Development of Immunochromatographic Assay for Determination of Tetracycline in Human Serum

Abstract

Determining antibiotic concentration in human blood provides useful pharmacokinetic information. Commonly used methods such as ELISA require a long time to obtain results and thus cannot be applied when information is needed immediately. In this study, a novel antibody-based lateral flow technique was developed for tetracycline detection in human serum. Contrary to tests developed to analyze food samples, the features of work with serum as analyzed probe were studied for the first time here. The application of labeled and unlabeled specific antibodies was compared. For this purpose, specific and anti-species antibodies were labeled with gold nanoparticles and used for antigen–antibody interaction on the membrane surface with observed staining in the test zone. For both schemes, optimal conditions were established to provide the best sensitivity. The developed assay has a limit of visual detection as low as 35 and 11 ng/mL for the direct and indirect labeled antibodies, respectively. The limit of instrumental detection is from 0.4 to 3.5 ng/mL for diluted and undiluted sera. The use of indirect antibody labeling showed a small increase in sensitivity compared to traditional direct antibody labeling. The developed method showed no cross-reactivity with antibiotics of other classes. The method was used to test samples of serum. The results showed high correlation with the data obtained by ELISA (R2 = 0.98968). The assay provides a quick assessment of the amount of antibiotics in the blood and keeps them under control throughout the duration of therapy.

Design of Multiplex Lateral Flow Tests: A Case Study for Simultaneous Detection of Three Antibiotics

“…Mouse anti-tetracycline, anti-chloramphenicol and anti-lincomycin monoclonal antibodies, as well as conjugates of TET, CAP and LIN with bovine serum albumin (BSA) were purchased in Eximio Biotec (Wuxi, China). The used antibodies did not interact with antibiotics from other chemical classes, as we have demonstrated earlier for individual ICAs of tetracycline [24], chloramphenicol [25] and lincomycin [26].…”

“…Then 1 mL of 50 mM potassium phosphate buffer, pH 7.4 with 0.1 M NaCl (PBS) containing 0.25% BSA, 0.25% Tween 20, 1% saccharose and 0.05% NaN 3 was added to the precipitate. The composition of the buffer was based on the previous studies described in [24]. An optical density of the obtained conjugate was established using a Libra S60 spectrophotometer (BioChrom, Cambridge, UK).…”

“…The LIN-BSA, TET-BSA and CAP-BSA conjugates (for the test zone) and mouse IgG (for the control zone) were applied to the working nitrocellulose membrane in an amount of 0.1 µL per 1 mm. The concentrations of LIN-BSA, TET-BSA and CAP-BSA conjugates were chosen on the base of our previous studies to provide sufficient coloration in the absence of analyte [24][25][26] and were equal to 1, 0.5 and 0.5 mg/mL respectively. The difference in the chosen concentrations for these commercial conjugates may be caused by their composition and/or sorption properties.…”

“…Test strips were brought to room temperature and immersed into the samples with various concentrations of antibiotics in 50 mM phosphate buffer, pH 7.4, with 0.25% Tween 20 [24] for 7 min.…”

Monitoring Antibiotics and Inflammatory Markers in Human Blood: Impact in Choice of Antibiotic Therapy and Used Methods

“…To evaluate the efficacy of antibiotic therapy, it is necessary to concentrate on the main indicators-the concentration of the administered antibiotic (in blood, serum, plasma, urine, cerebrospinal fluid, if necessary) [70]-as well as the content of inflammatory markers (C-reactive protein as an acute phase protein and procalcitonin as an express marker of bacterial infections) [71][72][73].…”