Chemicals
N,N’-Dicyclohexylcarbodiimide (DCC), N-hydroxysuccinimide (NHS), and Freund’s complete adjuvant (FCA) were purchased from Sigma (St. Louis, MO, USA). Tetramethylbenzidine (TMB) was obtained from Amresco Chemical Co. (Solon, OH, USA). Bovine serum albumin (BSA) and ovalbumin (OVA) were purchased from Roche (Florence, USA). Goat anti-rabbit IgG horseradish peroxidase (IgG-HRP) was obtained from Jackson ImmunoResearch Laboratories, Inc. (West Grove, Pennsylvania, USA). SAR (purity >98%) was purchased from Wuhu Delta Medical Technology Co., Ltd. (Wuhu, China). All chemicals and solvents were of analytical grades. A 96-well polystyrene immunoplate was purchased from Corning Costar Corp. (New York, USA).
The media for ELISA included the following components: phosphate-buffered saline (PBS, 0.02 mol/L phosphate buffer, pH 7.2, containing 0.15 mol/L NaCl); coating buffer (0.05 mol/L carbonate-hydrogen carbonate, pH 9.6); blocking buffer (0.02 mol/L PBS containing 0.5% gelatin); washing buffer (PBS-T, 0.02 mol/L PBS containing 0.05% Tween 20).
Animals
Two male rabbits (2.5 kg) were obtained from the Qinglong Mountain Animal Breeding Ground of the Nanjing Jiangning District (Nanjing, China, Certificate No. SCXK (Su) 2007-0008). Six male Sprague-Dawley rats (clean grade, 7–9 weeks old and weighing 250–280 g) were obtained from the Comparative Medicine Center of Yangzhou University (Yangzhou, China, Certificate No. SCXK (Su) 2007-0001). Protocols for the animal studies were approved by the Regulations of Experimental Animal Administration, State Committee of Science and Technology of the People’s Republic of China.
Preparation of the Immunogen for Sarsasapogenin
SAR was coupled to BSA based on a method reported by Naar with slight modifications. Fifty milligrams of SAR and 1 g of succinic anhydride were added to 1 mL pyridine and refluxed at 100 °C for 9 h. The reaction was monitored by thin-layer chromatography (TLC) using a chloroform:methanol:water mixture (8:1:0.1, v/v/v) as the developing solvent. The solution was extracted three times with chloroform (15 mL for each extraction). The combined extracts were washed three times with water (25 mL for each wash). The succinylated SAR (SAR-HS) was further purified by Sephadex LH-20 (chloroform:methanol=1:1).
Succinylated SAR was covalently attached to BSA by an active ester method. The active ester of SAR-HS was prepared by stirring 10 mg SAR-HS, 19 mg DCC, and 13 mg NHS in 1 mL dimethylformamide for 5 h at room temperature. The solution was added dropwise to 10 mL phosphate-buffered saline (PBS, 0.01 mol/L, pH 7.4) containing 30 mg BSA and incubated at 4 °C for 12 h with constant stirring to produce SAR-HS-BSA. The reaction mixture was put into a dialysis bag and dialyzed successively using deionized water for 72 h at 4 °C. Finally, the solution was lyophilized and stored at −20 °C before use. The coating antigen was prepared according to the conjugation protocol described above using SAR-HS and OVA.
Antiserum Production in Rabbits
Two male rabbits were used for the immunization of SAR-HS-BSA. Pre-immunization blood samples were taken. The emulsifying mixture of the conjugate (1 mg) and FCA was injected into each rabbit subcutaneously. The same dose of the conjugate emulsified with incomplete Freund’s adjuvant in the same ratio was used as a booster once every 14 days. Blood was collected from the marginal ear vein 1 week after each boost. The blood was centrifuged at 3000 rounds per minute for 15 minutes, and the serum was collected and stored at −20 °C. Ten days after the last injection, whole blood was collected from the carotid artery, and the serum was prepared using the same procedure as described above.
ELISA Procedure
The wells of a 96-well immunoplate were coated overnight with SAR-HS-OVA (10 μg/mL) dissolved in 50 mmol/L carbonate buffer (pH 9.6). The plate was washed three times with PBS-T and treated with 150 μL of PBS-T containing 0.5% gelatin for 1 hour to reduce non-specific adsorption. Fifty microliters of antibody was added to each well followed by the addition of a competitor and incubated for 2 hours. The plate was washed three times with PBS-T and incubated with 100 μL of HRP-conjugated anti-rabbit IgG (1:40,000 dilution) for 1 hour. Subsequently, the plate was washed three times with PBS-T, 100 μL of TMB peroxidase substrate solution was added to each well, and the plate was incubated for 20 minutes in the dark at room temperature. The reaction was stopped by the addition of 50 μL of 2 mol/L H₂SO₄. The activity of enzyme bound to the plate was spectrophotometrically measured by a microplate reader at 450 nm.
The cross-reactivities (CRs) of polyclonal antibodies (PAbs) against various compounds were evaluated and calculated using the method of Weiler and Zenk.
Sample Preparation
The calibration and quality control samples for SAR were prepared by adding standard SAR to negative control serum. A stock solution of standard SAR prepared in methanol (0.5 mg/mL) was serially diluted with methanol to obtain a series of working solutions. Ten microliters of each working solution was added to 200 μL of negative control serum to prepare the calibration samples spanning a concentration range of 1 ng/mL to 1000 ng/mL, and to prepare the quality control samples at 10, 100, and 500 ng/mL. All the samples were mixed on a vortex mixer for 30 seconds before application to the microtiter plate. The separated real rat serum samples obtained from the pharmacokinetic study were added to the microtiter plate for analysis.
Pharmacokinetic Evaluation
Rats were intragastrically administered with 100 mg/kg SAR. Blood samples were collected at 0.5, 1, 2, 4, 6, 8, 10, 12, 24, 48, and 72 hours after administration. Serum was separated by centrifugation at 8000 rounds per minute for 5 minutes. The separated serum was stored at −20 °C prior to use.
Relevance: 7
Summary Feedback: This article presents the development of a sensitive indirect competitive ELISA method for detecting sarsasapogenin in rat plasma. It details the creation of an immunogen, the production of antibodies, and their validation through pharmacokinetic studies, showcasing its practical application.
Publication Decision: Yes
Immunology, ELISA, Biochemical Techniques, Chemical Analysis, Immunogen Preparation
