Preparing method and detecting method for MRJR1 antibody pairs in honey and kit

An immunological detection method and antibody pair technology, which is applied in the field of determination of honey component content, can solve the problems that there is no fast and low-cost method for detecting the quality of honey, and it is impossible to distinguish true and false honey, and achieve high accuracy, high sensitivity, and catalytic efficiency. high effect

Inactive Publication Date: 2016-06-22
ZHEJIANG UNIV
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  • Description
  • Claims
  • Application Information

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Problems solved by technology

In July 2015, the State Food and Drug Administration officially responded on its official website: the current national standard for honey cannot identify the authenticity of honey, and will actively promote the revision of

Method used

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  • Preparing method and detecting method for MRJR1 antibody pairs in honey and kit
  • Preparing method and detecting method for MRJR1 antibody pairs in honey and kit
  • Preparing method and detecting method for MRJR1 antibody pairs in honey and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Screening of MRJP1-specific polypeptides

[0032] Log in to the international gene information database http: / / www.ncbi.nlm.nih.gov / , search and download the amino acid sequences encoded by the genes of the 9 members of the MRJPs family (MRJP1~MRJP9), the sequence number of MRJP1 is NM_001011579, and the sequence number of MRJP2 为NM_001011580、MRJP3的序列号为NM_001011601、MRJP4的序列号为NM_001011610、MRJP5的序列号为NM_001011599、MRJP6的序列号为NM_001011622、MRJP7的序列号为NM_001014429、MRJP8的序列号为NM_001011564、MRJP9的序列号为 NM_001024697. Input the amino acid sequence encoded by the above-mentioned sequence MRJP1~MRJP9 gene into GENTYX software for homology analysis, after the analysis is completed, the output is as follows figure 1 The homology analysis diagram shown ( figure 1 ).

[0033] according to figure 1 As a result of homology analysis, two specific polypeptides of MRJP1 were screened out: the polypeptide SGEYDYKNNYPSDID (SEQ ID NO: 2) located at positions 56-70 of the amino acid sequ...

Embodiment 2

[0034] Example 2: Preparation of MRJP1-specific polyclonal antibody

[0035] (1) Antigen preparation

[0036] Chemical synthesis was carried out according to the polypeptide sequences C-IKEALPHVPIFD-NH2 and C-SGEYDYKNNYPSDID-NH2. Conjugate 5 mg of synthetic polypeptide with KLH (the coupling agent is Sulfo-SMCC) as an immune antigen; mix another 5 mg of synthetic polypeptide with bovine serum albumin (BSA, a type of albumin in bovine serum, containing 583 amino acid residues base, molecular weight 66.430kDa) coupled (coupling agent glutaraldehyde) as the detection antigen. Dilute with PBS to a concentration of 1mg / mL, aliquot and store at -20°C.

[0037] (2) Immune rabbits

[0038] Two healthy New Zealand white rabbits were selected and immunized on days 1, 15, 29, and 43. The immunization procedure is as follows: Take 1 mL of antigen, add 1 mL of Freund’s complete adjuvant, emulsify, drop a drop of emulsified antigen solution into physiological saline, and use a syringe t...

Embodiment 3

[0041] Example 3: Purification of polyclonal antibodies by affinity chromatography

[0042] (1) Preparation of affinity chromatography column

[0043] 1 mL of thiol protein agarose-coupled resin (Sulfolink Resin) was coupled with 1 mg of synthetic MRJP1-specific polypeptide. 5 mg of synthetic MRJP1 polypeptide was linked to activated thiol protein agarose-coupled resin to prepare an antigen affinity column. The affinity column was equilibrated with 10 times the column volume of PBS, and the solution was drained.

[0044] (2) Antibody purification

[0045] Filter the polyclonal antibody serum with a 0.45 μm filter membrane, then pass it through an antigen affinity column, drain the solution, and collect the flow-through; equilibrate with 10 times the column volume of PBS buffer, drain the solution; add 5 mL of antibody eluent (Glycine 5g, dissolved in 100mL ultrapure water, adjusted to pH 2.7 with concentrated HCl, stored at 4°C), and the eluate was collected in 1mL / tube.

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Abstract

The invention discloses a preparing method and a detecting method for MRJR1 antibody pairs in honey and a kit. The amino acid sequence of the specific polypeptide M4 of MRJR1 is IKEALPHVPIFD, the IKEALPHVPIFD is shown in SEQ ID NO:1; the amino acid sequence of the specific polypeptide M5 of the MRJR1 is SGEYDYKNNYPSDID, and the SGEYDYKNNYPSDID is shown in SEQ ID NO:2; New Zealand white rabbits are immunized. An ELLSA double-antibody sandwich method includes the following steps that a caught antibody SP-1 is coated, sealed and washed, an antigen is added, an enzyme-labeled detecting antibody HRP-SP-2 of an is added, washing is carried out, a color rendering substrate is added, a color rendering stopping solution is added, and the light absorption value of 450 nm-630 nm of a sample is measured through an enzyme-labeling instrument. The antigen is to-be-detected honey or pure honey of a known honey source, and the quality of the to-be-detected honey is judged by comparing the light absorption value of the MRJR1 of the to-be-detected honey and the light absorption value of the MRJR1 of the pure honey of the known honey source. According to the preparing method, the detecting method and the kit, a new reliable rapid detecting method is provided for the quality of the honey and detection of adulteration amount, and the detecting method can be used for honey-product-quality control of honey-product-quality supervision departments and honey-product-quality control of trade processing enterprises.

Description

technical field [0001] The present invention relates to the determination technology of honey component content, in particular to a preparation method of endogenous royal jelly major protein MRJP1 specific diabody contained in honey and secreted by bees after sealing, and a diabody for detecting adulterated honey with diabody Sandwich method, etc. Background technique [0002] Honey is a natural sweet substance formed by bee worker bees after they collect the nectar, secretion or honeydew of plants, combine with their own secretions and fully brew in the comb. my country is a big country in honey production and export. In 2012, the national output reached 451,600 tons, accounting for 1 / 4 of the world's total honey production; in 2013, it exported 125,000 tons of honey, accounting for 1 / 4 of the world's honey trade volume. According to the description of the authenticity of honey in the industry standard GH / T18796-2012 "Honey" of the People's Republic of China on supply and ...

Claims

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Application Information

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IPC IPC(8): C07K16/18C07K16/06G01N33/68G01N33/543
CPCC07K16/18C07K16/06
Inventor 沈立荣王一然谌迪张一帆沈苗宏辛晓璇
Owner ZHEJIANG UNIV
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