阅读说明：本技术 一种人抑制素b的单克隆抗体及其应用和试剂盒 (Monoclonal antibody of human inhibin B, application and kit thereof ) 是由 汤久停 张跃峰 孙理智 李新远 刘功成 渠海 付光宇 于 2019-11-22 设计创作，主要内容包括：本发明涉及体外诊断技术领域,公开了一种人抑制素B的单克隆抗体及其应用和试剂盒。本发明所述单克隆抗体在人抑制素B单克隆抗体基础上切除Fc段。本发明以去除Fc段的人抑制素B单克隆抗体为酶标抗体,基于双抗体夹心法检测人抑制素B,可达到较高的特异性、灵敏度和回收率,同时可排除临床异样样本；以磁微粒作为载体,结合化学发光法检测,可极大的提高反应速度,在30min内有效检测人INHB。(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.)

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.

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.