Membrane strip biosensor system for point-of-care testing
A technology of biosensors and membrane strips, applied in the field of biosensors, can solve problems such as difficulties
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Embodiment 1
[0100] Example 1: Synthesis of antibody-colloidal gold conjugates
[0101] The acidity and antibody concentration of various reaction solutions were measured according to the standard protocol, and then the conjugates were synthesized under the optimal conditions (references: S.H.Paek et al., 1999, Anal.Lett., Vol.32, 335-360 ).
[0102] Briefly, dialyzed HBsAg-specific antibody solution (100 mg / ml, 0.8 ml) in neutral pH buffer was added to gold solution (8 ml), adjusted to pH 9.0, and reacted for 30 minutes. Then, 1 ml of a 5% casein solution (casein-PB) prepared by dissolving casein in 10 mM phosphate buffer (pH 7.4, PB) was added to the solution and reacted for 30 minutes. After the reaction solution was centrifuged at 15,000 rpm for 45 minutes, the supernatant was removed. Casein-PB was added to the remaining gold precipitate and the final volume of the conjugate was adjusted to 0.2 ml, stored at 4°C until use.
Embodiment 2
[0103] Example 2 Synthesis of antibody-enzyme conjugates
[0104] Conjugation between the analyte-specific antibody and the enzyme is performed by a chemical reaction using a cross-linking agent. After reacting the antibody with a 20-fold molar excess of SMCC for 4 hours at 4°C, the excess SMCC was removed by Sephadex G-15 gel chromatography, and the antibody was directly conjugated with the activated enzyme as described below. For enzyme activation, protein solutions were reacted in 5 mM EDTA-containing PB with 20-fold molar excess of SPDP for 1 hour at room temperature. To introduce sulfhydryl groups on the molecule, DTT (final concentration 10 mM) was added to the reaction mixture and reacted at 37° C. for another 2 hours. Excess reagents were removed on Sephadex G-15 gel columns. Two activation reagents, antibody and enzyme, were mixed at a molar ratio of 1:10 and reacted overnight at 4°C. Purification of these synthesized antibody-enzyme conjugates was performed using ...
Embodiment 3
[0105] Example 3 Signal generating shims with immobilized antibodies
[0106]Nitrocellulose (NC) membranes optimized for migration efficiency and pore size were used as signal generating spacers. An NC membrane (pore size: 12 mm, Millipore) was used for antibody immobilization. Physical adsorption and chemical methods can be used as immobilization methods, and the appropriate method is finally selected by considering the convenience and reproducibility of the method according to the results of the experiment. Antibodies were immobilized at predetermined positions on NC membrane strips (7 x 25 mm) by physical adsorption. 1 mg / ml antibody (1.5 ml) diluted with PB containing 140 mM NaCl was spotted on one position (10 mm from the bottom) of the membrane using a microdispenser, followed by reaction at room temperature for 1 hour. The strips with immobilized antibodies were soaked in 0.5% casein (pH 7.6, casein-Tris) dissolved in 100 mM Tris buffer for 1 hour to block the residua...
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