Nano-antibody combination for detecting carcino-embryonic antigen by double-antibody sandwich method

A double-antibody sandwich and nanobody technology, applied in the field of immunology, to achieve the effect of improved accuracy, low detection limit, and good matching degree

Pending Publication Date: 2020-04-14
深圳市国创纳米抗体技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The excellent antigen affinity guarantees the application prospect of the nanobody in the differential diagnosis of the target antigen, but in specific applications, even the nanobody with high antigen affinity still has great technical problems in the actual operation of antigen detection
For example, in the immuno-sandwich assay, the overlapping degree of antigen-binding sites between the first antibody and the second antibody, as well as a series of problems caused by the too small molecular weight of the nanobody itself are still the key to obtaining an efficient detection kit. technical difficulties

Method used

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  • Nano-antibody combination for detecting carcino-embryonic antigen by double-antibody sandwich method
  • Nano-antibody combination for detecting carcino-embryonic antigen by double-antibody sandwich method
  • Nano-antibody combination for detecting carcino-embryonic antigen by double-antibody sandwich method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Preparation of anti-CEA monovalent nanobody

[0027] Refer to Chinese invention patent application CN106749667A. The variable region sequence of the monovalent Nanobody is shown in SEQ ID NO.1.

Embodiment 2

[0028] Example 2. Preparation of anti-CEA bivalent nanobody

[0029] 2.1VHH1-LHC-VHH1-pMES4 vector construction:

[0030] Due to the longer hinge region (LHC) of the antibody, design two downstream primers and perform two rounds of PCR. The operation is as follows:

[0031] 2.1.1 The first round of PCR was performed using monovalent Nanobody DNA as a template, and the primer sequences were as follows:

[0032] 11F1: AACTGCAGGAGAGCGGTGGCGGTC

[0033] 11R1:CAAGTGACCGTTAGCAGCGAACCGAAAACCCCCGAAACC

[0034] GCAGCCGCAGCCTCAACCGCAACCGCAGCCG

[0035] The PCR reaction conditions and program are: 95°C for 5 minutes; 30 cycles of 95°C for 30 seconds, 55°C for 30 seconds, 72°C for 30 seconds; 72°C for 7 minutes. Use the agarose gel recovery kit to recover the band of about 300bp ( figure 1 : M is Trans 2K DNAMarker; 1 is negative control, 2-10 is the first round of PCR product).

[0036] 2.1.2 Use the recovered product of the first round of PCR as a template for the second round of ...

Embodiment 3

[0046] Example 3. Affinity testing of anti-CEA Nanobodies

[0047] 3.1 Chip antigen coupling: prepare CEA antigen with different pH sodium acetate buffer (pH 5.5, pH 5.0, pH 4.5, pH 4.0) to make 20 μg / mL working solution, and prepare 50 mM NaOH regeneration solution at the same time, use Biacore T100 The template method in the instrument of the protein interaction analysis system is used to analyze the electrostatic binding between the antigens at different pH conditions and the surface of the chip (GE Company), and the most neutral one is selected based on the signal increase of 5 times RL. The pH system and adjust the antigen concentration as the coupling conditions as needed. The chip was coupled according to the template method that comes with the instrument: select the blank coupling mode for channel 1, select the Target coupling mode for channel 2, and set the target to the designed theoretical coupling amount. The coupling process takes approximately 60 minutes.

[00...

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Abstract

The invention discloses a nano-antibody combination for detecting carcino-embryonic antigen by a double-antibody sandwich method. The nano antibody combination comprises an anti-carcino-embryonic antigen bivalent nano antibody serving as a capture antibody and an anti-carcino-embryonic antigen monovalent nano antibody serving as a detection antibody. The invention further discloses an applicationof the nano antibody combination in preparation of a carcino-embryonic antigen detection kit. The nano antibody combination of the anti-cancer embryo antigen bivalent nano antibody and the anti-cancerembryo antigen monovalent nano antibody provided by the invention has good matching degree, shows excellent P / N value, lowest detection limit and accuracy in CEA antigen detection, and can meet detection of CEA in clinical samples.

Description

technical field [0001] The invention discloses a nanobody combination, which belongs to the field of immunology. Background technique [0002] Carcinoembryonic antigen (CEA, also known as CEACAM-5 or CD66e) is a glycoprotein with a molecular weight of about 180 kDa. CEA is a member of the immunoglobulin superfamily and contains seven domains linked to the cell membrane via glycosylphosphatidylinositol (GPI) anchors. The seven domains include a single N-terminal Ig variable domain and six domains homologous to Ig constant domains (A1-B1-A2-B2-A3-B3). CEA, originally classified as a protein expressed only in fetal tissues, has now been identified in several normal adult tissues. Overexpression of CEA has been observed in many types of cancer, including colorectal, pancreatic, lung, gastric, hepatoma, breast and thyroid cancers. Therefore, CEA has been identified as a tumor-associated antigen. CEA is readily cleaved from the cell surface and shed from the tumor into the blo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/30G01N33/535G01N33/543G01N33/574G01N21/76
CPCC07K16/3007G01N33/57473G01N33/54306G01N33/535G01N21/76C07K2317/569C07K2317/35
Inventor 林景涛宋海鹏于建立张霞王欢陈晓恒
Owner 深圳市国创纳米抗体技术有限公司
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