A kind of anti-activated blood coagulation factor V monoclonal antibody and its preparation method and application

A monoclonal antibody and coagulation factor technology, applied in the biological field, can solve the problems of no anti-activated FV monoclonal antibody preparation report, lack of quantitative detection of activated FV and other problems, and achieve the effect of high affinity, strong specificity and good application prospects.

Active Publication Date: 2021-04-30
吴剑波 +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] However, there is still a lack of effective methods for quantitative detection of activated FV. The development of anti-activated FV monoclonal antibodies is of great significance for establishing a specific and sensitive FVa activity detection method to assist in the diagnosis of abnormal FV diseases.
There is no report on the preparation of anti-activated FV monoclonal antibody

Method used

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  • A kind of anti-activated blood coagulation factor V monoclonal antibody and its preparation method and application
  • A kind of anti-activated blood coagulation factor V monoclonal antibody and its preparation method and application

Examples

Experimental program
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Embodiment 1

[0021] Embodiment 1 Preparation of monoclonal antibody against activated FV of the present invention

[0022] 1. Screening of fully human FVa single chain antibody in phage antibody library

[0023] The FVa recombinant protein (product of HTI Company) was used as an antigen to screen the fully human phage antibody library. Dilute the FVa antigen with coating diluent (0.05mol / L sodium carbonate-sodium bicarbonate, pH 9.6) to 10ug / mL, add 100ul to each well of the ELISA plate, and place in a 4°C refrigerator for overnight coating. The blocking solution was mixed with PBST (PBS, 0.05% Tween 20) and 1% BSA, and 200ul was added to each well, and blocked for 1 hour. Wash 5 times with PBST, 3 min each time, add fully human Fab-based phage display library (from Shanghai Boyu Biotechnology Company phage display library), and incubate overnight at room temperature. Discard the liquid in the immunoplate, wash with PBST 5 times, each time for 3 minutes, and the phage tightly bound to th...

experiment example 1

[0040] Experimental example 1 Antigen-binding activity assay of the monoclonal antibody of the present invention

[0041] 1. Experimental method

[0042] The purified monoclonal antibody 1C11 prepared in Example 1 was used for gradient concentration affinity detection by ELISA method.

[0043] ELISA method: Dilute the FVa antigen with coating diluent to 1ug / mL, add 100ul of the diluted antigen to each well of the ELISA plate, and place it in a 4°C refrigerator overnight. Add 150ul of blocking solution to each well, and block at 37°C for 40min. Add 200ul of washing solution to each well and wash 3 times, each time 3min. Add the monoclonal recombinant phage to be tested and incubate at 37°C for 2h. Wash 3 times with washing solution, 3 min each time. Add goat anti-human-HRP (dilution ratio 1:2000) working solution, incubate at 37°C for 1 hour, add 200ul washing solution to each well and wash 3 times, 3 minutes each time. Add 100ul of temporarily prepared TMB substrate solut...

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Abstract

The invention discloses an anti-activated coagulation factor V monoclonal antibody, which is characterized in that the amino acid sequence of its light chain is shown in SEQ ID NO: 3, and the amino acid sequence of its heavy chain is shown in SEQ ID NO: 4. The invention also discloses the preparation method and application of the aforementioned monoclonal monomer. The anti-activated blood coagulation factor V monoclonal antibody of the invention has strong binding ability to activated blood coagulation factor V, and can be used for detection of activated blood coagulation factor V.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an anti-activated blood coagulation factor V monoclonal antibody and its preparation method and application. Background technique [0002] Coagulation factor V (factor V, FV) is a single-chain glycoprotein with a molecular weight of 333KD, which exists in plasma almost in an inactive state under physiological conditions. Under the action of thrombin, plasma FV is transformed into activated coagulation factor V (activated factor V, FVa) with coagulation activity. FVa in plasma is an important cofactor in the blood coagulation process, and plays a vital role in regulating the blood coagulation process. The mechanism is as follows: figure 1 shown. During the coagulation process, FVa, FXa, calcium ions and phospholipids together form a prothrombin complex, which can increase the activation efficiency of prothrombin by 300,000 times. FVa can be inactivated by cleavage of ac...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/36C12N15/13C12N15/85C12N5/10G01N33/68G01N33/577
CPCC07K16/36G01N33/86G01N2333/7456
Inventor 吴剑波杨燕
Owner 吴剑波
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