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Monoclonal antibody against Italian bee mrjp1 and colloidal gold detection card

A technology of monoclonal antibody and colloidal gold, which is applied in the field of biotechnology and immunoassay to achieve the effect of good specificity and high sensitivity

Active Publication Date: 2020-12-25
BEE RES INST CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Standards and authenticity evaluation methods for native honey have not yet been formulated or promulgated

Method used

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  • Monoclonal antibody against Italian bee mrjp1 and colloidal gold detection card
  • Monoclonal antibody against Italian bee mrjp1 and colloidal gold detection card
  • Monoclonal antibody against Italian bee mrjp1 and colloidal gold detection card

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Preparation of Monoclonal Antibody to Royal Jellybumin 1

[0036] 1. Antigen acquisition

[0037] Royal jelly is rich in apalbumin 1. Based on the method reported in the literature, the protein of MRJP1 with high purity was obtained and used as an antigen to immunize mice to obtain highly specific antibodies. SDS-PAGE was used to detect the purity of the antigen obtained, and the results are shown in figure 1 ; In the figure, Lane1 is the purified protein of MRJP1, and Lane2 is the Marker. Depend on figure 1 It can be seen that the antigen used in the present invention has high purity and no interfering protein. Then put the antigen with the required purity into the dialysis belt, and dialyze with 1×PBS at 4°C (change the medium twice), 4 o Concentrate the dialysis product by ultrafiltration.

[0038] 2. Antibody preparation

[0039] (1) Mice immunization and serum titer evaluation

[0040] 1. Immunize Balb / c mice, adult females about 8 weeks old, mix a...

Embodiment 2

[0168] Example 2 Establishment of Double Antibody Sandwich ELISA Experiment

[0169] 1. Experimental principle

[0170] Coat the microwell plate with the purified antibody to make a solid phase carrier, add the specimen or standard, biotinylated anti-MRJP1 antibody, and HRP-labeled avidin to the microwell coated with the anti-MRJP1 antibody in sequence, and then thoroughly After washing, the color was developed with the substrate TMB. TMB is converted into blue under the catalysis of peroxidase, and into the final yellow under the action of acid. The depth of the color is positively correlated with the MRJP1 in the sample. Measure the absorbance (OD value) at a wavelength of 450 nm with a microplate reader to calculate the sample concentration.

[0171] Standard curve graph and standard curve linear range

[0172] 1) Initial screening of antibody pairing

[0173] Specific steps are as follows:

[0174] A Coating: Coat the antibody to be tested at 2 µg / mL, at 37 °C for 2 ...

Embodiment 3

[0186] Example 3 Establishment of MRJP1 colloidal gold detection experiment

[0187] 1 Experimental protocol

[0188] 1.1 Materials

[0189] The royal jelly and honey samples used in this experiment were all purchased from the bee farm.

[0190] 1.2 Instruments and reagents

[0191] 1.2.1 Reagents: MRJP1 monoclonal antibody pair was independently developed, goat anti-mouse IgG-HRP secondary antibody was purchased from Jackson, nitrocellulose membrane was from Millipore, USA, chloroauric acid was purchased from Tianjin Chemical Plant, and trisodium citrate was purchased from Guangzhou Reagent Factory, laboratory water is ultrapure water, and all chemical reagents are of analytical grade.

[0192] 1.2.2 Instruments: Xiangyi TGL-16 high-speed refrigerated centrifuge, Thermo Carbon Dioxide Incubator 3111 for CO2 incubator, contact film spraying machine for American Imagene Technology Inc, NanoDrop 2000C spectrophotometer for American Thermo Fisher.

[0193] 1.3 Method

[0194...

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Abstract

The invention relates to the field of biotechnology and immunodetection, in particular to an anti-apis mellifera MRJP1 monoclonal antibody and a colloidal gold detection card. The invention provides ahybridoma cell strain 1F 61D 7 and a hybridoma cell strain 6F 124B 4 which stably secrete the anti-apis mellifera MRJP1 monoclonal antibody, the secreted antibody only recognizes apis mellifera MRJP1protein, does not recognize apis cerana MRJP1 protein, has the advantages of good specificity, high sensitivity and the like, and has no cross reaction with other family related proteins. The colloidal gold detection card and test strip of the apis mellifera MRJP1 are further developed from the antibody, the colloidal gold detection card and test strip have the advantages of high precision, smallbatch difference and the like, rapid detection of the apis mellifera MRJP1 in a bee product is achieved, and the colloidal gold detection card and test strip are particularly suitable for high-efficiency and large-batch detection of whether apis mellifera honey is mixed into Chinese honey (local honey) or not, and are particularly suitable for high-efficiency and large-batch detection of whetherapis mellifera honey is mixed into Chinese honey (local honey) or not

Description

technical field [0001] The invention relates to the fields of biotechnology and immune detection, in particular to a monoclonal antibody against Italian bee MRJP1 and a colloidal gold detection card. Background technique [0002] Italian bees mainly adopt the method of chasing flowers and chasing nectar for off-site breeding. They tend to produce single-flower honey, and the amount of honey produced is large. It is the main source of commercial honey in my country. Due to the mismatch between consumption demand and output, there is a certain degree of adulteration in the honey currently circulating in the market. There are many forms of honey adulteration, which can be mainly divided into honey adulteration, honey source adulteration and blending syrup, etc. Among them, blending fructose syrup is the most important form of honey adulteration. How to quickly identify honey adulteration has become the current It is a major industrial problem that the bee product consumption i...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/20C07K16/18G01N33/68G01N33/577G01N33/58C12R1/91
CPCC07K16/18G01N33/577G01N33/587G01N33/68G01N2333/43565
Inventor 杨术鹏李熠周金慧
Owner BEE RES INST CHINESE ACAD OF AGRI SCI
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