Method for preparing tussah silk fibroin protein monoclonal antibody

A technology of tussah silk fibroin and monoclonal antibody, applied in anti-animal/human immunoglobulins, material testing products, measuring devices, etc., can solve the problem of weak antigen binding specificity, low polyclonal antibody purity, and physical and chemical properties. Inhomogeneous and other problems, to achieve the effect of excellent extraction effect, short production cycle and high sensitivity

Active Publication Date: 2017-10-20
ZHEJIANG SCI-TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Polyclonal antibodies to tussah silk fibroin protein have appeared at present. The specificity of the antibody depends on the determinant of the antigen molecule. It is very difficult to separate the antibodies, and polyclonal antibodies have problems such as low purity, uneven physical and chemical properties, and weak binding specificity to antigens.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031]A) Weigh 10 g of tussah silk and add it to deionized water with a bath ratio of 1:25, adjust the pH to 2.5 with hydrochloric acid, degumming in a water bath at 100°C for 60 minutes, and add NaHCO after the water bath is over 3 Adjust the pH to neutral and repeat the operation 3 times. The degummed tussah silk was soaked in 200 mL deionized water for 2 h, and then dried at 60 °C.

[0032] B) Weigh 10 g FeCl 2 , add 50 mL of deionized water to dissolve, then add 15 mL of tetrasodium iminodisuccinate dropwise, mix well and dilute to 100 mL.

[0033] C) Weigh 1 g of the silk fibroin sample obtained in step A), add 15 mL of the mixed solution prepared in step B), stir and incubate on a 60°C water bath for 60 min. After it was taken out and cooled, the solution was treated under 30 kHz ultrasonic wave for 30 min, and at the same time, the pH was slowly adjusted to 8.5 with acetic acid. Put the neutralized solution into a dialysis bag (molecular weight cut-off 8000) for dial...

Embodiment 2

[0042] A) Weigh 10 g of tussah silk and add it to deionized water at a bath ratio of 1:25, adjust the pH to 3 with hydrochloric acid, degumming in a water bath at 100°C for 60 minutes, and add NaHCO after the water bath is over 3 Adjust the pH to neutral and repeat the operation 3 times. The degummed tussah silk was soaked in 200 mL deionized water for 2 h, and then dried at 60 °C.

[0043] B) Weigh 10 g FeCl 2 , add 50 mL of deionized water to dissolve, then add 15 mL of tetrasodium iminodisuccinate dropwise, mix well and dilute to 100 mL.

[0044] C) Weigh 1 g of the silk fibroin sample obtained in step A), add 15 mL of the mixed solution prepared in step B), stir and incubate on a 60°C water bath for 60 min. After taking it out and waiting for cooling, the solution was treated under 50 kHz ultrasonic wave for 30 min, and at the same time, the pH was slowly adjusted to 9 with acetic acid. Put the neutralized solution into a dialysis bag (molecular weight cut-off 8000) for...

Embodiment 3

[0053] A) Weigh 10 g of tussah silk and add it to deionized water at a bath ratio of 1:25, adjust the pH to 3 with hydrochloric acid, degumming in a water bath at 100°C for 60 minutes, and add NaHCO after the water bath is over 3 Adjust the pH to neutral and repeat the operation 3 times. The degummed tussah silk was soaked in 200 mL deionized water for 2 h, and then dried at 60 °C.

[0054] B) Weigh 10 g FeCl 2 , add 50 mL of deionized water to dissolve, then add 15 mL of tetrasodium iminodisuccinate dropwise, mix well and dilute to 100 mL.

[0055] C) Weigh 1 g of the silk fibroin sample obtained in step A), add 15 mL of the mixed solution prepared in step B), stir and incubate on a 60°C water bath for 60 min. After it was taken out and cooled, the solution was treated under 40kHz ultrasonic wave for 30min, and at the same time, the pH was slowly adjusted to 8.5 with acetic acid. Put the neutralized solution into a dialysis bag (molecular weight cut-off 8000) for dialysis,...

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PUM

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Abstract

The present invention relates to THE archaeological detection field, and discloses a method for preparing a tussah silk fibroin protein monoclonal antibody, a FeCl2 and tetrasodium iminodisuccinate mixed solution system is used for extraction of tussah silk fibroin, the tussah silk fibroin as a complete antigen is injected into the body of rabbits, a rabbit with high immunizing potency is selected, splenic lymphocytes and myeloma cells of the rabbit with the high immunizing potency are fused, after obtained hybridoma is cultured, a bacterial strain with positive secreted antibodies, strong antibody secreting ability and good cell growth conditions can be selected by indirect ELISA method, the bacterial strain is cloned by limiting dilution method until the secreting positive rate of the antibody with grown hybridoma is 100%, the selected cells are used for large-scale production, and then a chromatographic column is used for purification of the monoclonal antibody. The antibody prepared by the method has strong specificity, and in the application process, the operation is simple and quick, and the detection accuracy is high.

Description

technical field [0001] The invention relates to the field of archaeological detection, in particular to a method for preparing tussah silk fibroin monoclonal antibody. Background technique [0002] Silk is a natural polymer protein fiber, mainly divided into two categories: domestic silkworm silk and tussah silk. my country is the first country to utilize tussah silkworms and breed tussah silkworms, but its origin has been shrouded in fog, and a more scientific method is urgently needed to identify unearthed silk fabrics. The conventional detection methods of silk mainly include Fourier transform infrared spectroscopy, Raman spectroscopy, X-ray diffraction, etc. However, silk will be affected by many factors if it is kept in the environment of tombs or ruins for a long time, resulting in problems such as protein degradation and macromolecular chain breaks. , it is difficult to detect the existence of silk fibroin by some traditional methods. Therefore, how to use advance...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18G01N33/68G01N33/577
CPCC07K16/18G01N33/68
Inventor 王秉李津梁军龙陈茹茹陈博逸
Owner ZHEJIANG SCI-TECH UNIV
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