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Reaction carrier and kit for detecting Epstein Barr (EB) virus replication and transcription activator (Rta)-immunoglobulin G (IgG) antibody

A technology of Epstein-Barr virus and reaction carrier, which is applied in the field of reaction carrier and kit for detecting EB virus Rta-IgG antibody, and achieves the effects of high accuracy, easy operation and low cost

Active Publication Date: 2013-06-05
同昕生物技术(北京)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no kit for the quantitative detection of Epstein-Barr virus Rta-IgG antibody that can meet the above requirements on the market

Method used

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  • Reaction carrier and kit for detecting Epstein Barr (EB) virus replication and transcription activator (Rta)-immunoglobulin G (IgG) antibody
  • Reaction carrier and kit for detecting Epstein Barr (EB) virus replication and transcription activator (Rta)-immunoglobulin G (IgG) antibody
  • Reaction carrier and kit for detecting Epstein Barr (EB) virus replication and transcription activator (Rta)-immunoglobulin G (IgG) antibody

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Embodiment 1. Preparation of genetically recombinant Epstein-Barr virus Rta protein

[0055] Preparation: Inoculate the CHO / RTA cell strain obtained above with the preservation number: CGMCC6955 into a cell culture dish, and the complete culture solution of the cells must be supplemented with 0.1mmol / L hypoxanthine and 0.016mmol / L thymine, 10% Fetal bovine serum (Hangzhou Sijiqing Bioengineering Co., Ltd.), after the cells reached a density of about 90%, replaced with a serum-free medium without aminomethylpterate (MTX), collected the cell culture medium after 1-2 days, Proteins in cell culture medium were extracted by trichloroacetic acid precipitation.

[0056] Purification: Purify the target protein in the cell supernatant with GE's streptavidin sepharose high performance. According to the operation manual of BCA-100 Protein Quantification Kit (Beijing Saichi Biology, product number 300001-B), the test was carried out to quantitatively measure the expressed Epstein-...

Embodiment 2

[0058] Embodiment 2. The composition of kit of the present invention:

[0059] 1. ELISA plate coated with Rta protein antigen: the high-purity gene recombinant Rta protein is coated, and the coating concentration can be 0.05-0.25 μg / ml;

[0060] 2. The working dilution of HRP-labeled goat anti-human IgG can be 1:5000—1:80000;

[0061] 3. EB virus Rta-IgG calibrator solution: 6 bottles, 1ml / bottle, the concentrations are 0U / ml, 12.5U / ml, 25U / ml, 50U / ml, 100U / ml, 200U / ml;

[0062] 4. Sample diluent: 50mM phosphate buffer containing 0.3M NaCl, 25ml / bottle, 2 bottles;

[0063] 5. Substrate chromogenic solution: composed of A liquid and B liquid, the substrate chromogenic liquid A is carbamide peroxide, 7ml / bottle, 1 bottle; the substrate chromogenic liquid B is tetramethylbenzidine, 7ml / bottle, 1 bottle;

[0064] 6. Termination solution: 2mol / l sulfuric acid;

[0065] 7. Concentrated washing solution: pH value is 7.4, containing 0.05% Proclin300 in the final concentration in...

Embodiment 3

[0066] Embodiment 3. Preparation of kit

[0067] 1. Preparation of microtiter plates coated with high-purity gene recombinant Rta protein

[0068] (1) Coating protein is high-purity gene recombinant Rta protein

[0069] The high-purity gene recombinant Rta protein prepared in Example 1 was used as the coating protein.

[0070] (2) Preparation of antigen-coated microtiter plates

[0071] Dilute the above Epstein-Barr virus Rta protein 1:10000 with coating buffer (pH value 9.6, 0.1mol / L carbonate buffer), add to the wells of the microplate, 100 μl per well, and react at 37°C for 2 hours , shake off the coating solution, pat dry, add 200 μl of blocking solution (containing phosphate buffer with a final concentration of 2% bovine serum albumin) to each well, react at 37°C for 2 hours, shake off the blocking solution, pat dry, and dry , stored in vacuum packaging in aluminum foil bags.

[0072] 2. Preparation of working concentration enzyme solution

[0073] HRP-labeled goat a...

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Abstract

The invention discloses a reaction carrier and a kit for detecting an Epstein Barr (EB) virus replication and transcription activator (Rta)-immunoglobulin G (IgG) antibody. The reaction carrier for immunological detection of the EB virus Rta-IgG antibody is coated with gene recombinant EB virus Rta protein, and the gene recombinant EB virus Rta protein is obtained by expression of Chinese hamster ovary (CHO) cell line CHO / RTA with the preservation number of China General Microbiological Culture Collection Center (CGMCC) 6955. The kit comprises the reaction carrier. Clinical experiments show that the kit for the quantitative detection of the EB virus Rta-IgG is easy and convenient to operate, high in sensitivity and good in specificity.

Description

technical field [0001] The invention relates to the technical field of medical immunology and in vitro serological diagnosis, in particular to a reaction carrier and a kit for detecting Epstein-Barr virus Rta-IgG antibody. Background technique [0002] Nasopharygeal carcinoma (NPC) refers to malignant tumors that occur on the top and side walls of the nasopharyngeal cavity. Take first place. [0003] Years of research have found that nasopharyngeal carcinoma is most closely related to Epstein-Barr virus. Epstein-Barr virus is activated to infect nasopharyngeal cells, and when it enters a cleavage and replication state from the latent period, it will cause abnormal division and proliferation of nasopharyngeal cells and cause cancer. The serum of nasopharyngeal carcinoma patients has a spectrum of antibodies against various Epstein-Barr virus antigens. Generally, the risk of developing nasopharyngeal carcinoma is assessed by detecting serum Epstein-Barr virus antibodies, whic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68
Inventor 贾凤芹李全焦守恕
Owner 同昕生物技术(北京)有限公司
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