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Human encephalitis B virus antibody solid phase competition ELISA detection kit, and preparation method and detection method thereof

A technology of Japanese encephalitis virus and detection kit, which is applied in the direction of biological testing, measuring devices, material inspection products, etc., and can solve the problems of inability to detect the antibody titer of the tested sample, inability to judge the infection or the recovery status of the infection period, There are no problems such as detection of antibody titer, and the effects of eliminating species source differences, low cost, and easy operation are achieved.

Inactive Publication Date: 2018-09-07
合肥知恩生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the ELISA method for the detection of human JE virus antibody is mostly indirect ELISA. This method can only be used for specific JE serum samples, and can only judge the negative or positive of serum samples. There is no standard for detecting antibody titers, so it cannot The antibody titer of the tested sample is tested, and the results cannot be used to judge the infection or the period of infection and recovery after immunization

Method used

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  • Human encephalitis B virus antibody solid phase competition ELISA detection kit, and preparation method and detection method thereof
  • Human encephalitis B virus antibody solid phase competition ELISA detection kit, and preparation method and detection method thereof
  • Human encephalitis B virus antibody solid phase competition ELISA detection kit, and preparation method and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1. Preparation of Human Japanese Encephalitis Virus Antigen

[0044] The cultivated Vero cells, when the cell growth density of Vero (African green monkey kidney cells: commonly used cells for virus cultivation) reached about 80%, the human Japanese encephalitis virus (10 5 TCID 50 ), after melting on ice, make a 5-fold dilution with OPTI-MEM, take 2.5mL to infect 75cm 2 Incubate the cells in the cell bottle at 37°C for 1 hour, shake gently 2-3 times every 15 minutes, then discard the virus infection solution, add 10mL OPTI-MEM, and finally place at 37°C, 5% CO 2 cultured in a constant temperature incubator. After 3 days, the cells were damaged. After the diseased cells floated, the cell bottle was placed at -80°C, freeze-thawed three times, and then centrifuged at 4°C, 8000rpm for 30 minutes to remove cell debris, and then the supernatant was placed at 4°C, Centrifuge at 35000rpm (horizontal rotor) for 3 hours, discard the supernatant, suspend the precipitate ...

Embodiment 2

[0045] Embodiment 2. Preparation of serum

[0046] (1) Preparation of human Japanese encephalitis virus positive serum

[0047] In this study, positive serum of human Japanese encephalitis virus was used as the serum samples of infected patients obtained from clinical diagnosis and treatment. Part of it is hyperimmune serum obtained from a small amount of blood samples obtained from volunteers vaccinated against Japanese encephalitis virus. After the serum is prepared, it is stored in -80°C.

[0048] (2) Preparation of monkey anti-human Japanese encephalitis virus serum

[0049] Select healthy male monkeys weighing 20-25 kg, emulsify the ultra-purified human Japanese encephalitis virus with an equal volume of complete Freund's adjuvant, and inject it subcutaneously at multiple points on the back, with a dose of 3 mg / monkey; 15 days after the first immunization , human Japanese encephalitis virus was emulsified with an equal volume of incomplete Freund's adjuvant, and the mon...

Embodiment 3

[0050] Embodiment 3. Optimization of reaction conditions

[0051] The coating concentration of the inactivated antigen of human Japanese encephalitis virus, the dilution ratio of monkey anti-human Japanese encephalitis virus positive serum, the dilution ratio of HRP-labeled anti-monkey IgG secondary antibody and the concentration of blocking solution were optimized by matrix method. The OD ratio of negative and positive serum is the largest, and when the OD value of the complete monkey competition serum well (this well is only added with diluted monkey anti-human Japanese encephalitis virus positive serum and the same amount of PBST) is about 1.5, determine the reaction conditions: coating ELISA plate (50 μL / well), the antigen concentration is 10 ng / μL, blocked with 2% BSA, 37 ° C, 1 hour (100 μL / well); monkey competition serum was diluted 1:1000 (50 μL / well), HRP-labeled The anti-monkey IgG enzyme-labeled secondary antibody was diluted 1:3000 (50 μL / well).

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Abstract

The invention relates to a medical detection kit, and concretely relates to a human encephalitis B virus antibody solid phase competition ELISA detection kit, and a preparation method and a detectionmethod thereof. The kit includes an ELISA plate coated with a human encephalitis B virus inactivation antigen, human encephalitis B virus positive serum, monkey anti-human encephalitis B virus positive control serum, and human encephalitis B virus negative control serum. The kit can detect the serums of human encephalitis B virus infected patients regardless of race, so high-flux detection of clinic patient serum samples is achieved, and the antibody titer of the samples can be detected as needed. The solid phase competition ELISA kit has the advantages of simplicity in operation, easiness inknowing, no high-end devices, easiness in promotion, and low cost, and is suitable for clinic serum sample detection and laboratory research application.

Description

technical field [0001] The invention relates to a test kit for medical detection. Specifically, the invention is a solid-phase competition ELISA detection kit for human Japanese encephalitis virus antibody and its preparation method and detection method. Background technique [0002] Human Japanese encephalitis virus (JEV) is abbreviated as JE virus, which belongs to the Flavivirus genus of the Arboviridae Flaviviridae family. The main symptoms of infected patients are persistent high fever, headache, vomiting, lethargy, and convulsions. Severe patients may die with high fever, convulsions, cerebral edema, respiratory or circulatory failure, and some patients will have sequelae after recovery. The key to preventing Japanese encephalitis is to eliminate the gathering places of mosquitoes, and to exterminate overwintering mosquitoes and newborn mosquitoes. Vaccination of the human body can improve the resistance to JE virus infection, and it also has a good effect on the pre...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/569
CPCG01N33/56983G01N33/6854G01N33/6893G01N2333/185
Inventor 刘宗梁李燕华曹齐树
Owner 合肥知恩生物技术有限公司
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