Hybridoma cell strain DC 1F5 secreting anti-abscisic acid monoclonal antibody and application of hybridoma cell strain DC 1F5
A hybridoma cell line and monoclonal antibody technology, which is applied in the direction of analytical materials, anti-plant immunoglobulin, biochemical equipment and methods, etc., can solve problems such as application limitations, and achieve high sensitivity, good affinity, and high specificity Effect
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Embodiment 1
[0032] Example 1: Synthesis of Abscisic Acid Complete Antigen
[0033] Take 4.5 mg of abscisic acid, add 5.0 mg of EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and 3.7 mg of NHS (N-hydroxysuccinimide) , dissolved in DMF (N,N-dimethylformamide), stirred at room temperature, and activated for 6 hours; another 15mg of BSA (bovine serum albumin) was dissolved in 3mL of 0.05M, pH9.6 CBS (carbonate buffer solution) solution; the above activation solution was added dropwise to the BSA solution, stirred and reacted overnight at room temperature, and the immunogen was taken out and dialyzed in PBS for 3 days to obtain the complete abscisic acid antigen, which was stored at -20°C.
[0034] Abscisic acid has the chemical structural formula shown in the following formula (a), and the complete antigen of abscisic acid has the chemical structural formula shown in the following formula (b):
[0035]
Embodiment 2
[0036] Embodiment 2: Preparation of anti-abscisic acid monoclonal antibody hybridoma cell line
[0037]1. Animal immunization: healthy BALB / c mice aged 6-8 weeks were selected for immunization. The complete antigen of abscisic acid (1 mg / mL) was emulsified with an equal amount of Freund's adjuvant, and the BALB / c mice were immunized by subcutaneous multi-point injection. Freund's complete adjuvant was used for the first immunization, and the first immunization dose was 100 μL per mouse; Freund's incomplete adjuvant was used for booster immunization, and the booster immunization dose was 50 μL per mouse; no adjuvant was used for sprint immunization, and it was mixed evenly with normal saline and directly injected into the abdominal cavity. Injection, the immunization dose during the sprint immunization is half of the previous immunization dose; the interval between each immunization is three weeks; the immunization process includes 1 first immunization, 4 booster immunizations ...
Embodiment 3
[0043] Example 3: Preparation and identification of anti-abscisic acid monoclonal antibody
[0044] BALB / c mice aged 8-10 weeks were taken, and each mouse was intraperitoneally injected with 1 mL of paraffin oil; 7 days later, each mouse was intraperitoneally injected with 1×10 6 For hybridoma cells, ascites fluid was collected from the 7th day, and the ascites fluid was purified by octanoic acid-saturated ammonium sulfate method, and the obtained monoclonal antibody was stored at -20°C.
[0045] The mouse monoclonal antibody subtype identification kit was used to identify the immunoglobulin subtype of the monoclonal antibody obtained from ascites purification, and the subtype was IgG2b, as shown in Table 1.
[0046] Table 1. Subtype Identification of Abscisic Acid Monoclonal Antibody
[0047] Antibody isotype subclass OD value IgA 0.133 IgG1 0.113 IgG2a 0.233 IgG2b 2.022 IgG3 0.106 IgM 0.073
[0048] Determination of the I...
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