Double-labeling time-resolved fluorescence immunoassay method and kit for HIV (human immunodeficiency virus) antibody and HIV-1p24 antigen

A time-resolved fluorescence, hiv-1p24 technology, applied in the direction of analytical materials, biological material analysis, measurement devices, etc., can solve the problems of shortening the HIV detection window period, low HIV detection sensitivity, poor stability, etc., and achieve simple detection methods and high sensitivity High and stable effect

Inactive Publication Date: 2014-06-18
GUANGZHOU FENGHUA BIOENG
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The method of the present invention can solve the technical problems of low sensitivity, poor stability and cumbersome operation in HIV detection in the prior art. In particular, HIV antibodies and antigens can be quantitatively distinguished, the window period of HIV detection can be shortened, and it can also be used for short-term anti-inflammatory Evaluation of the value of the effect of viral therapy

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  • Double-labeling time-resolved fluorescence immunoassay method and kit for HIV (human immunodeficiency virus) antibody and HIV-1p24 antigen
  • Double-labeling time-resolved fluorescence immunoassay method and kit for HIV (human immunodeficiency virus) antibody and HIV-1p24 antigen
  • Double-labeling time-resolved fluorescence immunoassay method and kit for HIV (human immunodeficiency virus) antibody and HIV-1p24 antigen

Examples

Experimental program
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Effect test

Embodiment 1

[0032] Embodiment 1: the preparation of kit

[0033] The kit of this embodiment 1 includes: a solid phase carrier coated with HIV antigen and HIV-1p24 monoclonal antibody at the same time, calibrator, biotin-labeled HIV-1p24 monoclonal antibody, HIV antigen labeled with lanthanide 1, Lanthanide 2-labeled Streptavidin, Assay Buffer, Wash Concentrate, and Enhancement Solution. Its specific preparation method is as follows:

[0034] ① Preparation of antigen and antibody solid-phase carrier: HIV recombinant antigen (including HIV gp120, gp41, gp36 epitopes, which can detect HIV-1 type, HIV-2 type, HIV-10 subgroup) and HIV-1p24 monoclonal antibody Coating buffer (you can use pH9.6, 50mmol / L carbonic acid buffer; pH4.5, 20mmol / L phosphate buffer; pH7.8, 50mmol / L Tris-HCl buffer or pH4.5, 50mmol / L citrate buffer, etc.) were diluted to 0.1, 0.5, 1, 2, 5, 10μg / mL concentration as coating solution, and HIV antigen coating solution and HIV- 100 μl / well of 1p24 antibody coating solutio...

Embodiment 2

[0045] Example 2: Detection and Analysis of HIV Antibody and HIV-1p24 Antigen

[0046] ① Reagent preparation

[0047] Antigen and antibody solid-phase carrier: Equilibrate the required amount of antigen and antibody solid-phase carrier prepared in Example 1 to room temperature (20-25°C). The remaining antigens and antibody solid phase carriers were placed in ziplock bags in time to be sealed and stored at 2-8°C.

[0048] Washing liquid: Mix 40ml of concentrated washing liquid and 960ml of purified water in a clean container in Example 1, and use it as a working washing liquid for subsequent use.

[0049] Marker working solution: the Eu prepared in Example 1 3+ Labeled HIV recombinant antigen and Sm 3+ Labeled streptavidin and experimental buffer were added into the same clean disposable container at a volume ratio of 1:1:20 and mixed well, prepared within 30 minutes before use, and used up in the current experiment.

[0050] Biotin-labeled antibody working solution: Add bi...

Embodiment 3

[0054] Embodiment 3: detection result analysis

[0055] (1) Detection performance of HIV antibody

[0056] ① Conformity rate of negative reference products: 20 national negative reference products for HIV antibody detection, no more than 2 positive reactions (≥18 / 20).

[0057] ②Conformity rate of positive reference products: 18 national HIV1 antibody positive reference products were detected, all of which were positive reactions (18 / 18), and the detection fluorescence values ​​of samples P11 and P12 were P12≥P11; 2 HIV2 antibody positive reference products , were positive (2 / 2).

[0058] ③Minimum detection limit: 6 copies of the national minimum detection limit reference product were tested, no less than 3 copies were positive and the matrix serum S1 was negative.

[0059] ④Linearity: After testing the reference products of the enterprise, after statistical analysis of the measured values ​​of 5 samples from L1 to L5, the linear correlation coefficient r between the measured...

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Abstract

The invention discloses a double-labeling time-resolved fluorescence immunoassay method and kit for HIV (human immunodeficiency virus) antibody-HIV-1p24 antigen. The analytical method mainly comprises the steps of preparing a solid phase carrier coated with an HIV recombinant antigen and an HIV-1p24 monoclonal antibody simultaneously; preparing biotin-labeled HIV-1p24 monoclonal antibody; preparing lanthanide 1-labeled HIV recombinant antigen; preparing lanthanide 2-labeled streptavidin; adding a calibrator containing HIV standard antibody and HIV-1p24 standard antigen or a sample to be tested into the solid phase carrier coated with the antigen and the antibody, adding the biotin-labeled HIV-1p24 antibody, incubating, washing, then adding the lanthanide 1-labeled HIV antigen and the lanthanide 2-labeled streptavidin, incubating again, washing, and adding enhancement solution for fluorescence detection. The analytical method overcomes the difficulty that the antigen and the antibody cannot be distinguished in the existing joint detection for HIV antigen and antibody, realizes simultaneous and quantitative detection of the HIV antibody and the HIV-1p24 antigen and therefore shortens the window phase of HIV detection.

Description

technical field [0001] The present invention relates to the analysis and detection technology of human immunodeficiency virus (human immunodeficiency virus, HIV), in particular to an HIV antibody and HIV-1p24 antigen double-labeled time-resolved fluorescent immunoassay method and kit. Background technique [0002] Human immunodeficiency virus (HIV), first discovered in the United States in 1981, is a lentivirus that infects cells of the human immune system and is a type of retrovirus. The virus destroys the immune ability of the human body, leading to the loss of immune system resistance, which leads to the occurrence of various diseases and cancers in the human body, and eventually leads to AIDS (acquired immunodeficiency syndrome, acquired immunodeficiency syndrome, AIDS) . [0003] The HIV viral genome consists of two identical positive-strand RNAs, each about 9.2-9.8kb in length, encoding at least nine proteins, which can be divided into three categories: structural pro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/571G01N33/533
CPCG01N33/577G01N33/533G01N33/56983G01N33/571G01N2333/16
Inventor 谭玉华李奕辉陈建起范主桥
Owner GUANGZHOU FENGHUA BIOENG
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