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Hybrid tumor cell strain 7D2 of human cardiac troponin I as well as monoclonal antibody and application thereof

A hybridoma cell line, cardiac troponin technology, applied in the field of cellular immunity, can solve problems such as deficiency

Active Publication Date: 2018-01-09
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In the study, it was found that cTnI was initially released into the blood in the form of a monomer, but as the degree of myocardial injury increased, in order to prevent cTnI from being degraded, cTnI would exist in the form of a complex, and then due to the influence of various factors, the complex It is decomposed into monomers again, so the antibody used to detect cTnI should be able to recognize various complex forms of cTnI in the blood with the same efficiency, and there is a serious lack of such monoclonal antibody resources in the prior art

Method used

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  • Hybrid tumor cell strain 7D2 of human cardiac troponin I as well as monoclonal antibody and application thereof
  • Hybrid tumor cell strain 7D2 of human cardiac troponin I as well as monoclonal antibody and application thereof
  • Hybrid tumor cell strain 7D2 of human cardiac troponin I as well as monoclonal antibody and application thereof

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Experimental program
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Embodiment 1

[0024] The preparation of hybridoma cell line and the monoclonal antibody secreted thereof comprises the following steps:

[0025] 1. Antigen

[0026] Monoclonal antibody 7D2 is a monoclonal antibody prepared by immunizing Balb / c mice with a complete antigen coupled with amino acids 13-24 on cTnI and BSA. The preparation method of the complete antigen containing amino acids 13-24 on cTnI is as follows: : Using DiscoveryStudio4.0 software to simulate the cTnI-T-C structure, analyze the epitope on the cTnI subunit, and select the 13th-24th amino acid as the epitope antigen. The 13-24 amino acids on cTnI were synthesized and coupled with BSA to form a complete antigen. This step was synthesized by Hangzhou Zhongpei Company.

[0027] The monoclonal antibody 7H4 antibody is a monoclonal antibody prepared by immunizing Balb / c mice with the whole protein complex cTnI-T-C, wherein cTnI-T-C is purchased from Hytest Company of Finland.

[0028] 2. Preparation of hybridoma cell lines: ...

Embodiment 2

[0058] Using the monoclonal antibody 7D2 as the capture antibody and 7H4 as the detection antibody to establish a double antibody sandwich ELISA detection method, it can be used to develop a rapid detection kit for myocardial infarction. The establishment process of the double antibody sandwich ELISA method is as follows:

[0059] 1. Selection of paired antibodies

[0060] (1) Coating: Dilute antibody 7D2 to 2 μg / mL with coating solution, add 100 μL to 96-well enzyme-linked plate. overnight at 4°C. Wash three times with PBS-T buffer, 3min each time.

[0061] (2) Blocking: add 5% skimmed milk powder to the enzyme-linked plate, add 200 μL to each well, and block for one hour at 37°C. Wash three times with PBS-T buffer, 3min each time.

[0062] (3) Add antigen cTnI: Dilute the antigen to 1000ng / mL, 500ng / mL, 250ng / mL, 125ng / mL, 62.ng / mL, 31.25ng / mL, 15.625ng / mL with PBS buffer. Add 100 μL to each well. Incubate at 37°C for one hour. Wash three times with PBS-T buffer, 3min...

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Abstract

The invention discloses a hybrid tumor cell strain 7D2 of human cardiac troponin I as well as a monoclonal antibody and application thereof. The hybrid tumor cell strain 7D2 is preserved in CCTCC (China Center For Type Culture Collection) on August 23th, 2017, and the preservation number is CCTCC NO C2017120. The monoclonal antibody 7D2 secreted by the hybrid tumor cell strain 7D2 is a cTnI rat derived monoclonal antibody with high affinity and high specificity; the cTnI in three forms of free cTnI, dipolymer compounds cTnI-C and tripolymer compounds cTnI-T-C can be combined, so that the monoclonal antibody can be used for detecting the cTnI content in serum; the foundation is laid for the development of a kit for fast detecting myocardial infarction.

Description

technical field [0001] The invention relates to the technical field of cellular immunity, more specifically, to the hybridoma cell line 7D2 of human cardiac troponin I, monoclonal antibody and application. Background technique [0002] Cardiac troponin is a key protein that regulates heart contraction. Trimeric complex composed of three subunits of cardiac troponin I (cTnI), cardiac troponin C (cTnC) and cardiac troponin T (cTnT). cTnI is the inhibitory subunit of actin, which regulates the interaction between actin and myosin by inhibiting the activity of adenosine triphosphate (ATPase) of actin. The molecular weight of cTnI is about 24kDa, and it is composed of 209 amino acids. It is a protein rich in α-helix, and its theoretical isoelectric point is 9.87. There are three subtypes of TnI (Troponin I): There are fast skeletal muscle type and slow skeletal muscle type in skeletal muscle troponin I (sTnI), which have similar molecular weight (20KD), but the amino acid betwe...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/18G01N33/68G01N33/577C12R1/91
Inventor 王宏余娟李文丽
Owner JINAN UNIVERSITY
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