CN116621978A - Antibody and kit for beta-CTX detection and application thereof - Google Patents
Antibody and kit for beta-CTX detection and application thereof Download PDFInfo
- Publication number
- CN116621978A CN116621978A CN202211243489.6A CN202211243489A CN116621978A CN 116621978 A CN116621978 A CN 116621978A CN 202211243489 A CN202211243489 A CN 202211243489A CN 116621978 A CN116621978 A CN 116621978A
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- ctx
- beta
- kit
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
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Abstract
The invention discloses an antibody for beta-CTX detection, a kit and application thereof, belonging to the fields of in vitro diagnosis reagents, bioengineering technology and medical instrument intersection, wherein the kit comprises a luminous reagent, a solid-phase reagent, a calibrator and a quality control product, wherein the luminous reagent is an acridine ester marked beta-CTX antibody solution, the solid-phase reagent is a suspension of magnetic particles coated with the beta-CTX antibody, the kit is detected by a double-antibody sandwich method principle, and can be carried with a full-automatic luminous detector for automatic detection, and the kit has the characteristics of high sensitivity, strong specificity, wide detection range and high detection efficiency.
Description
Technical Field
The invention relates to the fields of in-vitro diagnostic reagents, bioengineering technology and medical instrument crossing, in particular to an antibody and a kit for beta-CTX detection and application thereof.
Background
Bone turnover is a self-renewing process in which old bone is continuously absorbed by osteoclasts and new bone is continuously formed by osteoblasts, and bone growth and structural integrity can be maintained only when bone tissue is continuously subjected to bone modeling and bone reconstruction. Bone turnover is critical for repairing skeletal fatigue damage and maintaining body mineral balance. Bone turnover biochemical markers or bone turnover markers (bone turnover markers, BTMs) are metabolites or enzymes produced during bone turnover, and are divided into bone formation indicators reflecting osteoblast activity and bone formation status and bone resorption indicators representing osteoclast activity and bone resorption level. The BTMs have important reference values for diagnosis and identification of various bone diseases, fracture risk prediction, drug efficacy evaluation and the like. In recent years, along with the research progress of osteoporosis and metabolic bone diseases, especially evidence-based medical evidence of BTMs at home and abroad is continuously emerging, and the clinical application of BTMs is becoming widespread.
The BTMs widely used at present comprise beta-CTX, P1NP, OC and b-ALP, and the main clinical application in China is beta-CTX and P1NP, OC, ALP. Among them, β -CTX (β -Cross caps) is currently internationally recognized as a representative bone resorption marker. beta-CTX is a type I collagen degradation fragment consisting of 8 amino acids, formed by the conversion of alpha-aspartic acid of the C-terminal peptide to the beta form. An increase in serum beta-CTX levels indicates an increase in bone resorption in the patient, and the serum beta-CTX levels return to normal following bone resorption inhibition treatment. Due to circadian rhythm and food effects, β -CTX levels may show significant changes over different time periods, and detection differences may be minimized by standardized sample collection in the fasted state of the morning.
In research and clinical practice, most applications of beta-CTX measurement are performed on blood samples. The specific differences in serum and plasma beta-CTX methods lead to difficulties in standardizing the beta-CTX commercial assay. On the other hand, the existing detection means have various defects such as higher detection cost, lower sensitivity and the like. In China, the beta-CTX detection is mainly clinically applied to the reagent of Roche company, but the reagent is expensive and is unfavorable for popularization in the basic level. Therefore, a beta-CTX detection kit with high sensitivity, strong specificity, wide detection range and high detection efficiency is to be developed and used for monitoring the anti-absorption treatment effect of osteoporosis or other bone diseases.
Disclosure of Invention
The invention aims to solve the problems of developing a beta-CTX detection kit with high sensitivity, strong specificity, wide detection range and high detection efficiency, which is used for monitoring the anti-absorption treatment effect of osteoporosis or other bone diseases.
To solve the above problems, the first aspect of the present invention provides an antibody for detection of β -CTX.
The antibody for beta-CTX detection is a monoclonal antibody produced by hybridoma cells, and the preparation method comprises the following steps:
antigen synthesis: coupling macromolecular carrier protein on the basis of polypeptide with polypeptide sequence shown in SEQ ID No.1, wherein the macromolecular carrier protein is one or more of BSA and OVA, so as to prepare recombinant immunogen with antigenicity and immunogenicity;
SEQ ID No.1：Cys Glu Lys Ala His Asp Gly Gly Arg；
the molecular weight of the polypeptide alone is usually too small to stimulate a sufficient immune response, and when coupled to a carrier protein such as KLH, BSA, OVA with many epitopes, the recombinant antigen is more beneficial to stimulating T in mice in subsequent experiments than the polypeptide H Cells, in turn, induce B lymphocytes to produce an immune response.
Animal immunization: the recombinant immunogen and the adjuvant prepared by the macromolecular carrier protein are used for performing primary immunization on mice through subcutaneous injection, boosting immunization is performed after the primary immunization, and armpit injection impact immunization without the adjuvant is performed after the final boosting immunization;
animal immunity utilizes the humoral immune response mechanism of organisms, the primary immune response and the secondary immune response are started successively through continuous stimulation of antigens, and the actual immune effect is measured by the antibody titer in serum in actual operation. There is a lag phase in the initiation of primary responses after antigen stimulation of the body, in which young B cells undergo clonal selection, proliferation, and then differentiation into memory cells or plasma cells; after a lag phase, serum titers increase exponentially to a peak and then decrease; in the primary immune reaction, igM is secreted firstly, then through antibody subtype conversion, the proportion of the IgG is increased, the initiation of the secondary immune reaction depends on the number of memory B cells and memory T cells, and the memory cells can be activated when encountering the same antigen again, thereby leading to the occurrence of the secondary immune reaction; when the number of memory cells is much greater than that of young B cells, the secondary response starts faster and is stronger; the secondary reaction has shorter lag phase than the primary reaction, higher peak plasma cell and serum titers, and longer duration; meanwhile, the secreted antibody is subjected to affinity maturation and subtype conversion, so that the quality is higher; adjuvants are needed for animal immunization, and the adjuvants mainly have the effects of delaying the release of antigens, enhancing phagocytosis of phagocytes and presentation of antigens.
Cell fusion: extracting spleen cells of the mice, mixing the spleen cells with myeloma cells, adding PEG, and inducing cells to carry out cell fusion;
PEG is an artificially synthesized drug and has the following functions in cell fusion: coagulating the cells; the phospholipid bilayer of the cell membrane at the contact position is destroyed, so that fusion occurs between the cell membranes at the contact position, and cytoplasm is communicated, so that a hit binuclear or polynuclear fusion cell is formed.
Monoclonal antibody preparation: coating the polypeptide-OVA conjugate shown in SEQ ID No.1 and the OVA conjugate of the polypeptide shown in SEQ ID No.2 by 1 mug/ml, detecting and selecting double positive clones by an indirect ELISA method, performing subcloning, ensuring that the monoclonal antibody is obtained by amplification culture and purification;
SEQ ID No.2：Cys Glu Lys Ala His Asp Gly Gly Arg Glu Lys Ala His Asp Gly Gly Arg；
antibody labeling: uniformly mixing the monoclonal antibody with biotin, and reacting to obtain a biotin-labeled monoclonal antibody;
the antibody labeling means that a label is covalently connected to an antibody, specifically reacts with an object to be detected to form a multi-component complex, and the test result is directly observed in a microscopic way or automatically measured by means of a fluorescent microscope, a ray measuring instrument, an enzyme-labeled detector, an electron microscope, a luminous immunoassay instrument and other precise instruments, and biotin can be combined with molecules such as protein, fluorescein and the like without affecting the biological activity of the latter, so that the antibody labeling agent is an ideal labeling agent. One antibody molecule can be coupled with a plurality of biotin molecules, and the biotin molecules can be combined with enzyme or fluorescein, so that a biological amplification system is formed, and the detection sensitivity is remarkably improved.
Antibody pairing screening: and (3) incubating, coating and sealing unlabeled monoclonal antibodies, adding a polypeptide-OVA conjugate of the polypeptide shown in SEQ ID No.2 for incubation, adding a biotin-labeled monoclonal antibody for incubation, adding SA-HRP for incubation and color development, detecting, and selecting positive pairing with strong specificity and high sensitivity.
Typically, an antigen molecule has multiple epitopes, and the antigen is injected into an animal to immunize the animal, and different antibodies are produced against the different epitopes. Antibodies are produced with specificity and specificity, i.e. one antibody molecule will specifically bind to only one epitope, two antibodies against different epitopes, possibly binding to the antigen molecule at the same time, if both antibodies bind to the same antigen molecule at the same time, the two antibodies are paired antibodies. The antibody pairing is commonly applied to double-antibody sandwich ELISA experiments, can carry out qualitative and quantitative detection on target antigens, and has the advantages of high sensitivity, high specificity and high stability.
In a second aspect, the invention provides a kit for the detection of beta-CTX.
Further, the kit comprises the monoclonal antibody prepared by the beta-CTX antibody preparation method.
Further, the kit for beta-CTX detection provided by the invention is a chemiluminescent immunoassay kit comprising a luminescent reagent, a solid phase reagent, a standard substance and a quality control substance.
Further, the solid phase reagent of the kit is magnetic particle suspension coated with the beta-CTX monoclonal antibody, and the concentration of the magnetic particles is 0.05-0.5 mug/ml.
Preferably, the magnetic particles in the solid phase reagent of the kit are superparamagnetism nanometer magnetic microspheres with active chemical groups, and the surface active groups are any one of carboxyl, tosyl, epoxy and amino.
Further, the luminescent reagent of the kit is beta-CTX monoclonal antibody solution marked with chemiluminescent reagent, the concentration is 0.05-1.0 mug/ml, and the beta-CTX antibody in the luminescent reagent and the beta-CTX antibody in the solid phase reagent are two monoclonal antibodies which can be paired aiming at different antigen targets.
Preferably, the luminescent reagent of the kit is any one of acridinium ester, isoluminol, alkaline phosphatase, peroxidase and terpyridyl ruthenium.
Further, the formula of the diluent of the kit comprises buffer salt, inorganic salt, surfactant, protein protectant, chemical protectant and preservative, wherein Ph is 6-8, and the buffer salt is one or more of HEPES, phosphate buffer and TRIS buffer; the inorganic salt is one or more of sodium chloride, potassium chloride, magnesium chloride and zinc sulfate; the surfactant is one or more of Tween 120 triton 405, NP-40 and sodium dodecyl sulfate; the protein protectant is one or more of HSA, BSA, gelatin, polidocanol and alpha cyclodextrin, the chemical protectant is one or more of polyvinylpyrrolidone, biolipidure, glycol and dextran sulfate sodium salt, and the preservative is one or more of proclin300, gentamicin, levofloxacin and phenoxyethanol.
Further, the calibrator is a liquid calibrator with 2 different concentration points, wherein the concentration of the liquid calibrator is in the range of 0-2000pg/ml, and the liquid calibrator is prepared from dimer polypeptide of the protein shown in SEQ ID No. 2; the quality control product is a liquid quality control product with 2 different concentration points, wherein the concentration of the liquid quality control product is prepared from dimer polypeptide obtained by expressing the sequence 2 protein and is in the range of 0-1000 pg/ml.
In a third aspect, the invention provides the use of a kit for the detection of beta-CTX.
The kit is detected by a double-antibody sandwich method principle, and the main reaction flow is that an instrument sample needle automatically absorbs 30 mu l of serum sample in a reaction tube, a reagent needle respectively absorbs 40 mu l of solid phase reagent and 60 mu l of luminous reagent in the reaction tube, the reagent needle is fully mixed and incubated for 15 minutes, an analyzer is automatically transferred into a cleaning station to be repeatedly cleaned for 3 times, finally the analyzer is transferred into a measuring pool, pre-excitation liquid and excitation liquid are pumped in through a pipeline, a PMT in the measuring pool collects generated optical signals in the reaction tube and uploads the optical signals to software, and final measured concentration is given through a calibration curve input in the instrument.
The invention has the beneficial effects that: (1) The beta-CTX detection kit provided by the invention is a chemiluminescent immunoassay kit, excitation light is not needed during analysis, the detection lower limit is low, and the sensitivity is high; (2) The beta-CTX detection kit provided by the invention uses the magnetic microsphere as a solid phase carrier, is convenient to clean, removes non-specific binding, and is beneficial to improving detection accuracy; (3) The beta-CTX detection kit provided by the invention also has the characteristics of strong specificity, good repeatability and high stability.
Drawings
FIG. 1 shows correlation detection results in an embodiment of the present invention;
FIG. 2 shows the linear detection results according to an embodiment of the present invention;
FIG. 3 shows the results of thermal stability testing in an embodiment of the present invention;
fig. 4 shows the results of an on-board stability test in an embodiment of the present invention.
Detailed Description
In order that the above-recited objects, features and advantages of the present invention will become more readily apparent, a more particular description of embodiments of the invention will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings. It should be noted that the following examples are only for illustrating the implementation method and typical parameters of the present invention, and are not intended to limit the scope of the parameters described in the present invention, so that reasonable variations are introduced and still fall within the scope of the claims of the present invention.
It should be noted that endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and that such range or value should be understood to include values approaching such range or value. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.
The embodiment of the invention provides an antibody for beta-CTX detection, a kit and application thereof, wherein the antibody is prepared by coating polypeptides shown as SEQ ID No.1 and SEQ ID No.2 by carrier proteins and then preparing a screened beta-CTX monoclonal antibody. The kit is a chemiluminescent immunoassay kit comprising a luminescent reagent, a solid phase reagent, a standard substance and a quality control substance, wherein the solid phase reagent is suspension with the concentration of magnetic particles coated with the beta-CTX monoclonal antibody of 0.05-0.5 mug/ml; the luminescent reagent is beta-CTX monoclonal antibody solution with the concentration of 0.05-1.0 mug/ml marked with chemiluminescent reagent; the calibrator is a liquid calibrator with 2 different concentration points, wherein the concentration of the liquid calibrator is in the range of 0-2000pg/ml, and the liquid calibrator is prepared from dimer polypeptide of the protein shown in SEQ ID No. 2; the quality control product is a liquid quality control product with 2 different concentration points, wherein the concentration of the liquid quality control product is in the range of 0-1000pg/ml, and the liquid quality control product is prepared from dimer polypeptide obtained by expressing the sequence 2 protein.
EXAMPLE 1 preparation of beta-CTX monoclonal antibodies
1. Polypeptide synthesis
The polypeptide shown in SEQ ID No.1 and SEQ ID No.2 is synthesized, the polypeptide synthesis is entrusted to the relevant CXO company, and the final purity is more than or equal to 95%.
SEQ ID No.1：Cys Glu Lys Ala His Asp Gly Gly Arg
SEQ ID No.2：Cys Glu Lys Ala His Asp Gly Gly Arg Glu Lys Ala His Asp Gly Gly Arg
2. Recombinant immunogen preparation
5mg/ml of carrier protein (BSA and OVA) solution was prepared with PBS (ph=7.4), mixed with sulfoo-SMCC (sulfoo-SMCC, thermo) added at a molar ratio of 1:10 per 5mg of carrier protein, reacted for 30 minutes at room temperature or overnight at 4 ℃, excess crosslinker was removed using a desalting column equilibrated with PBS (ph=7.4), and the polypeptide dry powder was reconstituted to 5mg/ml to activate the post carrier: the polypeptides were mixed in a molar ratio of 1:10, reacted for 30 minutes at room temperature, or reacted overnight at 4 ℃. The reaction was dialyzed against PBS (ph=7.4) 3 times, changing the solution every 4 hours, to give the conjugate.
3. Immunization of animals
The immunogen is SEQ ID No.1-BSA conjugate, and the immunized mice are female Balb/c mice with 6-8 weeks of age and health. At the time of primary immunization, the immunization dose is 100 ug/mouse, freund's complete adjuvant is used for mixing, and subcutaneous multipoint injection is carried out; boosting was performed 3 weeks later, 50 ug/mouse, mixed with Freund's incomplete adjuvant, injected subcutaneously at multiple points, and thereafter boosted again at 2 week intervals; 3 booster immunizations were performed after the first immunization. The orbital blood collection was performed 1 week after the first, third and fourth, and the ELISA plate was coated with 1ug/ml of each of the SEQ ID No.1-OVA conjugate and the SEQ ID No.2-OVA conjugate, and the titer of the mouse antiserum was detected by the indirect ELISA method. After 10 days of the last booster, 100 ug/mouse without adjuvant was used for impact underarm injection; fusion experiments were performed 3 days after the impact.
4. Cell fusion and monoclonal antibody preparation experiments
The impacted mice were sacrificed at cervical amputation and sterilized by immersion in 75% ethanol. In an ultra clean bench, the spleens of mice were removed under aseptic conditions, pressed with sterilized ground glass flakes and gently ground to isolate spleen cells, spleen cells were counted. SP2/0 cells and splenocyte 1:5, 500g was centrifuged at room temperature for 5 minutes, and the supernatant was discarded. 1.5ml of PEG was added and the PEG and cells were thoroughly and homogeneously mixed, and this process was completed within 3 minutes. 50ml of fresh DMEM medium was added, centrifuged at 500g for 5 minutes at room temperature, and HAT medium was resuspended and gently mixed. 100 ul/hole is added into a 96-well plate, the 96-well plate is put into an incubator for culture, HT culture medium is replaced when cells in the 96-well plate grow to 1/3 bottom area after 4-5 days, SEQ ID No.1-OVA conjugate and SEQ ID No.2-OVA conjugate are coated by 1ug/ml, double positive clones are detected and selected by an indirect ELISA method, 3-4 subclones are carried out, expanded culture is ensured when monoclonal is formed, ascites cell injection is carried out, and monoclonal antibodies are obtained by purification.
5. Antibody labelling
The monoclonal antibody is diluted to the concentration of 1mg/ml by 1xPBS, biotin (EZ-Link NHS-PEG4-Biotin, thermo) is added into each 2mg of antibody in the molar ratio of 1:5 (antibody: biotin) and is quickly mixed, after the mixture is uniformly mixed, a centrifuge tube containing the mixed solution is placed on a vertical stirrer for reaction for 30min or overnight at 4 ℃, and the biotinylated antibody is dialyzed 3 times by 1xPBS, and the solution is changed once every 4 hours. Obtaining the biotin-labeled monoclonal antibody.
6. Antibody pairing screening
The unlabeled monoclonal antibody is coated by incubating for 1h at 37 ℃ with 2 mug/ml x100 μl per well; 200. Mu.L of 2% BSA was blocked, incubated at 37℃for 1h, PBST was washed 3 times, 1ug/ml of SEQ ID No.2-OVA conjugate was added to each well, each pair was provided with a negative control without antigen, incubated at 37℃for 1h, and PBST was washed 3 times. Adding biotin-labeled monoclonal antibody (1:200), incubating for 1h at 37 ℃, and washing with PBST for 3 times; SA-HRP 1:2000, 100 μl, incubation at 37deg.C for 30min, PBST wash 4 times; and (3) color development, namely reading an OD value at a wavelength of 450nm, wherein the OD value is 2.1 times greater than that of a negative control hole, judging positive pairing, and selecting an antibody with high sensitivity and strong specificity to perform sample test on the preparation of the chemiluminescent reagent.
Example 2 beta-CTX chemiluminescent kit preparation
Preparing a diluent: sequentially dissolving a buffering agent, inorganic salt, a metal chelating agent, a protein protecting agent, a preservative and a surfactant into purified water, regulating the pH value to be within the range of 6.0-8.0, carrying out constant volume according to the preparation amount, and filtering through a 0.22 mu m filter membrane for later use;
preparing a solid phase reagent: coating the surface of magnetic particles (purchased from JSR company in Japan) with a beta-CTX antibody to prepare an intermediate product, and then adding a proper amount of the intermediate product of the magnetic particles into a diluent to prepare working solution with the concentration of 0.05-0.5 mg/ml;
preparing a luminescent reagent diluent: sequentially dissolving a buffering agent, inorganic salt, a metal chelating agent, a protein protecting agent, a preservative and a surfactant into purified water, regulating the pH value to be within the range of 5.0-7.0, carrying out constant volume according to the preparation amount, and filtering through a 0.22 mu m filter membrane for later use;
preparing a luminescent reagent: marking acridinium ester (NSP-SA-NHS, sigma) on a beta-CTX antibody according to a certain proportion to prepare an intermediate product, and then adding a proper amount of the luminescent reagent intermediate product into a luminescent reagent diluent to prepare a luminescent reagent working solution with the concentration of 0.05-1.0 mug/ml;
preparing a calibrator diluent: sequentially dissolving a buffering agent, inorganic salt, a metal chelating agent, a protein protecting agent, a preservative and a surfactant into purified water, regulating the pH value to be within the range of 6.0-8.0, carrying out constant volume according to the preparation amount, and filtering through a 0.22 mu m filter membrane for later use;
preparing a calibrator: adding beta-CTX recombinant antigen into calibrator diluent according to a certain proportion to prepare calibrator solutions with the concentrations of 200pg/ml and 5000pg/ml respectively, and respectively marking the calibrator solutions as CAL1 and CAL2;
preparing a quality control product: the beta-CTX recombinant antigen is added into the calibrator diluent according to a certain proportion to prepare quality control substance solutions with the concentrations of 200pg/ml and 2000pg/ml respectively, which are respectively marked as QC1 and QC2.
Example 3 correlation detection
The gradient dilution recombinant beta-CTX antigen is detected and reassigned by a Roche beta-CTX detection kit, 2 points in the range of 0-6000pg/mL are selected as calibration points, and a self-assembled light-emitting reagent and a solid-phase reagent are used for detecting and calibrating an MS-i3080 chemiluminescent immunoassay analyzer, and the test conditions are as follows: one step method, 60. Mu.L of luminescent reagent, 40. Mu.L of solid phase reagent, 30. Mu.L of sample, incubation for 15min at 37 ℃.
40 samples are collected, the theoretical value covers the linear range of 0-6000pg/mL, and the samples are tested by using the Roche beta-CTX detection kit according to the specification conditions. And the test conditions are the same as above, and the test result is recorded. And calculating the test correlation coefficient R2 of the kit and the Roche kit.
The results of fig. 1 show that the detection results of the kit and the rogowski kit have better correlation, and the correlation coefficient R2 is 0.9915.
Example 4 sensitivity detection
The blank sample was prepared from 5% bovine serum and was continuously assayed 25 times under the same conditions as above. Recording test results, respectively calculating an average value AV and a standard deviation SD, and calculating a lowest detection line LOB=AV+2SD; 5 low-concentration samples with the concentration of about 10pg/ml are respectively added by taking 5% bovine serum as a matrix, each sample is continuously measured for 5 times, the test conditions are the same, and the test results are recorded.
TABLE 1 sensitivity test results
| Sensitivity of | Batch 1 (pg/ml) | Batch 2 (pg/ml) | Batch 3 (pg/ml) |
| LOB | 0 | 0 | 0 |
| LOD | 9.89 | 9.58 | 9.93 |
The results in Table 1 show that the LOB of the kit is 0 and the lowest limit of detection LOD is less than 10pg/ml.
EXAMPLE 5 specific assay
1. Mu.g/mL of osteocalcin, parathyroid hormone and bone ALP samples were tested using self-assembling luminescent and solid phase reagents under the same conditions as above, and the test results were recorded and shown in Table 2.
TABLE 2 results of specific assays
As shown in Table 2, the detection results showed that none of them was higher than 10pg/mL, i.e., no cross reaction was detected.
Example 6 repeatability test
Samples with concentrations of 200pg/ml and 2000pg/ml were tested continuously for 10 times under the same test conditions, and test results were recorded. The average value AV and standard deviation SD were calculated, and the lowest detection line cv=sd/AV was calculated, and the detection results are shown in table 3.
Table 3 repeatability test
The result shows that the continuous test of samples with the concentration of 200pg/ml and 2000pg/ml by using the kit can meet the requirement that CV is less than or equal to 5 percent and can be controlled within 2 percent.
Example 7 Linear detection
The high concentration sample near the upper limit of the linear range and the low concentration sample near the lower limit of the linear range are mixed into 8-11 diluted concentrations, each diluted concentration is tested 3 times, the test conditions are the same, and the test results are recorded, and the results are shown in figure 2.
The result shows that the linear regression coefficient in the linear regression equation meets the requirement that r is more than or equal to 0.99.
Example 8 thermal stability detection
The calibrator and 40 samples were tested simultaneously with the reagent placed at 37℃for 7 days and equilibrated at 2-8℃for 1 day and the reagent placed at 2-8℃under the same test conditions, and the test results were recorded as shown in Table 4 and FIG. 3.
Table 4 thermal stability test
The results in table 4 and fig. 3 show that the two-point calibration values and target values of the two groups of reagents are within 5%, and the results of the samples measured after calibration are basically no deviation.
Example 9 on-board stability detection
After the beta-CTX detection kit is opened, the kit is put into an instrument, the instrument is in a starting state to keep the temperature of a reagent disk, quality control products are measured 3 times in each working day, the average value of the results is obtained, continuous monitoring is carried out for 28 days, the measured concentration is compared with the target value of the quality control products, the testing conditions are the same, and the testing results are recorded, wherein the results are shown in figure 4.
The results show that the accuracy of the quality control product can still meet the requirement that the deviation is less than or equal to +/-10% after the airborne continuous monitoring is carried out for 28 days and the calibration is not carried out.
Example 10 detection
1. Normal reference value range establishment
The beta-CTX detection kit provided by the invention is used for respectively detecting the beta-CTX in serum of 1250 normal human samples of different ages. The normal reference value range is set up as shown in table 5.
TABLE 5 statistical analysis of serum beta-CTX detection results for normal humans
2. Reference range verification
The beta-CTX detection kit is used for detecting 60 physical examination healthy crowd samples, analyzing sample results, removing outliers (if any, alternatively supplementing reference individuals) by using a Dixon method, and calculating data exceeding a reference interval. 58 cases are met, the coincidence rate is 96.67%, and the reference range is applicable.
Although the present disclosure is described above, the scope of protection of the present disclosure is not limited thereto. Various changes and modifications may be made by one skilled in the art without departing from the spirit and scope of the disclosure, and these changes and modifications will fall within the scope of the invention.
Claims (10)
1. A method for preparing a beta-CTX monoclonal antibody, which is characterized by comprising the following steps:
antigen synthesis: coupling macromolecular carrier protein on the basis of polypeptide with polypeptide sequence shown in SEQ ID No.1, wherein the macromolecular carrier protein is one or more of BSA and OVA, so as to prepare recombinant immunogen with antigenicity and immunogenicity;
SEQ ID No.1：Cys Glu Lys Ala His Asp Gly Gly Arg；
animal immunization: the recombinant immunogen and the adjuvant prepared by the macromolecular carrier protein are used for performing primary immunization on mice through subcutaneous injection, boosting immunization is performed after the primary immunization, and armpit injection impact immunization without the adjuvant is performed after the final boosting immunization;
cell fusion: extracting spleen cells of the mice, mixing the spleen cells with myeloma cells, adding PEG, and inducing cells to carry out cell fusion;
monoclonal antibody preparation: coating the polypeptide-OVA conjugate shown in SEQ ID No.1 and the OVA conjugate of the polypeptide shown in SEQ ID No.2 by 1 mug/ml, detecting and selecting double positive clones by an indirect ELISA method, performing subcloning, ensuring that the monoclonal antibody is obtained by amplification culture and purification;
SEQ ID No.2：Cys Glu Lys Ala His Asp Gly Gly Arg Glu Lys Ala His Asp Gly Gly Arg；
antibody labeling: uniformly mixing the monoclonal antibody with biotin, and reacting to obtain a biotin-labeled monoclonal antibody;
antibody pairing screening: and (3) incubating, coating and sealing an unlabeled monoclonal antibody, adding a polypeptide-OVA conjugate of the polypeptide shown in SEQ ID No.2 for immune reaction, washing, adding a biotin-labeled monoclonal antibody, washing, adding SA-HRP for incubation and color development, reading an OD value at 450nm, and selecting positive pairing with strong specificity and high sensitivity.
2. A kit for the detection of β -CTX comprising the monoclonal antibody produced by the method for producing β -CTX antibody of claim 1.
3. The kit for beta-CTX assay according to claim 2, wherein the kit is a chemiluminescent immunoassay kit comprising luminescent reagents, solid phase reagents, standards and quality control.
4. The kit for beta-CTX assay according to claim 3, wherein the solid phase reagent is a suspension of magnetic particles coated with beta-CTX monoclonal antibody, and the concentration of the magnetic particles is 0.05-0.5 μg/ml.
5. The kit for beta-CTX assay according to claim 4, wherein the magnetic particles are superparamagnetic nanospheres having active chemical groups, and the surface active groups are any one of carboxyl, tosyl, epoxy and amino groups.
6. The kit for detecting beta-CTX according to claim 3, wherein the luminescent reagent is a beta-CTX monoclonal antibody solution labeled with a chemiluminescent reagent at a concentration of 0.05-1.0 μg/ml, and the beta-CTX antibody in the luminescent reagent and the beta-CTX antibody in the solid phase reagent are two monoclonal antibodies capable of pairing against different antigen targets.
7. The kit for beta-CTX assay according to claim 6, wherein the chemiluminescent reagent comprises any one of acridinium ester, isoluminol, alkaline phosphatase, peroxidase, ruthenium terpyridyl.
8. The kit for beta-CTX assay according to claim 3, wherein the diluent formulation of the kit consists of one or more of buffer salts, inorganic salts, surfactants, protein protectants, chemical protectants, preservatives, ph of 6-8, buffer salts of HEPES, phosphate buffer, TRIS buffer; the inorganic salt is one or more of sodium chloride, potassium chloride, magnesium chloride and zinc sulfate; the surfactant is one or more of Tween 120 triton 405, NP-40 and sodium dodecyl sulfate; the protein protectant is one or more of HSA, BSA, gelatin, polidocanol and alpha cyclodextrin, the chemical protectant is one or more of polyvinylpyrrolidone, biolipidure, glycol and dextran sulfate sodium salt, and the preservative is one or more of proclin300, gentamicin, levofloxacin and phenoxyethanol.
9. The kit for beta-CTX assay according to claim 3, wherein the calibrator is a liquid calibrator at 2 different concentration points in the range of 0-2000pg/ml prepared from the dimeric polypeptide of the protein shown in SEQ ID No. 2; the quality control product is a liquid quality control product with 2 different concentration points, wherein the concentration of the liquid quality control product is prepared from dimer polypeptide obtained by expressing the sequence 2 protein and is in the range of 0-1000 pg/ml.
10. A method for detecting a kit for β -CTX detection as claimed in any one of claims 2 to 9, wherein the detection is performed by the principle of the double antibody sandwich method, and the main reaction procedure is that an instrument sample needle automatically sucks 30 μl of serum sample into a reaction tube, a reagent needle respectively sucks 40 μl of solid phase reagent and 60 μl of luminescent reagent into the reaction tube, and after fully mixing, incubation is performed for 15 minutes, an analyzer is automatically transferred into a cleaning station for 3 times of repeated cleaning, and finally transferred into a measuring cell, a pre-excitation liquid and an excitation liquid are pumped through a pipeline, and PMT in the measuring cell collects generated optical signals in the reaction tube and uploads the signals to software, and final measured concentration is given through a calibration curve input in the instrument.
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| CN202211243489.6A CN116621978A (en) | 2022-10-12 | 2022-10-12 | Antibody and kit for beta-CTX detection and application thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117434276A (en) * | 2023-12-12 | 2024-01-23 | 南京羿检医学科技有限公司 | THSD7A antibody detection kit and application thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117434276A (en) * | 2023-12-12 | 2024-01-23 | 南京羿检医学科技有限公司 | THSD7A antibody detection kit and application thereof |
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