Fluorescence Polarization Immunoassay Research Articles

The many faces of brucellosis: diagnostic and management approach.

This review aims to highlight the multifaceted nature of brucellosis, emphasizing the latest advancements in its diagnosis and management. Given the global prevalence and potential complications of brucellosis, understanding recent advancements in diagnostic techniques and treatment strategies is crucial for clinicians. Recent literature reveals significant progress in diagnostic methods, including the application of fluorescence polarization immunoassay and time-resolved fluorescence resonance energy transfer technologies as well as the invention of artificial Brucella antigens, which offer enhanced sensitivity and specificity. Advances in molecular diagnostics and serological tests have improved early detection rates, however their interpretation remains challenging. Evolving treatment regimens such as the use of hydroxychloroquine as part of triple therapy and the use of nano-delivery systems in therapies have shown promise, in hopes of reducing relapse rates and managing chronic cases. The findings underscore the necessity for clinicians to adopt a comprehensive approach to diagnosing and managing brucellosis. Integrating advanced diagnostic tools with tailored therapeutic strategies can significantly improve patient outcomes. Future research should focus on optimizing these diagnostic techniques and exploring novel therapeutic agents.

Morphine synovial fluid concentrations after intravenous regional limb perfusion in horses during standing sedation.

Addition of morphine to the perfusate while performing intravenous regional limb perfusion (IVRLP) may be helpful in treating painful infectious orthopaedic conditions of the distal limb. The main objective of this study was to determine synovial morphine concentrations following IVRLP with morphine alone or in combination with amikacin. Randomised cross-over in vivo experiment. Six horses underwent IVRLP with 0.1 mg/kg morphine sulphate diluted to 60 mL using 0.9% NaCl (M group) or combined with 2 g amikacin and 0.9% NaCl (MA group) with a 2-week washout period between treatments. Synovial fluid was collected from the radiocarpal joint (RCJ) at 10, 20, 30, 120, 240, 480, 720 and 1440 min after IVRLP. The tourniquet was removed after the 30-min sample was collected. Synovial concentrations of morphine and major metabolites were measured using liquid chromatography-tandem mass spectrometry. Amikacin concentrations were quantified by a fluorescence polarisation immunoassay. Measurable concentrations of morphine were apparent in the RCJ of all horses. Median CMAX of morphine in the M group was 4753.1 (2115.7-14 934.5) ng/mL and 4477 (3434.3-7363) ng/mL in the MA group (p = 0.5). Median CMAX of synovial amikacin was 322.6 (157.5-1371.6 μg/mL). Limitations include small sample size. Investigators were not blinded to the treatments and a third treatment group where amikacin alone was administered via IVRLP to the study population was not included. IVRLP using morphine is a feasible technique and synovial morphine concentrations were measurable following IVRLP and were not affected when used concurrently with amikacin. Administration of morphine via IVRLP may be beneficial as an analgesic technique for orthopaedic conditions of the distal limb while limiting potential serious systemic side-effects.

Development of fluorescence polarization immunoassay to detect dibutylphthalate (DBP) in environmental water samples

Development of fluorescence polarization immunoassay to detect dibutylphthalate (DBP) in environmental water samples

Faster perfusate instillation time results in more systemic leakage of amikacin sulfate when performing intravenous regional limb perfusion in horses.

To evaluate if a difference in synovial amikacin concentrations exists in the radiocarpal joint (RCJ) following different durations of instillation of an IV regional limb perfusion (IVRLP) perfusate. 7 healthy horses. Horses received 2 IVRLPs with 2 g amikacin diluted to 60 mL with 0.9% NaCl via the cephalic vein in a crossover study design with a wash-out period between procedures. Instillation of the perfusate was administered over a 1-minute (technique 1) and 5-minute (technique 5) period. Concentrations of amikacin within the RCJ were measured at time (T) 5, 10, 15, 20, 25, and 30 minutes after instillation of the perfusate. Systemic concentrations of amikacin were measured at T0, 5, 10, 15, 20, 25, 29 minutes, and 1 minute after tourniquet removal (T31). Amikacin concentrations were determined by fluorescence polarization immunoassay. The median maximum concentration (CMAX) of amikacin within the RCJ for technique 1 was 338.4 µg/mL (range, 60 to 4,925 µg/mL), while the median CMAX for technique 5 was higher at 694.8 µg/mL (range, 169.2 to 3,410 µg/mL; P = .398). There was a higher amikacin blood concentration over time for technique 1 compared to technique 5 (P = .004). Administration of perfusate at different rates did not significantly affect synovial concentration of amikacin within the RCJ when performing IVRLP. However, increased systemic leakage was noted when the perfusate was administered over 1 minute, which might affect synovial concentrations in a larger group of horses.

An acute herb-drug interaction of Magnoliae Officinalis Cortex with methotrexate via inhibiting multidrug resistance-associated protein 2.

Magnoliae Officinalis Cortex (MOC), an herbal drug, contains polyphenolic lignans mainly magnolol (MN) and honokiol (HK). Methotrexate (MTX), a critical drug for cancers and autoimmune deseases, is a substrate of multidrug resistance-associated protein 2 (MRP2) and breast cancer resistance protein (BCRP). This study investigated the effect of coadministration of MOC on the pharmacokinetics of MTX and relevant mechanisms. Sprague-Dawley rats were orally administered MTX alone and with single dose (2.0 and 4.0 g/kg) and repeated seven doses of MOC (2.0 g/kg thrice daily for 2 days, the 7th dose given at 0.5 h before MTX). The serum concentrations of MTX were determined by a fluorescence polarization immunoassay. The results showed that a single dose of MOC at 2.0 g/kg significantly increased the AUC0-t and MRT of MTX by 352% and 308%, and a single dose at 4.0 g/kg significantly enhanced the AUC0-t and MRT by 362% and 291%, respectively. Likewise, repeated seven doses of MOC at 2.0 g/kg significantly increased the AUC0-t and MRT of MTX by 461% and 334%, respectively. Mechanism studies indicated that the function of MRP2 was significantly inhibited by MN, HK and the serum metabolites of MOC (MOCM), whereas BCRP was not inhibited by MOCM. In conclusion, coadministration of MOC markedly enhanced the systemic exposure and mean residence time of MTX through inhibiting the MRP2-mediated excretion of MTX.

Synthesis of Steroid Tracers by an Oxime Ligation Method and Their Use in Fluorescent Polarisation Immunoassay

Synthesis of Steroid Tracers by an Oxime Ligation Method and Their Use in Fluorescent Polarisation Immunoassay

Development of a Fluorescence Polarization Immunoassay for the Quantification of Tylosin in Honey

Development of a Fluorescence Polarization Immunoassay for the Quantification of Tylosin in Honey

Therapeutic monitoring of anti-seizure medications in low- and middle-income countries: a systematic review

Background The treatment gap for epilepsy is large in low- and middle-income countries (LMICs) and the effectiveness and safety of the available anti-seizure medication (ASMs) is not fully understood. We systematically reviewed available evidence on therapeutic drug monitoring (TDM) of ASM in LMIC. Methods We searched four main databases (PubMed, Psych-Info, CINAHL and Embase) up to 31st December 2020, with eligible articles screened using a PRISMA checklist and a set of exclusion and inclusion criteria. Full texts were examined to evaluate the extent and practice of TDM in LMICs. Analyses were performed using Stata 13 and descriptive statistics were used to pool median distribution of TDM across studies. Results Of the 6,309 articles identified in the initial search, 65 (1.0%) met the eligibility criteria. TDM of ASMs was mostly done to assess toxicity (42.8%), but rarely to monitor adherence (9.0%). TDM differed by economic status and infrastructural status with majority of the studies coming from Europe (53.8%) and upper-middle-income countries (87.6%). First generation ASMs (82.3%) were more likely to be monitored than second generation ASMs (17.6%) and carbamazepine was the most frequently monitored drug. Fluorescence Polarization Immunoassay (FPIA) was the most common technique used for TDM (41.5%) followed by High-Performance Liquid Chromatography (HPLC) (16.9%). In addition, FPIA was the cheapest method of TDM based on approximated costs ($1000, TDx system). Assay validation and quality control were reported variably, and reference ranges used during TDM of ASMs were relatively uniform. Conclusions TDM is mostly done to evaluate ASM toxicity, but rarely for other reasons such as evaluating adherence or assessing clinical efficacy. There is a need for more investment in comprehensive and targeted TDM in LMICs when initiating treatment, switching therapies, adding or removing ASM and evaluating treatment response and safety of both first generation and second generation ASMs.

Fluorescence Polarization Assays for Organic Compounds in Food Safety.

Elevated concentrations of toxic organic compounds observed in food products pose serious dangers to human health. Both natural and artificial pollutants can cause food contamination. The stages of food production, packaging, transportation, and storage can also largely cause the appearance of undesirable substances in food products. The health consequences of ingesting food containing toxic contaminants range from mild gastroenteritis to deaths resulting from dysfunctional internal organs and neurological syndromes. The World Health Organization (WHO) sets recommendations for the content of such chemicals in food, including a minimum allowable concentration considered safe for human consumption. However, the control of food products from chemical pollutants is necessary. Moreover, fast, sensitive, and inexpensive methods are needed to detect them at the point of need. Currently, immune analysis methods are most widely used to determine pollutants in food. The development of fluorescence polarization immunoassay (FPIA) methods in a competitive format is a powerful and modern tool for detecting organic molecules in various matrices, thereby making FPIA methods useful for food safety applications. Due to the availability of portable devices for measuring the fluorescence polarization signal, FPIA methods can be used at the point of need. The variety of fluorescent labels and recognizing elements (receptors, monoclonal and polyclonal antibodies, and nanobodies) permits fluorescence polarization (FP) assays to detect significantly lower limits of organic substances. The FP assay is a homogeneous, fast, and quantitative method. The development of various formats of FP assays makes them promising in determining food pollutants. This review summarizes publications on FP analyses for detecting organic contaminants (pesticides, hormones, toxins, antibiotics, and other pharmaceuticals) in food products during 2018-2023. Further, it demonstrates the prospects for using this method to determine pollutants at the point of need and for detecting high molecular weight substances, fungi, and bacterial infections during food safety inspections.

Detection of Dibutyl Phthalate in Surface Water by Fluorescence Polarization Immunoassay.

Dibutyl phthalate (DBP) is widely used as a plasticizer in the production of polymeric materials to give them flexibility, strength and extensibility. However, due to its negative impact on human health, in particular reproductive functions and fetal development, the content of DBP must be controlled in food and the environment. The present study aims to develop a sensitive, fast and simple fluorescence polarization immunoassay (FPIA) using monoclonal antibodies derived against DBP (MAb-DBP) for its detection in open waters. New conjugates of DBP with various fluorescein derivatives were obtained and characterized: 5-aminomethylfluorescein (AMF) and dichlorotriazinylaminofluorescein (DTAF). The advantages of using the DBP-AMF conjugate in the FPIA method are shown, the kinetics of binding of this chemical with antibodies are studied, the analysis is optimized, and the concentration of monoclonal antibodies is selected for sensitivity analysis-16 nM. The calibration dependence of the fluorescence polarization signal for the detection of DBP was obtained. The observed IC50 (DBP concentration at which a 50% decrease in the fluorescence polarization signal occurs, 40 ng/mL) and the limit of detection (LOD, 7.5 ng/mL) values were improved by a factor of 45 over the previously described FPIA using polyclonal antibodies. This technique was tested by the recovery method, and the high percentage of DBP discovery in water ranged from 85 to 110%. Using the developed method, real water samples from Lake Onega were tested, and a good correlation was shown between the results of the determination of DBP by the FPIA method and GC-MS. Thus, the FPIA method developed in this work can be used to determine DBP in open-water reservoirs.

Fluorescence polarization immunoassay for the determination and screening of medicines.

Modern clinical medicine with its achievements has helped a person to get rid of many infectious and non-communicable diseases, restore health, preserve and improve the quality of life of people with chronic dis- eases. Tens of thousands of medicines are used in medical practice. However, paradoxical as it sounds, having created medicines for almost all diseases, humanity has not become healthier and the need for medicines is only growing every year. More than 20 % of people receiving medications during therapy have various complications. Therefore, the search for medicines does not stop, but only increases at the present time. Another important problem is the detecting drugs in environmental objects and food products. Most drugs that get into wastewater from pharmaceutical factories and farms, even after treatment at wastewater treatment plants, are still diagnosed in the water. The method of fluorescence polarization is extremely widespread in clinical and biomedical fields. Thanks to the introduction into laboratory diagnostics of devices capable of measuring the signal of fluorescence polarization on microplates, polarization fluorescent analysis is used not only in the traditional format: the detection of drugs in human physiological fluids, environmental objects and food, but also in high-tech screening of drugs, significantly speeding up and facilitating the process of identifying new drugs.

Rapid determination of domoic acid in seafood by fluorescence polarization immunoassay using a portable analyzer

Monitoring phycotoxin accumulation in marine products such as edible shellfish is a regulatory requirement in many countries. Therefore, a simple and rapid onsite quantification method is sought. Herein, we present a fluorescence polarization immunoassay (FPIA), a well-known one-step immunoassay, using a portable fluorescence polarization analyzer for domoic acid (DA), widely referred to as the primary toxin of amnesic shellfish poisoning (ASP). To establish FPIA for DA, the matrix effect of methanol, which is widely used to extract DA from shellfish, on FPIA was investigated. To validate this method, we performed a spike recovery test using oysters containing DA at a concentration equivalent to the regulatory limits of North America and the European Union (20 mg/kg). The recovery rate was found to be 79.4–114.7%, which is equivalent to that of the commercially available enzyme-linked immunosorbent assay (ELISA). We expect that this FPIA system will enable the quantitative onsite analysis of DA and significantly contribute to the safety of marine products.Graphical

BPA Endocrine Disruptor Detection at the Cutting Edge: FPIA and ELISA Immunoassays.

BPA is a chemical commonly used in the production of polymer-based materials that can have detrimental effects on the thyroid gland and impact human reproductive health. Various expensive methods, such as liquid and gas chromatography, have been suggested for detecting BPA. The fluorescence polarization immunoassay (FPIA) is an inexpensive and efficient homogeneous mix-and-read method that allows for high-throughput screening. FPIA offers high specificity and sensitivity and can be carried out in a single phase within a timeframe of 20-30 min. In this study, new tracer molecules were designed that linked the fluorescein fluorophore with and without a spacer to the bisphenol A moiety. To assess the influence of the C6 spacer on the sensitivity of an assay based on the respective antibody, hapten-protein conjugates were synthesized and assessed for performance in an ELISA setup, and this resulted in a highly sensitive assay with a detection limit of 0.05 g/L. The lowest limit of detection was reached by employing the spacer derivate in the FPIA and was 1.0 μg/L, working range from 2 to 155 μg/L. The validation of the methods was conducted using actual samples compared to LC-MS/MS, which served as the reference method. The FPIA and ELISA both demonstrated satisfactory concordance.

Effects of a 10% dimethyl sulfoxide solution on radiocarpal joint amikacin pharmacokinetics during intravenous regional limb perfusion in standing sedated horses.

To determine the effect of a 10% dimethyl sulfoxide (DMSO) solution on the peak concentration (CMAX ) of amikacin in the radiocarpal joint (RCJ) during intravenous regional limb perfusion (IVRLP) compared with 0.9% NaCl. Randomized crossover study. Seven healthy adult horses. The horses underwent IVRLP with 2 g of amikacin sulfate diluted to 60 mL using a 10% DMSO or 0.9% NaCl solution. Synovial fluid was collected from the RCJ at 5, 10, 15, 20, 25, and 30 minutes after IVRLP. The wide rubber tourniquet placed on the antebrachium was removed after the 30 min sample. Amikacin concentrations were quantified by a fluorescence polarization immunoassay. The mean CMAX and time to peak concentration (TMAX ) of amikacin within the RCJ were determined. A one-sided paired t-test was used to determine the differences between treatments. The significance level was p < .05. The mean ± SD CMAX in the DMSO group was 1361.8 ± 593 μg/mL and in the 0.9% NaCl group it was 860 ± 481.6 μg/mL (p = .058). Mean TMAX using the 10% DMSO solution was 23 and 18 min using the 0.9% NaCl perfusate (p = .161). No adverse effects were associated with use of the 10% DMSO solution. Although there were higher mean peak synovial concentrations using the 10% DMSO solution no difference in synovial amikacin CMAX between perfusate type was detected (p = .058). Use of a 10% DMSO solution in conjunction with amikacin during IVRLP is a feasible technique and does not negatively affect the synovial amikacin levels achieved. Further research is warranted to determine other effects of using DMSO during IVRLP.

A Sensitive Fluorescence Polarization Immunoassay for the Rapid Detection of Okadaic Acid in Environmental Waters

In this study, a homogeneous fluorescence polarization immunoassay (FPIA) for the detection of hazardous aquatic toxin okadaic acid (OA) contaminating environmental waters was for the first time developed. A conjugate of the analyte with a fluorophore based on a fluorescein derivative (tracer) was synthesized, and its interaction with specific anti-OA monoclonal antibodies (MAbs) was tested. A MAbs–tracer pair demonstrated highly affine immune binding (KD = 0.8 nM). Under optimal conditions, the limit of OA detection in the FPIA was 0.08 ng/mL (0.1 nM), and the working range of detectable concentrations was 0.4–72.5 ng/mL (0.5–90 nM). The developed FPIA was approbated for the determination of OA in real matrices: river water and seawater samples. No matrix effect of water was observed; therefore, no sample preparation was required before analysis. Due to this factor, the entire analytical procedure took less than 10 min. Using a compact portable fluorescence polarization analyzer enables the on-site testing of water samples. The developed analysis is very fast, easy to operate, and sensitive and can be extended to the determination of other aquatic toxins or low-molecular-weight water or food contaminants.

A Robust Homogeneous Fluorescence Polarization Immunoassay for Rapid Determination of Erythromycin in Milk.

Erythromycin (ERY) is one of the most common macrolides applied in veterinary medicine to treat diseases or as a feed additive for animal growth promotion. Long-term irrational use of ERY could lead to residues in animal-derived food and the emergence of drug-resistant strains, posing potential threats to human health. In this study, a highly sensitive, specific, robust, and rapid fluorescence polarization immunoassay (FPIA) for the determination of ERY in milk has been described. Herein, to achieve high sensitivity, five tracers of ERY with different fluorescein structures were synthesized and paired with three monoclonal antibodies (mAbs). Under the optimized conditions, the combination of mAb 5B2 and tracer ERM-FITC achieved the lowest IC50 value in the FPIA with 7.39 μg/L for ERM. The established FPIA was used to detect ERY in milk, revealing a limit of detection (LOD) of 14.08 μg/L with recoveries of 96.08-107.77% and coefficients of variations (CVs) of 3.41-10.97%. The total detection time of the developed FPIA was less than 5 min from the addition of samples to the result readout. All the above results showed that the proposed FPIA in this study was a rapid, accurate, and simple method for the screening of ERY in milk samples.

Effects of Addition of Blocking Agents on Fluorescence Polarization Immunoassay of Okadaic Acid Using a Microfluidic Device

マイクロ流体デバイスを利用した分析では，流路壁面への試料等の吸着が問題になる場合がある．特にポリジメチルシロキサン（PDMS）製デバイスを用いた免疫分析では，抗体や測定対象等の吸着が測定性能に大きく影響する．そこで本研究では，下痢性貝毒であるオカダ酸（OA）を測定対象として，蛍光偏光免疫分析によるオカダ酸の検出感度における二種類のブロックキング剤添加の影響について検討した．また，得られた検量線から抗体とトレーサーに対する解離定数を算出した．

Determination of Nicotinic Acid in Food and Pharmaceuticals by a Simple and Rapid Fluorescence Polarization Immunoassay (FPIA)

A simple and rapid fluorescence polarization immunoassay (FPIA) based on polyclonal antibodies is reported for the first time for the determination of nicotinic acid. First, six nicotinic acid analogues were coupled with fluorescein isothiocyanate isomer I (FITC) to obtain tracers. The influences of tracer structure on the performance of FPIA as well as the structure-activity relationships were explored. Subsequently, the tracer concentration and antibody dilution ratio on the performance of FPIA were investigated. For the optimal conditions, the detection range of FPIA was from 1.1 to 145.6 μg mL−1 and its IC20 value as the limit of detection (LOD) was 1.1 μg mL−1. The cross-reactivity of antibodies with common vitamins was evaluated. The intra-assay recovery was from 92.0% to 101.2% in food and tablets, with inter-assay recoveries between 92.0% and 103.8%. The developed FPIA provided good sensitivity and reproducibility and therefore provided efficient and economic screening for nicotinic acid in food and tablets.

Antiseizure Effects of Scoparone, Borneol and Their Impact on the Anticonvulsant Potency of Four Classic Antiseizure Medications in the Mouse MES Model-An Isobolographic Transformation.

Numerous botanical drugs containing coumarins and terpenes are used in ethnomedicine all over the world for their various therapeutic properties, especially those affecting the CNS system. The treatment of epilepsy is based on antiseizure medications (ASMs), although novel strategies using naturally occurring substances with confirmed antiseizure properties are being developed nowadays. The aim of this study was to determine the anticonvulsant profiles of scoparone (a simple coumarin) and borneol (a bicyclic monoterpenoid) when administered separately and in combination, as well as their impact on the antiseizure effects of four classic ASMs (carbamazepine, phenytoin, phenobarbital and valproate) in the mouse model of maximal electroshock-induced (MES) tonic-clonic seizures. MES-induced seizures were evoked in mice receiving the respective doses of the tested natural compounds and classic ASMs (when applied alone or in combinations). Interactions for two-drug and three-drug mixtures were assessed by means of isobolographic transformation of data. Polygonograms were used to illustrate the types of interactions occurring among drugs. The total brain content of ASMs was measured in mice receiving the respective drug treatments with fluorescent polarization immunoassay. Scoparone and borneol, when administered alone, exerted anticonvulsant properties in the mouse MES model. The two-drug mixtures of scoparone with valproate, borneol with phenobarbital and borneol with valproate produced synergistic interactions in the mouse MES model, while the remaining tested two-drug mixtures produced additivity. The three-drug mixtures of scoparone + borneol with valproate and phenobarbital produced synergistic interactions in the mouse MES model. Verification of total brain concentrations of valproate and phenobarbital revealed that borneol elevated the total brain concentrations of both ASMs, while scoparone did not affect the brain content of these ASMs in mice. The synergistic interaction of scoparone with valproate observed in the mouse MES model is pharmacodynamic in nature. Borneol elevated the brain concentrations of the tested ASMs, contributing to the pharmacokinetic nature of the observed synergistic interactions with valproate and phenobarbital in the mouse MES model.

Development of Fluorescence Polarization Immunoassay of Sulfathiazol and its Application for Honey Testing

A fluorescence polarization immunoassay has been developed for the detection of the antibacterial sulfathiazole. The latter belongs to the sulfonamide class of antibacterial, which is used in beekeeping for the prevention and treatment of infectious diseases of bees. The assay conditions, which ensure the achievement of high analytical characteristics, are determined. The duration of the analysis is 5 min; the limit of sulfathiazole detection is 3 ng/mL. A study of the assay selectivity concerning other sulfonamides demonstrated the high specificity only towards sulfathiazole. The optimal method of sample preparation before the control honey contamination was proposed. The effectiveness of the developed analysis for the detection of sulfathiazole in honey samples was confirmed. The developed fluorescent polarization immunoassay is a rapid and efficient analytical system for rapid and sensitive control of honey quality and safety.