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AFP-antigen-based AFP nanobody A65

A nano-antibody and antigen technology, applied in the biological field, can solve the problems of expensive antibody and antigen standard products, and achieve the effect of small molecular weight, good specificity and high application value

Active Publication Date: 2018-05-04
NANCHANG DAJIA TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

And antibody and antigen standards are very expensive, and some toxins are dangerous

Method used

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  • AFP-antigen-based AFP nanobody A65
  • AFP-antigen-based AFP nanobody A65
  • AFP-antigen-based AFP nanobody A65

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1. Construction of camel-derived immune single domain heavy chain antibody library

[0023] 1) Immunity of alpacas:

[0024] A healthy adult alpaca was selected and immunized by intradermal and subcutaneous multi-point injections on the back. For the first immunization, Freund's complete adjuvant was used, and the dose of immunogen was 1 mg; on the 28th day, the second immunization was performed with Freund's incomplete adjuvant. The dose of immunogen is 0.5 mg, three immunizations on the 49th day, Freund's incomplete adjuvant, the immunogen dose is 0.25 mg; on the 70th day, four immunizations, Freund's incomplete adjuvant, the immunogen dose is 0.25 mg. On the seventh day after the third booster immunization, the peripheral blood of the alpaca was collected for the construction of a phage display library.

[0025] 2) Separation of camel-derived white blood cells: add lymphocyte separation solution to the centrifuge tube, then add blood samples, centrifuge at 7...

Embodiment 2

[0049] Example 2. Affinity panning and identification of AFP Nanobodies

[0050] 1) Affinity panning of AFP Nanobody: In the first round of panning, the anti-AFP antigen was diluted with PBS (pH 7.4), and coated with a microtiter plate at a final concentration of 100 μg / mL, and left overnight at 4°C. After washing with PBST (10 mM PBS, 0.5% Tween-20 (v / v)) 3 times, 300 μL of 3% BSA-PBS was added to block for 1 hour at 37°C. After 1 hour, the blocking solution was discarded, washed 5 times with PBST, and 100 μL camel-derived immune single-domain heavy chain antibody library (titer about 1.0×10 11 cfu), incubated at 37°C for 1 hour. Aspirate unbound phage, wash with PBST 10 times, add 100 μL of 0.2M Glycine-HCl (pH 2.2) to elute, shake gently for 15 min, and immediately neutralize with 15 μL of 1M HCl (pH 9.1). Take 10 μL of the eluted phage to determine the titer, and the rest is used to infect 20 mL of E.coli TG1 strain grown to the logarithmic phase for amplification. The ...

Embodiment 3

[0061] Example 3. The sequencing of the gene encoding the AFP Nanobody and the determination of its amino acid sequence

[0062] The positive phage clones identified by double-antibody sandwich ELISA were subjected to DNA sequencing, and the amino acid sequence could be obtained according to the DNA sequencing results and the codon table.

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PUM

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Abstract

The invention belongs to the field of biotechnology, and relates to a camel-derived immune nanobody (single-domain heavy chain antibody, VHH) specifically binding to alpha-fetoprotein and an application thereof. The amino acid sequence of the antibody is SEQ ID NO.: 1, and the nucleotide encoding the amino acid is also involved. The alpha-fetoprotein nanobody of the present invention can be used as an antibody in the immunological detection of AFP for the purpose of non-disease diagnosis and treatment. Compared with Scfv antibody and Fab antibody, camel-derived immune VHH has higher specificity, higher affinity, more stable structure, acid, alkali and high temperature resistance, and high detection sensitivity, so its immune detection stability has been greatly improved. Environmental tolerance has also been improved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a nanobody (variable domain of heavy chain of heavy chain antibody, VHH) specifically binding to alpha-fetoprotein (Alpha-Fetal Protein, AFP) and its application. Background technique [0002] Alpha-fetoprotein is a glycoprotein produced by fetal liver or adult liver. It is commonly found in the blood of pregnant women and infants, but its content is very small in the blood of healthy adults. At present, it is mainly used for the early diagnosis and curative effect of primary liver cancer It is an important serological marker for primary liver cancer (PHC) for evaluation and prognosis evaluation. Alpha-fetoprotein as a marker is overexpressed in various tumors, especially hepatocellular carcinoma (HCC). With further in-depth research on the structure and function of AFP gene, it plays an increasingly important role in the early diagnosis and biological treatment of HCC. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/18C07K14/47C07K1/22C12N15/13G01N33/68
Inventor 付金衡何庆华许杨涂追陈静
Owner NANCHANG DAJIA TECH
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