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AFP nanometer antibody A80 based on AFP antigen

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: 2015-11-11
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 nanometer antibody A80 based on AFP antigen
  • AFP nanometer antibody A80 based on AFP antigen
  • AFP nanometer antibody A80 based on AFP antigen

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 (pH8.5);

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

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

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

[0055] E Quantification of biotinylated proteins with NanoDrop.

[0056] 2) panning

[0057] With streptavidin (100 μg / mL streptavidin dissolved in 0.1M NaHCO 3 (pH8.6)) coated microtiter plate, 37 ℃ 2h. After washing 3 times with PBST (10mMPBS, 0.5% Tween-20 (v / v)), add 300 μL of 3% OVA-PBS to block at 37°C for 1 hour (the blocking solution should contain 0.1 μg / mL streptavidin to bind Biotin contained in the BSA blocking solution), and the phage was pre-bound...

Embodiment 3

[0068] Example 3. The sequencing of the gene encoding the AFP Nanobody and the determination of its amino acid sequence

[0069] 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 technical field of biology, and relates to a camel source immunity nanometer antibody (single-domain heavy-chain antibody, VHH) specifically bound with AFP and application of the antibody. The amino acid sequence of the antibody is SEQ ID NO.:1. The invention further relates to nucleotides for encoding amino acids. The AFP nanometer antibody can be used as the antibody to be applied to immunological detection of AFP not for disease diagnosis and treatment. Compared with a Scfv antibody and a Fab antibody, the camel source immunity VHH has the higher specificity and affinity, more stable in structure, resistant to acid, base and high temperature, high in detection flexibility and the like, so that the immunity detection stability is greatly improved, and meanwhile the environment resistance is greatly improved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a nanobody (single domain heavy chain antibody, Variable domain of heavy chain of heavy chain antibody, VHH) specifically binding to alpha-fetoprotein (Alpha-Fetal Protein, AFP) and an application thereof. 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 d...

Claims

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Application Information

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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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