Micro-molecule indirectly labeled dual-antigen sandwich method for determining hepatitis C virus total antibody

A hepatitis C virus, double antigen sandwich technology, applied in the direction of measuring devices, analytical materials, instruments, etc., to achieve the effect of improving sensitivity, good specificity, and improving detection sensitivity

Active Publication Date: 2006-12-27
SUZHOU SYM BIO LIFESCI CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there are no reports at home and abroad that the technology of detecting HCV total antibody by the double-antigen sandwich method using small molecules as indirect labels is used to detect human serum HCV total antibody

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Preparation of Example 1 Biotin-labeled HCV Antigen

[0042] Proceed as follows:

[0043] (1) Dialyze the HCV recombinant antigen against pH 7.4 100mM PBS (containing 3M urea) for 24 hours at room temperature (+20~+25°C), and change the medium twice, 2,000mL each time. Adjust the concentration to about 1 mg / mL.

[0044] (2) Take the required amount of Biotin-cap-NHS, dissolve it with DMF to 40 mg / mL, and immediately add it to the dialyzed HCV recombinant antigen solution according to the mass ratio of Biotin-cap-NHS and antigen of 1:2 , mix quickly. Let stand at room temperature (+20~+25°C) for 1 hour.

[0045] (3) Dialysis against pH 7.8, 50mM TSA (containing 3M urea) at room temperature (+20~+25°C) with sufficient stirring for 24 hours, and the medium was changed twice, 1,000mL each time. Purified biotin-labeled HCV antigen was obtained.

Embodiment 2E

[0046] Embodiment 2 Preparation of Eu-labeled streptavidin

[0047] Proceed as follows:

[0048] (1) Dissolve 1 mg of streptavidin in 2,000 μL of pure water, and dialyze against pH 9.6 100 mM CBS at room temperature (+20 to +25°C) for 24 hours, and change the medium twice, 2,000 mL each time.

[0049] (2) Take 1mg Eu-DTTA and dissolve it with 1000μL pH 9.5 100mM CBS. Add the completely dissolved Eu-DTTA into the dialyzed streptavidin solution (about 1 mg / mL), shake while adding, and let stand at room temperature (+20~+25°C) for 72 hours. The mass ratio of Eu-DTTA to streptavidin is about 1:1.

[0050] (3) Sephacryl S-200 HR (1.6×50cm) was used to separate Eu-SA from free Eu-DTTA, and eluted with pH 7.850mM TSA. The separation process was monitored by a nucleic acid and protein detector. Purified Eu-labeled streptavidin was obtained.

Embodiment 3

[0051] Example 3 Preparation of HCV antigen-coated microwell plate

[0052] Proceed as follows:

[0053] (1) Pipette an appropriate amount of HCV recombinant antigen into the required volume of coating buffer, mix well to avoid the generation of air bubbles, and prepare the HCV antigen coating solution;

[0054] (2) Add 100 μL of HCV antigen coating working solution to each well of the microwell plate, and let it stand at room temperature (+20~+25℃) for 24 hours;

[0055] (3) Wash twice; inject 300 μL of blocking buffer into each well, and let stand at room temperature (+20~+25°C) overnight (about 16 hours);

[0056] (4) Blot up the blocking buffer, add 300 μL of 4% (w / v) sucrose solution to each well, blot dry, put the plate in a spin dryer to spin dry for 5 minutes; turn on the freeze dryer to make the vacuum degree <50Pa, keep After 3 hours, stop the machine, put the dried reaction plate into a tin-platinum bag, heat seal it and store it at 2-8°C.

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PUM

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Abstract

The invention relates to a method for testing the total antibody of hepatitis C virus (HCV), based on dual-antibody interlayer method of small-molecule indirect mark, wherein said method is characterized in that: it uses small molecule matter as mark; uses dual-antigen interlayer method to test the total antibody of sample; and said method comprises: using small molecule material to mark HCV antigen; using HCV antigen to pack solid material; using single generate material to mark small molecule antibody or other material that combined with small molecule; the HCV antibody of sample can be reacted with HCV antigen of solid material surface and small molecule mark, to form dual-antigen interlayer composite; then reacting the small molecule with single report molecule, to make the composite with detected signal generate material. The invention can keep activity of antigen, with signal amplifying function and better specificity of dual-antigen interlayer mode, while it can detect variable kinds of antibodies, to improve the detecting sensitivity and specificity.

Description

1. Technical field: [0001] The invention relates to the field of in vitro immunodiagnosis, in particular to a method for measuring hepatitis C virus (HCV) total antibody based on small molecule indirect labeling double-antigen sandwich immunoassay. 2. Background technology: [0002] Hepatitis C is a worldwide disease, the global HCV infection rate is about 3% (170 million people); the prevalence rate of hepatitis C in my country is 3.2%, and the number of infected people exceeds 41 million. Because most patients with acute HCV infection have no symptoms or no obvious symptoms, and the virus is difficult to remove from the body, about 70-80% of the cases will turn into chronic HCV infection, which can lead to chronic inflammation, necrosis and fibrosis of the liver, and some patients may develop chronic HCV infection. The development of liver cirrhosis and even hepatocellular carcinoma (HCC) is extremely harmful to the health and life of patients. HCV infection has become a ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N33/543
Inventor 吴冯波欧阳海桥唐晓燕
Owner SUZHOU SYM BIO LIFESCI CO LTD
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