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Hepatitis B virus PreS1 antigen enzyme-linked immunoassay kit employing one-step method

A hepatitis B virus, enzyme-linked immunosorbent technology, applied in measuring devices, instruments, scientific instruments and other directions, can solve problems such as insufficient difference between batches of ELISA plates, and achieve the effects of simple and fast operation, low cost, and good repeatability

Inactive Publication Date: 2013-04-17
WUHAN KANGZHU BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The technical problem to be solved by the present invention is to solve the problem of insufficient batch-to-batch difference in traditional enzyme-labeled plates, and to design and establish a one-step detection kit for PreS1 antigen

Method used

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  • Hepatitis B virus PreS1 antigen enzyme-linked immunoassay kit employing one-step method
  • Hepatitis B virus PreS1 antigen enzyme-linked immunoassay kit employing one-step method
  • Hepatitis B virus PreS1 antigen enzyme-linked immunoassay kit employing one-step method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] 1. The production steps of preparing the one-step hepatitis B virus pre-S1 protein ELISA kit:

[0034] 1. Biotinylated antibody preparation:

[0035] (1) Purification of coated antibody (HBsAb): immunize guinea pigs with recombinant antigen to obtain anti-PreS1 antiserum or immunize Balb / C mice to obtain positive mouse splenocytes fused with myeloma cells to form hybridoma cells, and screen positive clones to establish strains. Ascites is obtained; the antiserum or ascites is purified by salting out, ion exchange chromatography, or affinity chromatography to obtain pure anti-PreS1 antibody with a purity of more than 95%. The potency is greater than 100,000.

[0036] (2) Biotin-conjugated anti-PreS1 antibody:

[0037] Dissolve biotin (BNHS) in N, N-dimethylamide (DMF) to 1 mg / Ml by conventional method, and dilute the purified anti-PreS1 antibody to 1-2 mg with 0.1 mol / L NaHCO3 with a pH value of 9.0 / mL, according to the volume ratio of BNHS and antibody is 1:8 or the...

Embodiment 2

[0080] Operation steps of one-step hepatitis B virus pre-S1 protein ELISA kit

[0081] 1. The operation steps are as follows:

[0082] (1) Take out the coated strip well or plate and return to room temperature.

[0083] (2) Dosing: Dilute the concentrated washing solution (20X) with distilled water or deionized water for later use (1ml of 20 times thick washing solution + 19ml of distilled water is the working solution).

[0084] (3) Adding samples: add 50ul of the serum to be tested in each well, each plate is provided with a positive control well (50ul), a negative control well (50ul), a blank control well (add distilled water 50ul), and then except the blank control well, Add 50ul of enzyme labeling solution to each well.

[0085] (4) Incubation: seal the plate with a sealing film and incubate at 37°C for 30 minutes.

[0086] (5) Washing: Carefully peel off the sealing film, dry the liquid in the hole, wash it with washing liquid for 5 times, and buckle dry.

[0087] (6...

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Abstract

The invention provides a hepatitis B virus PreS1 antigen enzyme-linked immunoassay kit employing a one-step method. The concentration of HBs-Ag-PreS1 protein in blood serum of a sufferer can be exclusively detected. The kit comprises an elisa plate enveloped reaction strip, an enzyme labeling pre-S1 antibody, a positive control solution, a negative control solution, a bottle of 20-fold wash buffer, a bottle of substrate buffer A, a bottle of substrate buffer B and a bottle of stop buffer (2NH2SO4), wherein the elisa plate enveloped reaction strip is prepared from a primary ingredient avidin which is enveloped in advance and then added to a biotin anti-hepatitis B virus PreS1 monoclonal antibody or an anti-HBS antibody. The kit is good in uniformity, specificity and sensitivity, rapid and convenient to collect, can obtain an experiment result about 30 minutes by the one-step method, and is time-saving (the experiment can be fastest finished by 6-8 hours in a PCR (polymerase chain reaction) method) compared with the PCR detection method. The positivity detected by clinical PreS1 protein and the HBV-DNA positivity detected by PCR have good coincidence rate.

Description

technical field [0001] The invention belongs to the technical field of biological preparations. In particular, it relates to a one-step hepatitis B virus pre-S1 (PreS1) antigen (Ag) enzyme-linked immunoassay kit and a preparation method. Background technique [0002] my country is a high-incidence area of ​​hepatitis B virus. Hepatitis B has become a social public problem that endangers the health of our people. Serum markers of hepatitis B virus (HBVM) as a routine item for hepatitis B virus diagnosis can no longer meet clinical requirements. HBV - DNA, viral surface antigen (HBsAg), hepatitis B virus pre-S1 (PreS1Ag) antigen are all used as evidence of HBV infection and replication in humans. [0003] HBV is a hepadnavirus, composed of an incomplete double-stranded DNA, about 3200 amino acids, with 4 open reading frames: S region, C region, P region, and X region. The S gene region can be further divided into three segments, which are the pre-S1 (preS1) region, the pre-S2...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/576
Inventor 张年
Owner WUHAN KANGZHU BIOTECH
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