Newcastle disease immune body immune colloidal gold fast detecting reagent kit and its application
A chicken Newcastle disease and kit technology, applied in the field of immunization applications, can solve the problems of easy to disperse, poor biological safety, and inability to implement, and achieve the effects of high biological safety and low production cost.
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Embodiment 1
[0026] Preparation of HN Protein of Chicken Newcastle Disease Virus
[0027] For the molecular biology methods used, please refer to the literature: Sambrook J, Fritsch EF, Mani Artis T, editors (translated by Jin Dongyan, Li Mengfeng, etc.), Molecular Cloning Experiment Guide, Second Edition, Beijing Science Press, 1992 method provided in.
[0028] 1. Cloning and sequencing of HN gene
[0029] The HN gene involved in the present invention is obtained by the applicant's own clone according to the HN gene sequence of NDV La Sota line included in GenBank, and the sequence has 98% homology with the known sequence. The specific method of cloning and sequencing of the HN gene is: use chicken Newcastle disease (NDV) Losota strain, purchase from the Lanzhou Veterinary Research Institute of the Chinese Academy of Agricultural Sciences, or refer to references: Sun Shuna, Newcastle disease virus F gene and HN gene clone expression and its Preliminary establishment of ELISA antibody de...
Embodiment 2
[0038] Preparation of rabbit anti-mouse IgG antibody:
[0039] 1. Extraction of Balb / C mouse IgG
[0040] The extraction and purification of IgG are carried out according to the methods described in Shen Guan et al. (Shen Guan et al., Modern Immunology Experimental Technology (Second Edition) [M], Hubei Science and Technology Press, 2002): Take 10.0mL healthy Balb / C small After mouse serum was mixed with an equal amount of 0.8% sodium chloride solution, saturated (NH 4 ) 2 SO 4 Solution 20.0mL, made to 50% saturation (NH 4 ) 2 SO 4 solution. Take it out after standing at 4°C for 30min; centrifuge at 3,000r / min for 30min at 4°C, discard the supernatant, add 20.0mL of 0.8% sodium chloride solution to the precipitate; after the precipitate dissolves, slowly add 10.0mL of saturated (NH 4 ) 2 SO 4 solution, made into 33% saturation (NH 4 ) 2 SO 4 solution. Take it out after standing at 4°C for 30min; centrifuge at 3,000r / min for 30min at 4°C. Discard the supernatant, ...
Embodiment 3
[0054] Preparation of nitrocellulose membrane
[0055] 1 Preparation of coating buffer: 0.01M pH7.2 PB buffer containing 3% methanol was used as coating buffer, filtered through 0.22μ membrane, set at 4°C for later use, and valid for one week. 1000ml 3% methanol 0.01M pH7.2 PB buffer formulation: KCl 0.2g, Na 2 HPO 4 12H 2 O 2.9g, KH 2 PO 4 0.2g, methanol 30ml, distilled deionized water to 1000ml.
[0056] 2 Preparation of nitrocellulose membrane: Dilute the HN protein to 50-100 μg / ml with coating buffer, adjust the machine, and mark the line as T line, that is, the detection line (T line) is close to the end of the gold standard pad, and the distance from the gold standard The end of the pad is about 5mm; dilute the rabbit anti-mouse IgG antibody to 50-100μg / ml with the coating buffer, adjust the machine, draw the line C, which is the quality control line, and the C line is close to the absorbent pad, about 3mm away from the absorbent pad . The distance between the tw...
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