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Hepatitis b surface antigen and antibody detection method

A hepatitis B and surface antigen technology, applied in the field of new fluorescent detection reagents, can solve the problems of inability to suppress the virus and low activity

Inactive Publication Date: 2008-12-31
BEIJING FANBO BIOCHEM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although neotopes are definitely important, or they play an important dominant immune role, sometimes the antibodies formed are completely incapable of suppressing the virus due to monomeric immunity, or, perhaps, are too inactive (as measured by neutralization of viral infection)

Method used

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  • Hepatitis b surface antigen and antibody detection method
  • Hepatitis b surface antigen and antibody detection method
  • Hepatitis b surface antigen and antibody detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Synthesis of embodiment 1 fluorescent HRP substrate

[0078] According to the attached figure 1 The reaction scheme shown synthesizes ADHP.

[0079] 25g of Resorufin (Resorufin) was suspended in 100mL of acetic anhydride at room temperature. During stirring, 3 mL of triethylamine and 60 g of stannous chloride were slowly added to this suspension. The reaction mixture was heated and stirred at 100-130°C until the mixture changed from a red suspension to a yellow-orange solution. The reaction mixture was cooled to 30-40°C and poured into ice water. Filter with suction and wash with a large amount of water to obtain a precipitate. The crude product was further purified by recrystallization to obtain 20 g of pure compound O, O, N-triacetyldihydroresorufin.

[0080] Suspend 10 g of O, O, N-triacetyldihydroresorufin in methanol at room temperature. With stirring, slowly add 10% K to this suspension 2 CO 3 , the mixture was stirred rapidly at room temperature until t...

Embodiment 2

[0081] Example 2. Synthesis of HRP-labeled polypeptides

[0082] In this example, a covalent linker between a synthetic polypeptide mainly containing HBsAg (hepatitis B surface antigen) determinant and horseradish peroxidase (HRP) was prepared. The selection of the polypeptide amino acid sequence with the HBsAg determinant has been reported by A.R.Neurath, N.Strick and N.R.Oleszko in the following document: "Localization of a Hepatitis B Surface Antigen Determinant Deduced FromResults of Chemical Modifications", J. Virol. Methods, 3, 115-125, 1981. Wherein the amino acid sequence is as follows:

[0083] P135-155A:

[0084] Pro-Ser-Cys-Cys-Cys-Thr-Lys-Pro-Thr-Asp-Gly-Asn-Cys-Thr-Cys-Ile-Pro-Ile-Pro-Ser-Ser;

[0085] P135-155B:

[0086] Pro-Ser-Cys-Cys-Cys-Thr-Lys-Pro-Ser-Asp-Gly-Asn-Cys-Thr-Cys-Ile-Pro-Ile-Pro-Ser-Ser;

[0087] P135-143: Pro-Ser-Cys-Cys-Cys-Thr-Lys-Pro-Thr; or

[0088] P143-155: Ser-Asp-Gly-Asn-Cys-Thr-Cys-Ile-Pro-Ile-Pro-Ser-Ser.

[0089] The horserad...

Embodiment 3

[0092] Example 3. Fluorescence assay of HRP-polypeptide linker activity

[0093] Taking the HRP-polypeptide linker P135-155A-HRP as an example, the fluorescence values ​​of different concentrations of the HRP-polypeptide linker were measured.

[0094] Take the DMSO stock solution of 5mM ADHP, with 50mM containing 1mM H 2 o 2 Dilute to 100 μM in sodium phosphate buffer (pH 7.4). These assay solutions were incubated at room temperature for 30 min in the presence of different concentrations of HRP-polypeptide conjugates. An automatic microplate reader was used to detect the fluorescence at an excitation wavelength of 530±25nm and an emission wavelength of 590±35nm. Fluorescence without the HRP control response was background fluorescence, and the background fluorescence value was subtracted from each value. figure 2 It was shown that ADHP can detect very low concentrations of HRP-tagged polypeptides.

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Abstract

The invention discloses a covalent conjugate of polypeptide and horseradish peroxidase (HRP), the covalent conjugate is reacted with a novel fluorescent substrate of the HRP to generate very strong fluorescent signals, the HRP-labeled polypeptide can be used for the diagnosis of hepatitis B surface antigen or antibody, and the conjugate is a product formed by the covalent conjugation of the polypeptide with less than 50 amino acids and the horseradish peroxidase (HRP). The HRP-labeled polypeptide with the covalent bond connection is easily identified and combined by the antibody, the existence of the hepatitis B surface antibody or antigen in a sample can be measured through various modes. The HRP-labeled polypeptide conjugate of the invention is reacted with the fluorescent substrate ADHP of the HRP, thereby being used for the fluorescence detection of the hepatitis B surface antigen and antibody.

Description

technical field [0001] The present invention relates to a novel fluorescence detection reagent for detecting hepatitis B surface antigen or antibody, a kit containing the reagent, and a fluorescence detection method for rapidly measuring hepatitis B surface antigen and antibody by using the reagent or kit; The fluorescent detection reagents used are horseradish peroxidase (HRP)-labeled polypeptide and N-acetyl-3,7-dihydroxyphenazine (ADHP). The detection method of the invention can be used for fluorescent detection of hepatitis B surface antigen and antibody. Background technique [0002] Antigen (Ag) is a substance that causes a special reaction after the invasion of a foreign substance is detected. Antigens can be proteins, polysaccharides, nucleic acids, fats, synthetic polymers and microorganisms, etc., and antigens can be characterized by a certain extremely small size. Antigens can be as small as 1000 Daltons or as large as 1 million Daltons. [0003] After the fore...

Claims

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Application Information

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IPC IPC(8): G01N33/52G01N33/576
Inventor 杜池敏郑超斌第五振军
Owner BEIJING FANBO BIOCHEM
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