Estriol hapten and antibody and application thereof in fast sensor method for uncojugated estriol detection
An estriol and hapten technology, applied in the field of biosensors, can solve the problems of difficulty in distinguishing structural analogs, insufficient antibody specificity, insufficient sensitivity, etc., so as to improve the ability to catalyze hydrogen peroxide, improve specific performance, The effect of increasing sensitivity
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Embodiment 1
[0053] This example provides a preparation method of estriol immune hapten and competition hapten.
[0054] The synthetic route map of estriol immune hapten is shown in figure 1 shown. Take 0.5g (1.7mM) estriol in a 25mL round bottom flask, add 3mL of anhydrous DMF, stir and dissolve under nitrogen protection at 0°C. Then add 45.0 mg (1.9 mM) NaH, stir for 20 min, and finally add 0.17 mg (1.7 mM) succinic anhydride, and continue the reaction at 0° C. for 30 min. (Refer to ZhaoDangetal, 2015) Adjust the pH of the reaction solution to about 3 with 1M HCl. Spot the silica gel plate to monitor the reaction, developer methanol: chloroform = 1:5 (volume ratio, the same below), raw material Rf = 0.7, product Rf = 0.1. 200-300 mesh silica gel passes through the column, developer methanol: chloroform = 1:10 After evaporating the solvent to dryness, the quality of the product (estriol immune hapten) obtained is 0.24g, and the mass spectrometry identification figure is shown in figu...
Embodiment 2
[0056] Example 2 Polymer horseradish peroxidase (MHRP) was labeled on the estriol monoclonal antibody.
[0057] Screening of estriol monoclonal antibody: the mice after 5 times of immunization were killed, and spleen cells were taken to fuse with mouse SP2 / 0 cells (myeloma cells and spleen cells at a ratio of 5-10:1). Positive hybridoma cells were screened by indirect ELISA and indirect competition ELISA, and the wells showing positive and competitive inhibition reactions were positive wells, and were used for further subcloning. Under sterile conditions, the cells in the positive wells were eluted, and the cells were transferred to a 96-well culture plate pre-plated with feeder cells using an elbow pipette. Each original positive well clone was counted by a cell counting plate and limitedly diluted to each well. 1 cell. One 96-well cell culture plate was limitedly diluted for each positive original clone. After the cells adhere to the wall and cover 1 / 2 to 1 / 3 of the bottom...
Embodiment 3
[0060] This embodiment provides a disposable one-step electrodeposition immunobiological sensor for detecting histamine, which uses an electrode with a Prussian blue-chitosan-nano-gold composite film on the surface.
[0061] Nano gold and Prussian blue (Fe4[Fe(CN) 6 ] 3 ) molar concentration ratio is 1:1000, and the chitosan content is 0.02%, and the preparation method is as follows:
[0062] Dissolve 0.05g chitosan (deacetylation degree ≥ 95%, viscosity 100-200mpa.s) in 100mL 1.0% acetic acid solution, stir at room temperature for 3 hours to completely dissolve chitosan, and prepare 0.05% chitosan sugar solution. 0.0406gFeCl 3 , 0.0822gK 3 [Fe(CN) 6 ], 0.7455g KCl and 1mL concentrated hydrochloric acid were added to 100mL of the above-mentioned chitosan solution, and ultrasonically dispersed at room temperature until a stable dark green dispersion was obtained; then the prepared 1mL nano-gold colloid was added to the above-mentioned solution that had been mixed , and th...
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