US 12,282,016 B2
Labeling method for improving signal intensity of time-resolved fluorescence
Renyao Jin, Hangzhou (CN); Yanling Song, Hangzhou (CN); Xiaoxia Liu, Hangzhou (CN); Jiacheng Yang, Hangzhou (CN); and Lu Zhai, Hangzhou (CN)
Assigned to ZHEJIANG GONGSHANG UNIVERSITY, Hangzhou (CN)
Filed by Zhejiang Gongshang University, Hangzhou (CN)
Filed on Dec. 28, 2021, as Appl. No. 17/563,550.
Claims priority of application No. 202110715114.4 (CN), filed on Jun. 26, 2021; and application No. 202110715118.2 (CN), filed on Jun. 26, 2021.
Prior Publication US 2022/0412964 A1, Dec. 29, 2022
Int. Cl. G01N 33/533 (2006.01); G01N 21/64 (2006.01)
CPC G01N 33/533 (2013.01) [G01N 21/6408 (2013.01); G01N 21/6428 (2013.01); G01N 2021/6439 (2013.01)] 2 Claims
OG exemplary drawing
 
1. A labeling method for improving signal intensity of time-resolved fluorescence, comprising the following steps:
weighing and dissolving 4-7 mg of 2-S-(4-aminobenzene)-1,4,7 triazacyclononane-1,4,7-triacetic acid (NOTA) into 1 mL of a 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) solution at pH 7.4 with a concentration of 0.01 mol·L−1 to obtain an NOTA chelating agent solution, wherein the NOTA chelating agent solution is a first solution;
adding 500-700 μL of a glutaraldehyde solution with a concentration of 20 mmol·L−1 to the first solution for reaction in dark place over a night at room temperature to obtain a second solution;
weighing and dissolving 20-30 mg of purified lyophilized powder of a monoclonal antibody into 3 ml of the HEPES solution at pH 7.4 with the concentration of 0.01 mol·L−1, and performing magnetic stirring for mixing evenly at room temperature, thus to obtain a third solution;
dropwisely adding the second solution to the third solution, then adjusting a pH value to 9.0, and stirring the mixed second and third solutions for reaction for 4-6 h in dark place at 4° C., thus to obtain a fourth solution;
loading the fourth solution to a dialysis bag having a molecular weight cut-off of 8 KDa for dialysis by the HEPES solution at pH 7.4 with the concentration of 0.01 mol·L−1, replacing the HEPES solution once every 4 h and replacing 4-6 times in total, and then absorbing a reaction solution in the dialysis bag to obtain a fifth solution;
weighing 0.11-0.15 g of EuCl3.6H2O and preparing a EuCl3 solution with 10 mL of ultrapure water, thus to obtaining a sixth solution; and
taking and adding 200-400 μL of the sixth solution to the fifth solution for reaction for 4-6 h in dark place at room temperature, then placing the mixed fifth and sixth solution after reaction to another dialysis bag having a molecular weight cut-off of 8 KDa for dialysis, and replacing the HEPES solution once every 4 h and 4-5 times in total; centrifuging the dialyzed solution for 3-5 times with a 30 KDa ultrafiltration centrifugal tube at 7000-9000 rpm, and redissolving a remaining solution with 5-10 ml of the HEPES solution at pH 7.4 with the concentration of 0.01 mol·L−1; wherein the obtained reaction solution is an olaquindox antibody complex immunolabelled by time-resolved fluorescence.