HBV-DNA kit for quantitative enzyme-linked measurment of heaptitis B core antigen

A technology of core antigen and ELISA, which is applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of unable to reflect the real state of the virus, the immune response of patients, the existence of hepatitis B virus, and the complicated operation. Achieve the effect of stable specificity, easy operation and high sensitivity

Inactive Publication Date: 2007-10-03
BEIJING BIOKIT
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  • Abstract
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  • Application Information

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Problems solved by technology

However, the "two-and-a-half" reagents cannot reflect the existence of hepatitis B virus well and cannot be quantified; although the HBV DNA fluorescent quantitative PCR kit can quantify the copy number of HBV DNA, it cannot reflect the true state of the virus and the immune response of patients, and the price expensive and complex

Method used

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  • HBV-DNA kit for quantitative enzyme-linked measurment of heaptitis B core antigen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1: Preparation of Hepatitis B Core Antigen ELISA Quantitative Assay Kit for HBV DNA

[0056] 1. Preparation and purification of anti-HbsAg polyclonal antibody:

[0057] Immune rabbits (purchased from Beijing Experimental Animal Research Center) with HbsAg (purchased from Beijing Medico Biotechnology Co., Ltd.) to obtain antiserum; add saturated ammonium sulfate to the antiserum to a final concentration of 33%, and stir for 1 hour; Centrifuge at 4000g for 15 minutes, remove the supernatant; re-dissolve the precipitate with the same volume of saturated ammonium sulfate as above and remove the precipitate; centrifuge at 4000g for 15 minutes, discard the supernatant, dissolve and dialyze with an appropriate phosphate buffer to obtain the crude polyclonal antibody, and then DEAE column chromatography removes impurity proteins (DEAE fiber chromatography column is purchased from PHAMACIAL company), pre-equilibrates the DEAE column with PB buffer, then elutes, collects ...

Embodiment 2

[0083] Embodiment 2: The usage method of HBcAg quantitative determination HBV DNA

[0084] 1. Detection

[0085] 1. Take out the HBV surface antibody polyantibody pre-coated reaction strip from the kit, place it at room temperature for a period of time, until the temperature of the strip is consistent with room temperature, wash twice with distilled water, and pat dry.

[0086] 2. Add 100 microliters of each concentration of HBV DNA serum standard, test serum, negative serum, and positive serum to the reaction wells, set a blank well for each experiment, add 100 microliters of buffer solution, and react at 45°C for 30 minutes.

[0087] 3. Discard the liquid in the well, wash 7 times with distilled water, and pat dry.

[0088] 4. Add shell-opening agent: add 3 drops of shell-opening agent to each well (not add to blank well) and incubate at 45°C for 30 minutes (note: do not pour this solution away).

[0089] 5. Take out the HBV core antibody (HBcAb) polyclonal antibody pre-co...

Embodiment 3

[0100] Embodiment 3: HBcAg ELISA quantitative determination of HBV DNA application and fluorescent quantitative PCR detection of HBV DNA comparison

[0101] 1. Sources of clinical specimens:

[0102] The inventor collected 152 positive sera from the Huainan Institute of Liver Diseases and Beijing You'an Hospital, including 79 cases of major three yang, 73 cases of small three yang, and 43 completely negative sera; a total of 195 serums. These sera were then measured simultaneously with the method of the present invention and a commercial fluorescent quantitative HBV DNA PCR detection kit.

[0103] 2. Determination method:

[0104] HBcAg ELISA for quantitative determination of serum HBV DNA was operated according to the instruction manual (self-made kit) (the method was the same as in Example 2), and the fluorescent quantitative HBV DNA PCR assay was operated according to the manual of the fluorescent quantitative PCR detection kit produced by Guangzhou Zhongshan Daan Gene Tec...

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Abstract

The present invention discloses HBV-DNA kit for quantitative enzyme-linked immunological measurement of hepatitis B core antigen and its preparation process, and belongs to the field of molecular biology and immunological diagnosis reagent. The kit includes: fluorescent quantitative HBV-DNA serum standard, HBsAb polyclonal antibody pre-coated standard enzyme reaction strip, HBcAb polyclonal antibody pre-coated standard enzyme reaction strip, diductor, enzyme labeling core antibody, substrate developer, positive contrast, negative contrast and terminating liquid. The present invention can measure HBV-DNA content in serum specifically and accurately, and has the advantages of simple operation, high sensitivity, high specificity, high stability, etc.

Description

technical field [0001] The invention relates to a hepatitis B core antigen ELISA quantitative assay kit for HBV-DNA and a preparation method thereof, belonging to the field of molecular biology and immunological diagnostic reagents. Background technique [0002] Hepatitis B virus (HBV) is the main pathogenic factor of hepatitis, and there are about 150 million HBV carriers in the world. Hepatitis B core antigen (HBcAg) exists in hepatitis B virus particles, not in the blood in a free state, and is a direct indicator of hepatitis B virus activity. But for a long time, clinicians have judged the condition of hepatitis B patients based on the test results of the "two-and-a-half" five kits, and used the HBV DNA PCR fluorescent quantitative kit to measure the serum hepatitis B virus DNA to understand the efficacy of drugs. However, the "two-and-a-half" reagents cannot reflect the existence of hepatitis B virus well and cannot be quantified; although the HBV DNA fluorescent quant...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/576G01N33/543G01N33/533
Inventor 陈禹保
Owner BEIJING BIOKIT
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