阅读说明：本技术 针对破伤风类毒素的检测抗体对及其应用 (Detection antibody pair aiming at tetanus toxoid and application thereof ) 是由 胡业勤 陈雯 雷念潮 杨晓明 段凯 李新国 李茜 于 2019-11-26 设计创作，主要内容包括：本发明属于生物技术领域,具体的,涉及针对破伤风类毒素的检测抗体对及其应用,所述的单克隆抗体,所述的抗体为抗体2B12和1B23,2B12重链可变区(VH)氨基酸序列如SEQ ID NO.1所示,轻链可变区(VL)氨基酸序列如SEQ ID NO.2所示；1B23的重链可变区(VH)氨基酸序列如SEQ ID NO.3所示、轻链可变区(VL)氨基酸序列如SEQ ID NO.4所示。本发明还涉及编码所述单克隆抗体2B12、1B23的核酸序列以及由其制备的检测破伤风类毒素的试剂盒。(The invention belongs to the technical field of biology, and particularly relates to a detection antibody pair aiming at tetanus toxoid and application thereof, wherein the monoclonal antibody is an antibody 2B12 and 1B23, the amino acid sequence of a heavy chain variable region (VH) of 2B12 is shown as SEQ ID No.1, and the amino acid sequence of a light chain variable region (VL) is shown as SEQ ID No. 2; the heavy chain variable region (VH) amino acid sequence of 1B23 is shown in SEQ ID NO.3, and the light chain variable region (VL) amino acid sequence is shown in SEQ ID NO. 4. The invention also relates to nucleic acid sequences encoding the monoclonal antibodies 2B12 and 1B23 and a kit for detecting tetanus toxoid by the nucleic acid sequences.)

1. A panel of monoclonal antibodies, used alone or in combination, for the detection of Tetanus Toxoid (TT), said antibodies being:

(1) the heavy chain variable region (VH) amino acid sequence of the monoclonal antibody 1B12 is shown in SEQ ID NO.1, and the light chain variable region (VL) amino acid sequence is shown in SEQ ID NO. 2;

(2) the heavy chain variable region (VH) amino acid sequence of the monoclonal antibody 1B23 is shown in SEQ ID NO.3, and the light chain variable region (VL) amino acid sequence is shown in SEQ ID NO. 4.

2. The monoclonal antibody of claim 1, wherein the constant region (Fc) region of antibodies 2B12 and 1B23 is murine Fc.

3. The monoclonal antibody of claim 1 or 2, wherein the antibodies 2B12 and 1B23 are murine antibodies.

4. A nucleic acid fragment encoding the monoclonal antibody of any one of claims 1-3.

5. Use of a monoclonal antibody according to any one of claims 1 to 3 in the manufacture of a test reagent for the detection of tetanus toxoid.

6. A test kit for the detection of tetanus toxoid, the kit comprising:

(1) detecting an effective amount of 1B12 antibody and/or 1B23 antibody;

(2) necessary chromogenic or quantitative reagents.

7. The kit of claim 6, wherein the kit is an enzyme-linked immunosorbent assay (ELISA) kit.

8. The kit of claim 7, wherein the kit is characterized in that

(1) monoclonal antibody 1B12 was used as a coating or capture antibody;

(2) 1B23 antibody labeled with a quantitative tag was used as the detection antibody or secondary antibody.

9. The kit of claim 8, wherein the quantitative label is preferably horseradish peroxidase or biotin.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a detection antibody pair for tetanus toxoid and application thereof.

Background

Tetanus is caused by infection with Clostridium tetani. Clostridium tetani infects wounds, and under anoxic conditions, it produces exotoxins that act on the central nervous system of humans. In 1974, the world health conference officially proposed an expanded immunization program to prevent seven diseases including tetanus, and then China effectively pursued the vaccination of tetanus vaccine. Tetanus toxoid is chemically treated to lose its toxicity and maintain its antigenicity, induce the body to produce immune response and react specifically with tetanus toxoid. Therefore, the quality of the toxoid should be evaluated based on its immunological activity. Generally, the immunogenicity is determined by animal experiments, and the antigenicity is determined by a flocculent unit method. The determination of the flocculent unit is a method for conventionally determining the binding force of the toxoid and the corresponding antitoxin, and the result is judged by naked eyes with certain errors. It is therefore desirable to monitor the quality of tetanus toxoid during production in a simple, rapid, sensitive, low-impact method. Enzyme-linked immunosorbent assay (ELISA) has the advantages of sensitivity, rapidness and strong tolerance, and can be used for quality control of the toxoid in the production process of tetanus toxoid.

Disclosure of Invention

The invention firstly relates to a group of monoclonal antibodies for detecting Tetanus Toxoid (TT) individually and/or jointly, wherein the antibodies are as follows:

(1) the monoclonal antibody 1B12 which is a heavy chain variable domain antibody,

the amino acid sequence of the heavy chain variable region (VH) is shown as SEQ ID NO.1, SEQ ID NO. 1:

the light chain variable region (VL) amino acid sequence is shown in SEQ ID NO.2, SEQ ID NO. 2:

(2) the monoclonal antibody 1B23 which is a heavy chain variable domain antibody,

the amino acid sequence of the heavy chain variable region (VH) is shown as SEQ ID NO.3, SEQ ID NO. 3:

the light chain variable region (VL) amino acid sequence is shown in SEQ ID NO.4, SEQ ID NO. 4:

the invention also relates to nucleic acid sequences encoding the monoclonal antibodies 1B12, 1B 23.

The constant region (Fc) region of the 1B12 and 1B23 monoclonal antibodies is murine Fc.

The monoclonal antibodies 1B12 and 1B23 are all murine antibodies.

The invention also relates to application of the monoclonal antibodies 1B12 and 1B23 in preparation of a detection reagent for tetanus toxoid.

The invention also relates to a detection kit for detecting tetanus toxoid, comprising:

(1) detecting an effective amount of 1B12 antibody and/or 1B23 antibody;

(2) necessary chromogenic or quantitative reagents.

Preferably, the kit is an enzyme-linked immunosorbent assay (ELISA) kit.

More preferably, the

The enzyme linked immunosorbent assay kit is a double-antibody sandwich enzyme linked immunosorbent assay kit, wherein,

(1) monoclonal antibody 1B12 was used as a coating antibody or a capture antibody;

(2) using 1B23 antibody labeled with quantitative label as detection antibody or secondary antibody;

the quantitative label is preferably horseradish peroxidase or biotin.

Drawings

FIG. 1, linear range in the detection of the double-antibody ELISA.

Detailed Description

The main reagents and sources used by the invention are as follows:

the amino acid sequences of the light chain and the heavy chain of the anti-TT-1B12 monoclonal antibody are respectively shown as SEQ ID NO.1 and SEQ ID NO.2, and the amino acid sequences of the light chain and the heavy chain of the anti-TT-1B23 monoclonal antibody are respectively shown as SEQ ID NO.3 and SEQ ID NO. 4;

the 96-well enzyme label plate is purchased from Xiamen Yunpeng science and technology Limited; tetanus toxoid standards were purchased from NIBSC (No. 04/150);

other reagents are all domestic analytical purifications unless specified otherwise.