Abstract
In this study, a polyclonal antibody with high avidity and specificity to the potent hypocholesterolaemic agent rosuvastatin (ROS) has been prepared and used in the development of highly sensitive enzyme-linked immunosorbent assay (ELISA) for determination of ROS in plasma. ROS was coupled to keyhole limpt hemocyanin (KLH) and bovine serum albumin (BSA) using carbodiimide reagent. ROS-KLH conjugate was used for immunization of female 8-weeks old New Zealand white rabbits. The immune response of the rabbits was monitored by direct ELISA using ROS-BSA immobilized onto microwell plates as a solid phase. The rabbit that showed the highest antibody titer and avidity to ROS was scarified and its sera were collected. The IgG fraction was isolated and purified by avidity chromatography on protein A column. The purified antibody showed high avidity to ROS; IC50 = 0.4 ng/ml. The specificity of the antibody for ROS was evaluated by indirect ELISA using various competitors from the ROS-structural analogues and the therapeutic agents used with ROS in a combination therapy. The proposed ELISA involved a competitive binding reaction between ROS, in plasma sample, and the immobilized ROS-BSA for the binding sites on a limited amount of the anti-ROS antibody. The bound anti-ROS antibody was quantified with horseradish peroxidase-labeled second anti-rabbit IgG antibody (HRP-IgG) and 3,3',5,5'-tetramethylbenzidine (TMB) as a substrate for the peroxidase enzyme. The concentration of ROS in the sample was quantified by its ability to inhibit the binding of the anti-ROS antibody to the immobilized ROS-BSA and subsequently the color intensity in the assay wells. The assay enabled the determination of ROS in plasma at concentrations as low as 40 pg/ml.
Highlights
Rosuvastatin (ROS); (3R,5S,6E)-7-[4-((4-fluorophenyl)-6(1-methylethyl)-2-[methyl amino]-5pyrimidinyl]-3, 5-dihydroxy-6-heptenoic acid (Figure 1), is an effective 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor and it is widely prescribed in the treatment of patients with hypercholesterolaemia [1]
A 50 μl of anti-ROS antibody sample was dispensed in each well and plate was incubated for 1.5 h at 37°C, the plates were washed with PBST, and 50 μl of horseradish peroxidase-labeled second anti-rabbit IgG antibody (HRP-IgG) (1/5,000 in phosphate buffered saline (PBS)) was added to each well
In competitive enzyme-linked immunosorbent assay (ELISA), 50 μl of ROS sample was mixed with antibody solution and 50 μl of the mixture was dispensed into microplate wells that have been previously coated and blocked
Summary
Rosuvastatin (ROS); (3R,5S,6E)-7-[4-((4-fluorophenyl)-6(1-methylethyl)-2-[methyl (methylsulphonyl) amino]-5pyrimidinyl]-3, 5-dihydroxy-6-heptenoic acid (Figure 1), is an effective 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor and it is widely prescribed in the treatment of patients with hypercholesterolaemia [1]. The protein content of the dialyzate was determined by BCA reagent kit, and used as the pure anti-ROS antibody sample. ELISA procedures and data analysis Aliquot (50 μl) of either ROS-BSA conjugate or BSA protein solution (5 μg/ml in PBS) was dispensed in each well of the microwell plate. A 50 μl of anti-ROS antibody sample (rabbit serum or purified IgG) was dispensed in each well and plate was incubated for 1.5 h at 37°C, the plates were washed with PBST, and 50 μl of HRP-IgG (1/5,000 in PBS) was added to each well. In competitive ELISA, 50 μl of ROS sample (standard ROS solution or plasma that have been tenfold diluted with PBS) was mixed with antibody solution and 50 μl of the mixture was dispensed into microplate wells that have been previously coated and blocked. The concentrations of ROS in the samples were obtained by interpolation on the standard curve
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