Anti-human-CKMB (Hybrid Creatine Kinase Isoenzymes) antibody and application thereof

An antigen, CDR-VH2 technology, applied in the field of biotechnology and medicine, can solve problems such as poor specificity, and achieve the effect of high affinity and strong binding protein activity

Active Publication Date: 2020-06-30
DONGGUAN PENGZHI BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, immunosuppressive method is mostly used to measure CKMB in clinical laboratories. This method is simple and rapid, but has poor specificity.

Method used

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  • Anti-human-CKMB (Hybrid Creatine Kinase Isoenzymes) antibody and application thereof
  • Anti-human-CKMB (Hybrid Creatine Kinase Isoenzymes) antibody and application thereof
  • Anti-human-CKMB (Hybrid Creatine Kinase Isoenzymes) antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0141] 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 that secretes the Anti-CKMB 10C5 monoclonal antibody is an existing hybridoma cell line, which is resuscitated for future use.

[0142] 1. Primers

[0143] Amplify Heavy Chain and Light Chain 5'RACE Primers:

[0144] SMARTER II A Oligonucleotide:

[0145] 5'-AAGCAGTGGTATCAACGCAGAGTACXXXXX-3';

[0146] 5'-RACE CDS Primer (5'-CDS): 5'-(T) 25 VN-3'(N=A,C,G,orT; V=A,G,orC);

[0147] Universal Primer A Mix (UPM): 5'-CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT-3';

[0148] Nested Universal Primer A (NUP): 5'-AAGCAGTG...

Embodiment 2

[0179] Although the antibody of sample 1 obtained in Example 1 (with sequences such as the heavy chain and light chain shown in SEQ ID NO: 11 and 12) has the ability to bind CKMB, the affinity and antibody activity are not ideal, so the applicant passed the The antibody's light chain CDRs and heavy chain CDRs were mutated.

[0180] After analysis, the complementarity determining region (WT) of the heavy chain:

[0181] CDR-VH1 is G-V(X1)-S-I(X2)-T-T-G-T(X3)-D-R;

[0182] CDR-VH2 is I-F(X1)-Y-S-G-T-L(X2)-T-Y-N-P-S-V(X3)-T-S;

[0183] CDR-VH3 is R-E-R(X1)-T-Y-Y-P(X2)-Y-G-Y-F-D-V-F(X3)-G;

[0184] Complementarity-determining regions of the light chain:

[0185] CDR-VL1 is H-G(X1)-S-N(X2)-N-I-N-I(X3)-W-L-S;

[0186] CDR-VL2 is K-G(X1)-S-D-I(X2)-H-T;

[0187] CDR-VL3 is Q-A(X1)-Q-S-Y-A(X2)-W-T-W(X3)-G;

[0188] Among them, X1, X2, and X3 are mutation sites.

[0189] Table 1 Mutation sites related to antibody activity

[0190]

[0191]

[0192] After the mutation, the ...

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Abstract

The invention relates to a novel separated binding protein comprising a CKMB (Hybrid Creatine Kinase Isoenzymes) antigen binding structural domain, and researches the aspects of preparation, application and the like of the binding protein. The binding protein has high activity, has a very high affinity with a human CKMB protein, and can be widely applied to the field of detection of the CKMB protein.

Description

technical field [0001] The invention relates to the fields of biotechnology and medical technology, in particular to an anti-human CKMB antibody and its application. Background technique [0002] There are four isoenzyme forms of creatine kinase isoenzymes (CK): muscle type (MM), brain type (BB), hybrid type (MB) and mitochondrial type (MiMi). The MM type mainly exists in various muscle cells and is a dimer composed of two identical subunits; the BB type mainly exists in brain cells; the MB type mainly exists in cardiomyocytes; the MiMi type mainly exists in cardiac muscle and in skeletal muscle mitochondria. CKMB (hybrid creatine kinase isoenzyme) is usually divided into two isoforms, MB1 and MB2. In cardiomyocytes, CKMB mainly exists in the form of MB2. Once cardiomyocytes are damaged, MB2 will be released, making CKMB in serum The level rises rapidly in a short period of time, usually within 6 hours of onset, and reaches the peak at about 24 hours, and gradually decreas...

Claims

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Application Information

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IPC IPC(8): C07K16/40C12N15/13G01N33/573
CPCC07K16/40G01N33/573C07K2317/565C07K2317/92G01N2800/324
Inventor 崔鹏何志强孟媛钟冬梅周全兴梁碧游辉马秋燕李蔚芝
Owner DONGGUAN PENGZHI BIOTECH CO LTD
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