ELISA detection kit for detecting Apis cerana Smith honey and Apis mellifera honey

A detection kit and kit technology, applied in biological testing, measuring devices, material inspection products, etc., can solve the problems of lack of detection methods, loss of bee honey producers and consumers, etc., and achieve good specificity and sensitivity, Reasonable design and good thermal stability

Active Publication Date: 2019-10-25
ZHEJIANG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Driven by profit, it is very rampant in the market to impersonate Italian bee honey or mix it with Chinese bee honey, coupled with the lack of relevant detection methods, it leads to more unscrupulous counterfeiters, causing losses to producers and consumers of Chinese bee honey
At present, there are no relevant detection kits on the market. In order to curb this kind of counterfeiting and adulteration and meet the industry's demand for rapid and sensitive detection, it is very necessary to develop relevant detection kits.

Method used

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  • ELISA detection kit for detecting Apis cerana Smith honey and Apis mellifera honey
  • ELISA detection kit for detecting Apis cerana Smith honey and Apis mellifera honey
  • ELISA detection kit for detecting Apis cerana Smith honey and Apis mellifera honey

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1 The preparation of the kit is realized through the following steps.

[0039] (1) Selection of target antigen and antibody preparation scheme: MRJP2 does not show polymorphism in Western honeybees, but it has a certain level of polymorphism in Apis mellifera, and the genetic difference of MRJP2 at the interspecies level is relatively large. Obviously, the MRJP2 protein was chosen as the target protein for related antibody preparation. The properties of MRJPs are similar, and it is difficult to achieve high-purity separation of a single royal jelly protein component in honey and royal jelly. We adopted the method of prokaryotic expression to obtain MRJP2 from Zhongfeng and Italian bee.

[0040] (2) Structural analysis and sequence optimization of MRJP2 proteins from Chinese and Italian pheasants: TMHMM Server V2.0 online analysis software was used to analyze the transmembrane domain, and both MRJP2 from Chinese and Italian pheasants had no transmembrane stru...

Embodiment 2

[0052] Example 2 In this example, the prokaryotic expression conditions of Zhongfeng MRJP2 and Italian bee MRJP2 were determined.

[0053] The transformed Escherichia coli was cultured under different induction conditions, two groups with final IPTG concentrations of 0.2 mM and 1 mM were set, and the culture temperature was 4 h at 37° C. or 16 h at 15° C. The target was expressed under each condition, and the bacterial solution under each condition was collected by centrifugation and precipitated, and then crushed, and the supernatant could be precipitated to prepare samples for soluble SDS-PAGE analysis ( Figure 4 ), the prokaryotic-expressed Chinese bee MRJP2 protein was soluble under the induction condition of 15°C, while the prokaryotic-expressed Italian bee MRJP2 protein existed in the form of inclusion bodies under all induction conditions.

[0054] Determine the optimal induction conditions for prokaryotic expression of MRJP2 in Chinese and Italian bees. See the induct...

Embodiment 3

[0056] In this example, we verified the specificity and sensitivity of the prepared specific Chinese bee MRJP2 antibody and the specific Italian bee MRJP2 antibody.

[0057] Antibody specificity and sensitivity were verified with the purified MRJPs of Chinese honey and Italian honey. The concentration of the MRJPs of the Chinese bee honey and the MRJPs of the Italian bee honey is 2 μg / mL, and the antibody is diluted (the initial concentration of the antibody is 1 mg / mL), and the results are shown in Image 6 , the dilution factor of the honey antibody of the Chinese bee is from 2000 times to 16000 times. The dilution factor of Italian bee honey antibody ranges from 5000 times to 160000 times. The titer of anti-antibodies to the specificity protein is greater than 160,000, and when the titer is greater than 20,000, there is no cross-reaction with the protein (judgment standard: OD450nm of the protein / OD450nm of the blank control is less than 2.1). The specific antibody tite...

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Abstract

The invention provides an ELISA detection kit for detecting Apis cerana Smith honey and Apis mellifera honey. The MRJP2(major royal jelly protein 2) is selected as the target protein for Apis cerana Smith honey and Apis mellifera honey detection, the Apis cerana Smith MRJP2 and the Apis mellifera MRJP2 are obtained through a Prokaryotic expression purification, and preparing and purifying to obtain the specificity and identity antibody of the Apis cerana Smith MRJP2 and the Apis mellifera MRJP2, and the Apis cerana Smith honey and the Apis mellifera detection kit is developed by using a doubleantibody sandwich method. The ELISA detection kit disclosed by the invention has high sensitivity, the developing condition can be observed through the naked eyes, 1% of Apis mellifera honey doping in the Apis cerana Smith can be identified within 45min, the convenient and fast detection requirement is satisfied, and the detection kit is a reliable tool for Apis cerana Smith honey and Apis mellifera honey detection.

Description

technical field [0001] The invention belongs to the field of detection of bee products, and relates to an ELISA detection kit for Chinese bee honey and Italian bee honey. [0002] technical background [0003] As a unique honey variety in China, Zhongfeng honey is deeply loved by Chinese people, but its output is low. In the domestic market, the retail price of Zhongfeng honey is generally 80-200 yuan / 500g, which is three to five times the price of domestic Italian honey. times. Driven by profit, it is very rampant to impersonate Italian bee honey or mix it with Chinese bee honey in the market. In addition, the lack of relevant detection methods makes counterfeiters more unscrupulous, causing losses to producers and consumers of Chinese bee honey. At present, there are no relevant detection kits on the market. In order to curb this kind of counterfeiting and adulteration and meet the industry's demand for rapid and sensitive detection, it is very necessary to develop relevan...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/58G01N33/543G01N33/535C12N15/12C12N15/70C07K14/435
CPCC07K14/43572C12N15/70C12N2800/22G01N33/535G01N33/543G01N33/581G01N33/68
Inventor 胡福良张言政郑火青张翠平
Owner ZHEJIANG UNIV
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