Anti-major-royal-jelly-protein 3 monoclonal antibody and enzyme-linked immunoassay kit for detecting major royal jelly protein 3
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A technology of enzyme-linked immunosorbent reagent and monoclonal antibody, applied in the direction of anti-animal/human immunoglobulin, biological test, measuring device, etc.
Active Publication Date: 2018-04-10
BEE RES INST CHINESE ACAD OF AGRI SCI
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At present, there is no internationally recognized method that can be widely promoted and can faithfully reflect the freshness of royal jelly, which is also a defect of the current quality standard of royal jelly
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Embodiment 1
[0021] Example 1 Monoclonal antibody preparation of royal jellybumin 3
[0022] 1. Sequence analysis of Major royal jelly protein 3 gene and selection of antigen sequence
[0023] Major royal jelly protein 3 gene encodes 544 amino acids, no transmembrane region, 1-20aa signal peptide sequence, good hydrophilicity, and the sequence is different from other high homology proteins of MRJPs family.
[0024] After transmembrane region analysis, signal peptide analysis, hydrophobicity analysis, disordered sequence analysis, antigenicity analysis, homology analysis and structural domain analysis, the amino acid sequence described in SEQ ID No.1 was finally selected as the antigenic sequence in Escherichia coli Antigens were expressed and prepared in .
[0025] 2. Antigen preparation and purification
[0026] 1. A small amount of expression
[0027] 1.1 Sequencing to identify the correct recombinant plasmid to transform the expression host.
[0028] 1.2 Pick a single colony contain...
Embodiment 2
[0180] The preparation of the enzyme-linked immunosorbent kit of embodiment 2 double antibody sandwich ELISA method
[0181] 1. Experimental principle
[0182] Coat the microwell plate with the purified antibody to make a solid phase carrier, add the specimen or standard, biotinylated anti-MRJP3 antibody, and HRP-labeled avidin to the microwells coated with the anti-MRJP3 antibody in sequence, and 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. There is a positive correlation between the depth of the color and the MRJP3 in the sample. Measure the absorbance (OD value) at a wavelength of 450 nm with a microplate reader, and calculate the sample concentration.
[0183] 1. Standard curve diagram and standard curve linear range
[0184] 1) Standard curve setting
[0185] The setting principle of the standard curve is based on the detectio...
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Abstract
The invention discloses an anti-major-royal-jelly-protein 3 monoclonal antibody and an enzyme-linked immunoassay kit for detecting major royal jelly protein 3. According to the present invention, 112positive cell lines are obtained by using a sequence represented by SEQ ID No.1 as antigen through cell fusion, 14 fusion cell lines are obtained through cross reaction detection, the 7 cell lines with high affinity are selected through three rounds of subcloning, and subjected to antibody epitope detection and pairing experiment, and the standard curve drawing and sample determination results show that the two cell lines such as 3G4 and 2D2 have good linear relationship and high sensitivity; the enzyme-linked immunoassay kit for detecting major royal jelly protein 3 through a double antibodysandwich ELISA method is further constructed through the two cell lines such as 3G4 and 2D2; and the enzyme-linked immunoassay kit can specifically detect the MRJP3 and other related proteins, does not have cross reaction, has the detection range of 0.78-50 ng/mL, has the detection sensitivity of 194 pg/mL, and further has advantages of high precision and low batch difference.
Description
technical field [0001] The present invention relates to the preparation and application of royal jellybumin monoclonal antibody, in particular to the preparation of royal jellybumin 3 monoclonal antibody, and the present invention further relates to the detection of double antibody sandwich ELISA constructed with the prepared royal jellybumin 3 monoclonal antibody An ELISA kit for royal jelly major protein 3 (MRJP3) and an application thereof belong to the detection field of royal jelly major protein 3. Background technique [0002] Royal jelly is a natural functional bee product that has the functions of nourishing health care and prolonging life. The composition of royal jelly is complex and rich in nutrition, among which the protein content is 12.0%-15.0%. Because royal jelly is not easy to preserve, it is easy to deteriorate. Therefore, the quality of royal jelly is closely related to its freshness. At present, there is no internationally recognized method that can be...
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