CN112062847A

CN112062847A - INHB antibody and application thereof

Info

Publication number: CN112062847A
Application number: CN202010343886.5A
Authority: CN
Inventors: 李因来; 储光亮; 何燕燕
Original assignee: Hangzhou Biogenome Biotechnology Co ltd
Current assignee: Hangzhou Biogenome Biotechnology Co ltd
Priority date: 2020-04-27
Filing date: 2020-04-27
Publication date: 2020-12-11

Abstract

The invention discloses an INHB antibody which is of an IgG type and is prepared from monoclonal cell strains A-1 and B-2, wherein the cell strain A-1 is preserved in the China general microbiological culture Collection center with the preservation date of 2019, 1 month and 16 days and the preservation number of CGMCC No. 17090; the cell strain B-2 is preserved in the China general microbiological culture Collection center (CGMCC), the preservation date is 2019, 1 month and 16 days, and the preservation number is CGMCC No. 17091. The monoclonal antibody comprises A100 and B100, the antibody A100 is used as a coating antibody, the B100 is used as a labeled antibody, a DAS-ELISA method is established, and the INHB content in natural serum is detected. The invention has the beneficial effects that: the monoclonal antibody can be specifically combined with INHB, and has high recognition capability and efficiency. The monoclonal antibody can be effectively applied to medicine preparation, and the targeting property and specificity of the medicine are increased.

Description

INHB antibody and application thereof

Technical Field

The invention relates to the field of genetic engineering, and particularly relates to an INHB antibody and application thereof.

Background

Inhibin is a water-soluble protein secreted by the gonads, and consists of alpha and beta subunits, wherein the beta subunit is divided into beta A subunit and beta B subunit, and the alpha subunit and the beta subunit are connected through disulfide bonds. Inhibin has two forms, inhibin A and inhibin B, inhibin A is composed of alpha subunit and beta subunit, and inhibin B is composed of alpha subunit and beta subunit. The two β subunits (β a, β a β B, β B) constitute activin (activin), whose action is opposite to that of inhibin. Inhibin is mainly produced by ovarian granulosa cells and testicular trophoblasts (sertoli's cells), and plays an important role in the development of sperm, the maturation of follicles, and the development of embryos, thereby regulating reproductive and developmental processes. Statins, in addition to inhibiting follicle-stimulating hormone (FSH) secretion, also act as paracrine and autocrine agents in a variety of tissues. mRNA of inhibin alpha subunit exists in tissues and organs such as brain, pituitary, spleen and adrenal gland, and has a wide range of physiological activities.

Inhibin B (inhibin B, INH-B) is a testis-derived glycoprotein, and serum inhibin B level in adult male body is significantly negatively correlated with FSH, and plays a negative feedback role on FSH. Shortly after birth, serotonin B levels gradually rise and a negative correlation between inhibin B and FSH is maintained. At the age of 20-30, the level of inhibin B reaches a peak and the level of inhibin B decreases gradually with age.

The important position of inhibin in reproductive system and the activity universality of inhibin open a wide prospect for clinical application of inhibin. Due to abnormally elevated serum inhibin levels in patients with reproductive tumors, particularly granulomatous tumors, mucinous carcinomas, and trophoblastic tumors. Therefore, measurement of serum inhibin levels is a possible indicator of primary and recurrent granulocytic tumors and can be used for early diagnosis of reproductive tumors and for diagnosis of primary, recurrent and metastatic tumors. The monoclonal antibodies are used in combination, a monoclonal antibody combination mode with better specificity is preferably selected, and ELISA reagent boxes for detecting inhibin and subunits of the inhibin are respectively assembled, so that the kit can be used for diagnosing ovarian granulocytic tumors, mucus cancers and trophoblastic tumors.

At present, no research is carried out on the use of a single marker for predicting OR (ovarian reserve function) to obtain satisfactory sensitivity and specificity, and the increase of the detection threshold of the predictive marker can increase the specificity but reduce the sensitivity and vice versa, so that the combined detection of the markers can improve the prediction of OR. Research shows that serum AMH and INHB levels are in positive correlation, and the prediction of OR can be improved by jointly applying the serum AMH and the INHB levels in clinical work. The OR decline is a progressive process, and the early detection and diagnosis of the OR decline and the timely intervention can improve the reproductive potential of women and enhance the pregnancy ability and the pregnancy outcome. Serum basal AMH and INHB levels are closely related to OR, both of which are currently valuable predictors.

The current clinical detection methods of INHB comprise an enzyme-linked immunosorbent assay (ELISA), an electrochemiluminescence method and a chemiluminescence method, and the methods have respective advantages and disadvantages. The ELISA method has multiple detection steps, long time consumption and more influence factors in the operation process, and is easy to cause false positive and false negative results. Therefore, the method is gradually replaced by a chemiluminescence method at present, but the method is a totally closed system, is high in price, needs special training of instrument operators, is high in maintenance and detection cost, is not suitable for single-person and small-batch detection, and is not beneficial to wide development of INHB detection at home at present.

Chinese patent CN201510108832X discloses a kit suitable for rapidly detecting AMH and INHB by using a double-label time-resolved fluoroimmunoassay method and a using method thereof, and the INHB and the AMH are simultaneously detected by using the fluorescent label method, so that the detection steps are reduced, the sample adding time is shortened, the usage amount of a blood sample is reduced, but the detection sensitivity is not high, the actual detection time is long, the requirements of the fluoroimmunoassay on environment and operation are high, and the detection success rate of the kit is not particularly considerable.

Chinese patent CN201710568452.3 discloses an immunochromatography reagent strip for fluorescence quantitative detection of INHB and a preparation method thereof, and the immunochromatography method is adopted, and the detection method has the advantages of high sensitivity, simple operation and low cost. The inhibin B with the concentration as low as 10pg/mL in the blood sample can be detected, a detection instrument does not need professional operators, and the detection result can be obtained within 15 minutes. In the practical use process, the sensitivity is general, the detection result is only qualitative and cannot be quantitative, and the usability in clinic is basically 0.

Disclosure of Invention

In order to detect the content of INHB in natural serum, an INHB antibody and application thereof are provided. The invention is realized by the following technical scheme:

the invention provides an INHB antibody, wherein a monoclonal antibody is of an IgG type and is prepared from monoclonal cell strains A-1 and B-2, the cell strain A-1 is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, No. 3 of No.1 Xilu of the North Wenying district of the Chaoyang region in Beijing city, the preservation date is 2019, 1 month and 16 days, the preservation number is CGMCC No.17090, and the monoclonal antibody is classified and named as an INHB-resistant monoclonal antibody hybridoma cell; the cell strain B-2 is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, No. 3 of Xilu No.1 of Beijing, Chaoyang, the preservation date is 2019, 1 month and 16 days, the preservation number is CGMCC No.17091, and the cell strain is named as an INHB-resistant monoclonal antibody-secreting hybridoma by classification.

Preferably, the monoclonal antibody comprises A100 and B100, wherein A100 is prepared from A-1 and B100 is prepared from B-2. The invention uses solid phase polypeptide synthesis method to artificially synthesize 2 segments of inhibin alpha, beta B subunit. And screening out hybridoma cell strains aiming at alpha and beta B subunits, wherein the sequences of 2 subunits are as follows:

(1)INHα(1-29),29AA

S-T-P-L-M-S-W-P-W-S-P-S-A-L-R-L-L-Q-R-P-P-E-E-P-A-A-H-A-N

(2)INHβB(1-28),28AA

G-L-E-C-D-G-R-T-N-L-C-C-R-Q-Q-F-F-I-D-F-R-L-I-G-W-N-D-W

the invention also provides application of the INHB antibody, wherein an antibody A100 is used as a coating antibody, and an antibody B100 is used as a marker antibody, and a DAS-ELISA method is established to detect the INHB content in natural serum.

Preferably, the classification of a100 is IgG2 b.

Preferably, the B100 is classified as IgG 2B.

Compared with the prior art, the invention has the beneficial effects that:

(1) the monoclonal antibody can be specifically combined with INHB, and has high recognition capability and high efficiency.

(2) The monoclonal antibody can be effectively applied to medicine preparation, and increases the targeting property and specificity of the medicine.

Detailed Description

The present invention will be described in more detail with reference to examples. It is to be understood that the practice of the present invention is not limited to the following examples, and that various changes or modifications may be made without departing from the scope of the invention; and the methods in the following examples are conventional in the art unless otherwise specified.

Example 1:

first, obtaining hybridoma cell and preparing monoclonal antibody thereof

1. Animal immunization

The immunogen is a recombinant protein, purchased from Jinsbane technology (Nanjing) Inc., under the trade designations PE1934 and PE 1935. BALB/c female mice, 6 weeks old, weighing 18-20g, were immunized with the recombinant protein. That is, 100 mu g/one INHB recombinant protein is mixed with Freund's complete adjuvant, after full emulsification, 0.3 mL/one INHB is injected into dorsoventral subcutaneous multi-point injection, every 2 weeks later, 80 mu g/one antigen and Freund's incomplete adjuvant are taken to be fully emulsified, 0.3 mL/one INH is injected into abdominal cavity, after 4 th week of immunization, eyeball blood sampling is carried out on the mouse, serum titer is detected, titer is taken to reach 1: 10⁵And the above mice are performed with spleen strengthening immunity, when spleen is immunized, the amount of recombinant antigen is 30 mu g/mouse, and spleen cells are taken for cell fusion after 3 days.

2. Preparation of feeder layer cells

Taking BALB/c mouse abdominal cavity macrophages and thymocytes as feeder cells, and the specific operation method is as follows: BALB/c mice were bled and killed, the 75% alcohol body surface was sterilized, and then the abdominal skin was cut from the back abdomen with sterilized scissors and forceps, exposing the peritoneum. Taking 4ml of IMDM medium to the abdominal cavity by using an injector, slightly pressing the abdominal cavity by using fingers, repeatedly washing, and recovering a washing liquid. The thymus of the mouse was removed, crushed, washed with IMDM medium, and the wash solution was recovered. And mixing the two recovered solutions, centrifuging at 1200rpm for 3min, and removing the supernatant to obtain feeder layer cells.

3. Cell fusion

Taking immune mouse spleen cells and mouse myeloma cells (SP2/0) according to the ratio of 7: 1 in the bloodless stateMixing clear IMDM medium, centrifuging at 1300rpm for 3min, removing medium, adding 1mL 50% PEG (molecular weight 1500) in 37 deg.C water bath, fusing for 2min, terminating fusion with serum-free IMDM medium, centrifuging at 1300rpm for 3min, suspending precipitate with HAT medium, adding feeder cells, packaging into 96-well feeder cell plate, placing at 37 deg.C, and adding 5% CO₂The cell culture box.

4. Screening of hybridoma cells and cloning thereof

After culturing for 5 days in a cell culture box, when fused cells cover 10-30% of the bottom of a hole, screening positive holes secreting antibodies by using a conventional indirect ELISA method, namely using INHB recombinant protein as an antigen, diluting the protein with CB to 0.5 mu g/ml to coat a 96-hole enzyme label plate, coating the protein at 50 mu l/hole and at 4 ℃ overnight, after patting dry, blocking the protein with PBS buffer solution containing 1% BSA (200 mu l/hole), blocking the protein at 37 ℃ for 2 hours, and patting dry for later use; adding 50 μ l/well of cell culture supernatant to be detected into the ELISA plate, reacting at 37 deg.C for 30min, washing, drying, adding 50 μ l/well HRP-labeled goat anti-mouse IgG, incubating at 37 deg.C for 30min, washing, drying, adding 50 μ l/well TMB developing solution, developing at 37 deg.C in dark for 10min, adding 25 μ l/well 2M H₂SO₄The reaction was terminated and the value read at 0D 450. Determination of positive wells: OD450 value/negative control value is not less than 2.1. And selecting the cell wells with strong positive reaction, and cloning by a limiting dilution method. After cloning screening for three to four times, the monoclonal cell strain with 100 percent of positive rate is determined as a stable cell strain; after the enlarged culture, the culture medium is used for ascites preparation and liquid nitrogen preservation.

5. Preparation and purification of monoclonal antibody ascites

Taking F1 mouse of about 8 weeks old, injecting 0.3-0.5mL paraffin into abdominal cavity, injecting 8 × 10 into abdominal cavity 7-10 days later⁵The hybridoma cells can be injected for 7-10 days to show that the abdomen of the mouse is obviously enlarged, ascites is collected by an injection needle, the ascites is centrifuged for 3min at 8000rpm, and the supernatant is collected to be the monoclonal antibody ascites. Diluting 1 volume of ascites with 2 volumes of 0.06M acetic acid buffer solution with pH 4.8, adding octanoic acid (30 μ L/mL ascites) under stirring at room temperature, clarifying at 4 deg.C for 1h, centrifuging at 12000rpm for 20min, collecting supernatant, precipitating immunoglobulin with 50% saturated ammonium sulfateStanding at 4 deg.C for 2 hr, centrifuging at 3000rpm for 20min, dissolving the precipitate with 2 times volume of PBS solution, performing flow dialysis at 4 deg.C for 24 hr to obtain purified ascites antibody, and storing at-70 deg.C.

Second, screening preparation of paired antibodies

1. HRP labeling of antibodies

Dissolving 1mg HRP in 0.5ml distilled water, and adding 0.2ml NaIO₄The solution (0.06mol/L) is mixed evenly and placed under the condition of light shielding at 4 ℃ for 30 min. 0.2ml of ethylene glycol solution (0.16mol/L) was added thereto, and the mixture was stirred gently at room temperature (about 20 ℃ C.) for 30min in the absence of light. Adding 1mg antibody solution, mixing, placing into dialysis bag, and dialyzing in CBS solution under dark condition for 6 hr (or overnight at 4 deg.C). The reaction solution was removed from the dialysis bag and 50. mu.l of NaBH was added₄The solution (5mg/ml) was mixed well and placed at 4 ℃ in the dark for 2 h. Slowly adding equal volume (about 1.2ml) of saturated ammonium sulfate solution, mixing, placing at 4 deg.C in dark for 30min, centrifuging at 10000rpm for 10min to remove supernatant, re-dissolving the precipitate with 200 μ l PBS, and dialyzing in PBS for 12-18h, wherein the solution is changed once. Taking out the solution, measuring the volume, adding glycerol with the same volume, mixing well, and storing at-20 deg.C for use.

2. Screening for partner antibodies

The coated antibody was diluted to 4. mu.g/ml with CB, added to wells of an ELISA plate at 100. mu.l per well, and coated overnight at 4 ℃. The plate was blotted dry and 200. mu.l of blocking solution (1% BSA in PBS) was added to each well, blocked for 1-2h at 37 ℃ and blotted dry. Positive blood samples were diluted to 2ng/ml with PBS and loaded at 100. mu.l per well, with negative blood samples as controls. After incubation at 37 ℃ for 1h, the plates were washed 3 times in 0.05% PBST. The enzyme-labeled antibody was diluted to 1000-fold working solution with a 1% BSA-containing wash solution, 100. mu.l was added to each well, incubated at 37 ℃ for 1 hour, and then the plate was washed 4 times and patted dry. Mu.l of TMB developing solution was added to each well, and the mixture was incubated at 37 ℃ for 10 min. Add 50. mu.l of stop buffer to each well and read the OD on a microplate reader at 450 nm. The results are as follows: ELISA detection OD450 counts:

thirdly, performance evaluation and related parameters of paired antibody A100/B100

1. Specific detection of paired antibodies

The specificity of the A100/B100 pair was determined as described above, using 25 parts of positive serum and 25 parts of negative serum in total, with P/N ≥ 2 as positive, with the following results:

2. typing and identification of monoclonal antibodies

The antibody typing identification kit SBA cloning System-HRP (Southern Biotech, cat:5300-05) was used for identification according to the method described in the specification.

The specific method comprises the following steps:

coating: diluting the capture antibody to a concentration of 5-10. mu.g/mL with Borate Buffer (BBS) at pH8.2 or Phosphate Buffer (PBS) at pH7.4, adding 100. mu.L per well;

covering the enzyme label plate with a cover or a preservative film, and incubating for at least 12 hours in a humid environment at the temperature of 2-8 ℃;

sealing: spin-drying, washing with PBS or BBS containing 0.05% Tween for 3 times; spin-drying, adding PBS or BBS containing 1% BSA, 200 μ L per well;

standing the antibody-coated plate at room temperature for at least 1h to block free binding sites on the plate;

adding 100 mul hybridoma supernatant or antibody to be detected into each hole, covering, and incubating for 1h at room temperature by mild oscillation or overnight at 2-8 ℃;

sixthly, washing for 3 times by using a washing liquid;

seventhly, diluting HRP-labeled detection antibody 1:250-1:500 with BBS/BSA, adding 100 μ L of conjugate into a plate, covering the plate, and shaking at room temperature for 1h or overnight at 2-8 ℃;

washing 5 times with the lotion;

ninthly, preparing ABST substrate stock solution, namely dissolving 15mgABST powder in 1mL double distilled water, storing at 2-8 ℃ in a dark place, and stabilizing within 4 weeks;

preparing substrate solution for R, 525mg citric acid is added into 50mL double distilled water and stirred until complete dissolution; adjusting the pH value to 4.0 by NaOH; to 10mL of citrate buffer was added 0.2mL of stock solution of LABST and 10. mu.L of 30% H₂O₂；

Add 100. mu.L substrate buffer per well;

the plate reader was used to read at 405 nm.

The results are given in the table below.

ELISA detection OD405 counts:

	A100	B100
			Ig-κ	2.209	2.013
Ig-λ	0.051	0.049
			IgM	0.062	0.062
IgA	0.055	0.055
			IgG1	0.059	0.06
IgG2a	1.836	0.282
			IgG2b	3.5	3.5
IgG3	0.05	0.054

and (3) typing identification results:

Claims

1. the INHB antibody is characterized in that the monoclonal antibody is of an IgG type and is prepared from monoclonal cell strains A-1 and B-2, wherein the cell strain A-1 is preserved in the China general microbiological culture Collection center (CGMCC), the preservation date is 2019, 1 month and 16 days, and the preservation number is CGMCC No. 17090; the cell strain B-2 is preserved in the China general microbiological culture Collection center (CGMCC), the preservation date is 2019, 1 month and 16 days, and the preservation number is CGMCC No. 17091.

2. The INHB antibody of claim 1, wherein said monoclonal antibody comprises a100 and B100, wherein a100 is produced from a-1 and B100 is produced from B-2.

3. The application of the INHB antibody is characterized in that the monoclonal antibody utilizes an antibody A100 as a coating antibody and B100 as a marker antibody to establish a DAS-ELISA method and detect the INHB content in natural serum.

4. The use of an INHB antibody according to claim 3, wherein the typing of a100 is IgG2 b.

5. The use of an INHB antibody according to claim 2, wherein B100 is typed as IgG 2B.