat anti-isothiocyanic acid fluorescein antibody and the magnetic carboxyl bead;
(4) Preparing a magnetic particle reagent containing conjugates of fluorescein isothiocyanate antibody and carboxyl magnetic beads with the concentration of 1 to 2mg/mL by using a magnetic particle buffer solution.
In one embodiment, the calibrator and the quality control are prepared by the following method:
CXCL4 protein was diluted with calibrator and quality control buffer to give calibrators with the following concentration gradients: 0, 200, 1000, 2000, 4000, 8000ng/ml; and a first quality control product with the concentration of 700 to 1300ng/ml and a second quality control product with the concentration of 2800 to 5200 ng/ml.
In one embodiment, the anti-reagent buffer consists of: 0.1M Tris, 0.9wt.% sodium chloride, 1wt.% bovine serum albumin, 0.01wt.% tetracycline, 0.01wt.% neomycin sulfate, and a suitable amount of 4M hydrochloric acid at pH8 + -0.1.
In one embodiment, the magnetic particle buffer consists of: 0.1M Tris, 0.9wt.% sodium chloride, 1wt.% bovine serum albumin, 5wt.% methylcellulose, and an appropriate amount of 4M hydrochloric acid at pH8 ± 0.1.
In one embodiment, the buffer used to formulate the calibrator and quality control consists of: 0.1M Tris, 0.9wt.% sodium chloride, 2wt.% bovine serum albumin, 0.01wt.% tetracycline, 0.01wt.% neomycin sulfate, and an appropriate amount of 4M hydrochloric acid to pH 7.5 ± 0.1.
In one embodiment, the chemiluminescent kit is used in conjunction with a separately packaged luminescent substrate solution, or the chemiluminescent kit further comprises a luminescent substrate solution;
the luminophore solution contained (4-chlorobenzenethiol) (10-methyl-9, 10-acridylmethylene) disodium phosphate and was prepared by reacting (1: 4 to 1:10 to 50mg/L of (4-chlorobenzenethiol) (10-methyl-9, 10-acridanemethylene) phosphoric acid disodium salt; and is
The luminogenic substrate buffer comprises 0.1M Tris, 0.9wt.% sodium chloride, 0.3wt.% lucigenin, and 1wt.% sodium dodecyl sulfate.
In another aspect, the invention provides the use of a combination of a pair of antibodies FSA0013 and FSA0014 that specifically bind to CXCL4 protein for the preparation of a chemiluminescent kit for the diagnosis of depressive disorders by detecting CXCL4 protein,
the antibody FSA0013 is obtained by transiently co-transfecting recombinant plasmids respectively containing the heavy chain of the antibody FSA0013 and the light chain of the antibody FSA0013 into a Chinese hamster ovary cell ExpCHO-s expression system for expression, and then purifying by Protein A affinity chromatography to obtain the antibody FSA0013;
the recombinant plasmid containing the heavy chain coding sequence of the antibody FSA0013 is contained in Escherichia coli FSV0003-H, wherein the Escherichia coli FSV0003-H is preserved in China center for type culture Collection, the address of university of Wuhan, china, the preservation date is as follows: 2022, 11 months and 10 days, and the preservation number is CCTCC M20221754; and the recombinant plasmid containing the light chain coding sequence of the antibody FSA0013 is contained in Escherichia coli FSV0003-L, wherein the Escherichia coli FSV0003-L is preserved in China center for type culture Collection, the address of university of Wuhan, china, the preservation date: 2022, 11 months and 10 days, and the preservation number is CCTCC M20221755;
the antibody FSA0014 is obtained by transiently co-transfecting recombinant plasmids respectively containing the heavy chain of the antibody FSA0014 and the light chain of the antibody FSA0014 into a Chinese hamster ovary cell ExpicHO-s expression system for expression, and then purifying by using Protein A affinity chromatography to obtain the antibody FSA0014;
the recombinant plasmid containing the heavy chain coding sequence of the antibody FSA0014 is contained in Escherichia coli FSV0004-H, wherein the Escherichia coli FSV0004-H is preserved in China center for type culture Collection, the address of university of Wuhan, china, the preservation date is as follows: 2022, 11 months and 10 days, and the preservation number is CCTCC M20221752; and the recombinant plasmid comprising the light chain coding sequence of antibody FSA0014 is contained in Escherichia coli FSV0004-L, which Escherichia coli FSV0004-L is deposited at the China center for type culture Collection, the address of university of Wuhan, china, the date of deposition: 2022, 11 months and 10 days, and the preservation number is CCTCC M20221753;
the antibody FSA0013 is coupled with fluorescein isothiocyanate to form a conjugate of the fluorescein isothiocyanate and the antibody FSA0013; and is provided with
The antibody FSA0014 is conjugated with biotin to form a conjugate of biotin and the antibody FSA0014.
In one embodiment, the chemiluminescent kit further comprises the following components: anti-reagent C, magnetic particle reagent, calibrator and quality control material, wherein,
-said anti-agent C comprises a conjugate of alkaline phosphatase and streptavidin and is prepared by the following method:
(1) Activation of alkaline phosphatase:
diluting 25wt.% of glutaraldehyde to 5wt.% by using a pH9-10 carbonate buffer, dissolving alkaline phosphatase in 0.5ml of 5wt.% glutaraldehyde, combining with a water bath at 37 ℃ for 2-3 h, washing with absolute ethanol, discarding the supernatant, and adding 0.5ml of a pH9-10 carbonate buffer to dissolve the precipitate to obtain activated alkaline phosphatase;
(2) Coupling of alkaline phosphatase to streptavidin:
streptavidin was added to the mixture at a molar ratio of 1:2 to 1:10, adding the alkaline phosphatase solution activated in the step (1), and uniformly mixing for 4 to 5 hours at the temperature of 20 +/-5 ℃ to obtain a conjugate of the alkaline phosphatase and the streptavidin;
(3) Purification of alkaline phosphatase and streptavidin conjugate:
eluting with a bicarbonate buffer solution with the pH = 8-9, collecting eluates in parts, and adding the conjugate of the alkaline phosphatase and the streptavidin into an anti-reagent buffer solution to prepare an anti-reagent C;
-the magnetic particle reagent comprises a conjugate of a goat anti-fluorescein isothiocyanate antibody and a magnetic bead, and the magnetic particle reagent is prepared by the following method:
(1) Fully and uniformly mixing the concentrated solution of the carboxyl magnetic beads, taking out the carboxyl magnetic beads, placing the carboxyl magnetic beads in a magnetic field with the magnetic field strength of 3800mT for 15min, sucking the supernatant after the carboxyl magnetic beads are completely settled, adding a phosphoric acid buffer solution with the volume 5 times that of the carboxyl magnetic beads, uniformly mixing for 20 to 30min, placing the mixture in a magnetic field with the magnetic field strength of 3800mT, sucking the supernatant after the magnetic beads are completely settled for 15min, and repeating the cleaning step for 3 times to fully clean the carboxyl magnetic beads;
(2) And (3) fixing the volume of the carboxyl magnetic bead solution to 10-50mg/mL, wherein the volume is determined according to the following steps: the mass ratio of the goat anti-fluorescein isothiocyanate antibody is 100:1, adding the goat anti-fluorescein isothiocyanate antibody, and reacting for 18 hours at 2-8 ℃ in a uniformly mixed state to obtain a conjugate of the goat anti-fluorescein isothiocyanate antibody and the carboxyl magnetic beads;
(3) Adding a mixed sealing solution of 0.5 to 2M Tris solution, 5 to 12wt.% of bovine serum albumin, 7 to 15wt.% of casein solution and 1 to 10g of fish skin gel, sealing, uniformly mixing for 3 to 4 hours at 20 +/-5 ℃, washing for 3 times by using a phosphate buffer solution, and performing constant volume by using a magnetic particle buffer solution to obtain 10 to 20mg/mL of concentrated magnetic particle reagent containing the conjugate of the goat anti-isothiocyanic acid fluorescein antibody and the magnetic carboxyl bead;
(4) Preparing a magnetic particle reagent containing a conjugate of an goat anti-fluorescein isothiocyanate antibody and a carboxyl magnetic bead with the concentration of 1-2 mg/mL by using a magnetic particle buffer solution;
-the calibrator and the quality control are buffers of CXCL4 antigen at different concentrations, respectively, and the calibrator and the quality control are prepared by:
CXCL4 protein was diluted with calibrator and quality control buffer to give a calibrator with the following concentration gradient: 0, 200, 1000, 2000, 4000, 8000ng/ml; and a first quality control material with the concentration of 700 to 1300ng/ml and a second quality control material with the concentration of 2800 to 5200 ng/ml.
Advantageous effects
Compared with the prior art, the invention has the advantages that:
(1) According to the invention, the obvious difference of the CXCL4 protein expression quantity is found in plasma samples of healthy people and depressive disorder people, so that the CXCL4 protein is used as a marker to realize the differential diagnosis of the healthy people and the depressive disorder people on a molecular level.
(2) The invention screens a pair of rabbit antibodies with high specificity to CXCL4 protein from a plurality of antibodies, and the antibodies have very excellent binding specificity and affinity to CXCL4, and the affinity of the antibodies to the CXCL4 protein is more than 1000 times higher than that of the commercial antibodies sold on the market at present.
(3) The invention further develops a kit, the kit utilizes the principle of chemiluminescence, the differential diagnosis of the depressive disorder is realized by adopting the combination of an antibody pair with high specificity combined with CXCL4 and the carboxyl magnetic beads coupled by alkaline phosphatase, streptavidin conjugate and goat anti-fluorescein isothiocyanate antibody, and the result determination shows that the specificity of the kit is up to 92.5%, the sensitivity is up to 93.3% and the accuracy is up to 92.8%.
(4) The chemiluminescence kit developed by the invention realizes the differential diagnosis of depressive disorders on a molecular level through markers for the first time, so that the rapid, accurate and economic diagnosis of depressive disorders can be realized clinically, and the risk of misdiagnosis caused by simple symptom evaluation at present is reduced.
(5) The kit is suitable for the current clinical biochemical detection analytical instrument or digital detection platform, and can meet the requirement of rapid, accurate and economic diagnosis of large-scale clinical samples. Has important significance for the promotion of the current hospital digital diagnosis and the development of the digital medical industry.
Detailed Description
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention.
On biological preservation
The invention develops a pair of rabbit antibodies with high binding specificity and affinity for CXCL4 protein, which are named as antibody FSA0013 and antibody FSA0014 respectively.
The nucleotide sequences of the heavy chain and the light chain of the antibody FSA0013 respectively contain escherichia coli of corresponding recombinant plasmids, and the escherichia coli of the corresponding recombinant plasmids are respectively named as:
-FSV0003-H (the sequence contained in the recombinant plasmid contained in this E.coli corresponds to the sequence of the heavy chain of antibody FSA 0013), and
FSV0003-L (the sequence contained in the recombinant plasmid contained in this E.coli corresponds to the light chain sequence of antibody FSA 0013).
In the escherichia coli containing the corresponding recombinant plasmids in the nucleotide sequences of the heavy chain and the light chain of the antibody FSA0014 respectively, the escherichia coli of the corresponding recombinant plasmids are named as follows:
-FSV0004-H (the sequence contained in the recombinant plasmid contained in this E.coli corresponds to the sequence of the heavy chain of antibody FSA 0014), and
FSV0004-L (the sequence contained in the recombinant plasmid contained in this E.coli corresponds to the light chain sequence of antibody FSA 0014).
The 4 colibacillus are preserved in China center for type culture Collection, at the address of Wuhan university, wuhan, china, and the preservation date is as follows: 2022, 11/10, with the following deposition numbers:
escherichia coli FSV0003-H (Escherichia coli FSV 0003-H): the preservation number is CCTCC M20221754;
escherichia coli FSV0003-L (Escherichia coli FSV 0003-L): the preservation number is CCTCC M20221755;
escherichia coli FSV0004-H (Escherichia coli FSV 0004-H): the preservation number is CCTCC M20221752; and
escherichia coli FSV0004-L (Escherichia coli FSV 0004-L): the preservation number is CCTCC M20221753.
Herein, the term "CXCL4" refers to a chemokine (C-X-C motif) ligand 4 protein, which is also known as platelet factor 4 (PF 4). The number of CXCL4 in the UniProt protein database is P02776, see specifically https:// beta. UniProt. Org/uniprotkb/P02776/entry. CXCL4 and its full, alternate and abbreviated names are interchangeable herein.
In this context, the CXCL4 protein is used as a biomarker to distinguish between healthy and depressive disorders. An increase in the CXCL4 protein content in a human plasma sample indicates that the human has or is at risk of having a depressive disorder.
The chemiluminescence kit provided by the invention is used for detecting the content of CXCL4 protein in a plasma sample of a subject by using a chemiluminescence method, and the detection principle is as follows:
a Fluorescein Isothiocyanate (FITC) -labeled rabbit monoclonal anti-CXCL 4 antibody binds to CXCL4 in a sample, calibrator, or quality control to form an immune complex. And then adding magnetic particles connected with goat anti-FITC antibodies, and binding the antigen-antibody immune complexes on the magnetic particles through the specific binding of the anti-FITC antibodies and the FITC. Under the action of an external magnetic field, separating a compound formed by immunoreaction from other unbound substances, washing the compound, adding a biotin-labeled rabbit monoclonal anti-CXCL 4 paired antibody to form a sandwich compound with the compound, then adding a conjugate of alkaline phosphatase and streptavidin, further coupling with the biotin-labeled rabbit monoclonal anti-CXCL 4 paired antibody, and carrying out cascade amplification on a detection signal. Under the action of an external magnetic field, the 'sandwich' complex formed by the immunoreaction is separated from other unbound substances, and after washing, an enzymatic chemiluminescent substrate is added. The substrate is catalytically cracked under the action of enzyme to form an unstable excited intermediate, and when the excited intermediate returns to the ground state, photons are emitted to form a luminescence reaction, namely, a chemiluminescence apparatus is used for detecting the luminescence intensity of the reaction. The concentration of CXCL4 in the sample can be quantitatively measured by equation fitting in a manner that the luminescence intensity is in proportional relation to the content of CXCL4 in the sample in the detection wavelength range (230-700 nm).
In this context, the luminogenic substrate may not be placed within the kit, but rather packaged separately from the kit. Thus, in this context, the chemiluminescent kit is used in conjunction with a separately packaged solution of the luminogenic substrate. Alternatively, the chemiluminescent kit may also further comprise a luminescent substrate solution.
In this context, the chemiluminescent kit may be used in conjunction with a fully automated chemiluminescent detection instrument or automated/digital detection platform. A chemiluminescent kit typically includes a label indicating the intended use and/or method of use of its contents, e.g., a chemiluminescent kit may include instructions or inserts on how to detect CXCL4 protein expression using a CXCL4 antibody of the invention, and instructions or inserts indicating that an up-regulation of the level of CXCL4 protein expression compared to a reference value indicates that the subject suffers from a depressive disorder.
Examples
A further understanding of the invention may be obtained by reference to certain specific examples given herein.
Example 1 preparation of recombinant plasmids containing the light chain encoding CXCL4 antibody and the heavy chain of CXCL4 antibody
A200ug CXCL4 antigen (Cat. CXC-HM2L4, organism Caesar \20327) was used for immunization of a new Zealand white rabbit of 8 weeks old for health experiment at 2 weeks intervals for 5 times each time. Fresh blood was drawn from immunized rabbits, 10 ten thousand peripheral blood mononuclear cells (PBMC cells) were then isolated from the blood, and anti-CXCL 4-positive B cells were then further enriched and isolated from PBMCs using CXCL4 antigen in a flow cell sorter and seeded into 96-well plates (cat No. 3628, corning). The cells were further cultured, followed by RNA extraction using an RNA extraction kit (cat No. 74004, qiagen) for these anti-CXCL 4 positive B cells, followed by reverse transcription of RNA into cDNA using a reverse transcription kit (cat No. a3500, promega), followed by high fidelity enzymatic PCR amplification (cat No. KOD-201, toyobo) and DNA sequencing (Sanger, jinsler) for the gene regions encoding the antibody light and heavy chains. Then, the heavy chain sequence and the light chain sequence of the pair of antibodies (the antibodies are respectively named as FSA0013 and FSA 0014) obtained after sequencing are respectively inserted into a plasmid vector (pcDNA3.4, kinsry) to obtain 4 recombinant plasmids. Subsequently, the above recombinant plasmids were transformed into E.coli (JM 108, kiwi) to obtain 4 E.coli containing the recombinant plasmids for use.
The 4 E.coli containing recombinant plasmids were designated as:
FSV0003-H (the sequence contained in the recombinant plasmid contained in this E.coli corresponds to the heavy chain sequence of the anti-CXCL 4 antibody FSA 0013) and FSV0003-L (the sequence contained in the recombinant plasmid contained in this E.coli corresponds to the light chain sequence of the anti-CXCL 4 antibody FSA 0013); and
FSV0004-H (the sequence contained in the recombinant plasmid contained in this E.coli corresponds to the sequence of the heavy chain of anti-CXCL 4 antibody FSA 0014) and FSV0004-L (the sequence contained in the recombinant plasmid contained in this E.coli corresponds to the sequence of the light chain of anti-CXCL 4 antibody FSA 0014).
The 4 colibacillus are preserved in China center for type culture Collection, at the address of Wuhan university, wuhan, china, and the preservation date is as follows: 2022, 11/10, with the following deposition numbers:
escherichia coli FSV0003-H: the preservation number is CCTCC M20221754;
escherichia coli FSV0003-L: the preservation number is CCTCC M20221755;
escherichia coli FSV0004-H: the preservation number is CCTCC M20221752; and
escherichia coli FSV0004-L: the preservation number is CCTCC M20221753.
Example 2 preparation of anti-CXCL 4 antibodies FSA0013 and FSA0014
The 4 recombinant E.coli strains reserved for use in example 1 were subjected to amplification culture using LB sterile liquid medium (cat. No. B540111-0001, sangon Biotech) and then recombinant plasmids expressing the heavy and light chains of antibodies FSA0013 and FSA0014, respectively, were extracted from the E.coli after amplification culture using a plasmid extraction kit (cat. No. 12943, qiagen).
Recombinant plasmids containing the heavy chain of antibody FSA0013 and the light chain of antibody FSA0013, respectively, were transiently co-transfected into the Chinese hamster ovary cell ExpiCHO-s expression system (cat # A29133, gibco) for 7 days, followed by purification using Protein A affinity chromatography (cat # L00210, kinsery) to obtain antibody FSA0013.
Recombinant plasmids containing the heavy chain of antibody FSA0014 and the light chain of antibody FSA0014, respectively, were transiently co-transfected into Chinese hamster ovary cells ExpCHO-s expression system (cat # A29133, gibco) for 7 days, followed by purification using Protein A affinity chromatography (cat # L00210, kinsery) to give antibody FSA0014.
The purity of the resulting antibody was checked by SDS-PAGE (Biorad). In SDS-PAGE, the purity of the reduced band, or the purity of the sum of the reduced heavy and light chains, was calculated by peak area normalization using the ImageJ software system; the molecular weight of a non-reduction band is about 150 kDa, and the purity is more than 90%; the reduced heavy chain has a molecular weight of about 55 kDa, the light chain has a molecular weight of about 25 kDa, and the purity of the sum of the heavy and light chains is greater than 90%. The detection result meets the requirements of subsequent experiments.
Example 3 determination of the binding specificity of the antibodies FSA0013 and FSA0014 to CXCL4 at the protein level
The binding specificity of the antibodies FSA0013 and FSA0014 to CXCL4 at the protein level was determined by enzyme-linked immunosorbent assay (ELISA) using kits purchased from R & D Systems and determined as follows according to the supplier's manual:
coating the ELISA plate with 1ug/mL CXCL4-hFc, human total IgG, human IgG1, human IgG4, CXCL4-His, or His-tagged irrelevant protein;
wash plate 3 times with PBST and block plate with 5% PBS and milk mixed solution for 2 hours at room temperature;
wash the plate 3 times with PBST, then add diluted antibody (1 ug/ml) to ELISA plate 30 μ L per well, standing at room temperature for 60 min;
washing the plate 3 times with PBST, then adding an anti-rabbit IgG-Fc-HRP (Boster; BA 1061) to the ELISA plate, standing at room temperature for 60 minutes;
-adding TMB and terminating the reaction with 2M stop solution and reading the OD450;
final determination of the binding specificity of antibodies FSA0013 and FSA0014 for CXCL4 by OD450 readings.
The data of the results of the measurement are shown in table 1 below.
Table 1: binding capacity of antibodies FSA0013 and FSA0014 to different proteins at the same concentration (ELISA OD450 readings)
As a result, shown in the above table, antibodies FSA0013 and FSA0014 have high binding ability to both CXCL4-hFc protein and CXCL4-His, and neither bind to control human total IgG, human IgG1, human IgG4 and His-tagged unrelated proteins. The results show that the antibodies FSA0013 and FSA0014 to be detected have very excellent binding specificity for CXCL 4.
Example 4 determination of the affinity of the antibodies FSA0013 and FSA0014 for CXCL4 at the protein level
The affinity of the antibodies FSA0013, FSA0014 and the commercial anti-CXCL 4 antibody controls (cat 7952, R & D Systems) for CXCL4 at the protein level was determined by enzyme-linked immunoassay (ELISA) using kits purchased from R & D Systems and determined as follows according to the supplier's manual:
coating the ELISA plate with 1ug/mL CXCL 4-hFc;
wash plate 3 times with PBST and block plate with 5% PBS and milk mixed solution for 2 hours at room temperature;
wash the plate 3 times with PBST, then add two-fold diluted anti-CXCL 4 antibody (1 ug/ml starting concentration, 3-fold diluted, 12 concentration gradients) to the ELISA plate, 30 µ L per well, standing at room temperature for 60 min;
washing the plate 3 times with PBST, then adding an anti-rabbit IgG-Fc-HRP (Boster; BA 1061) to the ELISA plate, leaving it at room temperature for 60 minutes;
-adding TMB and terminating the reaction with 2M stop solution and reading the OD450;
final determination of EC50 affinity level of antibody for CXCL4 by OD450 reading.
The data of the results of the measurement are shown in table 2 below.
Table 2: affinity determination of anti-CXCL 4 antibodies for CXCL4 protein
As shown in the table above, the antibodies FSA0013 and FSA0014 have very strong affinity to CXCL4 protein (EC 50 < 30 pM), which is far better than that of the commercial anti-CXCL 4 antibody to CXCL4 protein (> 1480-fold).
Example 5 preparation of a chemiluminescent kit for the detection of CXCL4 antigen
Using the 2 anti-CXCL 4 antibodies FSA0013 and FSA0014 obtained in the example as a pair of detection antibodies, a chemiluminescent detection kit useful for detecting CXCL4 antigen was prepared.
A kit for detecting the expression level of CXCL4 protein by chemiluminescence comprises a magnetic microparticle reagent, an anti-reagent a, an anti-reagent B, an anti-reagent C, a calibrator, and a quality control.
The anti-reagent A is prepared byThe preparation method comprises the following steps:
(1) Coupling of fluorescein isothiocyanate to the CXCL 4-specific antibody FSA 0013:
fluorescein isothiocyanate (cat No. 53027, pierce) was prepared in a Tris buffer at a concentration of 1.0 to 5.0mg/mL, and the volume ratio was 1:100 to 1:10000, diluting fluorescein isothiocyanate to obtain a diluent, adding the CXCL4 specific antibody FSA0013 into the diluent according to the mol ratio of 1 to 2 to 1;
(2) Purification of fluorescein isothiocyanate and CXCL 4-specific antibody conjugates:
eluting with bicarbonate buffer solution with pH = 8-9, collecting eluate, and adding the obtained conjugate of fluorescein isothiocyanate and antibody FSA0013 into anti-reagent buffer solution (the formula is shown in Table 3) to obtain anti-reagent A.
Table 3-anti-reagent buffer used in the anti-reagent a preparation:
anti-reagent B was prepared by the following method:
(1) Conjugation of biotin to the CXCL 4-specific antibody FSA 0014:
biotin (cargo number B4501, sigma) is prepared into a carbonate buffer solution with pH of 8-9, the concentration is 1.0 to 5.0mg/mL, CXCL4 paired antibody FSA0014 is added into the biotin according to the molar ratio of 1 to 5 to 1, and the mixture is uniformly mixed for 1 to 2 hours at the temperature of 20 +/-5 ℃; obtaining a conjugate of biotin and an antibody FSA0014;
(2) Purification of biotin-CXCL 4 companion antibody conjugate:
the mother liquor containing the conjugate of biotin and antibody FSA0014 was obtained by ultrafiltration using an ultrafiltration tube (Millipore) of the corresponding volume and centrifugation at 3000 rpm for 10min, and anti-reagent B was prepared by adding an anti-reagent buffer (formulation shown in Table 4).
Table 4-anti-reagent buffer used during anti-reagent B preparation:
anti-reagent C was prepared by the following method:
(1) Activation of alkaline phosphatase:
25wt.% glutaraldehyde (cat No. 8206031000, sigma) was diluted to 5wt.% with pH9-10 carbonate buffer; weighing alkaline phosphatase (SAE 0063, sigma) and dissolving in 0.5ml 5wt.% glutaraldehyde, and combining in water bath at 37 ℃ for 2-3 h; washing with anhydrous ethanol (Cat. No. 1070172511, sigma), discarding the supernatant, adding 0.5ml of pH9-10 carbonate buffer solution to dissolve the precipitate to obtain activated alkaline phosphatase;
(2) Coupling of alkaline phosphatase to streptavidin:
streptavidin (cat # 11721674001, roche) was added at a molar ratio of 1:2 to 1:10, adding the alkaline phosphatase solution activated in the step (1), and uniformly mixing for 4 to 5 hours at the temperature of 20 +/-5 ℃; obtaining a conjugate of alkaline phosphatase and streptavidin;
(3) Purification of alkaline phosphatase and streptavidin conjugate:
and (3) eluting with bicarbonate buffer solution with the pH of 8 to 9, collecting eluates, and adding the obtained conjugate of alkaline phosphatase and streptavidin into anti-reagent buffer solution (the formula is shown in Table 5) to prepare the anti-reagent C.
Table 5-anti-reagent buffer used during anti-reagent C preparation:
the magnetic microparticle reagent is prepared by the following method:
(1) Mixing the concentrated solution of carboxyl magnetic beads (product number LSKMAG25CBX10, millipore) thoroughly; taking out the carboxyl magnetic beads, placing the carboxyl magnetic beads in a magnetic field with the magnetic field intensity of 3800mT for 15min, and sucking the supernatant after the carboxyl magnetic beads are completely settled; adding a phosphate buffer solution with the volume 5 times that of the carboxyl magnetic beads, and uniformly mixing for 20 to 30min; placing in a magnetic field with a magnetic field intensity of 3800mT, and sucking supernatant after the magnetic beads are settled for 15 min; repeating the cleaning step for 3 times to fully clean the carboxyl magnetic beads;
(2) Fixing the volume of the carboxyl magnetic bead solution to 10-50 mg/mL; according to the carboxyl magnetic beads: the mass ratio of goat-anti-fluorescein isothiocyanate antibody (cat # A150-112A, bethy Laboratories) is 100:1 adding an goat-anti-fluorescein isothiocyanate antibody, and reacting for 18h at 2-8 ℃ in a uniformly mixed state to obtain a conjugate of the goat-anti-fluorescein isothiocyanate antibody and a carboxyl magnetic bead;
(3) And (3) sealing: adding 0.5-2M Tris solution, 5-12 wt.% bovine serum albumin, 7-15 wt.% casein solution and 1-10 g of mixed confining liquid of fish skin gel, uniformly mixing for 3-4 h at 20 +/-5 ℃, and washing for 3 times by using phosphate buffer; obtaining a concentrated magnetic particle reagent containing a conjugate of an goat anti-fluorescein isothiocyanate antibody and a carboxyl magnetic bead, wherein the volume of the concentrated magnetic particle reagent is 10 to 20mg/mL by using a magnetic particle buffer solution; storing at 2 to 8 ℃ for later use;
(4) When in use, magnetic particle reagents containing conjugates of fluorescein isothiocyanate-goat antibodies and carboxyl magnetic beads at concentrations of 1 to 2mg/mL are prepared by using magnetic particle buffer solutions (the formula is shown in Table 6).
Table 6-magnetic particle buffer used during the preparation of magnetic particle reagents:
the calibrator and the quality control material are prepared by the following method:
CXCL4 protein was diluted using calibrator and quality control buffers (formula shown in table 7). Calibrator concentration gradient: 0, 200, 1000, 2000, 4000, 8000ng/ml; quality control 1 (700 to 1300 ng/ml); quality control 2 (2800 to 5200 ng/ml).
Table 7-buffers used during calibrator and quality control preparation:
in the course of detecting a sample in a chemiluminescent kit, a luminescent substrate is also required. The luminescent substrate was (4-chlorobenzenethiol) (10-methyl-9, 10-acridalmethylene) phosphate disodium salt (APS-5). The luminescent substrate may not be placed in a kit, but may be packaged separately in another container.
The luminescent substrate solution was prepared by the following method:
luminescence substrate buffer (formulation shown in table 8) was used as follows: 4 to 1: the luminescent substrate (4-chlorobenzenethiol) (10-methyl-9, 10-acridylmethylene) disodium phosphate was diluted 10 at an initial concentration of 50 mg/L.
Table 8-buffers used during the preparation of the luminogenic substrate:
example 6 differential diagnosis of a chemiluminescent kit directed to a CXCL4 marker for depressive disorder patients
The plasma samples of healthy people and patients with depressive disorders in the present example were collected from the hospital for mental disease control in city of certain province of China, and the collection of plasma samples was approved by the ethical committee of this hospital and satisfied the patient's informed obligation, among which 53 samples of healthy people and 45 samples of patients with depressive disorders.
Plasma sample collection was as follows (final volume at least 1 ml):
(1) The walls of the tubes are annotated with precise information (plasma, name, sex, age, race, date, etc.).
(2) Blood collection: extracting appropriate amount of blood of patient, placing in anticoagulation tube (EDTA/heparin anticoagulation tube), shaking gently for 5 times to mix whole blood and anticoagulant completely to avoid blood coagulation, placing on 4 deg.C ice, and standing for 30 min.
(3) Centrifuging: fresh blood, at 4 ℃ 500 g, was centrifuged for 10 min.
(4) Plasma: the supernatant (plasma) was removed with a pipette and transferred to an Eppendorf Tube or 15ml Falcon Tube.
Note that:
(1) Taking the supernatant, and keeping the distance between the suction head and the tunica albuginea layer to be 0.5-1ml to ensure that the tunica albuginea layer is not sucked.
(2) The plasma preparation process ensures that hemolysis cannot occur.
(3) And (3) storage: immediately quick freezing with liquid nitrogen, and storing at-80 deg.C (or directly storing at-80 deg.C). Repeated freeze thawing is avoided. If there is no liquid nitrogen, the sample is stored directly at-80 ℃.
(4) All specimens need dry ice for sending
Using the chemiluminescence kit prepared in example 5, which contains a combination of antibodies to FSA0013 and FSA0014, the amount of CXCL4 protein in a sample was determined by the following method:
(1) Sample adding: respectively mixing 30 μ L of CXCL4 calibrator, quality control material and sample;
(2) Incubation reaction: adding 30 μ L of the anti-reagent A and 30 μ L of the anti-reagent B, mixing uniformly, and reacting for 5min at 37 ℃; adding 30 μ L of the anti-reagent C and 30 μ L of the magnetic particle reagent, mixing uniformly, and reacting for 10min at 37 ℃;
(3) Cleaning: standing in a magnetic field with the magnetic field intensity of 3800mT, and discharging supernatant; adding 300 mu L of cleaning solution and mixing evenly; repeating the steps for 3 times;
(4) Adding a substrate: adding 200 mu L of luminescent substrate solution and mixing evenly;
(5) Measured value: and detecting the luminous intensity, and calculating to obtain the content of the CXCL4 protein in the sample to be detected.
Table 9-preparation of the cleaning solution used in step (3) of the above measurement method, the formulation is as follows:
the cleaning solution is diluted by 10 to 20 times and then used for cleaning in the step (3) of the measurement method.
Table 10 below shows the calibration curve for the CXCL4 protein concentration determination
TABLE 10-CXCL4 protein calibration Curve
Table 11 below shows the concentrations (ng/ml) of CXCL4 protein in plasma samples of healthy humans and patients with depressive disorder measured using the chemiluminescence kit prepared in example 5.
TABLE 11-1 data determined for groups 1-15 of healthy and depressive disorder population
TABLE 11-2 measurement data of groups 16-30 of healthy and depressive disorder population
TABLE 11-3 measurement data of healthy population group 31-53 and depressive disorder group 31-45
For the results of tables 11-1 to 11-3 above, the average of the data determined from 53 samples of healthy population and 45 samples of depressive disorder population was calculated and shown: the mean concentration of CXCL4 of samples of healthy people is 1305 (ng/ml), and the mean concentration of CXCL4 of samples of depressive disorder people is 2421 (ng/ml).
As can be seen from the results of tables 11-1 to 11-3 above, by using the combination of antibodies FSA0013 and FSA0014 in a paired combination, and in particular the chemiluminescence kits prepared therefrom, there was a significant difference in the protein concentration of CXCL4 in plasma samples from healthy and depressive disorder populations, where plasma CXCL4 protein levels were 1.86 times higher (p < 0.0001) than in healthy populations.
Moreover, based on the calibration curve of table 10, when a CXCL4 plasma protein concentration of 1800 ng/ml is used as the dividing line for differential diagnosis of health and depressive disorders, the specificity of this kit is as high as 92.5%, the sensitivity is as high as 93.3%, and the accuracy is as high as 92.8%.
The chemiluminescence kit provided by the invention realizes the purpose of diagnosing depressive disorder on a molecular level through a marker by adopting the combination of an antibody pair with high specificity combined with CXCL4 and the carboxyl magnetic beads coupled with alkaline phosphatase, streptavidin conjugate and goat anti-fluorescein isothiocyanate antibody through a chemiluminescence method.
Claims (12)
1. A chemiluminescent kit for diagnosing depressive disorders by specifically detecting CXCL4 protein, comprising the following components: an anti-reagent A, an anti-reagent B, an anti-reagent C, a magnetic particle reagent, a calibrator and a quality control material, wherein,
-said anti-agent a comprises a conjugate of fluorescein isothiocyanate and antibody FSA0013, said antibody FSA0013 being expressed by transient co-transfection of recombinant plasmids comprising the heavy chain of antibody FSA0013 and the light chain of antibody FSA0013, respectively, into the chinese hamster ovary cell ExpiCHO-s expression system, followed by purification using Protein a affinity chromatography to yield said antibody FSA0013;
the recombinant plasmid containing the heavy chain coding sequence of the antibody FSA0013 is contained in Escherichia coli FSV0003-H, wherein the Escherichia coli FSV0003-H is preserved in China center for type culture Collection, the address of university of Wuhan, china, the preservation date is as follows: 2022, 11 months and 10 days, with a preservation number of CCTCC M20221754; and the recombinant plasmid containing the light chain coding sequence of the antibody FSA0013 is contained in Escherichia coli FSV0003-L, wherein the Escherichia coli FSV0003-L is preserved in China center for type culture Collection, the address of university of Wuhan, china, the preservation date: in 2022, 11 months and 10 days, the preservation number is CCTCC M20221755;
-said anti-agent B comprises a conjugate of biotin and antibody FSA0014, said antibody FSA0014 being expressed by transient co-transfection of recombinant plasmids comprising the heavy chain of antibody FSA0014 and the light chain of antibody FSA0014, respectively, into the chinese hamster ovary cell ExpiCHO-s expression system, followed by purification using Protein a affinity chromatography to obtain said antibody FSA0014;
the recombinant plasmid containing the heavy chain coding sequence of the antibody FSA0014 is contained in Escherichia coli FSV0004-H, wherein the Escherichia coli FSV0004-H is preserved in China center for type culture Collection, the address of university of Wuhan, china, the preservation date is as follows: 2022, 11 months and 10 days, and the preservation number is CCTCC M20221752; and the recombinant plasmid containing the light chain coding sequence of the antibody FSA0014 is contained in Escherichia coli FSV0004-L, wherein the Escherichia coli FSV0004-L is preserved in China center for type culture Collection, the address of university of Wuhan, china, the preservation date: in 2022, 11 months and 10 days, the preservation number is CCTCC M20221753;
-said anti-agent C comprises a conjugate of alkaline phosphatase and streptavidin;
-the magnetic particle reagent comprises a conjugate of a goat anti-fluorescein isothiocyanate antibody and a carboxylic magnetic bead; and is
-the calibrator and the quality control are buffers of CXCL4 antigen at different concentrations, respectively.
2. The chemiluminescent kit according to claim 1, characterized in that the anti-agent a is prepared by the following method:
(1) Coupling of fluorescein isothiocyanate to antibody FSA 0013:
the preparation concentration of fluorescein isothiocyanate is 1.0-5.0 mg/mL by using Tris buffer solution, and then the volume ratio of the Tris buffer solution to the fluorescein isothiocyanate is 1:100 to 1:10000 of diluting fluorescein isothiocyanate to obtain a diluent, adding the FSA0013 into the diluent according to the molar ratio of 1;
(2) Purification of conjugate of fluorescein isothiocyanate and antibody FSA 0013:
and eluting the conjugate of the fluorescein isothiocyanate and the antibody FSA0013 by using a bicarbonate buffer solution with the pH = 8-9, collecting an eluent, and adding the obtained conjugate of the fluorescein isothiocyanate and the antibody FSA0013 into an anti-reagent buffer solution to prepare the anti-reagent A.
3. The chemiluminescent kit according to claim 1, characterized in that the anti-reagent B is prepared by the following method:
(1) Coupling of biotin to antibody FSA 0014:
preparing biotin with a carbonate buffer solution with pH of 8-9 to a concentration of 1.0-5.0 mg/mL, adding the antibody FSA0014 into the biotin according to a molar ratio of 1;
(2) Purification of conjugate of biotin with antibody FSA 0014:
and (3) carrying out ultrafiltration and centrifugation on the conjugate of the biotin and the antibody FSA0014 at the rotating speed of 3000 rpm for 10min to obtain mother liquor containing the conjugate of the biotin and the antibody FSA0014, and adding an anti-reagent buffer solution to prepare an anti-reagent B.
4. The chemiluminescent kit according to claim 1, characterized in that the anti-reagent C is prepared by the following method:
(1) Activation of alkaline phosphatase:
diluting 25wt.% of glutaraldehyde to 5wt.% by using a carbonate buffer solution with pH of 9-10, dissolving alkaline phosphatase in 0.5ml of 5wt.% of glutaraldehyde, combining with a water bath at 37 ℃ for 2-3 h, washing with absolute ethyl alcohol, removing supernatant, adding 0.5ml of a carbonate buffer solution with pH of 9-10 to dissolve precipitate, and obtaining activated alkaline phosphatase;
(2) Coupling of alkaline phosphatase to streptavidin:
streptavidin was added to the mixture at a molar ratio of 1:2 to 1:10, adding the alkaline phosphatase solution activated in the step (1), and uniformly mixing for 4-5 hours at 20 +/-5 ℃ to obtain a conjugate of the alkaline phosphatase and the streptavidin;
(3) Purification of alkaline phosphatase and streptavidin conjugate:
eluting with bicarbonate buffer solution with the pH = 8-9, collecting eluates, and adding the conjugate of alkaline phosphatase and streptavidin to the anti-reagent buffer solution to obtain the anti-reagent C.
5. A chemiluminescent kit according to claim 1 wherein the magnetic particulate reagent is prepared by the following method:
(1) Fully and uniformly mixing the concentrated solution of the carboxyl magnetic beads, taking out the carboxyl magnetic beads, placing the carboxyl magnetic beads in a magnetic field with the magnetic field intensity of 3800mT for 15min, absorbing the supernatant after the carboxyl magnetic beads are completely settled, adding a phosphoric acid buffer solution with the volume 5 times that of the carboxyl magnetic beads, uniformly mixing for 20-30 min, placing the mixture in a magnetic field with the magnetic field intensity of 3800mT, absorbing the supernatant after the magnetic beads are completely settled for 15min, and repeating the cleaning step for 3 times to fully clean the carboxyl magnetic beads;
(2) And (3) fixing the volume of the carboxyl magnetic bead solution to 10-50mg/mL, wherein the volume is determined according to the following steps: the mass ratio of the goat-anti-fluorescein isothiocyanate antibody is 100:1, adding the goat-anti-fluorescein isothiocyanate antibody, and reacting for 18 hours at 2-8 ℃ in a uniformly mixed state to obtain a conjugate of the goat-anti-fluorescein isothiocyanate antibody and the carboxyl magnetic beads;
(3) Adding a mixed sealing solution of 0.5-2M Tris solution, 5-12 wt.% of bovine serum albumin, 7-15 wt.% of casein solution and 1-10 g of fish skin gel for sealing, uniformly mixing for 3-4 h at 20 +/-5 ℃, washing for 3 times by using a phosphate buffer solution, and performing constant volume by using a magnetic particle buffer solution to obtain a 10-20 mg/mL concentrated magnetic particle reagent containing the conjugate of the goat anti-isothiocyanic acid fluorescein antibody and the carboxyl magnetic beads;
(4) Preparing a magnetic particle reagent containing conjugates of goat-anti-fluorescein isothiocyanate antibodies and carboxyl magnetic beads with the concentration of 1-2 mg/mL by using a magnetic particle buffer solution.
6. The chemiluminescent kit of claim 1 wherein the calibrator and the quality control are prepared by the following method:
CXCL4 protein was diluted with calibrator and quality control buffer to give a calibrator with the following concentration gradient: 0, 200, 1000, 2000, 4000, 8000ng/ml; and a first quality control substance with the concentration of 700-1300 ng/ml and a second quality control substance with the concentration of 2800-5200 ng/ml.
7. The chemiluminescent kit of any one of claims 2 to 4 wherein the anti-reagent buffer consists of: 0.1M Tris, 0.9wt.% sodium chloride, 1wt.% bovine serum albumin, 0.01wt.% tetracycline, 0.01wt.% neomycin sulfate, and a suitable amount of 4M hydrochloric acid at pH8 ± 0.1.
8. A chemiluminescent kit according to claim 5 characterized in that the magnetic particle buffer consists of: 0.1M Tris, 0.9wt.% sodium chloride, 1wt.% bovine serum albumin, 5wt.% methylcellulose, and an appropriate amount of 4M hydrochloric acid at pH8 ± 0.1.
9. The chemiluminescent kit of claim 6 wherein the buffer used to formulate the calibrator and quality control consists of: 0.1M Tris, 0.9wt.% sodium chloride, 2wt.% bovine serum albumin, 0.01wt.% tetracycline, 0.01wt.% neomycin sulfate, and an appropriate amount of 4M hydrochloric acid to pH 7.5 ± 0.1.
10. The chemiluminescent kit of claim 1 wherein the chemiluminescent kit is used in conjunction with a separately packaged luminogenic substrate solution, or the chemiluminescent kit further comprises a luminogenic substrate solution;
the luminophore solution contained (4-chlorobenzenethiol) (10-methyl-9, 10-acridalmethylene) disodium phosphate and was prepared by mixing the luminophore buffer solution with a luminophore buffer solution according to 1:4 to 1:10 (4-chlorobenzenethiol) (10-methyl-9, 10-acridylmethylene) phosphate disodium salt at an initial concentration of 50 mg/L; and is
The luminogenic substrate buffer comprises 0.1M Tris, 0.9wt.% sodium chloride, 0.3wt.% lucigenin, and 1wt.% sodium dodecyl sulfate.
11. Use of a combination of a pair of antibodies FSA0013 and FSA0014 that specifically bind to CXCL4 protein for the preparation of a chemiluminescent kit for the diagnosis of depressive disorders by detecting CXCL4 protein,
the antibody FSA0013 is obtained by transiently co-transfecting recombinant plasmids respectively containing the heavy chain of the antibody FSA0013 and the light chain of the antibody FSA0013 into a Chinese hamster ovary cell ExpCHO-s expression system for expression, and then purifying by Protein A affinity chromatography to obtain the antibody FSA0013;
the recombinant plasmid containing the heavy chain coding sequence of the antibody FSA0013 is contained in Escherichia coli FSV0003-H, wherein the Escherichia coli FSV0003-H is preserved in China center for type culture Collection, the address of university of Wuhan, china, the preservation date is as follows: 2022, 11 months and 10 days, and the preservation number is CCTCC M20221754; and the recombinant plasmid comprising the light chain coding sequence of antibody FSA0013 is contained in Escherichia coli FSV0003-L, which Escherichia coli FSV0003-L is deposited at the China center for type culture Collection, the address of university of Wuhan, china, the date of deposition: in 2022, 11 months and 10 days, the preservation number is CCTCC M20221755;
the antibody FSA0014 is obtained by transiently co-transfecting recombinant plasmids respectively containing the heavy chain of the antibody FSA0014 and the light chain of the antibody FSA0014 into a Chinese hamster ovary cell ExpicHO-s expression system for expression, and then purifying by using Protein A affinity chromatography to obtain the antibody FSA0014;
the recombinant plasmid comprising the heavy chain coding sequence of antibody FSA0014 is contained in Escherichia coli FSV0004-H, which Escherichia coli FSV0004-H is deposited at the China center for type culture Collection, the address of university of Wuhan, china, the date of deposition: 2022, 11 months and 10 days, and the preservation number is CCTCC M20221752; and the recombinant plasmid comprising the light chain coding sequence of antibody FSA0014 is contained in Escherichia coli FSV0004-L, which Escherichia coli FSV0004-L is deposited at the China center for type culture Collection, the address of university of Wuhan, china, the date of deposition: in 2022, 11 months and 10 days, the preservation number is CCTCC M20221753;
the antibody FSA0013 is coupled with fluorescein isothiocyanate to form a conjugate of the fluorescein isothiocyanate and the antibody FSA0013; and is
And the antibody FSA0014 is coupled with biotin to form a conjugate of biotin and the antibody FSA0014.
12. Use according to claim 11, characterized in that the chemiluminescent kit further comprises the following components: anti-reagent C, magnetic particle reagent, calibrator and quality control product, wherein,
-said anti-agent C comprises a conjugate of alkaline phosphatase and streptavidin and is prepared by:
(1) Activation of alkaline phosphatase:
diluting 25wt.% of glutaraldehyde to 5wt.% by using a carbonate buffer solution with pH of 9-10, dissolving alkaline phosphatase in 0.5ml of 5wt.% of glutaraldehyde, combining with a water bath at 37 ℃ for 2-3 h, washing with absolute ethyl alcohol, removing supernatant, adding 0.5ml of a carbonate buffer solution with pH of 9-10 to dissolve precipitate, and obtaining activated alkaline phosphatase;
(2) Coupling of alkaline phosphatase to streptavidin:
streptavidin was added at a molar ratio of 1:2 to 1:10, adding the alkaline phosphatase solution activated in the step (1), and uniformly mixing for 4-5 hours at 20 +/-5 ℃ to obtain a conjugate of the alkaline phosphatase and the streptavidin;
(3) Purification of alkaline phosphatase and streptavidin conjugate:
eluting with bicarbonate buffer solution with the pH = 8-9, collecting eluates in parts, and adding the conjugate of the alkaline phosphatase and the streptavidin into the anti-reagent buffer solution to prepare an anti-reagent C;
-the magnetic particle reagent comprises a conjugate of goat anti-fluorescein isothiocyanate antibody and a carboxylic magnetic bead, and the magnetic particle reagent is prepared by the following method:
(1) Fully and uniformly mixing the concentrated solution of the carboxyl magnetic beads, taking out the carboxyl magnetic beads, placing the carboxyl magnetic beads in a magnetic field with the magnetic field intensity of 3800mT for 15min, absorbing the supernatant after the carboxyl magnetic beads are fully settled, adding phosphoric acid buffer solution with the volume 5 times that of the carboxyl magnetic beads, uniformly mixing for 20-30 min, placing the mixture in a magnetic field with the magnetic field intensity of 3800mT, absorbing the supernatant after the magnetic beads are fully settled for 15min, and repeating the cleaning step for 3 times to fully clean the carboxyl magnetic beads;
(2) And (3) fixing the volume of the carboxyl magnetic bead solution to 10-50mg/mL, and according to the volume of the carboxyl magnetic beads: the mass ratio of the goat-anti-fluorescein isothiocyanate antibody is 100:1, adding the goat-anti-fluorescein isothiocyanate antibody, and reacting for 18 hours at 2-8 ℃ in a uniformly mixed state to obtain a conjugate of the goat-anti-fluorescein isothiocyanate antibody and the carboxyl magnetic beads;
(3) Adding a mixed sealing solution of 0.5-2M Tris solution, 5-12 wt.% bovine serum albumin, 7-15 wt.% casein solution and 1-10 g of fish skin gel for sealing, uniformly mixing for 3-4 h at 20 +/-5 ℃, washing for 3 times by using a phosphate buffer solution, and performing constant volume by using a magnetic particle buffer solution to obtain a 10-20 mg/mL concentrated magnetic particle reagent containing the conjugate of the goat anti-fluorescein isothiocyanate antibody and the carboxyl magnetic beads;
(4) Preparing a magnetic particle reagent containing a conjugate of goat anti-fluorescein isothiocyanate antibody and carboxyl magnetic beads with the concentration of 1-2 mg/mL by using a magnetic particle buffer solution;
-the calibrator and the quality control are buffers with different concentrations of CXCL4 antigen, respectively, and the calibrator and the quality control are prepared by the following method:
CXCL4 protein was diluted with calibrator and quality control buffer to give calibrators with the following concentration gradients: 0, 200, 1000, 2000, 4000, 8000ng/ml; and a first quality control substance with the concentration of 700-1300 ng/ml and a second quality control substance with the concentration of 2800-5200 ng/ml.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211612897.4A CN115656521A (en) | 2022-12-15 | 2022-12-15 | Chemiluminescence kit for diagnosing depressive disorder by detecting CXCL4 protein |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211612897.4A CN115656521A (en) | 2022-12-15 | 2022-12-15 | Chemiluminescence kit for diagnosing depressive disorder by detecting CXCL4 protein |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN115656521A true CN115656521A (en) | 2023-01-31 |
Family
ID=85023291
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211612897.4A Pending CN115656521A (en) | 2022-12-15 | 2022-12-15 | Chemiluminescence kit for diagnosing depressive disorder by detecting CXCL4 protein |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115656521A (en) |
-
2022
- 2022-12-15 CN CN202211612897.4A patent/CN115656521A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CA2978998C (en) | Method and kit for measuring component to be assayed in specimen | |
| US10126312B2 (en) | Diagnostic method for urinary tract infection | |
| US20110256636A1 (en) | Methods and reagents for diagnosing rheumatoid arthrtis | |
| US11768202B2 (en) | Method of detecting anti-Ri in a subject with a previous streptococcal infection | |
| CN105277690A (en) | Reagent kit and method for full-automatically measuring antiprotease 3 antibody IgG | |
| WO2008143890A2 (en) | Biomarkers for septic shock patients | |
| CN113804896A (en) | Magnetic particle chemiluminescence kit for measuring interleukin-8 content in human serum | |
| TR201809001T4 (en) | A method for diagnosing or monitoring renal function or diagnosing renal dysfunction. | |
| JP6048923B2 (en) | Detection method and detection kit for chronic hepatitis B | |
| CN112480244A (en) | Anti-allergic nano antibody composition, antibody determination method and spray | |
| CN109142750B (en) | Kit for determining anti-histone antibody IgG and detection method | |
| US9176147B2 (en) | Detection of B-cell activating factor as a biomarker for antibody mediated rejection in transplant recipients | |
| US20170097352A1 (en) | Immunoglobulin-bound extracellular vesicles and uses thereof | |
| WO2024001044A1 (en) | Biomarker combination related to lung cancer, kit containing same, and use thereof | |
| CN115353566B (en) | Antibody combination for detecting interleukin 1-beta and application thereof | |
| KR102039816B1 (en) | Biomarkers for diagnosis or prognosis of fibrosis in kidney allografts | |
| CN115656521A (en) | Chemiluminescence kit for diagnosing depressive disorder by detecting CXCL4 protein | |
| CN111381046A (en) | Calprotectin chemiluminescence immunoassay kit and preparation method thereof | |
| CN113817025B (en) | SLE epitope polypeptides in the identification of SLE and other autoimmune diseases | |
| WO2013053918A1 (en) | Diagnosis of intraocular lymphoma | |
| CN113831401B (en) | SLE epitope polypeptide and application thereof in SLE diagnosis | |
| WO2023004626A1 (en) | Myeloma biomarker serpinf2 and use thereof | |
| WO2023004624A1 (en) | Myeloma biomarker lgals3bp and use thereof | |
| CN117434276A (en) | THSD7A antibody detection kit and application thereof | |
| CN113933497A (en) | I type collagen amino-terminal extension peptide magnetic particle chemiluminescence immunoassay kit and detection method thereof |
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 | ||
| REG | Reference to a national code |
Ref country code: HK Ref legal event code: DE Ref document number: 40085275 Country of ref document: HK |