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B.garinii polyclonal antibody and application

A technology of Borrelia garzii and polyclonal antibody, which is applied in the direction of anti-bacterial immunoglobulin, resistance to vector-borne diseases, immunoassay, etc., can solve the problems of easy cross-contamination, long culture period, and horizontal comparison of the effectiveness of PCR methods and other issues, to achieve the effect of good specificity, strong application prospects, and high potency

Inactive Publication Date: 2017-02-22
杭州贝英福生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In terms of laboratory diagnosis, due to the difficulty in isolating Lyme spirochetes and the long culture period, they are rarely used at present; although PCR technology has the advantages of high specificity, simple operation, and short time consumption, it is prone to cross-contamination in experiments. And there is no standard DNA extraction method yet, and there is no horizontal comparison of the effectiveness of various PCR methods

Method used

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  • B.garinii polyclonal antibody and application
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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1. Preparation of Borrelia garzii polyclonal antibody

[0024] (1) Antigen preparation: Borrelia garinii B.garinii NMJW1 strain was inoculated into 50ml of BSK-II medium (Sigma-Aldrich, USA), and cultivated in a 33°C, 5% CO2 constant temperature incubator until the cell concentration was 2× 108cfu / ml, to obtain the culture solution; then the culture solution was centrifuged at 4°C, 3200rcf for 25min, the precipitate was collected and washed twice with 0.9% NaCl aqueous solution with a mass concentration of 0.9%; After suspending the bacteria, incubate in a water bath at 60°C for 1 hour, and shake it from time to time; centrifuge at 3200 rcf for 25 minutes at 4°C, collect the bacterial precipitate, wash with 0.9% NaCl aqueous solution three times, and obtain the antigen of Borrelia garzii;

[0025] (2) Animal immunization: resuspend the obtained Borrelia garzii with 10ml of 0.9% NaCl aqueous solution, and the concentration of the whole bacterial antigen suspensio...

Embodiment 2

[0028] Example 2. Immunofluorescence detection of Borrelia Lyme disease

[0029] (1) Borrelia garinii B.garinii NMJW1 bacterial strain is inoculated in 15ml BSK-II culture medium (Sigma-Aldrich Company of the U.S.A.), cultivated to bacterium concentration 5×107cfu / ml at 33 ℃, 5% CO2 constant temperature incubator , to obtain cell culture fluid;

[0030] (2) Centrifuge the bacterial cell culture solution in step (1) at 4° C. at 3200 rcf for 25 min, collect the precipitate and resuspend and wash it with 15 ml of 0.9% NaCl aqueous solution with a mass concentration of 15 ml, repeat twice, and finally obtain the bacterial cells by centrifugation;

[0031] (3) Resuspend the bacterial cells obtained in step (2) with 10 ml of 0.9% NaCl aqueous solution, take 10 μl of the suspension and spread it evenly on the immunohistochemical glass slide, and let it dry naturally for 10 min;

[0032] (4) Fix the immunohistochemical slides obtained in step (3) at 60° C. for 30 min;

[0033] (5) F...

Embodiment 3

[0040] Example 3. Immunofluorescence detection of Borrelia Lyme disease in the midgut of larval ticks

[0041] (1) Collect the larvae that fell after being full of blood, and randomly take 10 midgut tissues for dissection;

[0042] (2) After the midgut tissue was washed 3 times with PBS solution, it was placed on an immunohistochemical slide and dried naturally for 20 min;

[0043] (3) Fix the immunohistochemical slide obtained in step (2) at 60° C. for 30 min;

[0044] (4) Immunohistochemical slides were fixed in acetone for 5 minutes, and dried at room temperature for 20 minutes;

[0045] (5) Add 100 μl of PST blocking solution to the midgut tissue, place the immunohistochemical slide in a sealed black box and keep it moist, and incubate at 37° C. for 30 minutes; the PST blocking solution contains 5 g of bovine serum albumin per 100 ml, 50μl Tween-20 in PBS solution;

[0046] (6) Remove the PST blocking solution, add 100 μl of primary antibody working solution, and incuba...

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Abstract

The invention discloses a B.garinii polyclonal antibody and an application. A BSK-II culture medium is inoculated with a B.garinii strain for enlargement culture, and a somatic antigen is obtained after inactivation; after the obtained somatic antigen and an adjuvant are fully emulsified, a New Zealand white rabbit is immunized by using multi-point intradermal injection; the immunized New Zealand white rabbit is anesthetized with chloral hydrate, whole blood is solidified at the room temperature after being collected, and serum containing the B.garinii polyclonal antibody is subjected to centrifugal separation. The B.garinii polyclonal antibody prepared with the method has good specificity and high titer, can effectively detect Lyme disease spirochete and has brighter application prospect.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a polyclonal antibody against Borrelia garzii and the application of the antibody in the preparation of detection reagents for Lyme disease spirochete. Background technique [0002] Lyme disease (LD) is a natural focal disease discovered in the 1970s with ticks as the vector. The disease can cause multi-organ and multi-system infection. Its clinical symptoms can be divided into three phases. The first phase is typical Symptoms are chronic migratory erythema; the second stage usually manifests as nervous system symptoms or heart involvement; the third stage mainly manifests as joint pain, nervous system damage or chronic atrophic acrodermatitis, and a small number of patients can be transformed into chronic disease (Bao Fu Kai, Liu Aihua. 2007. Research progress of Borrelia burgdorferi and Lyme disease. Journal of Tropical Medicine. 11.1125-1127.; Nau R., Christen H., Eiff...

Claims

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Application Information

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IPC IPC(8): C07K16/12C07K16/06G01N33/569
CPCC07K16/06C07K16/1207G01N33/56911G01N2469/10Y02A50/30
Inventor 徐海君赵蕊
Owner 杭州贝英福生物科技有限公司
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