阅读说明：本技术 一种特异性识别内源标记物Brdu的单克隆抗体的筛选方法 (Screening method of monoclonal antibody for specifically recognizing endogenous marker Brdu ) 是由 郑沣 孙小函 侯圆圆 刘姗姗 杨颂 黄强 张高英 于 2019-09-16 设计创作，主要内容包括：本发明公开了一种特异性识别内源标记物Brdu的单克隆抗体的筛选方法,包括如下步骤：制作Brdu内源标记的动物模型,以及制作Brdu内源标记的细胞模型,用内源标记的动物模型及细胞模型对单抗制备中的ELISA阳性融合孔进行筛选,融合后去掉不能识别内源性Brdu标记的ELISA阳性孔,保留能识别内源标记的ELISA阳性融合孔,进行亚克隆操作,分离单克隆,对单克隆孔同法进行内源Brdu标记筛选,获得特异性单克隆细胞株。(The invention discloses a screening method of a monoclonal antibody for specifically recognizing an endogenous marker Brdu, which comprises the following steps: the method comprises the steps of manufacturing an animal model of Brdu endogenous marker and a cell model of Brdu endogenous marker, screening ELISA positive fusion holes in monoclonal antibody preparation by using the animal model and the cell model of endogenous marker, removing the ELISA positive holes incapable of recognizing endogenous Brdu marker after fusion, reserving the ELISA positive fusion holes capable of recognizing endogenous marker, performing subcloning operation, separating monoclonal, performing endogenous Brdu marker screening on the monoclonal holes by the same method, and obtaining specific monoclonal cell strains.)

1. A screening method of a monoclonal antibody specifically recognizing an endogenous marker Brdu is characterized by comprising the following steps:

s1: making an animal model of Brdu endogenous marker;

s2: preparing a cell slide, namely culturing the cell slide in an incubator for 40min by using a culture medium containing 100 mu M/L Brdu to prepare a Brdu endogenesis labeled cell model;

s3: screening fusion holes in monoclonal antibody preparation by using an endogenous labeled animal model and a cell model;

s4: removing ELISA positive holes which can not recognize endogenous Brdu markers, and reserving ELISA positive fusion holes which can recognize endogenous markers;

s5: performing subcloning operation on ELISA positive fusion holes capable of recognizing endogenous markers, separating monoclone, performing endogenous Brdu marker screening on the monoclone holes by the same method, and obtaining specific monoclonal cell strains.

2. The method for screening monoclonal antibodies specifically recognizing the endogenous marker Brdu as claimed in claim 1, wherein in S1, the animal model preparation step of Brdu endogenous marker is:

1) injecting Brdu 50mg/kg into the abdominal cavity of the rat for 4 times, wherein the interval is 2 hours each time;

2) after the last 24 hours, the animals were sacrificed and tissues were taken;

3) fixing, embedding, slicing and processing to manufacture an animal model with endogenesis screening.

3. The method for screening monoclonal antibodies specifically recognizing the endogenous marker Brdu as claimed in claim 1, wherein in S2, the Brdu cell model preparation step:

(1) cleaning the cover glass with a detergent, then filling the detergent with tap water, washing with pure water for 3-6 times, soaking in 75% alcohol, and standing for 10-15 min;

(2) clamping a cover glass with a pair of tweezers, and burning the alcohol on an alcohol lamp to dry, wherein the drying temperature is 50-60 ℃;

(3) placing the dried cover glass in a six-hole plate, cooling, and dropwise adding the cell suspension on the cover glass;

(4) after 5 hours, slightly supplementing 1ml of culture medium, placing the culture medium in a 5% CO2 incubator at 37 ℃ for 12 hours, and adhering cells on a cover glass for good growth;

(5) adding 1ml of culture medium containing 100 mu M Brdu into each hole in a dark place, and incubating for 40min in an incubator;

(6) and (3) changing the solution, fixing for 15-20min by 4% paraformaldehyde, changing the PBS buffer solution, and storing at 4 ℃ for later use.

4. The method as claimed in claim 1, wherein in step S3, the fusion well screening process is performed by ELISA, and then the preliminarily screened ELISA positive fusion wells are directly screened by IHC and ICC using the prepared tissue slices of the animal model and cell slide of the cell model with the endogenous marker Brdu, so as to obtain ELISA positive fusion wells capable of identifying the endogenous marker Brdu, and the non-identifiable ELISA positive wells are removed.

5. The method for screening monoclonal antibodies specifically recognizing Brdu as an endogenous marker according to claim 1, wherein fusion wells and monoclonal wells used for monoclonal antibody production are directly subjected to specific endogenous screening in S3, S4 and S5.

Technical Field

The invention relates to the technical field of bioscience, in particular to a screening method of a monoclonal antibody for specifically recognizing an endogenous marker Brdu.

Background

Exogenous antigens are usually different from endogenous antigens, and usually, the exogenous antigens are used for preparing antibodies, so that the antibodies capable of recognizing the endogenous antigens are expected, but most antibodies cannot recognize the endogenous antigens; in the general monoclonal antibody preparation process, the first step of screening after fusion is indirect ELISA, and the method has weak specificity, only can indicate that the antibody and the exogenous antigen are combined with each other, but is difficult to specifically obtain the antibody for recognizing the endogenous antigen; fusion well supernatants are very small, usually less than 200. mu.l, making it difficult to perform other screens besides ELISA, which results in a heavy and blind work of subsequent subcloning procedures.

Disclosure of Invention

The invention provides a screening method of a monoclonal antibody for specifically recognizing an endogenous marker Brdu, which aims to solve the problems in the background technology.

The invention provides a screening method of a monoclonal antibody for specifically recognizing an endogenous marker Brdu, which comprises the following steps:

s1: making an animal model of Brdu endogenous marker;

s2: preparing a cell slide, namely culturing the cell slide in an incubator for 40min by using a culture medium containing 100 mu M/L Brdu to prepare a Brdu endogenesis labeled cell model;

s3: screening fusion holes in monoclonal antibody preparation by using an endogenous labeled animal model and a cell model;

s4: removing ELISA positive holes which can not recognize endogenous Brdu markers, and reserving ELISA positive fusion holes which can recognize endogenous markers;

s5: performing subcloning operation on ELISA positive fusion holes capable of recognizing endogenous markers, separating monoclone, performing endogenous Brdu marker screening on the monoclone holes by the same method, and obtaining specific monoclonal cell strains.

Preferably, in S1, the Brdu endogenously labeled animal model preparation step:

1) injecting Brdu 50mg/kg into the abdominal cavity of the rat for 4 times, wherein the interval is 2 hours each time;

2) after the last 24 hours, the animals were sacrificed and tissues were taken;

3) fixing, embedding, slicing and processing to manufacture an animal model with endogenesis screening.

Preferably, in S2, the Brdu endogenously labeled cell model preparation step:

(1) cleaning the cover glass with a detergent, then filling the detergent with tap water, washing with pure water for 3-6 times, soaking in 75% alcohol, and standing for 10-15 min;

(2) clamping a cover glass with a pair of tweezers, and burning the alcohol on an alcohol lamp to dry, wherein the drying temperature is 50-60 ℃;

(3) placing the dried cover glass in a six-hole plate, cooling, and dropwise adding the cell suspension on the cover glass;

(4) after 5 hours, slightly supplementing 1ml of culture medium, placing the culture medium in a 5% CO2 incubator at 37 ℃ for 12 hours, and adhering cells on a cover glass for good growth;

(5) adding 1ml of culture medium containing 100 mu M Brdu into each hole in a dark place, and incubating for 40min in an incubator;

(6) and (3) changing the solution, fixing for 15-20min by 4% paraformaldehyde, changing the PBS buffer solution, and storing at 4 ℃ for later use.

Preferably, in S3, the fusion pore screening process includes performing preliminary screening by ELISA, using the prepared animal model tissue slices labeled with the endogenous marker Brdu and the cell slide of the cell model, and performing IHC and ICC screening on the preliminarily screened ELISA positive fusion pore to obtain the ELISA positive fusion pore capable of recognizing the endogenous marker Brdu, and removing the unidentifiable ELISA positive pore.

Preferably, in the S3, S4 and S5, fusion wells and monoclonal wells in the preparation of the monoclonal antibody are directly subjected to specific endogenous screening.

The screening method of the monoclonal antibody for specifically recognizing the endogenous marker Brdu has the advantages that:

the invention directly carries out endogenous detection on the fusion supernatant, can carry out specificity screening on the antibody in the fusion hole supernatant at the fusion hole stage, can carry out micro-operation and batch operation, greatly saves the workload of subsequent subcloning operation, improves the screening pertinence, greatly increases the probability of obtaining the specific antibody, can obtain the fusion hole capable of specifically identifying the endogenous antigen at the fusion screening stage, further carries out subcloning operation on the fusion hole in a pertinence manner, simply and quickly obtains the specific monoclonal antibody, and the method for directly carrying out endogenous screening on the fusion hole supernatant is not only suitable for IHC, but also suitable for monoclonal antibody screening of various applications such as IF, WB, FC and the like.

Detailed Description

The invention is further illustrated by the following examples.