Polycations as Aptamer-Binding Modulators for Sensitive Fluorescence Anisotropy Assay of Aflatoxin B1.

Fluorescence induced by the excitation of a fluorophore with plane-polarized light has a different polarization depending on the size of the fluorophore-containing reagent and the rate of its rotation. Based on this effect, many analytical systems have been implemented in which an analyte contained in a sample and labeled with a fluorophore (usually fluorescein) competes to bind to antibodies. Replacing antibodies in such assays with aptamers, low-cost and stable oligonucleotide receptors, is complicated because binding a fluorophore to them causes a less significant change in the polarization of emissions. This work proposes and characterizes the compounds of the reaction medium that improve analyte binding and reduce the mobility of the aptamer-fluorophore complex, providing a higher analytical signal and a lower detection limit. This study was conducted on aflatoxin B1 (AFB1), a ubiquitous toxicant contaminating foods of plant origins. Eight aptamers specific to AFB1 with the same binding site and different regions stabilizing their structures were compared for affinity, based on which the aptamer with 38 nucleotides in length was selected. The polymers that interact reversibly with oligonucleotides, such as poly-L-lysine and polyethylene glycol, were tested. It was found that they provide the desired reduction in the depolarization of emitted light as well as high concentrations of magnesium cations. In the selected optimal medium, AFB1 detection reached a limit of 1 ng/mL, which was 12 times lower than in the tris buffer commonly used for anti-AFB1 aptamers. The assay time was 30 min. This method is suitable for controlling almond samples according to the maximum permissible levels of their contamination by AFB1. The proposed approach could be applied to improve other aptamer-based analytical systems.

Optical Sensors for Bacterial Detection.

Analytical devices for bacterial detection are an integral part of modern laboratory medicine, as they permit the early diagnosis of diseases and their timely treatment. Therefore, special attention is directed to the development of and improvements in monitoring and diagnostic methods, including biosensor-based ones. A promising direction in the development of bacterial detection methods is optical sensor systems based on colorimetric and fluorescence techniques, the surface plasmon resonance, and the measurement of orientational effects. This review shows the detecting capabilities of these systems and the promise of electro-optical analysis for bacterial detection. It also discusses the advantages and disadvantages of optical sensor systems and the prospects for their further improvement.

Fluorescence Polarization Immunoassay of Human Lactoferrin in Milk Using Small Single-Domain Antibodies.

Due to its unique structure and properties, human breast milk lactoferrin (hLF) has many nutritional and health-promoting functions in infants, including protection against inflammation and bacterial infections. The lack of LF in breastmilk or formula can result in the weakening of the infant's immune system. Noncompetitive polarization fluorescence immunoassay (FPIA) is a promising method for hLF quantification in milk and dairy products, which does not require the separation of the bound and free protein and allows to avoid time-consuming sample preparation. The use of fluorescently labeled single-domain camelid antibodies (nanobodies) for protein recognition in FPIA makes it possible to quantify relatively large antigens, in particular, hLF. In this work, we used previously obtained fluorescein isothiocyanate (FITC)-conjugated anti-hLF5 and anti-hLF16 nanobodies, which selectively recognized two different human lactoferrin epitopes, but did not bind to goat lactoferrin. The kinetics of hLF interaction with the FITC-labeled nanobodies was studied. The dissociation constant (KD) for the anti-LF5 and antiLF16 nanobodies was 3.2 ± 0.3 and 4.9 ± 0.4 nM, respectively, indicating the high-affinity binding of these nanobodies to hLF. We developed the FPIA protocol and determined the concentration of FITC-labeled anti-hLF5 and anti-hLF16 nanobodies that provided the optimal fluorescence signal and stable fluorescence polarization value. We also studied the dependence of fluorescence polarization on the hLF concentration in the noncompetitive FPIA with FITC-anti-hLF5 nanobody. The detection limit for hLF was 2.1 ± 0.2 µg/ml and the linear range for determining the hLF concentration was 3-10 µg/ml. FPIA is commonly used to assay low-molecular-weight substances; however, the use of fluorescently labeled nanobodies allows quantification of high-molecular-weight proteins. Here, we demonstrated that FPIA with fluorescently labeled nanobodies can be used for hLF quantification in milk.

Modulation of Aptamer-Ligand-Binding by Complementary Oligonucleotides: A G-Quadruplex Anti-Ochratoxin A Aptamer Case Study.

Short oligonucleotides are widely used for the construction of aptamer-based sensors and logical bioelements to modulate aptamer-ligand binding. However, relationships between the parameters (length, location of the complementary region) of oligonucleotides and their influence on aptamer-ligand interactions remain unclear. Here, we addressed this task by comparing the effects of short complementary oligonucleotides (ssDNAs) on the structure and ligand-binding ability of an aptamer and identifying ssDNAs' features that determine these effects. Within this, the interactions between the OTA-specific G-quadruplex aptamer 1.12.2 (5'-GATCGGGTGTGGGTGGCGTAAAGGGA GCATCGGACA-3') and 21 single-stranded DNA (ssDNA) oligonucleotides complementary to different regions of the aptamer were studied. Two sets of aptamer-ssDNA dissociation constants were obtained in the absence and in the presence of OTA by isothermal calorimetry and fluorescence anisotropy, respectively. In both sets, the binding constants depend on the number of hydrogen bonds formed in the aptamer-ssDNA complex. The ssDNAs' having more than 23 hydrogen bonds with the aptamer have a lower aptamer dissociation constant than for aptamer-OTA interactions. The ssDNAs' having less than 18 hydrogen bonds did not affect the aptamer-OTA affinity. The location of ssDNA's complementary site in the aptamer affeced the kinetics of the interaction and retention of OTA-binding in aptamer-ssDNA complexes. The location of the ssDNA site in the aptamer G-quadruplex led to its unfolding. In the presence of OTA, the unfolding process was longer and takes from 20 to 70 min. The refolding in the presence of OTA was possible and depends on the length and location of the ssDNA's complementary site. The location of the ssDNA site in the tail region led to its rapid displacement and wasn't affecting the G-qaudruplex's integrity. It makes the tail region more perspective for the development of ssDNA-based tools using this aptamer.

Fluorescence polarization immunoassay for the determination of diclofenac in wastewater.

Pharmacologically active compounds are often detected in wastewater and surface waters. The nonsteroidal anti-inflammatory drug diclofenac (DCF) was included in the European watch list of substances that requires its environmental monitoring in the member states. DCF may harmfully influence the ecosystem already at concentrations ≤ 1 µg L-1. The fast and easy quantification of DCF is becoming a subject of global importance. Fluorescence polarization immunoassay (FPIA) is a homogeneous mix-and-read method which does not require the immobilization of reagents. FPIA can be performed in one phase within 20-30 min, making it possible to analyse wastewater without any complicated pre-treatment. In this study, new tracer molecules with different structures, linking fluorophores to derivatives of the analyte, were synthesized, three homologous tracers based on DCF, two including a C6 spacer, and one heterologous tracer derived from 5-hydroxy-DCF. The tracer molecules were thoroughly assessed for performance. Regarding sensitivity of the FPIA, the lowest limit of detection reached was 2.0 µg L-1 with a working range up to 870 µg L-1. The method was validated for real wastewater samples against LC-MS/MS as reference method with good agreement of both methods. Graphical abstract.

Fluorescence polarization immunoassay for rapid determination of dehydroepiandrosterone in human urine.

In this paper, five fluorescein-labeled dehydroepiandrosterone (DHEA) derivatives (tracers) with different chain lengths between the fluorescein and hapten were synthesized and featured so as to establish a fluorescence polarization immunoassay (FPIA) for DHEA detection in human urine samples with previously prepared polyclonal antibody against DHEA. The outcomes of the structure of tracer on FPIA sensitivity were investigated. Under the optimal condition, the fluorescence polarization value (FP) decreases linearly in DHEA concentration, ranging from 1.6 to 243.3 ng mL-1, with the limit of detection of 1.1 ng mL-1 and IC50 value of 25.1 ng mL-1. Moreover, the developed FPIA was time-saving as it could complete the detection within 3 min. FPIA and commercial enzyme-linked immunosorbent assay kit were both applied to analyze the spiked human urine samples with DHEA. Excellent recoveries (92.1-108.0%) and satisfactory correlation coefficient (R2 = 0.98) were acquired with the two methods, indicating that the developed FPIA was a fast and efficient screening immunoassay with accuracy and sensitivity for DHEA detection in human urine samples. Graphical abstract.

Development of enzyme-free single-step immunoassays for glycocholic acid based on palladium nanoparticle-mediated signal generation.

Palladium nanoparticles (PdNPs) are composed mainly of inert noble metals, and their outstanding properties have attracted wide attention. PdNPs are not only capable of mimicking the oxidase-like characteristics of natural bio-enzymes, but they also present a clear black band in the test zone. In this work, the synthesized PdNPs promoted a transformation of colorless tetramethylbenzidine (TMB) to a blue oxidation product of TMB, providing a Km value of 0.09 mM for TMB, and revealing the good catalytic performance of the synthesized PdNPs. For both signal generation and amplification, PdNPs effectively replaced natural bio-enzymes as a new labeling tag. Thus, the PdNP-based enzyme-free single-step immunoassays were successfully developed for efficient and sensitive detection of glycocholic acid (GCA). Under optimal conditions, a noticeable linear relationship was identified by the enzyme-linked immunosorbent assay (ELISA) over a range of 8-2390 ng/mL, while the visual limit of detection (vLOD) in the constructed lateral flow immunoassay (LFA) was 10 ng/mL for GCA. The recovery rate in spiked urine samples obtained by the ELISA ranged from 84.2 to 117.9%, which was consistent with the results in LFA. The present work demonstrates the potential of PdNPs as labeling matrices in enzyme-free single-step immunoassays.

Platinum nanoflowers with peroxidase-like property in a dual immunoassay for dehydroepiandrosterone.

Platinum nanoflowers (PtNFs) were utilized in a competitive enzyme-linked immunosorbent assay (ELISA) and in a lateral flow immunoassay (LFIA) for superior peroxidase-like activity and intense brown color, respectively. PtNFs were linked to the polyclonal antibody (pAb) to form the pAb-PtNFs probes for the dual immunoassay. Based on optimized pAb-PtNF probes, both enzyme-linked immunosorbent assay (PtNFs-ELISA) and lateral flow immunoassay (PtNFs-LFIA) perform very well. The absorbance at 450 nm decreases linearly in the DHEA concentration range 2.1 to 118.1 ng mL-1, and the limit of detection is 1.3 ng mL-1 and the IC50 value is 15.7 ng mL-1 of PtNFs-ELISA. The visual cut-off value of PtNFs-LFIA is 10.0 ng mL-1. The average recoveries from spiked samples range from 95.0 to 108.9% with a coefficient of variation below 12.2%. Excellent recoveries and correlation between the two methods were observed. Furthermore, the designed immunosensors exhibited good selectivity, confirming a broad development prospect in DHEA monitoring. Graphical Abstract.

Immunochemical approaches for detection of aflatoxin B1 in herbal medicines.

INTRODUCTION: Aflatoxin B1 (AFB1) is a toxic low-molecular-weight secondary metabolite of Aspergillus flavus and A. parasiticus. AFB1 was classified as a Group I carcinogen by the World Health Organisation for Research on Cancer in 1993. AFB1 is an unavoidable natural contaminant of some herbal medicine, able to cause serious health issues for humans consuming the related medicine. OBJECTIVE: Therefore, this study aimed to develop an efficient fluorescence polarisation immunoassay (FPIA) and a rapid, low-cost, and easy-to-use membrane-based flow-through immunoassay (MBA) for determination of AFB1 in herbal medicine Origanum vulgare L., Rubus idaeus L., Urtica dioica L. and Sorbus aucuparia L. RESULTS: A cut-off level of the developed MBA was 0.8 ppb. Validation of the developed test was performed with blank and spiked samples. Using three naturally contaminated or three artificially spiked samples. The FPIA showed a linear working range of 8.6 to 64 ppb, and a half maximal inhibitory concentration (IC50 ) of 24 ppb. CONCLUSION: The results were in good correlation with the enzymelinked immunosorbent assay (ELISA) results (the IC50 0.1 ppb). Both the sample preparation and analysis are simple, cost-effective and easy to perform on-site in non-laboratory environments. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) was used as a confirmatory technique.

Fluorescence Polarization-Based Bioassays: New Horizons.

Fluorescence polarization holds considerable promise for bioanalytical systems because it allows the detection of selective interactions in real time and a choice of fluorophores, the detection of which the biosample matrix does not influence; thus, their choice simplifies and accelerates the preparation of samples. For decades, these possibilities were successfully applied in fluorescence polarization immunoassays based on differences in the polarization of fluorophore emissions excited by plane-polarized light, whether in a free state or as part of an immune complex. However, the results of recent studies demonstrate the efficacy of fluorescence polarization as a detected signal in many bioanalytical methods. This review summarizes and comparatively characterizes these developments. It considers the integration of fluorescence polarization with the use of alternative receptor molecules and various fluorophores; different schemes for the formation of detectable complexes and the amplification of the signals generated by them. New techniques for the detection of metal ions, nucleic acids, and enzymatic reactions based on fluorescence polarization are also considered.

Ciprofloxacin and Clinafloxacin Antibodies for an Immunoassay of Quinolones: Quantitative Structure⁻Activity Analysis of Cross-Reactivities.

A common problem in the immunodetection of structurally close compounds is understanding the regularities of immune recognition, and elucidating the basic structural elements that provide it. Correct identification of these elements would allow for select immunogens to obtain antibodies with either wide specificity to different representatives of a given chemical class (for class-specific immunoassays), or narrow specificity to a unique compound (mono-specific immunoassays). Fluoroquinolones (FQs; antibiotic contaminants of animal-derived foods) are of particular interest for such research. We studied the structural basis of immune recognition of FQs by antibodies against ciprofloxacin (CIP) and clinafloxacin (CLI) as the immunizing hapten. CIP and CLI possess the same cyclopropyl substituents at the N1 position, while their substituents at C7 and C8 are different. Anti-CIP antibodies were specific to 22 of 24 FQs, while anti-CLI antibodies were specific to 11 of 26 FQs. The molecular size was critical for the binding between the FQs and the anti-CIP antibody. The presence of the cyclopropyl ring at the N1 position was important for the recognition between fluoroquinolones and the anti-CLI antibody. The anti-CIP quantitative structure⁻activity relationship (QSAR) model was well-equipped to predict the test set (pred_R² = 0.944). The statistical parameters of the anti-CLI model were also high (R² = 0.885, q² = 0.864). Thus, the obtained QSAR models yielded sufficient correlation coefficients, internal stability, and predictive ability. This work broadens our knowledge of the molecular mechanisms of FQs' interaction with antibodies, and it will contribute to the further development of antibiotic immunoassays.

Fluorescence Polarization Immunoassay for Determination of Enrofloxacin in Pork Liver and Chicken.

Enrofloxacin (ENR) is a widely used fluoroquinolone (FQ) antibiotic for antibacterial treatment of edible animal. In this study, a rapid and highly specific fluorescence polarization immunoassay (FPIA) was developed for monitoring ENR residues in animal foods. First, ENR was covalently coupled to bovine serum albumin (BSA) to produce specific polyclonal antibodies (pAbs). Three fluorescein-labeled ENR tracers (A, B, and C) with different spacers were synthesized and compared to obtain higher sensitivity. Tracer C with the longest arm showed the best sensitivity among the three tracers. The developed FPIA method showed an IC50 (50% inhibitory concentration) of 21.49 ng·mL-1 with a dynamic working range (IC20-IC80) of 4.30-107.46 ng·mL-1 and a limit of detection (LOD, IC10) of 1.68 ng·mL-1. The cross-reactivity (CR) of several structurally related compounds was less than 2%. The recoveries of spiked pork liver and chicken samples varied from 91.3% to 112.9%, and the average coefficients of variation were less than 3.83% and 5.13%, respectively. The immunoassay took only 8 min excluding sample pretreatment. This indicated that the established method had high sensitivity, specificity, and the advantages of simplicity. Therefore, the proposed FPIA provided a useful screening method for the rapid detection of ENR residues in pork liver and chicken.

Measurement of (Aptamer-Small Target) KD Using the Competition between Fluorescently Labeled and Unlabeled Targets and the Detection of Fluorescence Anisotropy.

Registration of fluorescence anisotropy (FA) allows for characterizing the interactions of ligands with aptamers and other receptors under homogeneous conditions without reagent immobilization, prolonged incubations, and product separation. We proposed an approach for aptamer affinity determination by FA taking into account the difference in label fluorescence before and after complexation. The detailed step by step scheme using a native and fluorescently labeled ligand was described and justified in the paper. The scheme ensures the exclusion of data with low reliability and establishes valid criteria for selecting optimal concentrations of reagents (labeled ligand and aptamer) used in the experiments. The approach was experimentally tested using ochratoxin A (OTA), its fluorescein-labeled derivative (OTA-Flu), and the aptamer binding them. We demonstrated that it allows minimizing the influence of fluorescence change to accurately determine the dissociation constant. On the basis of FA registration, the binding constants of the aptamer-OTA-Flu and the aptamer-OTA complexes were found to be equal to 245 + 33 and 63 + 18 nM, respectively. The value for the aptamer-OTA complexes was confirmed by the equilibrium dialysis technique. The resulting constant was 80 ± 9 nM. The versatility and methodological simplicity of the proposed protocol, as well as the short implementation time, are why it can be recommended as an effective tool for characterizing aptamer-ligand complexes.

Fluorescence polarization immunoassay for rapid screening of the pesticides thiabendazole and tetraconazole in wheat.

Fluorescence polarization immunoassays (FPIAs) for thiabendazole and tetraconazole were first developed. Tracers for FPIAs of thiabendazole and tetraconazole were synthesized and the tracers' structures were confirmed by HPLC-MS/MS. The 4-aminomethylfluorescein-labeled tracers allowed achieving the best assay sensitivity and minimum reagent consumption in comparison with aminofluorescein-labeled and alkyldiaminefluoresceinthiocarbamyl-labeled tracers. Measurements of fluorescence polarization were performed using a portable device. The developed FPIA methods were applied for the analysis of wheat. Fast and simple sample preparation technique earlier developed by authors for pesticides was adapted for thiabendazole and tetraconazole. The limits of detection of thiabendazole and tetraconazole in wheat were 20 and 200 µg/kg, and the lower limits of quantification were 40 and 600 µg/kg, respectively. The recovery test was performed by two methods-FPIA and HPLC-MS/MS. The results obtained by FPIA correlated well with those obtained by HPLC-MS/MS (r2 = 0.9985 for thiabendazole, r2 = 0.9952 for tetraconazole). Average recoveries of thiabendazole and tetraconazole were 74 ± 4% and 72 ± 3% by FPIA, and average recoveries of thiabendazole and tetraconazole were 86 ± 2% and 74 ± 1% by HPLC-MS/MS (n = 15). Graphical abstract ᅟ.

High-sensitivity immunochromatographic assay for fumonisin B1 based on indirect antibody labeling.

OBJECTIVE: To develop a high-sensitivity immunochromatographic test for fumonisin B1 in plant extracts. RESULTS: Unlike conventional immunochromatographic tests, this assay is performed in two stages: competitive reaction with free specific antibodies and identifying immune complexes by their interaction with the anti-species antibody-conjugated gold nanoparticles. The use of a new geometry for the test strip membranes and a novel reagent application method ensures the proper order of these stages without additional manipulations. The contact of the ready-to-use test strip with the liquid sample suffices in initiating all stages of the assay and obtaining test results. The developed test was used on corn extracts; its instrumental limit of fumonisin B1 detection was 0.6 ng ml-1 at 15 min of assay duration. CONCLUSIONS: The proposed approach is flexible and can be used for a wide range of low molecular compounds. The use of anti-species antibody-conjugated gold nanoparticles in immunochromatography significantly facilitates the development of test systems by eliminating the need to synthesize and characterize the conjugates with specific antibodies for each new compound to be detected.

Chemiluminescence Immunoassay for S-Adenosylhomocysteine Detection and Its Application in DNA Methyltransferase Activity Evaluation and Inhibitors Screening.

Aberrant methylation by DNA transferase is associated with cancer initiation and progression. For high-throughput screening of DNA methyltransferase (MTase) activity and its inhibitors, a novel chemiluminescence immunoassay (CLIA) was established to detect S-adenosylhomocysteine (SAH), the product of S-adenosylmethionine (SAM) transmethylation reactions. We synthesized two kinds of immunogens for SAH and characterized the polyclonal antibodies in each group. The antibody with higher titer was used to develop a competitive CLIA for SAH, in which SAH in samples would compete with SAH coated on microplate in binding with SAH antibodies. Successively, horseradish peroxidase labeled goat antirabbit IgG (HRP-IgG) was conjugated with SAH antibodies on the microplate. In substrate solution containing luminol and H2O2, HRP-IgG catalyzed luminol oxidation by H2O2, generating a high chemiluminescence signal. The method could detect as low as 9.8 ng mL(-1) SAH with little cross-reaction (3.8%) to SAM. Since higher DNA MTase activity leads to more production of SAH, a correlation between the chemiluminescence intensity and DNA MTase activity was obtained in the range from 0.1 to 8.0 U/mL of DNA MTase. The inhibition study showed that, in the presence of SAM as methyl donor, Lomeguatrib, 5-Azacytidine, and 5-Aza-2'-deoxycytidine could inhibit the DNA MTase activity with IC50 values of 40.57 nM, 2.26 µM, and 0.48 µM, respectively. These results are consistent with the published studies. The proposed assay does not depend on recognizing methylated cytosines in oligonucleotides (methyl acceptor) and showed the potential as an accessible platform for sensitive detection of DNA MTase activity and screening its inhibitors.

Heterologous strategy enhancing the sensitivity of the fluorescence polarization immunoassay of clinafloxacin in goat milk.

BACKGROUND: Clinafloxacin is used for the treatment of disease in food-producing animals, e.g. Brucella melitensis, which often occurs in goats; however, the clinafloxacin residue in goat milk may harm human health and result in the development of drug-resistant bacterial strains or allergies. Despite this, there is not a rapid, sensitive and accurate analytical method in goat milk for rapid screening or monitoring purposes. RESULTS: One homologous and five heterologous tracers were designed and compared for fluorescence polarization immunoassay (FPIA) optimization. Based on the combination of a heterologous tracer (PAZ-FITC, synthesized with pazufloxacin and FITC) and the antibody against clinafloxacin, a highly sensitive FPIA was established for the detection of clinafloxacin residue in goat milk for the first time. The IC50 value was 29.3 µg L(-1) for clinafloxacin in the heterologous format - six times lower than that of the combination of the homologous tracers and the antibody. The recoveries ranged from 86.8% to 104.5%, with the relative standard deviation ranging from 4.1% to 7.2%. Validation by high-performance liquid chromatography (HPLC) confirmed that the results obtained from the proposed FPIA were in agreement with those of HPLC. CONCLUSION: This proposed heterologous strategy for enhanced FPIA is sensitive and rapid enough for the high-throughput detection of clinafloxacin residue in goat milk.

Stereospecific recognition and quantitative structure-activity relationship between antibodies and enantiomers: ofloxacin as a model hapten.

In this study, ofloxacin stereoisomers were chosen as a simple model to investigate the stereospecific recognition of chiral haptens and antibodies. Three polyclonal antibodies were studied and showed a relatively high enantioselectivity and an excellent sensitivity. Comparative molecular field analysis and comparative molecular similarity indices analysis were employed to investigate the chiral recognition between the antibody and the ofloxacin enantiomer, and all the models yielded high correlation and predictive ability. It was found that the chiral discrimination was probably caused by steric hindrance; the antibody stereospecificity could be ascribed to the variation of the R1 and R3 groups of quinolones; the common structure of the quinolones is also essential in the hapten-antibody recognition. The recognition between the chiral haptens and the antibodies was co-affected by multiple interaction forces, and those forces were defined explicitly at the sub-structural level. An illustrative enhanced model with good simplicity and universality was also developed for a better understanding of the stereospecific recognition of ofloxacin enantiomers and antibodies for the first time. This work provides insights into the stereospecific recognition of chiral haptens and antibodies.

Design of a sensitive fluorescent polarization immunoassay for rapid screening of milk for cephalexin.

In this paper we describe the development of a sensitive, fast, and easily performed fluorescence polarization immunoassay for determination of cephalexin in milk. The experimental work was performed to increase sensitivity and specificity. Therefore, the structures of the tracers were varied by synthesis of both cephalexin (CEX) and cephalotin (CET) conjugates with a variety of fluorescent labels. Two rabbit antisera containing antibodies against cephalexin and cephalotin were tested in homologous and heterologous combinations with the tracers. For every working antibody-tracer combination, the analytical conditions and cross-reactivity for structural analogues-cephalosporins and other antibiotics that could also be present in milk-were determined. It was found that the highest sensitivity was achieved by use of the homologous pair CET-EDF-anti-CET antibody (limit of detection (LOD) 0.4 µg kg(-1) for standard solutions prepared in buffer), but this combination was not appropriate because of high cross-reactivity with CET. For subsequent experiments, therefore, CEX- EDF-anti-CEX antibody were chosen (LOD 0.8 µg kg(-1) for standard solutions prepared in buffer). Part of this manuscript is devoted to the variation of precipitation agents for pretreatment of milk before analysis; milk is an extremely complicated matrix. The optimum protein precipitation agent was methanol. This technique for cephalexin determination was characterized by a limit of detection of 1 µg kg(-1). The method was validated by using naturally contaminated and spiked milk samples. The results obtained corresponded very well with those obtained by HPLC, which was used as confirmation method.

Fluorescence polarization immunoassay using IgY antibodies for detection of valnemulin in swine tissue.

Immunoglobulin Y (IgY) is derived from egg yolk and has been identified as a cheap and high-yield immunoreagent. The application of IgY in immunoassays for the detection of chemical contaminants in food samples has rarely been reported. In this work, we describe a rapid and sensitive fluorescence polarization immunoassay (FPIA) for valnemulin (VAL) using IgY which was produced using a previously prepared immunogen. Three fluorophore-labeled VAL tracers were synthesized and the sensitivity of the best tracer (VAL-DTAF) in the optimized FPIA with antibody IgY100 demonstrated an IC50 value of 12 ng mL(-1) in buffer. After evaluation of several extraction procedures, acidified acetonitrile was selected to extract VAL from swine tissue. The recoveries of VAL in spiked swine tissue at three levels (50, 100, and 200 µg kg(-1)) were higher than 79% with coefficients of variation (CVs) lower than 12%. The limit of detection (LOD) of the FPIA in swine tissue was 26 µg kg(-1) and was lower than the maximum residue limit (MRL) of VAL set by the European Union. The study showed that IgY could be a good substitute for IgG when developing a high-throughput assay for chemical residues.