Dissolution and labeling of chemiluminescence reagent acridinium ester
Acridinium ester is a type of chemiluminescence reagent used in chemiluminescence immunoassay. This kind of analysis technology is commonly used in detection items such as sex hormones, thyroid hormones, bone metabolism, tumor markers, immunoglobulins, etc., so acridinium esters The related research and use are very popular.
Compared with electrochemiluminescence, the application of acridinium ester as a chemiluminescence reagent is more common in China and does not require very expensive equipment. Acridine ester chemiluminescence detection mainly has the advantages of high sensitivity, strong specificity, good reagent stability, long validity period, wide detection linear range, simple operation, stable and fast method, multiple detection items, and high degree of automation. It has gradually become the current immunoassay technology. Mainstream method.
Method for dissolving acridine ester
Acridinium esters are usually stored in the form of freeze-dried powder at low temperature and protected from light. The first step is to prepare it into a solution during use. Acridinium esters usually directly label proteins, antibodies or nucleic acids in the detection process, and the detection environment is generally an aqueous solution, so some acridinium esters will add some anti-hydrolysis and hydrophilic groups. So is it water to dissolve acridine esters?
The answer is no. The process of dissolving and labeling acridine esters is still strictly anhydrous. Aprotonated solvent DMF (N,N-dimethylformamide) or DMSO (dimethyl sulfoxide) should be used. Wait to dissolve. Before labeling, the carboxylic acid at the end of the acridine ester body is connected to NHS (N-hydroxysuccinimide) to activate it. It is very active, so it can only be dissolved with aprotonated solvents. The higher concentration of acridine esters dissolved in DMF is 4mM (about 2.7mg/mL), and the solubility in DMSO is about 10mg/mL.
Labeling group of acridinium ester
The NHS labeling group is usually used on the acridinium ester, which can be directly connected to the primary amino group on the protein, and the N-hydroxysuccinimide is removed at the same time. After the acridine ester labeling is completed, it exists in the form of amide bond or ester bond with the labeled substance, and it can stably exist in the weak acid buffer. The hydrolysis resistant group and the hydrophilic group can also be stabilized in the weak acid buffer. Neutral aqueous solution. The active site of acridinium ester itself is unstable in alkaline and oxidizing environment. After coupling, use a weakly acidic buffer and bubbling nitrogen to remove oxygen.
There are many types of acridine esters. There are 6 types developed and sold by Desheng, four of which are labeled with NHS esters, one acridine carboxylic acid is linked to the protein through CDCI condensing agent, and the other is acridine hydrazide through a linking group. The free amino group in the group couples the aldehyde-containing polysaccharide with the nucleic acid.
In chemiluminescence analysis, the luminescence intensity of acridine ester is influenced by various factors, such as reaction medium, temperature, time, and excitation light source energy. To achieve good detection results, it is necessary to comprehensively consider and optimize these factors. Meanwhile, attention should be paid to controlling and standardizing experimental conditions to ensure accurate and reliable results. Thoroughly studying these influencing factors will help promote the development of chemiluminescence analysis methods.