Double-antibody sandwich ELISA (enzyme-linked immuno sorbent assay) method for peanut allergen Arah3
A peanut allergen, double-antibody sandwich technology, applied in the field of immunodetection, can solve the problems of long cycle of screening and antibody preparation
Pending Publication Date: 2022-05-27
NANKAI UNIV
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Problems solved by technology
[0002] The development of immunoassay technology for traditional food allergen components is based on the screening and preparation of monoclonal or polyclonal antibodies, which requires the injection of high-purity allergen protein during animal immunization, and the screening and preparation of antibodies takes a long time
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[0021] Unless otherwise defined, technical terms used in the following embodiments have the same meanings as commonly understood by those skilled in the art to which the present invention belongs. The test reagents used in the following examples are conventional biochemical reagents unless otherwise specified; the experimental methods are conventional methods unless otherwise specified.
[0022] The present invention will be described in detail below with reference to the embodiments.
[0023] A double-antibody sandwich ELISA method for peanut allergen Arah3, comprising the following steps:
[0024] (1) Preparation of Nanobodies and Screening of Nanobodies
[0025] The purified plasmid and pHEN6c plasmid vector were double digested with PstI and BstII restriction endonucleases. The digested product was purified using the Qiaquick PCR purification kit. Under the action of T4 DNA ligase, the VHH gene fragment was ligated with pHEN6c plasmid vector to form a new recombinant pl...
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The invention provides a double-antibody sandwich ELISA (enzyme-linked immuno sorbent assay) method of peanut allergen Arah3, which comprises the following steps: (1) preparation and screening of a nano antibody: connecting an obtained VHH gene segment with a plasmid vector to form a recombinant plasmid, electrically transforming the recombinant plasmid into an escherichia coli competent cell to express the nano antibody, and purifying to obtain a nano antibody protein; and (2) construction of a double-antibody sandwich ELISA method: coating an elisa plate with the captured antibody, adding peanut total protein to enable the peanut total protein to be combined with the captured antibody, then adding a detection antibody, taking a mouse anti-HA monoclonal antibody as a primary antibody and an HRP-labeled goat anti-mouse monoclonal antibody as a secondary antibody, and performing color development. The double-antibody sandwich ELISA method for the peanut allergen Arah3 is based on a specific nano antibody, can develop a high-sensitivity rapid detection system for trace allergens in food, and is used for detecting the allergens in food components.
Description
technical field [0001] The invention belongs to the field of immune detection, in particular to a double-antibody sandwich ELISA method for peanut allergen Arah3. Background technique [0002] The development of immunodetection technology for traditional food allergen components is based on the screening and preparation of monoclonal or polyclonal antibodies, which requires injection of high-purity allergen proteins during animal immunization, and the screening and preparation of antibodies takes a long time. A kind of heavy chain antibody (Heavy Chain only Antibodies, HCAbs) naturally exists in the peripheral blood of camel-derived animals. Compared with traditional monoclonal antibodies, heavy chain antibodies naturally lack the light chain and the first constant region (CH1) of the heavy chain. The heavy chain variable region of heavy chain antibody is cloned and expressed to obtain the antigen recognition and binding domain of heavy chain antibody, which is called nanobo...
Claims
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IPC IPC(8): C07K16/16C07K1/22C07K1/16C12N15/70C12N15/13G01N33/68
CPCC07K16/16C12N15/70G01N33/68C07K2317/569G01N2333/415
Inventor王硕胡耀中陆旸张川张博崴
OwnerNANKAI UNIV