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Anti-human cardiac troponin I antibody and applications thereof

A technology for binding proteins and antigens, applied in the fields of biotechnology and medicine, can solve the problems of low activity and poor affinity, and achieve the effect of high affinity and strong activity of binding proteins

Active Publication Date: 2020-04-17
DONGGUAN PENGZHI BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Due to low activity and poor affinity, the existing cTnI antibodies cannot be well applied to the detection of cTnI protein. Therefore, there is a strong demand in this field for antibodies that can effectively and specifically bind to and detect cTnI

Method used

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  • Anti-human cardiac troponin I antibody and applications thereof
  • Anti-human cardiac troponin I antibody and applications thereof
  • Anti-human cardiac troponin I antibody and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0135] In this example, the restriction endonuclease and Prime Star DNA polymerase were purchased from Takara Company. MagExtractor-RNA extraction kit was purchased from TOYOBO Company. SMARTERTM RACE cDNA Amplification Kit was purchased from Takara Company. The pMD-18T vector was purchased from Takara Company. Plasmid extraction kit was purchased from Tiangen Company. Primer synthesis and gene sequencing were performed by Invitrogen. The hybridoma cell line secreting Anti-cTnI 6E3 monoclonal antibody is the existing hybridoma cell line of the applicant, and it is recovered for use.

[0136] 1. Primers

[0137] Amplify Heavy Chain and Light Chain 5'RACE Primers:

[0138] SMARTER II A Oligonucleotide:

[0139] 5'-AAGCAGTGGTATCAACGCAGAGTACXXXXX-3';

[0140] 5'-RACE CDS Primer (5'-CDS): 5'-(T) 25 VN-3'(N=A,C,G,orT; V=A,G,orC);

[0141] Universal Primer A Mix (UPM): 5'-CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT-3';

[0142] Nested Universal Primer A (NUP): 5'-AAGCAGTG...

Embodiment 2

[0170] Although the antibody of sample 1 obtained in Example 1 (with sequences such as light chain and heavy chain shown in SEQ ID NO: 11 and 12) has the ability to bind cTnI protein, the affinity and antibody activity are not ideal, so the applicant passed The antibody's light chain CDRs and heavy chain CDRs were mutated.

[0171] After analysis, the complementarity determining region (WT) of the heavy chain:

[0172] CDR-VH1 is G-F(X1)-I-F-S(X2)-G-Q(X3)-T-M-N;

[0173] CDR-VH2 is L-L(X1)-N-P-S-D-N(X2)-T-T-Y-N-N(X3)-K-F;

[0174] CDR-VH3 is S-S(X1)-F(X2)-G-S-W-A(X3)-Q;

[0175] Complementarity-determining regions of the light chain:

[0176] CDR-VL1 is A-S-N(X1)-S-I(X2)-D-Y-E(X3)-G-D-S-Y;

[0177] CDR-VL2 is V-A-S-L(X1)-V(X2)-D-S;

[0178] CDR-VL3 is Q-Q-T(X1)-Y-E-G(X2)-P-W(X3)-T;

[0179] Among them, X1, X2, and X3 are mutation sites.

[0180] Table 1 Mutation sites related to antibody activity

[0181]

[0182] To detect the antibody activity after the mutation, ...

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Abstract

The invention relates to a novel isolated binding protein containing a cTnI antigen binding domain, and researches of the preparation, the applications and the like of the binding protein. The bindingprotein is high in activity, has high affinity with human cTnI protein, and can be widely applied to the field of cTnI protein detection.

Description

technical field [0001] The invention relates to the fields of biotechnology and medical technology, in particular to an anti-human cardiac troponin I antibody and its application. Background technique [0002] Before the 1980s, the World Health Organization (WHO) has always regarded myocardial enzyme activity as one of the diagnostic criteria for acute myocardial infarction (AMI). In the late 1980s, researchers discovered that the sensitivity and specificity of troponin (Tn) was higher than that of phosphocreatine kinase (CK), phosphocreatine kinase isoenzyme (CK-MB), lactate dehydrogenation enzymes and biomarkers such as aspartate aminotransferase. Cardiac troponin (cTnI) exists only in the myocardium and is a marker of cardiomyocytes. Its abnormal changes can affect the systolic function of the heart, and can be used to diagnose myocardial necrosis and judge myocardial injury. It has become a sensitive and specific marker of myocardial cell injury. It is recognized as th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18C12N15/13C12N15/85G01N33/68
CPCC07K16/18C12N15/85G01N33/6893C07K2317/565C07K2317/569C07K2317/55C07K2317/622C07K2317/31G01N2800/324G01N2800/12G01N2800/325
Inventor 崔鹏何志强孟媛钟冬梅游辉范凌云
Owner DONGGUAN PENGZHI BIOTECH CO LTD
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