Immune colloidal gold test strips for detection of fruit spot fungus and its monoclonal antibody and hybridoma cell line
A technology for detecting fruit spot disease and test paper, which is applied in the field of bioengineering, can solve the problem of low sensitivity of melon bacterial fruit spot disease, achieve remarkable technical progress, and achieve high detection specificity and sensitivity
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[0027] At first introduce the reagent and instrument used in the embodiment of the present invention:
[0028] Main reagents:
[0029] PEG and TWEEN-20 were from Sigma; BSA was from Shanghai Broad Biotechnology Co., Ltd.; colloidal gold solution was from Shanghai Gold Standard Biotechnology Co., Ltd.; nitrocellulose membrane (NC membrane) 135S was from Millipore; goat anti-mouse, HRP-goat anti Mouse and goat anti-rabbit were from Sangon Bioengineering Co., Ltd.
[0030] Main instruments:
[0031] Microplate reader SpectraMaxM2 was purchased from Molecular Devices; Nanodrop2000C was purchased from ThermoScientific; spotting instrument AD6010 was purchased from BIO-DOT; scanner PhantomV9 was purchased from MICROTEK; constant temperature culture shaker SPH-100B was purchased from Shanghai Shiping; TM LP358BR5057 was purchased from BIO-RAD; single-person purification bench SW-SJ-2D was purchased from Suzhou Purification.
Embodiment 1
[0032] Example 1 Preparation of Monoclonal Antibody to Phytophthora spp.
[0033] 1. Immunogen Preparation
[0034] The 8 kinds of spot bacteria strains of watermelon were enriched in the brain-heart infusion liquid medium first, and then streaked on the solid medium, and single colonies were cultured in the brain-heart infusion liquid medium for PCR identification, and the 600nm At the OD value, the faster-growing strain was selected as the immune strain. Centrifuge and resuspend in sterilized normal saline 3 times, and adjust the bacterial concentration to 10 8 CFU / mL.
[0035] 2. Preparation process of monoclonal antibody
[0036] 1) Experimental animals: Three 8-week-old, weighing about 20 g female Balb / c mice were selected as experimental animals.
[0037] 2) Immunization method: each mouse was intraperitoneally injected with 0.4 mL of immunogen, and the same dose was boosted once every two weeks.
[0038] 3) Blood collection: Blood was collected from the tail vein a...
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