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CEA (carcinoembryonic antigen) resistant nanomater antibody with high appetency and application

A nano-antibody and affinity technology, which is applied in the fields of application, antibody, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, etc. It can solve the problems of short serum half-life, low affinity, easy aggregation, etc., and achieve superior application Effects of foreground, high specificity, and sensitivity

Active Publication Date: 2018-04-06
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

However, due to the low molecular weight of Nanobodies, some structural defects such as low affinity, easy aggregation, and short serum half-life hinder the further application of Nanobodies.

Method used

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  • CEA (carcinoembryonic antigen) resistant nanomater antibody with high appetency and application
  • CEA (carcinoembryonic antigen) resistant nanomater antibody with high appetency and application
  • CEA (carcinoembryonic antigen) resistant nanomater antibody with high appetency and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1. Construction and screening of anti-CEA nanobody phage display library

[0031] 1.1 Immunization of alpaca: Select a healthy adult alpaca, mix the recombinant protein CEA and Freund's adjuvant at a ratio of 1:1, and immunize the alpaca by subcutaneous multi-point injection on the back at 6-7μg / Kg , a total of four immunizations, immunization interval of 2 weeks. Afterwards, the peripheral blood of the alpaca was collected for the construction of a phage display library.

[0032] 1.2 Separation of camel-derived lymphocytes: analyze lymphocytes from the collected camel-derived anticoagulated whole blood according to routine procedures in this technical field, every 2.5×10 7 Add 1mL RNA isolation reagent to each living cell, take 1mL for RNA extraction, and store at -80°C.

[0033] 1.3 Extraction of total RNA: extract total RNA according to routine procedures in this technical field, and adjust the concentration to 1 μg / μL with RNase-free water (for the identif...

Embodiment 2

[0061] Example 2. Human kidney epithelial cell line transfected with adenovirus E1A gene expresses fusion nanobody 2D5-HAP

[0062] The binding site sequence of human alkaline phosphatase is used as the amino acid sequence of the chemiluminescent region (shown in SEQ ID NO.5), which is fused with the flexible polypeptide and the 2D5 nanobody to form a nanobody with a chemiluminescent region sequence, and synthesized The nucleotide coding sequence of the fusion protein (shown in SEQ ID NO.6), two restriction sites HindⅢ and EcoRI are added at the two ends of the coding sequence of the nucleotide, and the two restriction sites The point ligated to the vector pcDNA3.1(+). 293 cells in logarithmic growth were used for transfection after the plasmid was extracted without endotoxin. After the transfected cells were cultured for 36 hours, the cell culture medium was poured into a 50ml centrifuge tube, centrifuged at 12000g for 5 minutes, the supernatant was collected, filtered with ...

Embodiment 3

[0063] Example 3.2D5-HAP affinity activity with CEA antigen

[0064] 3.1 Chip antigen coupling

[0065] The antigen was prepared into a 20 μg / mL working solution with different pH sodium acetate buffers (pH 5.5, pH 5.0, pH 4.5, pH 4.0), and a 50 mM NaOH regeneration solution was prepared at the same time, and the Biacore T100 protein interaction analysis system was used in the instrument The template method is used to analyze the electrostatic binding between the antigen and the surface of the chip (GE company) under different pH conditions, and the signal increase amount reaches 5 times RL as the standard, select the most suitable neutral pH system and adjust it as needed The antigen concentration was used as the condition during coupling. 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 amou...

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PUM

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Abstract

The invention discloses a CEA (carcinoembryonic antigen) resistant nanomater antibody with high appetency. The nanomater antibody has unique three complementary determining regions CDR1, CDR2 and CDR3. The invention also discloses application of the nanomater antibody to tumor treatment medicine and tumor detection reagents. The CEA resistant nanomater antibody provided by the invention has specific recognition and combination capability on CEA; the appetency of the nanomater antibody can reach 10 to 11; seven kinds of CEA analogues do not take conjugation reaction; during the detection on theCEA in a serum sample, the sensitivity can reach 1ng / ml; the condition shows that the nanomater antibody provided by the invention has high specificity and sensitivity.

Description

technical field [0001] The present invention relates to an antibody, more particularly, the present invention relates to a Nanobody. 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 th...

Claims

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Application Information

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IPC IPC(8): C07K16/30C12N15/13C12N15/85C12N5/10A61K39/395A61P35/00G01N33/574
CPCA61K2039/505C07K16/3007C07K2317/565C07K2317/567C07K2317/569C07K2317/92G01N33/57473
Inventor 宋海鹏于建立刘原源李飞古一黄琪周宇航王欢陈晓恒王崇洋李婧婵
Owner 深圳市国创纳米抗体技术有限公司
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