Anti-infectious hematopoietic necrosis virus monoclonal antibody and its application
A technology of necrosis of hematopoietic organs and monoclonal antibodies, applied in the field of immunology, can solve the problems of lack of good antigens and antiserum, unsuitable for grassroots laboratories, and inability to meet rapid detection, etc., to achieve improved purity, good specificity, and potency high effect
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Embodiment 1
[0026] Example 1 Preparation of monoclonal antibodies against infectious hematopoietic necrosis virus
[0027] 1. Antigen purification
[0028] The antigen used was IHNV-UK (UK strain of infectious hematopoietic necrosis virus) deposited by the OIE Reference Laboratory in Weymouth, UK. Inoculate EPC (carp epithelioma cells) in the exponential growth phase, and use M199 (containing Earle’s balanced salt solution, 10% fetal calf serum and 200U / mL penicillin-streptomycin, pH7.2-7.6) at 17.5℃ to increase the value. The virus was collected, centrifuged at 8000r / min for 30min, and then centrifuged at 24000r / min for 2h. The pellet was resuspended in 0.5 mL of 0.01MPBS.
[0029] 2. Animal immunity
[0030] The immunized mice were SPF-grade 5-week-old female BALB / c mice. Each mouse was immunized by intraperitoneal injection with the purified virus as the antigen. The virus was mixed with Freund’s complete adjuvant 1:1, and injected intraperitoneally; after 2 weeks, the booster immunization ...
Embodiment 2
[0035] Example 2: Determination of the biological characteristics of monoclonal antibodies against infectious hematopoietic necrosis virus
[0036] 1. Determination of potency
[0037] Methods: The ELISA plate was coated with purified infectious hematopoietic necrosis virus at a concentration of 15 μg / mL, and the ascites titer of monoclonal antibodies was identified by indirect ELISA.
[0038] Results: The ascites titer of the monoclonal antibody of the present invention is 1:2.5×10 5 , Indicating that the hybridoma cell line has the ability to secrete high-titer antibodies.
[0039] 2. Subtype identification of monoclonal antibodies
[0040] Method: Using SouthernBiotech's typing kit (SBAClonotyping TM System / HRP) to identify the Ig subtype of the monoclonal antibody of the present invention according to its instructions.
[0041] Results: The subtype of the monoclonal antibody of the present invention is IgG1 type k chain.
[0042] 3. Specificity analysis of monoclonal antibodies
[00...
Embodiment 3
[0047] Example 3 Infectious hematopoietic organ necrosis virus double sandwich ELISA detection kit
[0048] 1. The composition of the double-sandwich ELISA test kit for infectious hematopoietic necrosis virus
[0049] The composition of the Infectious Hematopoietic Organ Necrosis Virus Double Sandwich ELISA Test Kit is as follows: 1) A solid-phase carrier containing polyclonal antibodies against infectious Hematopoietic Organ Necrosis Virus; 2) Monoclonal antibodies prepared in Example 1; 3) Horseradish Peroxidase labeled goat anti-mouse antibody (purchased from sigma company); 4) enzyme substrate reaction solution; 5) positive control and negative control; 6) washing solution; 7) enzyme reaction termination solution.
[0050] 1) Preparation of solid-phase carrier containing polyclonal antibody against infectious hematopoietic organ necrosis virus: immunize goats with infectious hematopoietic necrosis virus (ATCCVR-714) purified by gradient centrifugation. The serum is prepared by a...
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