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Anti-human abnormal prothrombin antibody and its application

An abnormal prothrombin and antibody technology, applied in applications, anti-enzyme immunoglobulins, instruments, etc., can solve the problem of no specific detection of human DCP antibodies, so as to avoid the interference of the source of abnormal prothrombin and reduce the difficulty , the effect of broad application prospects

Active Publication Date: 2022-05-10
XIAMEN INNOBIOMAX BIOTECHNOLOGY CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The current research mainly focuses on the application method of detecting DCP or the corresponding antibody by using the antibody that recognizes the specific epitope of DCP and the antibody of prothrombin. There is no report on the specific detection of human DCP antibody.

Method used

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  • Anti-human abnormal prothrombin antibody and its application
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  • Anti-human abnormal prothrombin antibody and its application

Examples

Experimental program
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Effect test

Embodiment 1

[0051] Example 1 Preparation of Abnormal Prothrombin Recombinant Antigen

[0052] According to the prothrombin sequence (Accession number: NM_000506.5) retrieved from Gene Bank, recombinant expression cloning was designed, and the full-length sequence is shown in SEQ ID NO:9. According to the differences between DCP and normal prothrombin mainly in the first 50 amino acids, and the reported antibody epitopes are generally linear epitopes, the commercial vectors pColdTF and pGEX-20T vectors were used to construct prothrombin 1-50aa respectively The expression plasmid of the E. coli expression system is used to express the target protein, because the protein cannot be modified by gamma carboxylation in E. coli, so the expression product is abnormal prothrombin, DCP corresponding 1-50aa, the specific nucleotide sequence Shown in SEQ ID NO:10.

[0053] Because the two expression vectors both use the same restriction site, the primers are the same. Primer5.0 is used to design prim...

Embodiment 2

[0064] Example 2 Preparation of Abnormal Prothrombin Monoclonal Antibody

[0065] 1. Preparation of the immunogen: the immunogen is the antigen 1 prepared in Example 1. Antigen 1 was diluted to 0.4mg / mL with 10mmol / L PBS, and the corresponding antigen was mixed with Freund's adjuvant in equal volumes according to the final concentration of 200ug / ml to form a water-in-oil emulsion. Complete Freund's adjuvant was used for the initial immunization, and incomplete Freund's adjuvant was used for the booster immunization.

[0066] 2. Basic immunization: BALB / c female mice aged 6-8 weeks were selected for subcutaneous multi-point immunization. The dose of immunogen injection was 500 μL / mouse / time, the immunization interval was 2 weeks, and the complete immunization procedure was 4 injections. 2 weeks after the 3rd and 4th doses of immunization, the isolated serum was collected by orbital blood collection method for indirect ELISA to determine the immune titer. After the mouse serum ...

Embodiment 3

[0097] Example 3 Monoclonal Antibody Epitope Identification

[0098] 1. Identification of 15 peptides

[0099] Antigen 1 was diluted with carbonic acid buffer (20mmol / L CB, pH 9.6) and coated on a polyvinyl chloride plate at 200ng / ml. The purified antibody was mixed with the synthesized DCP1-50aa polypeptide (Shenggong Synthetic, the length of the polypeptide is 15aa, synthesized by overlapping 5 amino acids successively, as shown in the underline in the table, the specific sequence information is shown in Table 1) Proportional 100ng / ml antibody: 50ug / ml polypeptide is incubated in equal volume for 30min, which is the test sample; the control sample is 100ng Antibody / ml: PBS, incubate for 30 minutes, add the incubated test sample and control sample to the plate coated with antigen 1 and incubate for 30 minutes, wash the plate 5 times, add GAM-HRP (1 / 5000 dilution) and incubate for 30 minutes , wash the plate 5 times, add substrate and incubate for 15 min. Read the value of t...

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Abstract

The invention belongs to the field of antibodies, and in particular relates to an anti-human abnormal prothrombin antibody and the use of the antibody in preparing a hepatocellular carcinoma detection kit. The anti-human abnormal prothrombin antibody of the present invention can specifically bind human abnormal prothrombin, can further avoid interference from prothrombin in serum or plasma from the raw material level, and can further avoid abnormal blood coagulation from other species in animal serum products The interference of zymogens avoids related risks from the source, reduces the difficulty of reagent development, and has a good application prospect.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an anti-human abnormal prothrombin antibody and its application. Background technique [0002] Normal prothrombin is in hepatocytes, with the participation of γ-glutamyl carboxylase and related coenzymes mainly dependent on vitamin K, the 6th, 7th, 14th, 16th, 19th, 20th, 25th, 26th, The 10 glutamic acid (Glu) residues at positions 29 and 32 are γ-carboxylated to γ-carboxyglutamic acid (Gla), making it an active normal prothrombin, and one or more Glu residues at any of the above sites Incomplete carboxylation of residues may form abnormal prothrombin (Des-γ-carboxyprothrombin, hereinafter referred to as DCP), and lose coagulation function. At present, the N-terminal is called the Glu-Gla region. Except for this region, normal prothrombin and abnormal prothrombin have no other differences in structure. They are composed of prothrombin fragment F1, prothrombin fragment F2 and C-termi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/40C12N15/13G01N33/574
CPCC07K16/40G01N33/57438C07K2317/56C07K2317/565
Inventor 王海虹熊君辉曾蓓蓓邹梦雯柯起沈张志阳柯少君温顺华
Owner XIAMEN INNOBIOMAX BIOTECHNOLOGY CO LTD
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