A New Immunoassay Method for the Quantitation of Hydroxymethylglutaryl Co-Enzyme A Reductase Inhibitors in Human Plasma

Abstract

This study described the development of a convenient and highly sensitive enzyme immunoassay (EIA) method for the determination of four hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase inhibitors in human plasma. These HMG-CoA reductase inhibitors were atorvastatin (ATR), pravastatin (PRV), fluvastatin (FLV) and rosuvastatin (ROS). The selection of these compounds for the present study was based on their therapeutic importance in the treatment of hypercholesterolemia, and the need for new highly sensitive analytical methodology that can assess the low concentrations of these compounds in plasma samples, and overcome the major drawbacks of the previously reported methods for their analysis in plasma. The proposed EIA is a competitive antibody-capture type. In this method, drug conjugated with bovine serum albumin protein (drug-BSA conjugate) was immobilized onto the inner surface of microwells of a 96-well assay plate and the plate was used as a solid phase. The drug (in its sample) was mixed with a pre-determined limited amount of a polyclonal antibody specific to the target analyte drug (anti-drug antibody), and the mixture was dispensed into the microwells that have been coated with the drug-BSA conjugate. The competitive binding reaction occurred between the free drug (in the sample solution) and the immobilized version of the drug (drug-BSA conjugate) for the binding sites that are available on the anti-drug antibody. The amount of the anti-drug antibody that has been bound to the plate wells was quantified by a second antibody specific to the anti-drug antibody and labeled with horseradish peroxidase enzyme (HRP-IgG) and 3,3’,5,5’-tetramethylbenzidine (TMB) as a chromogenic substrate for the peroxidase enzyme. TMB was converted by the action of HRP enzyme into a colored product. The concentration of the drug in its sample was quantified by its ability to inhibit the binding of the anti-drug antibody to the immobilized drug-protein conjugate, and subsequently the color development in the assay wells. Therefore, the intensity of the produced color was inversely correlated with the concentration of the drug in its sample solution. For development of the proposed EIA for each particular drug, two key reagents were prepared. The first one was the drug-BSA conjugate for its use as a solid-phase antigen, and the second reagent was the anti-drug antibody. Each of investigated HMG-CoA reductase inhibitors was conjugated via its carboxylic group with the amino terminals of the BSA and keyhole limpet hemocyanin (KLH) as carrier proteins by carbodiimide reagent. The obtained conjugates were characterized in terms of their degrees of conjugation (percentages of the drug molecules linked to each molecule of the protein). The degrees of conjugation were determined by ultraviolet-spectrophotometric analysis for the conjugates, and they were found to be in the range of 7.05 - 22.25 and 10.80 - 45.66% for the conjugates of the drugs with BSA and KLH, respectively. Drug-BSA conjugates were used for immobilization on the microwells of the assay plates, and the drug-KLH conjugates were used for immunization of animals. Female 2-3 months old New Zealand white rabbits were immunized by their repetitive subcutaneous multiple injections with drug- KLH emulsified in Freund`s adjuvant. During the regime of immunization, blood test bleeds were collected from the rabbits, and analyzed by the EIA for detecting the development and monitoring the progress of the animal’s immune response. The sera of the rabbits that showed the highest affinity for target drugs were collected as crude anti-drug polyclonal antibody samples. These antisera were purified by affinity chromatography using protein A column. The purified antibody was used in the development of the proposed EIA. For establishment of the assay protocol, the experimental conditions affecting the assay performance were investigated and optimized. Under the optimum conditions, calibration curves for the investigated drugs were constructed. The data showed good correlation coefficients (r) on the fourparameter curve fit (r = 0.998, 0.995, 0.999 and 0.996 for ATR, PRV, FLV and ROS respectively). The working ranges of the proposed EIA at relative standard deviation (RSD) values less than 5% were found to be 100 - 10000, 500 - 20000, 20 - 1000, and 40 - 2000 pg/ml for ATR, PRV, FLV, and ROS respectively. The limits of detection (LOD) of the proposed EIA were found to be 40, 200, 10, and 25 pg/ml for ATR, PRV, FLV and ROS respectively. The RSD at the LOD values were found to be 5.54, 6.25, 6.21 and 6.84% for ATR, PRV, FLV and ROS respectively. The precision of the proposed EIA was satisfactory; the RSD for the intra-assay precisions were 2.70 - 4.63, 2.61 - 3.70, 2.46 - 5.37, and 2.47 - 4.46 % for ATR, PRV, FLV, and ROS, respectively. For the inter-assay precisions, RSD values were 3.3 - 5.71, 3.99 - 4.17, 3.19 - 6.64, and 3.24 - 5.27 % for ATR, PRV, FLV, and ROS, respectively. The mean analytical recovery values for the drugs spiked in drugfree plasma samples were 99.3 ± 2.81, 101.0 ± 2.99, 100.9 ± 1.42, and 100.3 ± 3.22% for ATR, PRV, FLV and ROS, respectively. These acceptable recovery values indicated the accuracy of the proposed method for determination of the investigated drugs in plasma samples. The accuracy of the proposed EIA was compared with that of an established HPLC method. The statistical analysis for the results that have been obtained by the two methods proved the good agreement between the two methods as the correlation coefficients were 0.9945, 0.9994, 0.9970, and 0.9871 for ATR, PRV, FLV, and ROS, respectively.