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Double-resistant sandwich ELISA (enzyme linked immunosorbent assay) detection kit of human cardiac troponin I

A technique for cardiac troponin and detection kit, applied in the field of immune detection, can solve problems such as lack, and achieve the effects of low cost, good specificity and high uniformity

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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  • Double-resistant sandwich ELISA (enzyme linked immunosorbent assay) detection kit of human cardiac troponin I
  • Double-resistant sandwich ELISA (enzyme linked immunosorbent assay) detection kit of human cardiac troponin I
  • Double-resistant sandwich ELISA (enzyme linked immunosorbent assay) detection kit of human cardiac troponin I

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Embodiment 1

[0030] The preparation of hybridoma cell line and the monoclonal antibody secreted thereof comprises the following steps:

[0031] 1. Antigen

[0032] Monoclonal antibody 7D2 is a monoclonal antibody prepared by immunizing Balb / c mice with antigenic peptides coupled to BSA at positions 13-24 of cTnI. The antigenic peptides are prepared by simulating cTnI with Discovery Studio4.0 software -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.

[0033] 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.

[0034] 2. Preparation of hybridoma cell lines: The preparation methods of hybridoma cell line 7D2 and hybridoma cell l...

Embodiment 2

[0064] Using the monoclonal antibody 7D2 as the capture antibody and 7H4 as the detection antibody, a double-antibody sandwich ELISA detection method was established, and a rapid detection kit for myocardial infarction was developed. The establishment process of the double antibody sandwich ELISA method is as follows:

[0065] 1. Selection of paired antibodies

[0066] (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.

[0067] (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.

[0068] (3) Adding antigen: Dilute the antigen (cTnI) to 1000ng / mL, 500ng / mL, 250ng / mL, 125ng / mL, 62.5ng / mL, 31.25ng / mL, 15.625ng / mL respectively with PBS buffer. Add 100 μL to each well. Incubate at 37°C for one hour. Wash three times with PBS...

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Abstract

The invention discloses a double-resistant sandwich ELISA (enzyme linked immunosorbent assay) detection kit of human cardiac troponin I. A monoclonal antibody 7D2 is used as a capturing antibody; a monoclonal antibody 7H4 is used as a detection antibody, wherein a hybrid tumor cell strain 7D2 secreting the monoclonal antibody 7D2 is preserved in CCTCC (China Center For Type Culture Collection) onAugust 23th, 2017, and the preservation number is CCTCC NO: C2017120. A hybrid tumor cell strain 7H4 secreting the monoclonal antibody 7H4 is preserved in CCTCC on August 23th, 2017, and the preservation number is CCTCC NO: C2017121. The detection kit has the advantages that the sensitivity is high; the cost is low; a fast and efficient analysis means is provided for the detection of the human cardiac troponin I.

Description

technical field [0001] The invention relates to the field of immunoassay, more specifically, to an ELISA detection kit for human cardiac troponin I. 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 between the two There is about 40% difference in the sequence...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/577G01N33/535
Inventor 王宏余娟李文丽
Owner JINAN UNIVERSITY
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