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AFP-antigen-based AFP nanobody A80

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 effects of small molecular weight, good specificity and cost saving

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 A80
  • AFP-antigen-based AFP nanobody A80
  • AFP-antigen-based AFP nanobody A80

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 alpaca:

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

[0025] 2) Separation of camel-derived leukocytes: add lymphocyte separation solution to the centrifuge tube, then add blood samples, centrifuge at 700g for 20min; carefully suck the leukocytes suspended in the m...

Embodiment 2

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

[0050] 1) Biotinylation of AFP as a target molecule

[0051] A Dissolve 2 mg of target protein in 1 mL of 50 mM NaHCO 3 (pH 8.5);

[0052] B Before use, dissolve 1 mg of Sulfo-NHS-LC-biotin in 1 mL of water, shake and mix well. Add 74 μL of this solution to the target protein solution;

[0053] C Put the tube on ice for 2h;

[0054] D Dialyze to remove unreacted biotin, dialyze against 1 liter of TBS buffer, and replace the dialysate at least twice;

[0055] E Quantify biotinylated protein with Nano Drop.

[0056] 2) Panning

[0057] with streptavidin (100 μg / mL streptavidin in 0.1 M NaHCO 3 (pH 8.6)) coated microtiter plate, 37 ℃ 2h. After washing 3 times with PBST (10mM PBS, 0.5% Tween-20 (v / v)), add 300 μL of 3% OVA-PBS and block for 1 hour at 37°C (blocking solution should contain 0.1 μg / mL streptavidin). In order to bind the biotin contained in the BSA blocking solution), the phage was prev...

Embodiment 3

[0068] Example 3. Sequencing of AFP Nanobody Encoding Gene and Determination of Amino Acid Sequence

[0069] The positive phage clones identified by the double-antibody sandwich enzyme-linked immunosorbent assay method were subjected to DNA sequencing, and the amino acid sequence was obtained according to the DNA sequencing results and the codon table.

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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, in particular to a nanobody (Variable domain of heavy chain of heavy chain antibody, VHH) that specifically binds to 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 is very little in the blood of healthy adults. Currently, 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 prognostic evaluation. Alpha-fetoprotein as a marker is overrepresented in various tumors, especially in hepatocellular carcinoma (HCC). With the further in-depth study of the structure and function of AFP gene, it plays an increasingly important role in the early diagnosis and biological treatment of HCC. In patients with other gastroin...

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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