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This study describes the applicability of the fluorescence polarization assay (FPA) based on the use of FITC-labeled oligosaccharide tracers of defined structure for the measurement of active lysozyme in hen egg white. Depending on the oligosaccharide chain length of the tracer, this method detects both the formation of the enzyme-to-tracer complex (because of lectin-like, i.e., carbohydrate-binding action of lysozyme) and tracer splitting (because of chitinase activity of lysozyme). Evaluation of the fluorescence polarization dynamics enables simultaneous measurement of the chitinase and lectin activities of lysozyme, which is crucial for its detection in complex biological systems. Hen egg white lysozyme (HEWL), unlike human lysozyme (HL), formed a stable complex with the chitotriose tracer that underwent no further transformations. This fact allows for easy measurement of the carbohydrate-binding activity of the HEWL. The results of the lysozyme activity measurement for hen egg samples obtained through the FPA correlated with the results obtained using the traditional turbidimetry method. The FPA does not have the drawbacks of turbidimetry, which are associated with the need to use bacterial cells that cannot be precisely standardized. Additionally, FPA offers advantages such as rapid analysis, the use of compact equipment, and standardized reagents. Therefore, the new express technique for measuring the lysozyme activity is applicable for evaluating the complex biomaterial, including for the purposes of food product quality control. [ABSTRACT FROM AUTHOR]

Rapid and specific diagnosis is necessary for both the treatment and prevention of infectious diseases. Bacteria and viruses that enter the bloodstream can trigger a strong immune response in infected animals and humans. The fluorescence polarization assay (FPA) is a rapid and accurate method for detecting specific antibodies in the blood that are produced in response to infection. One of the first examples of FPA is the non-competitive test for detecting brucellosis in animals, which was followed by the development of other protocols for detecting various infections. Fluorescently labeled polysaccharides (in the case of brucellosis and salmonellosis) or specific peptides (in the case of tuberculosis and salmonellosis, etc.) can be used as biorecognition elements for detecting infections. The availability of new laboratory equipment and mobile devices for fluorescence polarization measurements outside the laboratory has stimulated the development of new fluorescence polarization assays (FPAs) and the emergence of commercial kits on the market for the detection of brucellosis, tuberculosis, and equine infectious anemia viruses. It has been shown that, in addition to antibodies, the FPA method can detect both viruses and nucleic acids. The development of more specific and sensitive biomarkers is essential for the diagnosis of infections and therapy monitoring. This review summarizes studies published between 2003 and 2023 that focus on the detection of infections using FPA. Furthermore, it demonstrates the potential for using new biorecognition elements (e.g., aptamers, proteins, peptides) and the combined use of FPA with new technologies, such as PCR and CRISPR/Cas12a systems, for detecting various infectious agents. [ABSTRACT FROM AUTHOR]

Brucellosis in animals is an infectious disease caused by bacteria of the genus Brucella. Known methods for diagnosing brucellosis face some challenges, due to the difficulties in isolating and standardizing the natural brucellosis antigen. In this work, we investigated the possibility of using the fluorescence polarization assay (FPA) with synthetic glycoconjugate biosensing tracers to detect antibodies against Brucella as a new methodology for diagnosing brucellosis. Based on the received results, the synthetic fluorescein-labeled trisaccharide tracer is most effective for Brucellosis detection. This tracer is structurally related to the immune determinant fragment of the Brucella LPS buildup of N-formyl-d-perosamine units, connected via α-(1→3)-linkage at the non-reducing end and α-(1→2)-linkage at the reducing end. The sensitivity and specificity in the case of the use of trisaccharide tracer 3b were 71% and 100% (Yuden's method) and 87% and 88% (Euclidean method), respectively, which is comparable with the diagnostic efficiency of traditionally used serological methods, such as the agglutination test (AT), complement fixation test (CFT), and Rose Bengal test (RBT). Given the known advantages of FPA (e.g., speed, compactness of the equipment, and standard reagents) and the increased specificity of the developed test system, it would be appropriate to consider its widespread use for the diagnosis of brucellosis in animals, including rapid testing in the field. [ABSTRACT FROM AUTHOR]

Brucellosis in animals is an infectious disease caused by bacteria of the genus Brucella. Known methods for diagnosing brucellosis face some challenges, due to the difficulties in isolating and standardizing the natural brucellosis antigen. In this work, we investigated the possibility of using the fluorescence polarization assay (FPA) with synthetic glycoconjugate biosensing tracers to detect antibodies against Brucella as a new methodology for diagnosing brucellosis. Based on the received results, the synthetic fluorescein-labeled trisaccharide tracer is most effective for Brucellosis detection. This tracer is structurally related to the immune determinant fragment of the Brucella LPS buildup of N-formyl-d-perosamine units, connected via α-(1→3)-linkage at the non-reducing end and α-(1→2)-linkage at the reducing end. The sensitivity and specificity in the case of the use of trisaccharide tracer 3b were 71% and 100% (Yuden’s method) and 87% and 88% (Euclidean method), respectively, which is comparable with the diagnostic efficiency of traditionally used serological methods, such as the agglutination test (AT), complement fixation test (CFT), and Rose Bengal test (RBT). Given the known advantages of FPA (e.g., speed, compactness of the equipment, and standard reagents) and the increased specificity of the developed test system, it would be appropriate to consider its widespread use for the diagnosis of brucellosis in animals, including rapid testing in the field.

Sebastian Vaas,1 Markus O Zimmermann,1 Theresa Klett,1 Frank M Boeckler1,2 1Department of Pharmacy and Biochemistry, Eberhard Karls Universität Tübingen, Laboratory for Molecular Design and Pharmaceutical Biophysics, Institute of Pharmaceutical Sciences, Tübingen, 72076, Germany; 2Institute for Bioinformatics and Medical Informatics (IBMI), Eberhard Karls Universität Tübingen, Tübingen, 72076, GermanyCorrespondence: Frank M Boeckler, Molecular Design and Pharmaceutical Biophysics, Institute of Pharmaceutical Sciences, Eberhard Karls Universität Tübingen, Auf der Morgenstelle 8 (Haus B), Tübingen, D-72076, Germany, Tel +49 7071 29 74567, Fax +49 7071 29 5637, Email frank.boeckler@uni-tuebingen.deIntroduction: Therapeutic peptides are a significant class of drugs in the treatment of a wide range of diseases. To enhance their properties, such as stability or binding affinity, they are usually chemically modified. This includes, among other techniques, cyclization of the peptide chain by bridging, modifications to the backbone, and incorporation of unnatural amino acids. One approach previously established, is the use of halogenated aromatic amino acids. In principle, they are thereby enabled to form halogen bonds (XB). In this study, we focus on the -R-CF2X moiety (R = O, NHCO; X = Cl, Br) as an uncommon halogen bond donor. These groups enable more spatial variability in protein–protein interactions. The chosen approach via Fmoc-protected building blocks allows for the incorporation of these modified amino acids in peptides using solid-phase peptide synthesis.Results and Discussion: Using a competitive fluorescence polarization assay to monitor binding to Mdm4, we demonstrate that a p53-derived peptide with Lys24Nle(&epsiv;NHCOCF2X) exhibits an improved inhibition constant Ki compared to the unmodified peptide. Decreasing Ki values observed with the increasing XB capacity of the halogen atoms (F ≪ Cl < Br) indicates the formation of a halogen bond. By reducing the side chain length of Nle(&epsiv;NHCOCF2X) to Abu(γNHCOCF2X) as control experiments and through quantum mechanical calculations, we suggest that the observed affinity enhancement is related to halogen bond-induced intramolecular stabilization of the α-helical binding mode of the peptide or a direct interaction with His54 in human Mdm4.Graphical Abstract: Keywords: halogen bonding, solid-phase peptide synthesis, unnatural amino acids, fluorescence polarization assay, oncological target, MdmX

In this study, the identity of our recently produced natural TetR protein was identified by using the LC-ESI-MS/MS technique, and its recognition mechanisms, including the binding pocket, contact amino acids, intermolecular forces, binding sites, binding energies, and affinities for 10 tetracycline drugs were studied. Then, it was evolved by site-mutagenesis of an amino acid to produce a mutant, and a fluorescence polarization assay was developed to detect the 10 drugs in milk. The sensitivities for the 10 drugs were improved with IC50 values decreasing from 30.8–80.1 ng/mL to 15.5–55.2 ng/mL, and the limits of detection were in the range of 0.4–1.5 ng/mL. Furthermore, it was found that the binding affinity for a drug was the critical factor determining its sensitivity, and the binding energy showed little influence. This is the first study reporting the recognition mechanisms of a natural TetR protein for tetracyclines and the development of a fluorescence polarization assay for the detection of tetracyclines residues in food samples. [ABSTRACT FROM AUTHOR]

The residues of aminoglycosides in foods of animal origin are a potential risk to consumers. There have been some immunoassays reported for the screening of aminoglycoside residues, but the method showing the broadest detection spectrum can only be used to detect two drugs. This is because a broad specific recognition reagent is not available. In the present study, the receptor of aminoglycosides (ribosomal protein S12 of Lysinibacillussphaericus) was expressed, and its affinities and recognition mechanisms for 10 aminoglycosides were studied by using surface plasmon resonance and molecular docking, respectively. Then the receptor was used as a recognition reagent to develop a fluorescence polarization assay on a 96-well microplate for the detection of the 10 drugs in pork muscle samples. The limits of detection for the 10 drugs ranged from 5.25 to 30.25 ng/g. The sensitivities for the 10 drugs were generally consistent with their respective receptor affinities and binding energies. After comprehensive comparison, the method performances were better than all the previously reported immunoassays for aminoglycosides. This is the first study reporting the recognition mechanisms of ribosomal protein S12 of Lysinibacillussphaericus for 10 aminoglycosides and the use of it as a recognition reagent to develop a pseudo-immunoassay for the multi-determination of aminoglycosides in food samples. [ABSTRACT FROM AUTHOR]

Background: Bovine tuberculosis (bTB) is a major bacterial disease that causes significant economic disruption across the globe. Aims: Our study was based on using a fluorescence polarization assay (FPA) that used fluorescein-labeled ESAT-6 protein to detect Mycobacterium bovis antibodies in bovine serum. Methods: The ESAT-6 protein was used in a FPA. Positive TB reactors were determined by the comparative intradermal test (CID) and interferon gamma test (IFN-γ). Antibodies against M. bovis were detected using a fluorescein isothiocyanate (FITC) labeled tracer and a whole culture FITC labeled tracer in the positive cattle. Results: Of the 192 animals tested for bTB, 37 were found to be positive by either the CID or IFN-γ assays. Using the mP values from five culturepositive serum samples, a cutoff value of more than >127 mp provided the best discrimination between positive reactors and negative bTB animals. The ESAT-6 results of FPA in comparison with CID results revealed sensitivity of 92.9% and specificity of 64.6%, and in comparison with results IFN-γ, showed sensitivity of 95.7% and specificity of 49%. FPA using FITC labelled ESAT-6 as a tracer has better sensitivity (95.7%) and specificity (49.1%) than IFN-γ test in humoral immune response in animals. Conclusion: This work revealed that the ESAT-6 protein as an antigen can be used in diagnosing bTB using a practical and sensitive humoral test. [ABSTRACT FROM AUTHOR]

The milk ring test is a detection assay for antibodies against Brucella in bovine milk. It has good sensitivity but tends to give false positive results. In this study, we standardized the application of the fluorescence polarization assay (FPA) for the detection of antibodies against B.melitensis in goat milk. We obtained negative serum and milk samples from healthy goat flocks in the northern zone of Nuevo León. Positive milk and negative, weak, and strong controls were obtained by mixing volumes of positive control serum with negative control milk. Milk samples were treated with citric acid, after which an FPA was performed. Results were then compared with the Rose Bengal test and the FPA in serum. Milk treatment allowed the quantification of antibodies in samples. Significant differences were found between the 2%, 4%, and 6% groups, compared with the control group (F3, 67 = 17.45, p < 0.0001) but not between the 2% and 4% groups (p = 0.0718). The cut-off value was 74.1 mP, with a sensitivity (Se) of 95% and a specificity (Sp) of 100%. Se and Sp values in field milk samples were 84% and 74.55%, respectively. Despite the FPA test on milk samples showed lower Se and Sp than the FPA test on serum samples, its cutoff may be adjusted. It may be recommended as a screening test in goat milk and become useful for the control and eradication of the disease. [ABSTRACT FROM AUTHOR]

Abstract Background Brucellosis is an infectious-allergic zoonotic disease caused by bacteria of the genus Brucella. Early diagnosis is the key to preventing, treating, and controlling brucellosis. Fluorescence polarization immunoassay (FPA) is a new immunoassay for relatively rapid and accurate detection of antibodies or antigens based on antigen–antibody interaction. However, there is no report on FPA-based detection of human brucellosis in China. Therefore, this study is to evaluate the value of FPA for the diagnosis of human brucellosis in China. Methods We recruited 320 suspected brucellosis cases who had the clinical symptoms and epidemiological risk factors between January and December, 2019. According to China Guideline for Human Brucellosis Diagnosis, the Rose Bengal test (RBT) was used for the screening test, and the serum agglutination test (SAT) was used as the confirmatory test. Brucellosis was confirmed only if the results of both tests were positive. Additionally, FPA and enzyme linked immune sorbent assay (ELISA) were compared with SAT, and their sensitivity, specificity, coincidence rate and consistency coefficient (Kappa value) as diagnostic tests were analyzed individually and in combination. The optimal cut-off value of FPA was also determined using the receiver operator characteristic (ROC) curve. Results The optimum cut-off value of FPA was determined to be 88.5 millipolarization (mP) units, with a sensitivity of 94.5% and specificity of 100.0%. Additionally, the coincidence rate with the SAT test was 96.6%, and the Kappa value (0.9) showed excellent consistency. The sensitivity and specificity of FPA and ELISA combined were higher at 98.0% and 100.0% respectively. Conclusions When the cut-off value of FPA test is set at 88.5 mP, it has high value for the diagnosis of brucellosis. Additionally, when FPA and ELISA are combined, the sensitivity of diagnosis is significantly improved. Thus, FPA may have potential in the future as a diagnostic method for human brucellosis in China. Graphic abstract

Since Chagas disease, melioidosis, and Legionnaires' disease are all potentially life-threatening infections, there is an urgent need for new treatment strategies. All causative agents, Trypanosoma cruzi , Burkholderia pseudomallei , and Legionella pneumophila , express a virulence factor, the macrophage infectivity potentiator (MIP) protein, emerging as a promising new therapeutic target. Inhibition of MIP proteins having a peptidyl-prolyl isomerase activity leads to reduced viability, proliferation, and cell invasion. The affinity of a series of pipecolic acid-type MIP inhibitors was evaluated against all MIPs using a fluorescence polarization assay. The analysis of structure-activity relationships led to highly active inhibitors of MIPs of all pathogens, characterized by a one-digit nanomolar affinity for the MIPs and a very effective inhibition of their peptidyl-prolyl isomerase activity. Docking studies, molecular dynamics simulations, and quantum mechanical calculations suggest an extended σ-hole of the meta -halogenated phenyl sulfonamide to be responsible for the high affinity.

In this study, the identity of our recently produced natural TetR protein was identified by using the LC-ESI-MS/MS technique, and its recognition mechanisms, including the binding pocket, contact amino acids, intermolecular forces, binding sites, binding energies, and affinities for 10 tetracycline drugs were studied. Then, it was evolved by site-mutagenesis of an amino acid to produce a mutant, and a fluorescence polarization assay was developed to detect the 10 drugs in milk. The sensitivities for the 10 drugs were improved with IC50 values decreasing from 30.8–80.1 ng/mL to 15.5–55.2 ng/mL, and the limits of detection were in the range of 0.4–1.5 ng/mL. Furthermore, it was found that the binding affinity for a drug was the critical factor determining its sensitivity, and the binding energy showed little influence. This is the first study reporting the recognition mechanisms of a natural TetR protein for tetracyclines and the development of a fluorescence polarization assay for the detection of tetracyclines residues in food samples.

The residues of aminoglycosides in foods of animal origin are a potential risk to consumers. There have been some immunoassays reported for the screening of aminoglycoside residues, but the method showing the broadest detection spectrum can only be used to detect two drugs. This is because a broad specific recognition reagent is not available. In the present study, the receptor of aminoglycosides (ribosomal protein S12 of Lysinibacillussphaericus) was expressed, and its affinities and recognition mechanisms for 10 aminoglycosides were studied by using surface plasmon resonance and molecular docking, respectively. Then the receptor was used as a recognition reagent to develop a fluorescence polarization assay on a 96-well microplate for the detection of the 10 drugs in pork muscle samples. The limits of detection for the 10 drugs ranged from 5.25 to 30.25 ng/g. The sensitivities for the 10 drugs were generally consistent with their respective receptor affinities and binding energies. After comprehensive comparison, the method performances were better than all the previously reported immunoassays for aminoglycosides. This is the first study reporting the recognition mechanisms of ribosomal protein S12 of Lysinibacillussphaericus for 10 aminoglycosides and the use of it as a recognition reagent to develop a pseudo-immunoassay for the multi-determination of aminoglycosides in food samples.

The milk ring test is a detection assay for antibodies against Brucella in bovine milk. It has good sensitivity but tends to give false positive results. In this study, we standardized the application of the fluorescence polarization assay (FPA) for the detection of antibodies against B.melitensis in goat milk. We obtained negative serum and milk samples from healthy goat flocks in the northern zone of Nuevo León. Positive milk and negative, weak, and strong controls were obtained by mixing volumes of positive control serum with negative control milk. Milk samples were treated with citric acid, after which an FPA was performed. Results were then compared with the Rose Bengal test and the FPA in serum. Milk treatment allowed the quantification of antibodies in samples. Significant differences were found between the 2%, 4%, and 6% groups, compared with the control group (F3, 67 = 17.45, p < 0.0001) but not between the 2% and 4% groups (p = 0.0718). The cut-off value was 74.1 mP, with a sensitivity (Se) of 95% and a specificity (Sp) of 100%. Se and Sp values in field milk samples were 84% and 74.55%, respectively. Despite the FPA test on milk samples showed lower Se and Sp than the FPA test on serum samples, its cutoff may be adjusted. It may be recommended as a screening test in goat milk and become useful for the control and eradication of the disease.

We evaluated hemolyzed, bacterially contaminated, and Nobuto filter paper-derived serum, collected from 50 Rocky Mountain elk (Cervus elaphus nelson) in 2017 and 2019, divided into eight treatments to determine antibody retention. Serum was analyzed on Brucella abortus-specific fluorescence polarization assay utilizing plates and tubes. Reference titers and serostatus were compared to serum held at 22 C for 4, 8, 12, and 16 d; frozen clotted blood; blood with 2% and 10% elk rumen content (held for 8 d at 22 C); and serum eluted from Nobuto filter paper. Using Cohen's kappa test of agreement, plate assay serostatus agreement was substantial or outstanding in all treatments. Serostatus agreement was outstanding in all treatments utilizing tubes. The mean change in score (treatment minus reference) showed significant negative bias in serosuspect or seropositive animals in the frozen, 2% rumen, and 10% rumen treatments on the plate assay, and the day 16 and 10% rumen treatments on the tube assay, that could ultimately result in an animal being misclassified into a serosuspect or seronegative category. Serum eluted from Nobuto filter paper produced inconsistent results and is not recommended as an alternative to serum derived from blood. Although the potential for misclassification of animals with low titers exists, analyzing hemolyzed and bacterially contaminated serum from Brucella abortus nonendemic areas can increase sample size and the potential to detect seropositive animals. [ABSTRACT FROM AUTHOR]

Background: Brucellosis is an infectious-allergic zoonotic disease caused by bacteria of the genus Brucella. Early diagnosis is the key to preventing, treating, and controlling brucellosis. Fluorescence polarization immunoassay (FPA) is a new immunoassay for relatively rapid and accurate detection of antibodies or antigens based on antigen–antibody interaction. However, there is no report on FPA-based detection of human brucellosis in China. Therefore, this study is to evaluate the value of FPA for the diagnosis of human brucellosis in China. Methods: We recruited 320 suspected brucellosis cases who had the clinical symptoms and epidemiological risk factors between January and December, 2019. According to China Guideline for Human Brucellosis Diagnosis, the Rose Bengal test (RBT) was used for the screening test, and the serum agglutination test (SAT) was used as the confirmatory test. Brucellosis was confirmed only if the results of both tests were positive. Additionally, FPA and enzyme linked immune sorbent assay (ELISA) were compared with SAT, and their sensitivity, specificity, coincidence rate and consistency coefficient (Kappa value) as diagnostic tests were analyzed individually and in combination. The optimal cut-off value of FPA was also determined using the receiver operator characteristic (ROC) curve. Results: The optimum cut-off value of FPA was determined to be 88.5 millipolarization (mP) units, with a sensitivity of 94.5% and specificity of 100.0%. Additionally, the coincidence rate with the SAT test was 96.6%, and the Kappa value (0.9) showed excellent consistency. The sensitivity and specificity of FPA and ELISA combined were higher at 98.0% and 100.0% respectively. Conclusions: When the cut-off value of FPA test is set at 88.5 mP, it has high value for the diagnosis of brucellosis. Additionally, when FPA and ELISA are combined, the sensitivity of diagnosis is significantly improved. Thus, FPA may have potential in the future as a diagnostic method for human brucellosis in China. [ABSTRACT FROM AUTHOR]

Brucellosis is a widespread zoonotic disease that is also a potential agent of bioterrorism. Current serological assays to diagnose human brucellosis in clinical settings are based on detection of agglutinating anti-LPS antibodies. To better understand the universe of antibody responses that develop after B. melitensis infection, a protein microarray was fabricated containing 1,406 predicted B. melitensis proteins. The array was probed with sera from experimentally infected goats and naturally infected humans from an endemic region in Peru. The assay identified 18 antigens differentially recognized by infected and non-infected goats, and 13 serodiagnostic antigens that differentiate human patients proven to have acute brucellosis from syndromically similar patients. There were 31 cross-reactive antigens in healthy goats and 20 cross-reactive antigens in healthy humans. Only two of the serodiagnostic antigens and eight of the cross-reactive antigens overlap between humans and goats. Based on these results, a nitrocellulose line blot containing the human serodiagnostic antigens was fabricated and applied in a simple assay that validated the accuracy of the protein microarray results in the diagnosis of humans. These data demonstrate that an experimentally infected natural reservoir host produces a fundamentally different immune response than a naturally infected accidental human host.

We evaluated serologic responses of cattle, bison, elk, and swine representing negative control, early vaccination (4–8 wk), late vaccination (21–28 wk) or booster vaccination, early after-experimental challenge (2–4 wk), and late after-experimental challenge (8–21 wk), in a brucellosis fluorescence polarization assay (FPA; n = 10 sera per species per treatment) using negative control sera from cattle, bison, elk, and swine (n = 5 per species). Sera from cattle shedding Brucella abortus strain RB51 in milk were also evaluated against the 20 negative control sera. The species of negative control sera used in the FPA could increase (p < 0.05) delta millipolarization (mP; delta mP = sample mP − negative control mP) results. In general, the species of negative control sera did not alter the interpretation of FPA results in control, vaccinated, or infected animals. Even after repeated RB51 vaccinations in bison, cattle, or elk, or in cattle shedding RB51 in milk, serologic results from the FPA remained negative. Species differences in FPA results were noted; elk developed robust humoral responses very quickly after infection that resulted in strong positive FPA results. In cattle and bison, humoral responses appeared to develop over a longer period of time, and greater delta mP values were detected at later times after infection. Sensitivity of the FPA for detecting infected animals was greatest for elk in early challenge samples and bison in late challenge samples. Our data suggest that species of origin of negative control sera does not influence interpretation of the FPA in natural hosts of Brucella abortus. [ABSTRACT FROM AUTHOR]

The phytohormone ethylene is widely involved in many developmental processes and is a crucial regulator of defense responses against biotic and abiotic stresses in plants. Ethylene-responsive element binding protein, a member of the APETALA2/ethylene response factor (AP2/ERF) superfamily, is a transcription factor that regulates stress-responsive genes by recognizing a specific cis-acting element of target DNA. A previous study showed only the NMR structure of the AP2/ERF domain of AtERF100 in complex with a GCC box DNA motif. In this report, we determined the crystal structure of AtERF96 in complex with a GCC box at atomic resolution. We analyzed the binding residues of the conserved AP2/ERF domain in the DNA recognition sequence. In addition to the AP2/ERF domain, an N-terminal α-helix of AtERF96 participates in DNA interaction in the flanking region. We also demonstrated the structure of AtERF96 EDLL motif, a unique conserved motif in the group IX of AP2/ERF family, might involve in the transactivation of defense-related genes. Our study establishes the structural basis of the AtERF96 transcription factor in complex with the GCC box, as well as the DNA binding mechanisms of the N-terminal α-helix and AP2/ERF domain. [ABSTRACT FROM AUTHOR]

The eight mammalian Src-family tyrosine kinases are dynamic, multi-domain structures, which adopt distinct “open” and “closed” conformations. In the closed conformation, the regulatory SH3 and SH2 domains pack against the back of the kinase domain, providing allosteric control of kinase activity. Small molecule ligands that engage the regulatory SH3-SH2 region have the potential to modulate Src-family kinase activity for therapeutic advantage. Here we describe an HTS-compatible fluorescence polarization assay to identify small molecules that interact with the unique-SH3-SH2-linker (U32L) region of Hck, a Src-family member expressed exclusively in cells of myeloid lineage. Hck has significant potential as a drug target in acute myeloid leukemia, an aggressive form of cancer with substantial unmet clinical need. The assay combines recombinant Hck U32L protein with a fluorescent probe peptide that binds to the SH3 domain in U32L, resulting in an increased FP signal. Library compounds that interact with the U32L protein and interfere with probe binding reduce the FP signal, scoring as hits. Automated 384-well high-throughput screening of 60,000 compounds yielded Z'-factor coefficients > 0.7 across nearly 200 assay plates, and identified a series of hit compounds with a shared pyrimidine diamine substructure. Surface plasmon resonance assays confirmed direct binding of hit compounds to the Hck U32L target protein as well as near-full-length Hck. Binding was not observed with the individual SH3 and SH2 domains, demonstrating that these compounds recognize a specific three-dimensional conformation of the regulatory regions. This conclusion is supported by computational docking studies, which predict ligand contacts with a pocket formed by the juxtaposition of the SH3 domain, the SH3-SH2 domain connector, and the SH2-kinase linker. Each of the four validated hits stimulated recombinant, near-full-length Hck activity in vitro, providing evidence for allosteric effects on the kinase domain. These results provide a path to discovery and development of chemical scaffolds to target the regulatory regions of Hck and other Src family kinases as a new approach to pharmacological kinase control.

Brucellosis is a zoonosis of economic and public health concern. While most diagnostic tests for brucellosis can only be performed in the laboratory, the Fluorescence Polarization Assay (FPA) was developed as a rapid point-of-care field test. This pilot project aimed to validate the use of FPA for rapid diagnosis of ruminant brucellosis on the field, and to compare the FPA performance with that of the more commonly used Rose Bengal Test (RBT). Blood samples were first collected from ruminants in a livestock market, and later from a nearby slaughterhouse in Port Bouët, Abidjan, Côte d'Ivoire. Samples collected in the livestock market were processed and tested with the FPA in a central laboratory, while samples collected in the slaughterhouse were processed immediately and the FPA was performed on site. To assess the FPA intra-test agreement, a portion of the serum samples tested at the slaughterhouse were re-tested with the FPA in the laboratory later the same day. To assess inter-test agreement, all serum samples were retested with the RBT. A total of 232 samples were tested with the FPA, 106 and 126 from the livestock market and slaughterhouse, respectively. Of these, 26 tested positive and 39 were doubtful for brucellosis. The FPA was repeated on 28 of the samples collected at the slaughterhouse, and comparison of results indicated a moderate intra-test agreement (Kappa = 0.41). The agreement improved when the doubtful category was treated as negative (Kappa = 0.65), and when cattle were excluded (Kappa = 0.56 to 0.61). The RBT was performed on 229 samples, and of these 10 tested positive. A comparison of FPA and RBT results indicated poor agreement (Kappa = 0.00); this improved to slight when only samples taken at the livestock market and tested in the laboratory were considered (Kappa = 0.14). The FPA did not perform well in tropical field conditions, possibly due to the high ambient temperatures in the slaughterhouse. Moreover, a difference in performance was noted in relation to the species tested, whereby the FPA seemed to perform better on sheep and goat samples, compared to cattle samples. These findings highlight that further adjustments are needed before implementing the FPA on the field.

Based on streptavidin coated nanospheres and T7 exonuclease assisted dual-cycle signal amplification, we developed a novel sensitive fluorescence polarization detection method for miRNA. When target miRNA was present in the system, hairpin probe hybridized with miRNA, forming a double-stranded structure. The 5′ end of hairpin probe was then recognized and digested by T7 exonuclease, releasing the non-degraded single strand DNA fragments and miRNA. The released target miRNA could trigger the next cycle of hybridization and digestion, releasing more non-degraded fragments from hairpin probe. The fragments could hybridize with a signal probe (with carboxyfluorescein modification at 5′-end and biotin modification at 3′-end). The formed blunt 5′-end of signal probe was then recognized and degraded by T7 exonuclease, releasing the fragments and the fluorophore carboxyfluorescein. The next cycle of hybridization and digestion of signal probe was triggered by the released fragment at the same time. The free carboxyfluorescein cannot connect with streptavidin coated nanospheres which were used as the fluorescence polarization signal amplifier. So, there was a big change of fluorescence polarization signal after adding miRNA into the detection system, due to the different fluorescence polarization signal between nanospheres-captured intact signal probe and free carboxyfluorescein. The detection limit of this method is about 0.001 nM, and it has a good selectivity. In addition, it was also applicable to determine target miRNA in total miRNA extracts and compare the expression level of target miRNA in different cells. Consequently, the proposed method is expected to be used for the potential cancer diagnosis and the related biomedical research. Image 1 • A simple fluorescence polarization assay for sensitive and selective detection of miRNA was developed. • Dual-cycle signal amplification based on T7 Exo was designed. • Using streptavidin-modified nanospheres as mass amplifiers, there was a big difference in FP signal with a low LOD. • It could be used to determine target miRNA in total miRNA extracts and compare the expression level in different cells. [ABSTRACT FROM AUTHOR]

Abstract This paper demonstrates the use of a smartphone-based sensor for fluorescence polarization (FP) analysis of biomolecules. The FP detection can rapidly sense ligand-analyte bindings by measuring molecule mobility, and thus, FP-based assays have been widely used for rapid diagnostics in clinics. Here, we implemented the FP detection apparatus using a 3D-printed compact holder and the built-in camera of a smartphone. The system offers accurate measurements of the degree of polarization by simultaneously detecting the fluorescence intensities parallel and perpendicular to the polarization of the excitation. The fluorescence signal of the sample is excited by a laser or light-emitting diode and separated by a polarization beam cube depending on the polarization. Parallel and perpendicular polarized emissions are projected onto two different regions of the sensor chip in the smartphone camera. A custom software app was developed to count the average intensity in the areas of interest and compute the degree of polarization. We validated the system by measuring the polarization of dye molecules dissolved in solutions with different viscosities. As an example of biomolecule sensing, a competitive FP immunoassay of Prostaglandin E 2 was demonstrated using the developed system and exhibited the limit of detection of 1.57 ng/mL. The smartphone-based FP assay platform can also be implemented for the detection of toxins, disease biomarkers, and pathogens in resource-limited settings. Highlights • A smartphone-based detector is designed for fluorescence polarization immunoassays. • A rapid and wash-free assay is developed for biomolecule analysis using smartphone. • The smartphone detector can quantify dinoprostone in 30 minutes. • The system has the potential for disease diagnosis in resource-limited settings. [ABSTRACT FROM AUTHOR]

A recognition motif is vital in determining the specificity and sensitivity of the fluorescence polarization assay (FPA) for detecting chemical contaminants in food. Four candidates (Gyrase, GyrBA, TopIV, and QepA) were prepared for this study. The applicability of QepA was confirmed through DNA cleavage assay, inhibition effects, and mechanism investigations using molecular docking, compared to other counterparts. Finally, a novel FPA based on QepA and a CIP-FITC tracer for the detection of fluoroquinolones (FQs) in eggs was developed. The limits of detection (LODs) for eight fluoroquinolones ranged from 2.2 to 5.1 ng g -1 , with enrofloxacin, danofloxacin, and difloxacin meeting the maximum residue limits (MRLs). The spiked recoveries ranged from 65.8 to 103.6% with coefficients of variation (CVs) of 5.4-12.8%. Therefore, a new recognition motif for FQs that did not belong to conventional antibodies was identified, and QepA-based FPA could be a potential tool for rapid, homogeneous, and sensitive monitoring of the residue of FQs in eggs.

Brucellosis caused by the bacteria of the genus Brucella is an important zoonosis and constitutes a serious public health hazard. Brucellosis is diagnosed mainly by the Rose Bengal plate test and indirect ELISA, both of which have poor specificity because false positive serological reactions occur when screening animals that have been vaccinated with B. abortus S19. Fluorescence polarization assay (FPA) was evaluated for screening samples from cattle and buffalo calves with persistent antibody titers induced by vaccination. The standardized FPA exhibited relative sensitivity and specificity of 0.94 and 0.95, respectively, and the area under the curve, kappa and accuracy were 0.98, 0.87 and 0.95, respectively. Comparison of competitive ELISA and FPA revealed that, FPA is more specific than competitive ELISA. The high specificity, sensitivity and 95% accuracy of FPA indicate that, it is suitable for testing vaccinated animals because it can distinguish between infected from vaccinated animals. [ABSTRACT FROM AUTHOR]

Canine brucellosis is an infectious disease of worldwide distribution that can affect dogs, wild canids and man. It is caused by Brucella canis, but dogs can also be infected by smooth Brucella such as B. abortus and B. suis. Due to the increasing importance of dogs in our society, to the scarcity of information about canine brucellosis in the country and its zoonotic character, the aims of the present study were (i) to conduct a survey on the infection by B. canis and smooth Brucella in dogs from the municipality of Araguaína, Tocantins, Brazil, and (ii) to evaluate the risk factors associated with these infections. Sera from 241 dogs were analyzed by agar gel immunodiffusion (AGID) to detect B. canisantibodies, and Buffered Acidified Plate Antigen test (BAPA) and fluorescence polarization assay (FPA) to detect antibodies to smooth Brucella. From the 241 tested dogs, 132 reacted in the AGID and 128 reacted in the BAPA, but only two were positive in FPA. The seroprevalences of B. canis and smooth Brucella infections in dogs in Araguaína were 54.77% (95% CI: 48.25 to 61.17%) and 0.83% (95% CI: 0.10 to 2.97%), respectively. The analysis of risk factors showed associations between B. canis infection and vaccination against leptospirosis, and between B. canis infection and use of manufactured food. In conclusion, data from the present study showed a low prevalence of infection by smooth Brucella and a widespread and high prevalence of infection by B. canis in the city of Araguaína, Tocantins, Brazil.

Esta investigação teve por objetivo validar o teste de polarização fluorescente (TPF) para o diagnóstico sorológico da brucelose bovina, determinando a sensibilidade (SE) e a especificidade relativas (SP) e verificando a reprodutibilidade do teste em quatro laboratórios no Brasil. Foram selecionadas 1.389 amostras de soro sanguíneo, as quais foram inicialmente submetidas aos testes do antígeno acidificado tamponado (AAT) e mercaptoetanol (2-ME). As mesmas amostras foram submetidas à reação de fixação de complemento (RFC) e ao TPF. Para a avaliação do TPF, foi adotada a combinação dos resultados do AAT, da RFC e do 2-ME, utilizados como população de referência (padrão-ouro). Para a determinação do ponto de corte do TPF que proporciona a melhor combinação de sensibilidade e especificidade, foi usada a análise TG-ROC. A concordância entre os resultados dos quatro laboratórios foi determinada com base no indicador kappa e no coeficiente de correlação de Pearson. Os pontos de corte do TPF situaram-se entre 85,2 e 93,6 mP, conforme o laboratório. A sensibilidade variou de 91,7 a 97,3%, e a especificidade situou-se na faixa de 82,6 a 98,3%. Na comparação entre os resultados do TPF dos quatro laboratórios, o indicador kappa ficou entre 0,69 e 0,95, o que indica, na maioria das situações, reprodutibilidade excelente, e o coeficiente de correlação variou entre 0,76 e 0,99. Os resultados indicaram que o TPF apresentou bom desempenho, na maioria das situações, com sensibilidade e especificidade elevadas. Em comparação com os testes convencionais, o TPF apresenta as vantagens de ser de execução mais rápida e mais fácil e não estar sujeito à ocorrência de prozona, como a RFC e o 2-ME, nem de atividade anticomplementar, como a RFC.The purpose of this research was the interlaboratorial validation of the polarization fluorescence assay (PFA) for the serodiagnosis of bovine brucellosis, verifying the relative sensitivity, the relative specificity and the reproducibility of the test in four Brazilian laboratories. Serum samples from 1,389 bovines were selected and submitted to the rose Bengal (RBT) and 2-mercaptoethanol tests in one of the laboratories. The same samples were tested by the complement fixation (CFT) test and by the PFA in the four laboratories participating of the research. The reference population (golden standard) used to evaluate the PFA was the combination of the results of RBT, CFT and 2-ME. TG-ROC analysis was used to obtain the cut-off that provided the best combination of sensitivity and specificity. The agreement between laboratories was obtained by the kappa statistic and Pearson correlation coefficient (r). The PFA cut-off values were from 85.2 to 93.6. The sensitivity of the PFA assay varied from 91.7% to 97.3%, and the specificity values varied from 82.6% to 98.3%. When comparing PFA results from the four laboratories, the kappa values was between 0.69 and 0.95, which indicates, in most situations, excellent reproducibility, and the correlation coefficient varied from 0.76 to 0.99. The results showed that the PFA had a good performance, with high sensitivity and specificity. Compared to the conventional tests, the PFA has the advantages of being easy and quick to perform, and it is not prone to the occurrence of prozone, as the CFT or the 2-ME, nor to the occurrence of anti-complementary effect, as the CFT.

[Display omitted] • Based on aggregation of Y-shaped DNA for FP assay is reported for the first time. • It combined DSN-assisted target recycling and CHA for dual amplification. • This method could distinguish single-base mismatch with high selectivity. • The result for the expression level of miRNA in different cell lines is satisfactory. MicroRNAs (miRNAs) have emerged as one of the promising biomarkers. Their aberrant levels are intimately connected to various diseases. However, Sensitive assay of low-abundance miRNAs remain a grand challenge. Herein, a novel sensitive cascade-amplified fluorescence polarization (FP) sensing platform for miRNA detection was developed based on aggregation strategy of Y-shaped DNA via streptavidin (SA). The miRNA-155 was employed as the model target analyte. In the presence of miRNA-155, the duplex-specific nuclease (DSN) cycling amplification and catalytic hairpin assembly (CHA) cycling amplification were triggered successively. Owing to the target-triggering dual cyclic amplification, numerous Y-shaped DNA nanostructure could be easily generated according to the assembly order of hairpin probes. Then, SA was added to the system, inducing the aggregation of Y-shaped DNA nanostructure to form a large-size of DNA/SA complexes. Taking advantages of cascade-amplified strategy, a significant enhancement of the FP signal was achieved and a sensitive assay for miRNA detection was realized with the detection limit of 1 × 10-13 M. Beyond that, the proposed method could distinguish single-base mismatch and achieved satisfactory result for the expression level assay of miRNA-155 in different cell lines, holding great potential in the applications in miRNA-related clinical diagnosis and biochemical researches. [ABSTRACT FROM AUTHOR]

The relative sensitivity of the fluorescence polarization assay (FPA) was estimated by testing blood sera from 1515 sheep, belonging to flocks infected with Brucella melitensis in Sicily, which were previously shown as positive to at least two of the following tests: rose bengal test (RBT), complement fixation test (CFT), competitive ELISA (c-ELISA). FPA specificity was estimated by testing sera from 719 sheep from B. melitensis -free flocks from Sicily and Sardinia. Based upon results of a receiver-operating-characteristic (ROC) curve, optimal FPA cut-off value in millipolarization units (mP) was chosen at the mean of negative controls tested in the same assay, plus 6, which corresponded to 96.0% sensitivity (95% confidence interval: 93.9, 97.3) and 100% (99.5, 100) specificity. Such a cut off value was the one yielding the maximum sum of sensitivity and specificity (196.0), and it was adopted for subsequent analyses. Maximum FPA sensitivity (98.8%; 91.7, 99.8) was obtained on sera which were positive to CFT and c-ELISA, but negative to RBT. Median FPA values (first, and third quartiles) (109.7, 53.7, 169.3) were greatest for sera which were positive to RBT, CFT, and c-ELISA. Sensitivity of c-ELISA, which was estimated as the proportion of positive results on RBT and CFT positive sera, was 99.8% (99.4, 100.0), whereas specificity was 100.0% (98.5, 100.0). Although FPA resulted as an accurate test, c-ELISA was characterized by a superior sensitivity and it is, therefore, proposed to be used, in parallel with RBT, to increase the testing sensitivity in regions and flocks which can be considered as at a great risk of infection. [ABSTRACT FROM AUTHOR]

Myt1 kinase is a member of the Wee-kinase family involved in G2/M checkpoint regulation of the cell cycle. So far, no peptide substrate suitable for activity-based screening has been reported, hampering systematic development of Myt1 kinase inhibitors. Myt1 inhibitors had to be identified by using either binding assays or activity assays with expensive proteinous substrates. Here, a peptide microarray approach was used to identify peptidic Myt1 substrates. Wee1 kinase was profiled for comparison using the same technology. Myt1 hits from peptide microarray experiments were verified in solution by a fluorescence polarization assay and several peptide substrates derived from cellular proteins were identified. Subsequently, phosphorylation site determination was carried out by MS fragmentation studies and identified substrates were validated by kinase inhibitor profiling. [ABSTRACT FROM AUTHOR]

We report on a fluorescence polarization assay for the detection of the target analyte ATP by making use of an aptasensor and of mass-amplifying CdTe-CdS quantum dots. The ATP aptamer was modified with digoxin antigen and hybridized with its complementary DNA that was modified with the CdTe-CdS quantum dots. Following the addition of digoxin antibody, the mass-amplifying aptasensor probe is formed as a result of the immuno reaction. In the presence of ATP, the polarization of fluorescence decreases because the digoxin antibody becomes dissociated due to the recognition of the ATP by the ATP aptamer. Under optimized conditions, the method has a linear response to ATP in the 10 to 350 μM concentration range, and the limit of detection is 3.7 μM. The method combines the specific recognition capability of aptamers with the sensitivity of an immunoreaction. It has good selectivity and sensitivity, and can be used to detect ATP in serum samples. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Abstract: The casitas B-lineage lymphoma (Cbl) proteins play an important role in regulating signal transduction pathways by functioning as E3 ubiquitin ligases. The Cbl proteins contain a conserved tyrosine kinase binding (TKB) domain that binds more than a dozen proteins, including protein tyrosine kinases (PTKs), in a phosphorylation-dependent manner. The cell surface expression levels of the PTKs are regulated by Cbl-mediated ubiquitination, internalization, and degradation. Dysfunction in this signaling cascade has resulted in prolonged activation of the PTKs and, therefore, has been implicated in inflammatory diseases and various cancers. Due to this negative regulatory function, Cbl has been largely ignored as a therapeutic target. However, recent studies, such as the identification of (i) gain of function c-Cbl mutations in subsets of myeloid cancer and (ii) c-Cbl as a prostate basal cell marker that correlates with poor clinical outcome, suggest otherwise. Here we report the development of a competitive high-throughput fluorescence polarization assay in a 384-well format to identify inhibitors of Cbl(TKB). The high-throughput screen readiness of the assay was demonstrated by screening the Prestwick Chemical Library. [Copyright &y& Elsevier]

The purpose of this research was the interlaboratorial validation of the polarization fluorescence assay (PFA) for the serodiagnosis of bovine brucellosis, verifying the relative sensitivity, the relative specificity and the reproducibility of the test in four Brazilian laboratories. Serum samples from 1,389 bovines were selected and submitted to the rose Bengal (RBT) and 2-mercaptoethanol tests in one of the laboratories. The same samples were tested by the complement fixation (CFT) test and by the PFA in the four laboratories participating of the research. The reference population (golden standard) used to evaluate the PFA was the combination of the results of RBT, CFT and 2-ME. TG-ROC analysis was used to obtain the cut-off that provided the best combination of sensitivity and specificity. The agreement between laboratories was obtained by the kappa statistic and Pearson correlation coefficient (r). The PFA cut-off values were from 85.2 to 93.6. The sensitivity of the PFA assay varied from 91.7% to 97.3%, and the specificity values varied from 82.6% to 98.3%. When comparing PFA results from the four laboratories, the kappa values was between 0.69 and 0.95, which indicates, in most situations, excellent reproducibility, and the correlation coefficient varied from 0.76 to 0.99. The results showed that the PFA had a good performance, with high sensitivity and specificity. Compared to the conventional tests, the PFA has the advantages of being easy and quick to perform, and it is not prone to the occurrence of prozone, as the CFT or the 2-ME, nor to the occurrence of anti-complementary effect, as the CFT. [ABSTRACT FROM AUTHOR]

Abstract: Calcineurin is a Ca2+/calmodulin-dependent serine/threonine protein phosphatase involved in many biological processes and developmental programs, including immune response. One of the most studied substrates of calcineurin is the transcription factor NFAT (nuclear factor of activated T cells) responsible for T-cell activation. Different anticalcineurin drugs, such as cyclosporine A and FK506, are the most commonly used immunosuppressants in transplantation therapies. Unfortunately, their mechanism of action, completely blocking the calcineurin phosphatase activity while also requiring continuous administration, bears severe side effects. During recent years, the family of regulators of calcineurin (RCAN) has been described and studied extensively as modulators of calcineurin signaling pathways. The RCAN1 region, spanning amino acids 198 to 218 and responsible for inhibiting the calcineurin–NFAT signaling pathway in vivo, has been identified. An RCAN1-derived peptide spanning this sequence interferes with the calcineurin–NFAT interaction without affecting the general calcineurin phosphatase activity. Here we report the development of an optimized in vitro high-throughput fluorescence polarization assay based on the disruption of the RCAN1198–218–CnA interaction for identifying molecules with immunosuppressant potential. This approach led us to identify dipyridamole as a disruptor of such interaction. Moreover, three small molecules with a potential immunosuppressive effect were also identified. [Copyright &y& Elsevier]

Aim: Determine the optimal cut-off and the use of Fluorescence Polarization Assay (FPA) to improve the detection of brucellosis in individual goats in Argentina. Methods: Sera from 96 goats from a flock with abortion clue to B. melitensis biovar 1 were used to assess the efficacy of the FPA to detect brucellosis in goats. FPA results were compared with those of the Buffered Antigen Plate Agglutination test (BPAT) confirmed by Seroagglutination in tube (SAT), the competitive enzyme-linked immunosorbent assay (c-ELISA) and the indirect enzyme-linked immunosorbent assay (i-ELISA). Sera from 554 goats free from brucellosis were tested with the BPAT, SAT, c-ELISA and i-ELISA to determine its Specificity. Vaccination had not been performed in the flocks evaluated. Results: The most appropriate cut-off was selected for the FPA by using MedCalc software. It was fixed at 87 mP giving a sensitivity and specificity of 98.1% (CI 89.9-99.7) and 92.8% (CI 90.4-94.7). The relative sensitivity compared with i-ELISA and c-ELISA was 97% and 92.9% respectively. The relative specificity compared with i-ELISA and c-ELISA was 97.5% and 98% respectively. The kappa measures of agreement between tests was higher than 0.75 Conclusion: The high correlation between FPA results and other serological methods with sera goats is indicative of the excellent performance of FPA technique in diagnosis of caprine brucellosis and we endorse it as a recommended method. [ABSTRACT FROM AUTHOR]

Abstract: Effective surveillance of bovine tuberculosis (BTB) in developing countries where reliable data on disease prevalence is scarce or absent is a precondition for considering potential control options. We conducted a slaughterhouse survey to assess for the first time the burden of BTB in Southern Chad. Altogether, 954 slaughter animals were consecutively sampled and tested using the single intra-dermal comparative cervical tuberculin (SICCT) test, a recently developed fluorescence polarization assay (FPA) and routine abattoir meat inspection after slaughter. Gross visible lesions were detected in 11.3% (CI: 9.4–13.5%) of the animals examined and they were mostly located in the lymph nodes and the lung. Significantly more Mbororo zebus (15.0%) were affected by lesions than Arab zebus (9.9%; OR=2.20, CI: 1.41–3.41%; p <0.001). Of all animals tested, 7.7% (CI: 6.2–9.6%) reacted positively to SICCT if OIE guidelines were applied. However, receiver operating characteristic (ROC) analysis using Mycobacterium tuberculosis complex (MTBC) infected animals as the positive population and lesion negative animals as the negative population, revealed a better SICCT performance if the cut-off value was decreased to >2mm. SICCT reactor prevalence rose to 15.5% (CI: 13.3–18.0%) and FPA did not perform better than SICCT, when this setting adapted cut-off was applied. [Copyright &y& Elsevier]

Abstract: A single-domain variable heavy chain (VHH) antibody fragment specific to the mycotoxin 15-acetyldeoxynivalenol (15-AcDON) was obtained after immunization of a llama (Llama glama) with the protein conjugate 15-DON-BSA plus TiterMax™ Classic adjuvant. After confirmation of a polyclonal response to DON toxin in both conventional (cIgG) and heavy chain antibody (HCAb) fractions, a VHH library was constructed from amplified cDNA by nested PCR. VHH fragments with binding affinity for the mycotoxin were selected by panning of the phagemid library against microtiter plates coated with 15-DON-OVA. The dominant clone (NAT-267) was expressed in E. coli and was purified as a VHH monomer (mNAT-267) at a final concentration of 1.3mgmL−1. Isolated NAT-267 VHH DNA was fused to the homopentamerization domain of the B subunit of verotoxin to generate the pentabody format of single-domain antibody (sAb). The VHH pentamer (pNAT-267) was expressed in E. coli and was purified at a final concentration of 1.0mgmL−1. Surface plasmon resonance (SPR) analysis of soluble mNAT-267 binding kinetics to immobilized 15-DON-Horse Radish Peroxidase (HRP) indicated a dissociation constant (K D) of 5μM. Competitive direct enzyme-linked immunosorbent assay (CD-ELISA) and fluorescence polarization assay (FPA) inhibition experiments with monomer and pentamer confirmed binding to 15-AcDON. Competitive inhibition FPAs with mNAT-267 and pNAT-267 determined IC50 values of 1.24 and 0.50μM, respectively, for 15-AcDON hapten. These values were similar to the IC50 value of 1.42μM for 15-AcDON given by polyclonal llama serum sampled 56 days after immunization. Competition formats for structurally related trichothecenes resulted in no cross-reactivity to: DON; 3-acetyldeoxynivalenol (3-AcDON); neosolaniol (NEO); diacetoxyscirpenol (DAS); and T-2 toxin. Our study confirmed that recombinant VHH fragments capable of binding low molecular weight haptens can be produced through the creation and panning of hyper-immunized single-domain (sdAb) libraries. [Copyright &y& Elsevier]

Abstract: Vascular endothelial growth factor (VEGF) is a homodimeric proangiogenic protein that induces endothelial cell migration and proliferation primarily through interactions with its major receptors, VEGFR-1 and VEGFR-2. Inhibitors of one or both of these VEGF-receptor interactions could be beneficial as therapeutics for diseases caused by dysfunctional angiogenesis (e.g., cancer). Others have reported small peptides that bind to the VEGF dimer at surface regions that are recognized by the receptors. Here we report the development of a fluorescence polarization assay based on the binding to VEGF of a derivative of one of these peptides that has been labeled with BODIPY-tetramethylrhodamine (BODIPYTMR). This 384-well format assay is tolerant to dimethyl sulfoxide (DMSO, up to 4% [v/v]) and has a Z′ factor of 0.76, making it useful for identifying molecules that associate with the receptor-binding surface of the VEGF dimer. [Copyright &y& Elsevier]

Abstract: The current method for goat brucellosis diagnosis is based on the World Organization for Animal Health (OIE) using the screening card test (CT), with antigen at 8% (CT8) or 3% (CT3) of cell concentrations, and the confirmatory complement fixation test (CFT). However, these tests do not differentiate antibodies induced by vaccination from those derived from field infections by Brucella species or other bacterial agents; in places like Mexico, where the prevalence of brucellosis and the vaccination rates are high, there is a considerable percentage of false positive reactions that causes significant unnecessary slaughter of animals. Furthermore, results of the fluorescence polarization assay (FPA) using the Brucella abortus O-polysaccharide (OPS) tracer in goats are poorer than those with cattle. The present study was undertaken to investigate a tracer prepared from the native hapten (NH) of the Rev. 1 strain of Brucella melitensis to improve FPA performance on goat brucellosis diagnosis. Evaluation of 48 positive samples and 96 negative samples showed that the NH tracer was more accurate (p <0.01) than the OPS tracer (97.2% vs. 93.8% accuracy, respectively). On the diagnostic performance evaluation, the NH tracer performed better (87.5% accuracy, 79.5% sensitivity, 84.3% specificity, and 163.8 performance index) than the OPS tracer (83.5%, 75.9%, 81.0%, and 156.9, respectively) using 1009 positive and 2039 negative Mexican field goat sera samples selected by test series approved by the OIE (card test 3% and CFT). We demonstrated a new application for the NH lipopolysaccharide on detecting antibodies against Brucella using the FPA, which may yield faster results (minutes vs. 24–72h) than the immunodiagnosis assays frequently used in bovine brucellosis. In addition, NH tracer produces similar or better performance results than the conventional OPS tracer, using the FPA in goat sera samples. [Copyright &y& Elsevier]