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Nanometer antibodies to human cystatin C and application thereof

A technology of nanobodies and human cysts, applied in the fields of application, virus/bacteriophage, protease inhibitor immunoglobulin with anti-peptide structure, etc., can solve the problems of low sensitivity, complicated operation, poor reproducibility, etc.

Inactive Publication Date: 2014-01-15
上海方恩医疗用品有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, the detection reagents for detecting cystatin C in the prior art, such as the preparation of monoclonal pairs for pairing, have difficulties, and the patent "a method for preparing paired monoclonal antibodies against cystatin C (201110254775.8)" aims to solve this problem. A difficulty offers a way
However, even so, this method is still complicated to operate, and the developed monoclonal antibodies still have some defects, such as low sensitivity, poor reproducibility, and difficult access to antibodies, etc.

Method used

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  • Nanometer antibodies to human cystatin C and application thereof
  • Nanometer antibodies to human cystatin C and application thereof
  • Nanometer antibodies to human cystatin C and application thereof

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preparation example Construction

[0099] In an example of the present invention, the monoclonal antibody can be prepared by the following preparation method, which includes the steps of: (1) providing mice pretreated with adjuvant; (2) intraperitoneally inoculating the mouse with the said hybridoma cells and secrete monoclonal antibody; (3) extract ascites and separate and obtain said monoclonal antibody. As a preferred method, the method of isolating monoclonal antibody from ascites is as follows: collect ascites, precipitate with ammonium sulfate and octanoic acid, and then purify with Protein G prepacked chromatography column to obtain high-purity cystatin C monoclonal antibody .

[0100] In addition, the hybridoma cells can also be cultured and expanded in vitro according to conventional animal cell culture methods, so as to secrete the monoclonal antibody.

[0101] Reagent test kit

[0102] Those skilled in the art understand that an antigen may contain multiple epitopes (antigenic determinants), theref...

Embodiment 1

[0131] Embodiment 1, cystatin C recombinant expression

[0132] 1. Polymerase chain reaction (PCR) amplification of the full-length cystatin C gene

[0133] (1) Template: 293T total RNA of human kidney epithelial cells.

[0134] (2) Primer design: using the full-length mature cystatin C gene 363bp, design primers for amplifying the cystatin C gene after removing the signal peptide sequence, the upstream and downstream primers respectively carry endonucleases BamHI and XhoI sequence of restriction enzyme sites.

[0135] Upstream primer: 5' GGATCC AGTCCCGGCAAGCCG-3' (SEQ ID NO: 12);

[0136] Downstream primer: 5'-C CTCGAG CTAGGCGTCCTGACAGGT-3' (SEQ ID NO: 13);

[0137] (3) Total cellular RNA was extracted, and reverse-transcribed into cDNA using primer Oligo(dT)18 and reverse transcriptase.

[0138] The mature cystatin C gene (363bp) was obtained by RT-PCR, and connected to the cloning vector (pEASY-T1simple [purchased from Beijing Quanshijin Biotechnology Co., Ltd.]). A...

Embodiment 2

[0180] Embodiment 2, animal immunization

[0181] (1) Immunization of BALB / c mice

[0182] The mouse species is BALB / c. Mix 100 μg of recombinant cystatin C protein with an equal volume of Freund's complete adjuvant, emulsify completely, and inject subcutaneously in the back and feet of the mouse for immunization. Complete Freund's adjuvant was used for the first immunization, followed by incomplete Freund's adjuvant. One week after the fourth immunization, blood was collected from the orbit, the serum was separated, and the titer of anti-cystatin C protein antibody was measured. As detected by ELISA, the antibody titer of the mice after four immunizations was 1:512,000.

[0183] (2) ELISA steps

[0184] 1) Coating: Dilute the antigen (natural cystatin C) with 0.05M carbonate coating buffer (pH=9.6) to a protein content of 1.0 μg / ml. Add 0.1 ml to the reaction well of each polystyrene plate, overnight at 4°C, discard the solution in the well, and wash 3 times with 200 μl / wel...

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Abstract

The invention relates to nanometer antibodies to human cystatin C and high-sensitivity quantitative-detection kits thereof. The invention reveals nanometer antibodies and monoclonal antibodies to different epitopes of cystatin C, and the antibodies have good compatibility with cystatin C. Through usage of the nanometer antibodies and the monoclonal antibodies, a series of kits capable of conveniently, rapidly and accurately detecting cystatin C can be prepared. The invention provides the nanometer antibodies which can be used for matched detection of cystatin C. With the kits provided by the invention, cystatin C antigens can be sensitively detected, and good repeatability and stable results are obtained; the kits can realize rapid detection and are convenient and fast to use, and the nanometer antibodies need low production cost and are easily available.

Description

technical field [0001] The invention belongs to the field of biotechnology; more specifically, the invention relates to a human cystatin C nanobody and a highly sensitive quantitative detection kit thereof. Background technique [0002] The natural missing light chain isolated from camel blood by immunologist Hamers Casterman of Free University of Brussels, Belgium, contains only two heavy chains. The single-domain antibody (VHH) composed of domains, because of its crystal structure with a diameter of 2.5nm and a length of 4nm, the concept of nanobody was proposed. Once the nanobody was discovered, some of its unique characteristics attracted the attention of researchers. As a small molecular antibody, nanobodies have many advantages. Including: 1. Small molecule, stable structure and good heat resistance. Studies have confirmed that nanobodies can maintain relatively good biological activity when stored at 37°C for a week or stored at high temperature (90°C). In additio...

Claims

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Application Information

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IPC IPC(8): C07K16/38C12N15/13C12N7/01G01N33/68C12N5/20
Inventor 姚刚许超季军捷
Owner 上海方恩医疗用品有限公司
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