.jpg)
Scheme 1. Acridinium NHS ester derivatives applied for the all-in-one immunoassay.
.jpg)
Scheme 2. Acridinium ester chemiluminescence reaction mechanism of an antigen-binding antibody conjugated with (a) acridinium C2, NHS ester or (b) Lumiwo acridinium NHS ester in the all-in-one immunoassay.
Several types of acridinium esters that can be conjugated with an antibody are available for All-in-One immunoassays. For example, two different acridinium ester derivatives shown in Scheme 1 can be labeled with an antibody. Therefore, an antigen-binding antibody conjugated with an acridinium ester derivative shown in Schemes 2 - 3 is formed in the all-in-one immunoassay. Scheme 2(a) shows that the high-energy intermediate (or activator) formed with the consecutive additions of (1) hydrogen peroxide (5 – 40 mM) in nitroc acid (25 – 100 mM) and (2) NaOH (0.1 – 0.5 N) containing trace concentration of Triton X-100 is separated from the antibody. Then, the high-energy intermediate emits bright blue chemiluminescence. Also, After sequential addition of the 2 reagents, a high-energy intermediate bound to the antibody is formed as shown in Scheme 2(b). The high-energy intermediate also emits blue chemiluminescence. For example, acridinium ester chemiluminescence in the All-in-One immunoassay operated based on the reaction mechanism shown in Scheme 2 is proportionally enhanced with the increase of D-dimer in human serum.
The wavelength of chemiluminescence emitted in the all-in-one immunoassay using an acridinium ester links to a luminescent dye (e.g., fluorescein, rhodamine B, BDP) shown in Scheme 3 is longer than that using the acridinium C2 NHS ester shown in Scheme 2. This is becuase the emission wavelength of luminescent dye in Scheme 3 is logner than that of the high-energy intermediated formed from the chemiluminescence reaction shown in Scheme 2. Therefore, the color of chemiluminescence emitted from an acridinium ester linked to a luminescent dye in the all-in-one immunoassay depends on the emission wavelength of the luminescent dye. In conclusion, Scheme 3 indicates that the all-in-one immunoassay can simultaneously analyze different types of target antigens in a sample. In other words, the all-in-one immunoassay, which uses multiple acridinium esters combined with different luminescent dyes and antibodies capable of selectively binding to target antigens, can be applied as a new assay method to simultaneously quantify multiple types of target antigens in a sample..
.jpg)
Scheme 3. Chemiluminescence emitted from acridinium ester linked with (a) fluorescein, (b) rhodamine B, and (c) BDP in All-in-One immunoassays.