CN110790836A - Monoclonal antibody of human inhibin B, application and kit thereof - Google Patents
Monoclonal antibody of human inhibin B, application and kit thereof Download PDFInfo
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- CN110790836A CN110790836A CN201911154445.4A CN201911154445A CN110790836A CN 110790836 A CN110790836 A CN 110790836A CN 201911154445 A CN201911154445 A CN 201911154445A CN 110790836 A CN110790836 A CN 110790836A
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- inhibin
- human inhibin
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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
- C07K16/26—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against hormones ; against hormone releasing or inhibiting factors
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- G—PHYSICS
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- 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
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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
- G01N33/68—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
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- G—PHYSICS
- G01—MEASURING; TESTING
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/575—Hormones
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/36—Gynecology or obstetrics
- G01N2800/367—Infertility, e.g. sperm disorder, ovulatory dysfunction
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
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- Hematology (AREA)
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Abstract
The invention relates to the technical field of in-vitro diagnosis, and discloses a monoclonal antibody of human inhibin B, application thereof and a kit. The monoclonal antibody provided by the invention cuts off an Fc segment on the basis of the human inhibin B monoclonal antibody. The invention takes the monoclonal antibody of the human inhibin B without Fc segment as an enzyme-labeled antibody, detects the human inhibin B based on a double-antibody sandwich method, can achieve higher specificity, sensitivity and recovery rate, and can eliminate clinical abnormal samples; magnetic particles are used as carriers, and a chemiluminescence method is combined for detection, so that the reaction speed can be greatly improved, and the human INHB can be effectively detected within 30 min.
Description
Technical Field
The invention relates to the technical field of in-vitro diagnosis, in particular to a monoclonal antibody of human inhibin B, application and a kit thereof.
Background
Inhibin (INH) is a heterodimeric glycoprotein, in which the α -subunit and a β A-subunit form inhibin-A (INHA), and the β B-subunit form inhibin-B (INHB), which also exist in the blood as biologically inactive free subunits.
INHB is a testis-derived glycoprotein hormone, and the serum inhibin B level in adult males is significantly negatively correlated with FSH, and plays a negative feedback role on FSH. Inhibin B levels reflect the function of the entire testicular tissue and are a direct product of the seminiferous tract, and maintenance of detectable levels of inhibin B in adult male serum requires the presence of spermatogenic cells, and thus inhibin B is considered a serum marker of male spermatogenesis. Soon after birth, INHB levels gradually rise, reaching a peak (210 + -31) pg/ml at 4-12 months, and decline to a low value of (81 + -12) pg/ml at 3-9 years of age; after puberty has started, levels of INHB gradually increase again, reaching another peak (167 ± 20) pg/ml at age 20-30, after which INHB levels gradually decrease with age.
The serotonin B determination can be used for evaluating the spermatogenic function of male infertility patients, diagnosing cryptorchidism and precocious puberty of children, distinguishing obstructive and non-obstructive azoospermia patients, predicting the aspiration of testicular sperms of non-obstructive azoospermia patients, monitoring the damage of radiotherapy and chemotherapy to the spermatogenic function of male and the like. Inhibin B can more accurately reflect the spermatogenic function and the damage degree of testis than Follicle Stimulating Hormone (FSH); the combination of the two has higher diagnostic sensitivity and specificity than that of either single use.
Currently, enzyme-linked immunosorbent Assay (ELISA) and Fluorescence Immunochromatography (FICA) are commonly used for measuring the inhibin B index. ELISA relies on the detection technology of colorimetric method or polarized light method to suffer from too many interference factors, its sensitivity, linearity and stability are not high, unfavorable to use in clinical medical institution; fluorescence labeling of FICA is susceptible to environmental interference; meanwhile, false positive results are easily generated in the detection process, misdiagnosis is easily caused, and mental stress is caused to patients in hospitals.
Disclosure of Invention
In view of the above, the present invention aims to provide a monoclonal antibody against human inhibin B, which has high sensitivity, specificity and recovery rate when applied to chemiluminescence detection of human inhibin B, and can eliminate clinical abnormal samples; also provides the application of the monoclonal antibody;
it is another object of the present invention to provide a chemiluminescence assay-based kit containing the human inhibin B monoclonal antibody, and a detection method using the same, such as a magnetic particle chemiluminescence kit.
In order to achieve the above purpose, the invention provides the following technical scheme:
a monoclonal antibody of human inhibin B, which is characterized in that an Fc segment is cut off on the basis of the human inhibin B monoclonal antibody.
Preferably, the human inhibin B monoclonal antibody cleaves the Fc region as follows:
dialyzing the human inhibin B monoclonal antibody, then incubating with activated papain, then stopping the reaction with iodoacetamide, dialyzing again, purifying the dialyzate with ion exchange resin and eluting with NaCl, collecting the first peak, and obtaining the monoclonal antibody with the Fc segment cut off.
More preferably, the human inhibin B monoclonal antibody cleaves the Fc region as follows:
dialyzing the human inhibin B monoclonal antibody with phosphate buffer solution, then incubating with activated papain, then stopping reaction with iodoacetamide in a dark and ice bath, dialyzing again with Tris-HCl, purifying the dialyzate with DEAE ion exchange resin, eluting with NaCl, collecting the first peak, and obtaining the monoclonal antibody with the Fc segment cut off.
The activated papain is activated by phosphate buffer solution, EDTA and cysteine, and can be purified after being activated. The activation process specifically comprises weighing papain, preparing into solution with phosphate buffer solution identical to dialysis, adding EDTA (final concentration 2mmol/L) and cysteine (final concentration 10mmol/L), mixing, and activating papain at 37 deg.C for 40 min; the purification process can be that the enzyme solution passes through a Sephadex G-25 column, is eluted by the phosphate buffer solution containing 2mmol/L EDTA, and the protein peak is collected to obtain the purified activated papain.
The incubation process can be divided into two stages, the human inhibin B monoclonal antibody after dialysis treatment is mixed with activated papain, incubation is carried out at 37 ℃, then equal amount of enzyme solution is added, and incubation is continued; the method specifically comprises the following steps: mixing the dialyzed antibody and activated papain in a weight ratio of 3: 1, incubating at 37 ℃ for 9h, adding an equivalent amount of enzyme solution, and continuing to incubate for 9 h;
during the Fc fragment cutting process, the phosphate buffer solution is preferably 0.02mol/L phosphate buffer solution (pH7.5), the Tris-HCl is preferably 0.01mmol/L Tris-HCl, and the NaCl is preferably 0.1mol/L NaCl;
in a specific embodiment of the present invention, the human inhibin B monoclonal antibody cleaves the Fc region as follows:
dialyzing human inhibin B monoclonal antibody with 0.02mol/L phosphate buffer solution (pH7.5) for 6 h;
weighing papain, preparing 25mg/mL solution with the same buffer solution, adding EDTA (final concentration 2mmol/L) and cysteine (final concentration 10mmol/L), mixing, and activating papain at 37 deg.C for 40 min;
passing the enzyme solution through a Sephadex G-25 column, eluting with the buffer solution containing 2mmol/L EDTA, and collecting protein peak to obtain purified activated papain;
mixing the dialyzed human inhibin B monoclonal antibody and the activated papain in a weight ratio of 3: 1, incubating at 37 ℃ for 9h, adding an equivalent amount of enzyme solution, and continuing to incubate for 9 h;
adding iodoacetamide with the final concentration of 20-30 mmol/L, and carrying out ice bath for 1h in a dark place to terminate the reaction;
dialyzing the papain reaction product with 0.01mmol/L Tris-HCl for 16 h; and purifying the dialysate by using a DEAE ion exchange resin column, performing gradient elution by using 0.1mol/L NaCl solution, and collecting a first peak to obtain the purified monoclonal antibody with the Fc fragment cut off.
In a particular embodiment of the invention, the human inhibin B monoclonal antibody is selected as the monoclonal antibody against the inhibin B α subunit.
The monoclonal antibody for cutting off the Fc segment is used for detecting the human inhibin B, has higher specificity and recovery rate, the sensitivity can reach a high level of 1.6pg/ml, and clinical abnormal samples can be eliminated. Based on the content, the invention provides the application of the monoclonal antibody in detecting the content of the human inhibin B or in preparing a kit for detecting the human inhibin B.
According to the application, the invention provides a kit for detecting the heat inhibin B, which comprises an enzyme-labeled monoclonal antibody and a sample pretreatment solution; the sample pretreatment solution contained 0.4% carbamide peroxide in PBS buffer.
The sample pretreatment solution of the present invention functions to convert methionine in human inhibin B to methionine sulfoxide, so that the antigen in the sample can be bound to the Fc fragment-removed capture antibody, while it cannot be detected without adding the sample pretreatment solution. Therefore, the invention also provides application of the monoclonal antibody and the sample pretreatment solution in detecting the content of the human inhibin B or in preparing a kit for detecting the human inhibin B.
The kit provided by the invention adopts a double-antibody sandwich method, a calibrator of inhibin B or a sample to be detected is added into a carrier coated with a human inhibin B monoclonal antibody, and simultaneously, a proper amount of sample pretreatment solution is added for incubation and washing; then adding a certain amount of enzyme-labeled monoclonal antibody, and incubating; and (3) binding the inhibin B in the calibrator or the sample to be detected with the inhibin B monoclonal antibody and the enzyme-labeled monoclonal antibody on the carrier to form a carrier-coated antibody-inhibin B-enzyme-labeled monoclonal antibody-enzyme compound, and washing to remove unbound substances. Adding a substrate, oscillating, forming an antibody-antigen-enzyme labeled monoclonal antibody compound through immune reaction, wherein the compound catalyzes a luminescent substrate solution to emit photons, and the luminescent intensity is in direct proportion to the content of INHB. Any detection means based on this principle, such as chemiluminescence detection means, may be employed.
Based on the principle, the kit also comprises one or more than two of the following components:
a carrier coated with a human inhibin B monoclonal antibody, an inhibin B calibrator, a washing solution and an enzyme chromogenic substrate; the enzyme chromogenic substrate corresponds to the enzyme-labeled monoclonal antibody one by one; the washing solution is a concentrated washing solution, and is phosphate buffer solution with pH7.5 and 2mol/L, wherein the phosphate buffer solution contains 2% Tween20 and 1% Procline 300.
In a specific embodiment of the present invention, the carrier coated with the human inhibin B monoclonal antibody is magnetic fine particles, preferably carboxylated magnetic beads, and is used in combination with a chemiluminescence method.
According to the kit provided by the invention, the invention also provides a method for detecting the content of the human inhibin B, and the kit provided by the invention is used for detecting by adopting a chemiluminescence method.
According to the technical scheme, the monoclonal antibody of the human inhibin B without the Fc segment is used as the enzyme-labeled antibody, the human inhibin B is detected based on the double-antibody sandwich method, the high specificity, sensitivity and recovery rate can be achieved, and clinical abnormal samples can be eliminated; magnetic particles are used as carriers, and a chemiluminescence method is combined for detection, so that the reaction speed can be greatly improved, and the human INHB can be effectively detected within 30 min.
Drawings
FIG. 1 shows the results of clinical correlations between assays using the methods of the invention and commercially available kits;
FIG. 2 shows the results of clinical correlation of a commercially available kit with the present invention (cleavage of Fc fragment);
FIG. 3 shows the results of clinical correlation of the commercial kit with a control (no Fc fragment cleaved);
FIG. 4 shows the results of clinical correlations between assays using the methods of the invention and commercially available kits.
Detailed Description
The invention discloses a monoclonal antibody of human inhibin B, application and a kit thereof, and a person skilled in the art can realize the monoclonal antibody by appropriately improving process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the monoclonal antibodies and uses and kits of the invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations and modifications, or appropriate alterations and combinations, of the monoclonal antibodies and uses and kits described herein may be made to practice and use the techniques of the invention without departing from the spirit and scope of the invention.
The enzyme used in the invention is one of horseradish peroxidase, alkaline phosphatase and acridine ester, and then luminol, isoluminol and strong base which are corresponding substrates are added to promote the luminescence of the enzyme.
The monoclonal antibody of human inhibin B, its application and kit are further described below.
Example 1: preparation of the kit of the invention
The kit comprises carboxylated magnetic beads coated with an anti-inhibin B β subunit monoclonal antibody, an enzyme-labeled anti-inhibin B α subunit monoclonal antibody with an Fc section removed, a sample pretreatment solution and an inhibin B calibrator.
1. Preparation of carboxylated magnetic beads against inhibin B β subunit antibody:
1.1 preparation of inhibin B immunogen:
extracting pig follicle fluid: sucking from pig ovarian follicle, and storing at-20 deg.C;
removing impurities: removing steroid hormone from the activated carbon; diluting with 0.2mol/L ammonium acetate, centrifuging, and concentrating with polyethylene glycol;
sephadex G-200 gel column; eluting with 0.2mol/L ammonium acetate, and collecting eluted protein;
sephadex G-100 gel column; eluting with 0.2mol/L acetic acid buffer solution, collecting eluted protein and evaluating.
Immunization: the present invention provides for repeated and multiple immunizations of mice by the protocol reported in Wong et al, 1993, immunization at the lymph node sites of mice, initial immunization with Freund's complete adjuvant followed by boosting with RIBI adjuvant. Finally expressed as inclusion bodies in E.coli, converted to native dimers and purified by reverse phase and ion exchange chromatography.
Monoclonal antibodies were prepared by fusing SP2/0 myeloma cells with lymph node B lymphocytes using PEG, primary antibody cloning and recloning of secretory cell lines in ClonaCell methylcellulose to give single clones, and purification of the antibodies by ProteinG affinity chromatography.
1.2 preparation of carboxylated magnetic beads against inhibin B β subunit antibodies
Washing the magnetic particles with coating buffer (50mmol/L, pH7.6 phosphate buffer) for 5 times, adding certain amount of EDC and NHS for activation, and shaking for 30 min; after washing, coating with a coating antibody diluted to 100-; washing the solid phase carrier, and then blocking with 0.02mol/LBis-Tris buffer containing 1% BSA; removing the blocking solution, adding a buffer solution containing 0.5% Tween-20, 1% BSA, 1% EDTA, 0.1% Procline300, 1% L7600, pH7.4, and 0.02mol/LBis-Tris, storing, and storing at 2-8 deg.C for later use;
2. preparation of enzyme-labeled modified anti-inhibin B α subunit monoclonal antibody
2.1 preparation of labeled antibodies for cleavage of FC fragments
2.1.1 dialyzing the anti-inhibin B β subunit antibody against 0.02mol/L phosphate buffer (pH7.5) for 6 h;
2.1.2 weighing papain, preparing a 25mg/mL solution by using the same buffer solution, adding EDTA (the final concentration is 2mmol/L) and cysteine (the final concentration is 10mmol/L), uniformly mixing, and activating the papain at 37 ℃ for 40 min;
2.1.3 passing the enzyme solution through a Sephadex G-25 column, eluting with the buffer solution containing 2mmol/L EDTA, and collecting protein peak to obtain activated papain;
2.1.4 mixing the dialyzed antibody and the activated papain according to the weight ratio of 3: 1, incubating for 9h at 37 ℃, then adding the same amount of enzyme solution, and continuing to incubate for 9 h;
2.1.5 adding iodoacetamide with the final concentration of 20-30 mmol/L, and carrying out ice bath for 1h in a dark place to terminate the reaction;
2.1.6 dialyzing the papain reaction product with 0.01mmol/L Tris-HCl for 16 h;
2.1.7 purifying the dialyzate by using a DEAE ion exchange resin column, carrying out gradient elution by using 0.1mol/L NaCl solution, and collecting a first peak to obtain the purified labeled antibody with the Fc fragment cut off.
2.2 preparation of enzyme-labeled modified anti-inhibin B α subunit monoclonal antibodies
2.2.1 weighing 5mg of HRP and dissolving in 1mL of distilled water;
2.2.2 adding 0.06mol/L NaIO into the supernatant4Aqueous solution (10ml double distilled water +128mg NaIO)4)0.5mL, mixing evenly, and placing for 30min at 4 ℃;
2.2.3 filling the solution into a dialysis bag, dialyzing the solution against 1mMPH4.4 sodium acetate buffer solution, and standing at 4 ℃ overnight;
2.2.4 adding 1mL of aqueous solution containing 5mg of the purified antibody in 2.1.7, mixing, placing into a dialysis bag, adding 0.05mol/L of carbonate buffer solution with pH of 9.5, slowly stirring, dialyzing for 6h, and allowing them to bind;
2.2.5 addition of NaBH40.2mL of solution (5mg/mL), mixing uniformly, and placing at 4 ℃ for 2 h;
2.2.6 slowly adding equal volume of saturated ammonium sulfate solution into the above solution, mixing, centrifuging at 4 deg.C for 30min, removing supernatant, re-dissolving the precipitate with a little 0.02mol/L PBS solution with pH of 7.4, placing into dialysis bag, dialyzing with the same liquid at 4 deg.C, and desalting overnight;
taking out and centrifuging for 2.2.7 days to remove insoluble substances to obtain enzyme-antibody conjugate, diluting with 0.02mol/L PBS (pH7.4PBS), subpackaging into 1 mL/bottle, and storing at low temperature.
3. Preparation of calibrator for inhibin B
3.1 construction of inhibin B prokaryotic expression vector
3.1.1 anti-inhibin B β subunit monoclonal antibody is prepared by coupling hapten artificial recombinant inhibin B α subunit peptide segment and carrier protein into immunogen, then carrying out in vivo immunoreaction, cell culture and purification, and hapten artificial recombinant inhibin B α subunit peptide segment is prepared by the following method:
3.1.2 designing an advantageous sequence according to inhibin B coding gene INHBB retrieved from GeneBank, and carrying out prokaryotic expression;
3.1.3 transfer the positive clones of the correct epitope into e.colibl21(DE3), culture in LB medium, shake (150rpm) at 37 ℃ until OD600 ═ 0.4-0.6;
3.2 expression and purification of recombinant human inhibin B
3.2.1 after the above culture is finished, adding inducer IPTG (to make the final concentration be 1mM) to induce expression for 6h at 30 ℃;
3.2.2 the thalli collected after centrifugation needs to be resuspended by 0.05mol of PB with pH7.4, crushed by ultrasonic disruption, and centrifuged to collect supernatant;
3.2.3 the collected supernatant is analyzed by SDS-PAGE gel, a target protein band with molecular weight is collected and purified, and a carrier contains His label, so that the carrier is purified by a nickel column to obtain the hapten artificial recombinant inhibin B α subunit peptide section.
3.3 inhibin B calibrator
Diluting the purified recombinant human inhibin B with HEPES-NaOH buffer solution containing 1% BSA, 50% calf serum, 50mmol/L and pH7.8 to obtain S0-S56 calibrators; the concentrations were 0, 25, 50, 150, 500, 1500pg/ml, respectively.
4. Preparation of concentrated washing solution
2mol/L phosphate buffer pH7.5 containing 2% Tween20 and 1% Procline 300.
5. Preparation of the substrate
Luminol and hydrogen peroxide.
6. Sample pretreatment liquid: the sample pretreatment solution was a PBS buffer solution (1LPBS formulation: 3.116g disodium hydrogen phosphate, 0.203g sodium dihydrogen phosphate, 8.78g sodium chloride) containing 0.4% urea peroxide.
Example 2: method for using kit of the invention
1. Reagent preparation
1.1 calibrant and magnetic microparticles: taking out the reagent and balancing to room temperature (20-25 ℃);
1.2 washing solution: 10mL of the concentrated wash solution and 990mL of purified water were mixed in a clean vessel and used as the working wash solution. Purified water is requested to be self-provided by users;
1.3 substrate: taken out and equilibrated to room temperature.
2. Test procedure
2.1 sequentially incubating 50 mul of inhibin B calibrator, a sample to be tested, 50 mul of sample pretreatment solution and 20 mul of magnetic particle antibody suspension for 15 min;
2.2 after the incubation is finished; washing with washing working solution for 5 times; then adding 100 mu L enzyme-labeled antibody;
2.3 the mixed solution is incubated for 15min under oscillation at 37 ℃;
2.4 after the incubation is finished, washing for 5 times by using a washing working solution;
2.5 Add 100. mu.L of substrate to each reaction site;
2.6 the solid phase carrier is shaken and mixed evenly for 18 seconds at room temperature, and then the detection is carried out, and the analysis result is carried out.
Example 3: clinical relevance of the invention to the market
3.1 materials: kangrun inhibin B (INHB) quantitative detection kit (enzyme linked immunosorbent assay), batch number: 20180726, respectively;
3.2 sample sources: physical examination sample 500 parts of Zhengzhou Antu bioengineering GmbH;
3.3 Experimental procedures: 500 physical examination samples are randomly selected, the health and the sample detection of the invention are carried out in parallel, and the clinical correlation analysis is carried out on the detection results of the invention and the manufacturers sold in the market. The results are shown in FIG. 1.
In the figure, Y (the invention) is 0.9608 (congo) +6.7785, R2 is 0.9887, and N is 500.
And (4) analyzing results: through the parallel examination of the invention and the Kangrun of 500 physical examination samples, the clinical relevance of the reagent and the Kangrun is evaluated, and the experimental result shows that the R2 value of the reagent and a commercial Kangrun inhibin B (INHB) quantitative detection kit (an enzyme-linked immunosorbent assay) reaches above 0.98, the reagent is highly relevant to a commercial product, and the detection result has high accuracy.
Example 4: performance testing of the kits of the invention
1. Minimum detectable amount: according to the sensitivity of the test kit of the experimental scheme recommended by CLSI EP-17A document, the lowest detected amount of inhibin B is not higher than 1.6pg/mL, and the functional sensitivity is 5 pg/mL.
2. Linearity: inhibin B is 1.6-1500 pg/ml, and the linear correlation coefficient r between the measured value and the theoretical value is more than 0.99.
3. The recovery rate is 85-115%.
4. Stability: the kit is placed at 37 ℃ for 10 days (the validity period is 18 months) from the date of finished product production; and within the residual effective period of the finished product, calculating the time of the 37 ℃ placement according to the effective period of the 37 ℃ placement for 10 days, namely 18 months, and detecting to obtain a result which meets the requirements specified by each project.
5. Repeatability: the repeatability of the test kit is detected according to the experimental scheme recommended by CLSI EP-05A file, the repeatability in analysis is not more than 10%, and the difference between batches is not more than 15%.
6. Specificity: the experimental protocol recommended by reference to CLSI EP-07A document detects the interference of the kit, and the interference rate is required to be within +/-10%.
① the following endogenous substances were tested at the indicated concentrations and no significant interference was found.
TABLE 1
② the following cross-over species were detected at the indicated concentrations and no significant interference was found.
TABLE 2
Example 5: comparison with monoclonal antibody without Fc fragment cleavage
Using a commercial inhibin B (INHB) quantitative detection kit (enzyme linked immunosorbent assay), batch No.: 20180726, detecting whether Fc is cut off or not, using non-cut Fc as a control kit (the control and the kit of the invention only differ whether the enzyme-labeled monoclonal antibody removes the Fc segment or not, and the monoclonal antibodies are from the same hybridoma cell, and are prepared by the method of the embodiment 1), detecting 65 physical examination samples in parallel by using antibodies of Kangrun and whether the FC segment is cut off or not, and carrying out clinical correlation analysis on the detection results. The results of the raw data are shown in Table 3, and the line graphs of clinical relevance are shown in FIGS. 2 and 3, respectively.
TABLE 3
In FIG. 2, the present invention excises FC and Compton comparisons: y (Fc excision) ═ 0.9829 (congo) +13.855, R2 ═ 0.9923, N ═ 65;
in fig. 3, the control and the compliance ratios are: y (control not cleaved Fc) 0.992 (congo) +15.807, R2 0.9772, N65;
and (4) analyzing results: through parallel examination on whether Fc is cut off and the recovery is carried out on 65 physical examination samples, the clinical relevance of the cut-off of Fc and the recovery is evaluated, and the experimental result shows that the R2 value of a cut-off Fc and commercial recovery inhibin B (INHB) quantitative detection kit (an enzyme-linked immunosorbent assay) reaches above 0.99 and is highly relevant to commercial products; the clinical relevance of the control and the commercial kit is obvious, the dispersion is large, and samples with large deviation (the samples with numbers 311, 313, 323, 348 and 357, the deviation is more than 20%) exist.
Example 6: effect of sample pretreatment solution
The samples tested by the commercial comfort kit are used as the standard to respectively examine whether the pretreatment solution is added, the related methods refer to the previous examples, the difference is only whether the sample pretreatment solution is added for treatment, and the clinical relevance of the comfort kit is examined, and the results are shown in Table 4 and FIG. 4.
TABLE 4
As can be seen from the above table and FIG. 4, no reactivity was observed without adding the pretreatment solution; the addition of the pretreatment solution solves this problem because the addition of the pretreatment solution to the sample converts methionine in human inhibin B to methionine sulfoxide, and the antigen in the sample binds to the Fc fragment removed capture antibody. And has good clinical relevance with health and lubrication, and R2 can reach 0.9946.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (9)
1. A human inhibin B monoclonal antibody, wherein an Fc region is cleaved from the human inhibin B monoclonal antibody.
2. The monoclonal antibody of claim 1, wherein the human inhibin B monoclonal antibody cleaves the Fc region as follows:
dialyzing the human inhibin B monoclonal antibody, then incubating with activated papain, then stopping the reaction with iodoacetamide, dialyzing again, purifying the dialyzate with ion exchange resin and eluting with NaCl, collecting the first peak, and obtaining the monoclonal antibody with the Fc segment cut off.
3. The monoclonal antibody of claim 2, wherein the human inhibin B monoclonal antibody cleaves the Fc region as follows:
dialyzing the human inhibin B monoclonal antibody with phosphate buffer solution, then incubating with activated papain, then stopping reaction with iodoacetamide in a dark and ice bath, dialyzing again with Tris-HCl, purifying the dialyzate with DEAE ion exchange resin, eluting with NaCl, collecting the first peak, and obtaining the monoclonal antibody with the Fc segment cut off.
4. The monoclonal antibody according to claim 2 or 3, wherein the activated papain is papain activated by phosphate buffer, EDTA, cysteine.
5. The monoclonal antibody of any one of claims 1-3, wherein the human inhibin B monoclonal antibody is directed against the inhibin B α subunit.
6. Use of the monoclonal antibody of any one of claims 1-5 in detecting human inhibin B content or in preparing a kit for detecting human inhibin B.
7. A kit for detecting thermostatin B, which comprises an enzyme-labeled monoclonal antibody of any one of claims 1 to 5 and a sample pretreatment solution; the sample pretreatment solution was PBS buffer containing 0.4% carbamide peroxide.
8. The kit according to claim 7, further comprising one or more than two of the following components:
a carrier coated with a human inhibin B monoclonal antibody, an inhibin B calibrator, a washing solution and an enzyme chromogenic substrate.
9. A method for detecting the content of human inhibin B, which comprises using the kit of claim 7 or 8 and carrying out the detection by chemiluminescence.
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| CN119470931A (en) * | 2025-01-07 | 2025-02-18 | 深圳市雅为泓源生物科技有限公司 | An improved inhibin B detection kit and monoclonal antibody preparation method |
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