Preparation method of bombyx mori silk fibroin monoclonal antibody

A monoclonal antibody, mulberry silk technology, applied in anti-animal/human immunoglobulins, material testing products, measuring devices, etc., can solve the problem of weak antigen binding specificity, very difficult separation of antibodies, and low purity of polyclonal antibodies. and other problems, to achieve the effect of high sensitivity, strong specificity, and improved extraction rate

Active Publication Date: 2017-11-24
ZHEJIANG SCI-TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, polyclonal antibodies to silk fibroin protein have appeared. The specificity of antibodies depends on the determinants of antigen molecules. Antigen molecules have many antigen determinants. Therefore, the antibodies produced by im

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment approach 1

[0035] A) Weigh 15g of mulberry silk into deionized water at a bath ratio of 1:50, add 2.25g of sodium carbonate, and degummed at a constant temperature of 95°C in a water bath for 60 minutes. After the end of the water bath, add acetic acid to adjust the pH to neutral. Repeat the operation twice . The degummed mulberry silk was added to 200 mL of deionized water, soaked for 2 hours, and then dried at 60°C.

[0036] B) Mix 500mL isooctane and 1000mL n-octanol, stir and mix well for later use, weigh 0.3mol sodium bis(2-ethylhexyl) succinate sulfonate, 0.3mol polyvinylpyridinium quaternary ammonium salt solution In 500mL of the aforementioned mixed solution.

[0037] C) Weigh 1.5g of sodium carbonate and 2.9g of sodium bicarbonate, add them to 800mL of deionized water, stir to dissolve and dilute to 1000mL, adjust the pH to 9.6, take 5g of dried silk in step A) and dissolve in 50mL of carbon In the acidic acid buffer solution, stir until it is completely dissolved, then add 50 mL o...

Embodiment approach 2

[0048] A) Weigh 15g of mulberry silk into deionized water at a bath ratio of 1:50, add 3g of sodium carbonate, and degummed at a constant temperature of 95°C in a water bath for 60 minutes. After the end of the water bath, add acetic acid to adjust the pH to neutral. Repeat the operation twice. The degummed mulberry silk was added to 200 mL of deionized water and soaked for 2 hours, and then dried at 60°C.

[0049] B) Mix 500mL isooctane and 1000mL n-octanol, stir and mix well for later use, weigh 0.4mol sodium bis(2-ethylhexyl) succinate sulfonate, 0.4mol polyvinylpyridine quaternary ammonium salt solution In 500mL of the aforementioned mixed solution.

[0050] C) Weigh 1.5g of sodium carbonate and 2.9g of sodium bicarbonate, add to 800mL of deionized water, stir to dissolve and dilute to 1000mL, adjust the pH to 9.6, take 5g of dried silk in step A) and dissolve in 50mL of carbon In the acidic acid buffer solution, stir until it is completely dissolved, then add 50 mL of the sol...

Embodiment approach 3

[0061] A) Weigh 15g of mulberry silk and add it to deionized water at a bath ratio of 1:50. Add 3.75g of sodium carbonate and degumming at a constant temperature of 95°C in a water bath for 60 minutes. After the water bath is over, add acetic acid to adjust the pH to neutral. Repeat the operation twice . The degummed mulberry silk was added to 200 mL of deionized water and soaked for 2 hours, and then dried at 60°C.

[0062] B. Mix 500mL isooctane and 1000mL n-octanol, stir and mix well for later use, weigh 0.5mol sodium bis(2-ethylhexyl) succinate sulfonate, 0.5mol polyvinylpyridinium quaternary ammonium salt solution In 500mL of the aforementioned mixed solution.

[0063] C) Weigh 1.5g of sodium carbonate and 2.9g of sodium bicarbonate, add them to 800mL of deionized water, stir to dissolve and dilute to 1000mL, adjust the pH to 9.6, take 5g of dried silk in step A) and dissolve in 50mL of carbon In the acidic acid buffer solution, stir until it is completely dissolved, then ad...

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PUM

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Abstract

The invention relates to the field of archaeological detection and discloses a preparation method of a bombyx mori silk fibroin monoclonal antibody. The preparation method comprises the following steps: extracting bombyx mori silk fibroin by adopting an isooctane, n-octyl alcohol, bi(2-ethylhexyl) sodium sulfosuccinate and polyvinyl pyridine quaternary ammonium salt mixed system, injecting the bombyx mori silk fibroin serving as a complete antigen into the bodies of rabbits, selecting the rabbits with high immunizing potency, fusing splenic lymphocytes and myeloma cells, cultivating the obtained hybridoma cells, selecting strains with positive secretory antibodies, high secretion ability and good cell growth state by an indirect ELISA method, cloning by a limited dilution method until the antibody secretion positive rate of hybridoma cell growth is 100 percent, performing big turn production on the screened cells, and purifying the obtained monoclonal antibody by a chromatographic column. The antibody prepared by the method has high specificity, and is simple and rapid in operation during the application process and high in detection accuracy.

Description

Technical field [0001] The invention relates to the field of archaeological detection, in particular to a method for preparing a mulberry silk fibroin monoclonal antibody. Background technique [0002] Silk is a kind of natural polymer protein fiber, mainly divided into two categories: tussah silk and mulberry silk. In view of the fact that the origin of mulberry silk has been shrouded in the mist, a more scientific method is urgently needed to identify the unearthed silk. The conventional detection methods of silk mainly include Fourier infrared spectroscopy, Raman spectroscopy, X-ray diffraction, etc. However, long-term use of silk in the environment of tombs or ruins will be affected by various factors, resulting in protein degradation and macromolecular chain breakage. It is difficult to detect the presence of silk fibroin by some traditional methods. Therefore, how to use the advanced methods of natural science to establish a silk fabric micro-mark detection technology sy...

Claims

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Application Information

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IPC IPC(8): C07K16/18G01N33/68G01N33/577
CPCC07K16/18G01N33/577G01N33/68G01N2333/43578
Inventor 王秉李津梁军龙陈茹茹陈博逸
Owner ZHEJIANG SCI-TECH UNIV
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