Tobramycin monoclonal antibody as well as preparation method and application of tobramycin monoclonal antibody
A monoclonal antibody, tobramycin technology, applied in biochemical equipment and methods, chemical instruments and methods, microorganism-based methods, etc., can solve the problems of time-consuming repeatability and specificity, and achieve good sensitivity, The effect of convenient and fast synthesis and simple preparation method
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Embodiment 1
[0026] Example 1: Obtaining of hybridoma cell line 1A10 and identification of the monoclonal antibody against tobramycin produced by it
[0027] 1. Obtaining the hybridoma cell line 1A10
[0028] 1. Preparation of complete tobramycin antigen Tob-BSA / Tob-HSA
[0029] Dissolve 20mg of Tob (tobramycin) in 1mL of PBS (0.01M phosphate buffer saline) at pH 7.4, slowly add 0.5mL of freshly prepared 1% glutaraldehyde solution into the small molecule solution, stir at room temperature to activate 8min. Another 10 mg of BSA (HSA) was dissolved in 1 mL of 0.05 M, pH 9.6 CBS solution. The above-mentioned activated tobramycin solution was slowly added dropwise into the BSA (HSA) solution, and stirred at room temperature for 1 h. The solution was pre-cooled at 4° C., and a certain amount of sodium borohydride was weighed to make the final concentration of sodium borohydride in the reaction system 4 mg / mL, and stirred for 2 h. Dialyze at 4°C for three days, and store at -20°C.
[0030] ...
Embodiment 2
[0044] Example 2. The application of the indirect competition ELISA method established with the antibody secreted by the hybridoma cell line 1A10
[0045] The monoclonal antibody prepared by the hybridoma cell line 1A10 through ascites in vivo was applied to the tobramycin ELISA addition and recovery test, and the specific steps were as follows:
[0046] (1) Use 0.05 μg / mL Tob-OVA diluted with carbonate buffer solution (CBS) as the coating material to coat a 96-well microplate, 100 μL per well, coat at 37°C for 2 h, then wash with PBST Wash the plate three times, each time with 250 μL per well, each time for 3 min, and pat dry.
[0047] (2) Block with CBS containing 0.1% casein, 200 μL per well, block for 2 h at 37°C, wash the plate three times with PBST washing solution, 250 μL per well for 3 min each time, and pat dry.
[0048] (3) Prepare 0, 0.625, 1.25, 2.5, 5, 10, 20, 40 μg / L standard solutions of tobramycin in phosphate buffered saline (PBS). Add the standard solution ...
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