Chemiluminescence Immunoassay for S-Adenosylhomocysteine Detection and Its Application in DNA Methyltransferase Activity Evaluation and Inhibitors Screening.

Aberrant methylation by DNA transferase is associated with cancer initiation and progression. For high-throughput screening of DNA methyltransferase (MTase) activity and its inhibitors, a novel chemiluminescence immunoassay (CLIA) was established to detect S-adenosylhomocysteine (SAH), the product of S-adenosylmethionine (SAM) transmethylation reactions. We synthesized two kinds of immunogens for SAH and characterized the polyclonal antibodies in each group. The antibody with higher titer was used to develop a competitive CLIA for SAH, in which SAH in samples would compete with SAH coated on microplate in binding with SAH antibodies. Successively, horseradish peroxidase labeled goat antirabbit IgG (HRP-IgG) was conjugated with SAH antibodies on the microplate. In substrate solution containing luminol and H2O2, HRP-IgG catalyzed luminol oxidation by H2O2, generating a high chemiluminescence signal. The method could detect as low as 9.8 ng mL(-1) SAH with little cross-reaction (3.8%) to SAM. Since higher DNA MTase activity leads to more production of SAH, a correlation between the chemiluminescence intensity and DNA MTase activity was obtained in the range from 0.1 to 8.0 U/mL of DNA MTase. The inhibition study showed that, in the presence of SAM as methyl donor, Lomeguatrib, 5-Azacytidine, and 5-Aza-2'-deoxycytidine could inhibit the DNA MTase activity with IC50 values of 40.57 nM, 2.26 µM, and 0.48 µM, respectively. These results are consistent with the published studies. The proposed assay does not depend on recognizing methylated cytosines in oligonucleotides (methyl acceptor) and showed the potential as an accessible platform for sensitive detection of DNA MTase activity and screening its inhibitors.

Determination of sodium benzoate in food products by fluorescence polarization immunoassay.

A rapid and sensitive fluorescence polarization immunoassay (FPIA), based on a polyclonal antibody, has been developed for the detection of sodium benzoate in spiked samples. The immunogen and fluorescein-labeled analyte conjugate were successfully synthesized, and the tracer was purified by TLC. Under the optimal assay conditions, the FPIA shows a detection range of 0.3-20.0 µg mL(-1) for sodium benzoate with a detection limit of 0.26 µg mL(-1) in the borate buffer. In addition, the IC50 value was 2.48 µg mL(-1), and the cross-reactivity of the antibodies with ten structurally and functionally related analogs were detected respectively. Four kinds of food samples (energy drink, candy, ice sucker, RIO(TM) cocktail) were selected to evaluate the application of FPIA in real systems. The recoveries were 96.68-106.55% in energy drink; 95.78-100.80% in candy, 86.97-102.70% in ice sucker, and 103.58-109.87% in benzoate contained sample RIO(TM) cocktail, and coefficients of variation of this method were all lower than 11.25%. Comparing with the detection results of HPLC, the developed FPIA has comparative performance in the real sample determination. The results suggest that the FPIA developed in this study is a rapid, convenient and simple method, which is suitable to be used as a screening tool for homogeneous detection of sodium benzoate in food products.

Indirect competitive immunoassay for detection of vitamin B2 in foods and pharmaceuticals.

An indirect immunoassay for the determination of vitamin B2 in food samples and vitamin tablets was developed. A carbodiimide-modified active ester method was used to synthesize the immunogen for vitamin B2. The coupling ratio of vitamin B2 to carrier protein in immunogen was 19.98:1. The titer of the polyclonal antibody was 1:64000, and the antibody showed high specificity in the presence of vitamin B2 photolytic products and other B group vitamins. The immunoassay showed detection limits (LODs) of 1.07 ng/mL in PBS, 24.6 ng/g in vitamin drink, and 0.50 mg/kg in milk powder. Recovery was 99.58-110.91% in milk powder and 70.20-100.5% in vitamin drink. Vitamin B2 samples were analyzed by high-pressure liquid chromatography (HPLC) and the immunoassay, and results showed good agreement. Finally, this method was applied to detect vitamin B2 in commercial milk powder and vitamin tablets, and the detected amount correlated well with the labeled amount.