Human urinary albumin latex enhanced secondary-antibody competitive immunity turbidity detection kit as well as production method and use method thereof
A detection kit and latex-enhanced technology, applied in the biological field, can solve problems such as difficult standardization of clinical results, incomplete preparation methods of latex-antibody conjugates, and inability to use common test results.
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Embodiment 1
[0054] Preparation of rabbit anti-mouse IgG secondary antibody (A) or goat anti-rabbit IgG (B)-latex conjugate
[0055] Add 0.2ml of commercially available carboxyl nano-latex particles with a particle size of 100nm and a concentration of 10%, add 0.8ml of pH 4.6, 0.02M phosphate buffer, add 20ul of dicyclohexylcarbodiimide with a concentration of 20mg / ml, and incubate on a shaking table at 37°C After 20 minutes of reaction and activation, add 0.5ml, PH7.5, 0.02M phosphate buffer and 0.02ml purified rabbit anti-mouse IgG antibody or 0.05ml goat anti-rabbit IgG (secondary antibody), and then incubate at 37°C for 60 minutes of ligation Minutes, after the end of the linking reaction, add pH 7.0, 0.02M phosphate buffer to make the total solution volume to 10ml, then add 0.1ml 0.2M glycine, 0.1ml 1% Tween 20, 0.1ml proclin 300, shake at 37°C The bed was incubated for another 30 minutes to block and terminate the reaction. Add proclin300 at 0.1% to prepare rabbit anti-mouse IgG (A) ...
Embodiment 2
[0057] Determination of mouse anti-human albumin monoclonal antibody or rabbit anti-human albumin polyclonal antibody titer:
[0058] Dilute mouse anti-human albumin monoclonal antibody or rabbit anti-human albumin polyclonal antibody with water ratio, use 0.02M, PH7.0 (containing NaCl0.15M) phosphate buffer as R1, use the above method to prepare rabbit antibody respectively The mouse IgG secondary antibody-latex conjugate is R2. On the Hitachi 7170S automatic biochemical analyzer, set the measurement program according to the following parameters, and measure the primary antibody with different dilutions; measurement wavelength: 546nm; R 1 :R 2 : Sample=100:100:20, that is, after adding 100ul R1, add 20ul sample, keep warm at 37°C for 5 minutes, then add 100ul R 2 , read the light absorption value at point 17 and point 34, and calculate the light absorption difference between point 34 and point 17: △A=A34-A17. The results of titer determination are shown in Table (2) and Fig...
Embodiment 3
[0061] 3.1 Preparation of Human Urine Albumin Latex Enhanced Secondary Antibody Competitive Immunoturbidimetric Detection Kit:
[0062] Prepare the secondary antibody competition immunoturbidimetric detection reagent according to the following formula
[0063] Kit A:
[0064] R1: PH7.0, 0.02M phosphate buffer
[0065] Rabbit anti-mouse IgG secondary antibody-latex (0.01-0.05%)
[0066] Glycine (0.002M)
[0067] Tween20 (0.01%)
[0068] Proclin 300 (0.1%)
[0069] R2: PH7.5, 0.02M phosphate buffer
[0070] Mouse anti-human albumin monoclonal antibody (1:200)
[0071] NaCl(0.15M)
[0072] Casein (0.01%)
[0073] Tween20 (0.001%)
[0074] Proclin 300 (0.1%)
[0075] Kit B
[0076] R1: PH7.0, 0.02M phosphate buffer
[0077] Goat anti-rabbit IgG secondary antibody-latex (0.01-0.05%)
[0078] Glycine (0.002M)
[0079] Tween20 (0.01%)
[0080] Proclin 300 (0.1%)
[0081] R2: PH7.5, 0.02M phosphate buffer
[0082] Rabbit anti-human albumin (1:500)
[0083] NaCl(0.15M) ...
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