Labeling principle of chemiluminescence substrate acridinium ester in diagnostic reagents in chemiluminescence immunoassay, we label the antibody protein with acridine ester at first, so that the analyte can be detected after the immune reaction. In that case,how to label the antibody is very critical.Acridine salts and related compounds have been widely proven to be very useful chemiluminescent labels, surpassing radioisotopes in stability, label specificity, and detection sensitivity.The acridine-NHS ester, also name as acridine succinimide ester, can reacts with the primary amino groups of proteins. Under alkaline conditions, NHS is substituted as a leaving group and the protein forms a stable amide bond with the acridine ester. After the reaction was completed, the excess acridinium salt was removed through a desalting column. In the presence of alkaline hydrogen peroxide, acridine-labeled proteins can emit light without enzymatic catalysis.
Therefore, the addition of the excitation solution causes the reaction system to release photons at about 430 nm immediately, and the protein concentration can be detected by counting the number of photons with a standard luminometer. Because this luminescence process is completed within 2 seconds, the sample must be placed directly in front of the photon detector inside the photometer. Proteins, polypeptides, antibodies, and nucleic acids can all be labeled with acridine esters.
What should be noted for chemiluminescence reagent is dissolving acridine succinimide ester with strictly anhydrousstrictly anhydrous DMF to prevent the acridine ester from being hydrolyzed. And it should be stored in a dry seal at -20°C when not in use.Different from traditional acridine ester,the structure of acridine succinimide ester has been modified to increase the steric hindrance and enhance the hydrolysis resistance.It can be stable at PH buffer solution at room temperature with PH 7.0.
If the acridine carboxylic acid without -NHS, it is necessary to add a condensing agent such as EDCI, etc., in order to have a coupling reaction with an amine group-containing protein. Acridine hydrazide, containing free amino group, is suitable for direct coupling of polysaccharide nucleic acid or protein containing aldehyde group at the end of acridine hydrazide.
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