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AFP-antigen-based AFP nanobody A83

A nano-antibody and antigen technology, applied in the biological field, can solve the problem of expensive antibody antigen standard products, achieve the effect of small molecular weight, unique structure, and avoid the preparation process

Active Publication Date: 2018-05-01
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 A83
  • AFP-antigen-based AFP nanobody A83
  • AFP-antigen-based AFP nanobody A83

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) Biotinylation of AFP as 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 1mg Sulfo-NS-LC-biotin in 1mL water, shake and mix. Add 74 μL of this solution to the target protein solution;

[0053] C. Place the tube on ice for 2 hours;

[0054] D. Dialyze to remove unreacted biotin, dialyze with PBS buffer, change the dialysate at least twice;

[0055] E. Quantification of biotinylated proteins using NaNo Drop.

[0056] 2) panning

[0057] With streptavidin (100 μg / mL streptavidin dissolved in 0.1M NaHCO 3 (pH 8.6)) coated microtiter plate, 4 ℃ overnight. After washing 4 times with PBST (10mM PBS, 0.5% Tween-20 (v / v)), add 300 μL of 3% BSA-PBS to block for 1 hour at 37°C (the blocking solution should contain 0.1 μg / mL streptavidin to Combined with the biotin contained in the BSA blocking solution), the phage was ...

Embodiment 3

[0067] Example 3. The sequencing of the gene encoding the AFP Nanobody and the determination of its amino acid sequence

[0068] 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): C07K16/18C12N15/13G01N33/68
Inventor 付金衡何庆华许杨涂追陈静
Owner NANCHANG DAJIA TECH
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