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Kit for detecting HIV-1 p24 antigen and application of kit

An hiv-1p24 and kit technology, which is applied in the field of kits for determining HIV-1p24 antigens, can solve the problems of cumbersome and complicated detection process, poor detection result accuracy, poor detection sensitivity, etc., and achieve accurate and accurate detection results. Enhanced, highly sensitive effect

Active Publication Date: 2017-05-31
石家庄凯达生物工程有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] One of the purposes of the present invention is to provide a test kit for detecting HIV-1 p24 antigen, so as to solve the problems of poor detection sensitivity and poor accuracy of detection results in current HIV-1 p24 antigen detection
[0006] The second object of the present invention is to provide the application of the test kit for detecting HIV-1 p24 antigen, so as to solve the problem that the existing detection technology is not only less accurate, but also the detection process is complicated, time-consuming and labor-intensive.

Method used

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  • Kit for detecting HIV-1 p24 antigen and application of kit
  • Kit for detecting HIV-1 p24 antigen and application of kit
  • Kit for detecting HIV-1 p24 antigen and application of kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1 immunization mouse obtains monoclonal coating antibody and monoclonal antibody

[0032] (1) Immunized mice

[0033]Select female Balb / c mice aged 6-8 weeks, and immunize Balb / c mice with p24 antigen fragments from HIV-1BC subtype or HIV-1AE subtype, and the number of immune mice is 5 times (traditional immunization method 3 times), multi-point subcutaneous, muscle, and intraperitoneal immunization, 50ug / monkey, the time interval gradually increased, from 15 days to 40 days, and finally attacked 2 times (traditional attack once), the antigen was 30ug / bird, and finally Two days after the first challenge to the mice, the spleens of the mice were taken for cell fusion to prepare monoclonal antibody hybridoma cell lines.

[0034] (2) Preparation of immune spleen cells and myeloma cells

[0035] The immunized Balb / c mice were taken, and the eyeballs were enucleated and bled. The mice were killed, the spleen was aseptically removed, and the spleen lymphocyte su...

Embodiment 2

[0059] The preparation of embodiment 2 kit

[0060] 1. Preparation of ELISA plate

[0061] (1) Configure each solution:

[0062] Coating solution: take 1.59g of Na 2 CO 3 and 2.93 g of NaHCO 3 , after dissolving with a small amount of water for injection, then dilute to 1000mL with water for injection, and its pH value is 9.6;

[0063] Washing solution (20 times concentrated phosphate buffer): weigh 58.0g Na 2 HPO 4 12H 2 O, 5.93g NaH 2 PO 4 2H 2 O, 148g NaCl, 10mL Tween-20 and 1g of 2-chloroacetamide, after mixing, dissolve with water for injection, and set the volume to 1000mL, which is 20 times concentrated phosphate buffer solution; pH value is 7.4;

[0064] Blocking solution: Weigh 12.11g of Tris, add HCl to adjust the pH value to 7.2, 10g BSA, 20g sucrose, 20g sodium caseinate and 0.1g sodium azide, mix and dissolve with water for injection, and dilute to 1000mL.

[0065] (2) Using a 96-well microplate as the bottom plate, dilute the mouse anti-HIV-1 p24 monoc...

Embodiment 3

[0086] Qualitative and quantitative detection method of embodiment 3 HIV-1 p24 antigen

[0087] (1) Take the kit prepared in Example 2 out of the refrigerator and place it at room temperature for 30 minutes;

[0088] (2) Dilute washing solution: dilute 50mL washing solution to 1000mL with distilled water to obtain 1 times washing solution;

[0089] (3) Qualitative determination:

[0090] A. Take an ELISA plate, number the wells corresponding to the samples to be tested in sequence, 1 blank well, 3 negative control (NC) wells and 2 positive control (PC) wells (10IU / mL) on each plate. If the assay sample is cell culture supernatant, cell culture medium should be used as a negative control for 3 wells;

[0091] B. Add 25 μL of sample diluent to each reaction well, and do not add sample diluent to the blank well;

[0092] C. According to the number, add 100 μL of the sample to be tested, 100 μL of the diluted negative control, and positive control (10IU / mL) into the reaction we...

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Abstract

The invention discloses a kit for detecting an HIV-1 p24 antigen. The kit comprises an enzyme linked immunosorbent assay (ELISA) plate, a sample diluent, an enzyme conjugate, a negative control substance, a positive control substance, a substrate solution, a developing solution, a stop solution and a cleaning solution, wherein the ELISA plate is coated with an anti-mouse HIV-1 p24 monoclonal coating antibody, and the anti-mouse HIV-1 p24 monoclonal coating antibody is a 2-C6 antibody; the sample diluent contains a biotin-labeled anti-mouse HIV-1 p24 monoclonal antibody, and the biotin-labeled anti-mouse HIV-1 p24 monoclonal antibody is a biotin-labeled 11-A4 antibody. In addition, the invention also discloses application of the kit. Experiments show that the kit is easy to operate and accurate and clear in detection result, and the antigen sensitivity reaches 1.25IU / mL and is remarkably higher than the detection sensitivity of a conventional ordinary HIV-1 p24 detection kit when the kit is used for detecting the HIV-1 p24 antigen.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a kit for measuring HIV-1 p24 antigen and its application. Background technique [0002] AIDS is Acquired Immunodeficiency Syndrome (AIDS), which is caused by human immunodeficiency virus (HIV) infection. The HIV virion is spherical, with a diameter of 100-120 nm. The outer layer of the virus is a lipoprotein envelope, in which are embedded virus-specific glycoproteins gp120 and gp41; gp41 is a transmembrane protein, and gp120 is located on the surface, forming the spikes on the surface of the envelope. , and binds to gp41 through non-covalent interaction. Inside the virus is a nucleocapsid with 20-sided symmetry, and the core of the virus contains viral RNA, reverse transcriptase and nucleocapsid protein. The viral genome is two identical positive-strand RNAs, each about 9.2-9.8kb in length. The most specific capsid protein, p24, is encoded by the gag gene. p24 is the main core ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577
CPCG01N33/56988
Inventor 王强王从印张红中王惠芬姜少灏苏彦辉房桂珍张剑刘东刚
Owner 石家庄凯达生物工程有限公司
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