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Restricted movement among populations decreases genetic variation, which may be the case for the Montezuma quail ( Cyrtonyx montezumae ), a small game bird that rarely flies long distances. In the northern limit of its distribution, it inhabits oak-juniper-pine savannas of Arizona, New Mexico, and Texas. Understanding genetic structure can provide information about the demographic history of populations that is also important for conservation and management. The objective of this study was to determine patterns of genetic variation in Montezuma quail populations using nine DNA microsatellite loci. We genotyped 119 individuals from four study populations: Arizona, Western New Mexico, Central New Mexico, and West Texas. Compared to other quail, heterozygosity was low ( H ¯ 0 = 0.22 ± 0.04) and there were fewer alleles per locus ( Ā = 2.41 ± 0.27). The global population genetic differentiation index R ST = 0.045 suggests little genetic structure, even though a Bayesian allocation analysis suggested three genetic clusters ( K = 3). This analysis also suggested admixture between clusters. Nevertheless, an isolation-by-distance analysis indicates a strong correlation ( r = 0.937) and moderate evidence ( P = 0.032) of non-independence between geographical and genetic distances. Climate change projections indicate an increase in aridity for this region, especially in temperate ecosystems where the species occurs. In this scenario, corridors between the populations may disappear, thus causing their complete isolation., Competing Interests: The authors declare that they have no competing interests., (© 2023 Sánchez-Murrieta et al.)

Background: Tuberculosis, one of the oldest diseases affecting human beings, is still considered as a world public health problem by the World Health Organization., Method & Material: Therefore, there is a need for new and more powerful analytical methods for early illness diagnosis. With this idea in mind, the development of a High Fundamental Frequency (HFF) piezoelectric immunosensor for the sensitive detection of tuberculosis was undertaken. A 38 kDa protein secreted by Mycobacterium tuberculosis was first selected as the target biomarker. Then, specific monoclonal antibodies (MAbs) were obtained. Myc-31 MAb, which showed the highest affinity to the analyte, was employed to set up a reference enzyme-linked immunosorbent assay (ELISA) with a limit of detection of 14 ng mL-1 of 38 kDa antigen., Results & Discussion: For the development of the HFF piezoelectric immunosensor, 100 MHz quartz crystals were used as transducer elements. The gold electrode surface was functionalized by covalent immobilization of the target biomarker through mixed self-assembled monolayers (mSAM) of carboxylic alkane thiols. A competitive immunoassay based on Myc-31 MAb was integrated with the transducer as sensing bio-recognition event. Reliable assay signals were obtained using low concentrations of antigen for functionalization and MAb for the competitive immunoassay. Under optimized conditions, the HFF immunosensor calibration curve for 38 kDa determination showed a limit of detection as low as 11 ng mL-1 of the biomarker. The high detectability attained by this immunosensor, in the picomolar range, makes it a promising tool for the easy, direct and sensitive detection of the tuberculosis biomarker in biological fluids such as sputum., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

A Love Wave (LW) immunosensor was developed for the detection of carbaryl pesticide. The experimental setup consisted on: a compact electronic characterization circuit based on phase and amplitude detection at constant frequency; an automated flow injection system; a thermal control unit; a custom-made flow-through cell; and Quartz /SiO2 LW sensors with a 40 μm wavelength and 120 MHz center frequency. The carbaryl detection was based on a competitive immunoassay format using LIB-CNH45 monoclonal antibody (MAb). Bovine Serum Albumin-CNH (BSA-CNH) carbaryl hapten-conjugate was covalently immobilized, via mercaptohexadecanoic acid self-assembled monolayer (SAM), onto the gold sensing area of the LW sensors. This immobilization allowed the reusability of the sensor for at least 70 assays without significant signal losses. The LW immunosensor showed a limit of detection (LOD) of 0.09 μg/L, a sensitivity of 0.31 μg/L and a linear working range of 0.14-1.63 μg/L. In comparison to other carbaryl immunosensors, the LW immunosensor achieved a high sensitivity and a low LOD. These features turn the LW immunosensor into a promising tool for applications that demand a high resolution, such as for the detection of pesticides in drinking water at European regulatory levels.

Two immunoassays, a Lateral Flow ImmunoAssay (LFIA) based on colloidal gold nanoparticle labels and an indirect competitive chemiluminescence enzyme-linked immunosorbent assay (CL-ELISA), were developed and a high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was optimized to assess the possible release of bisphenol A (BPA, 4,4'-isopropylidenediphenol) from different plastic baby bottles treated with simulating solutions. Coating conjugate concentration, anti-BPA antibody dilution, incubation time of the primary and secondary antibodies, and tolerance to different organic solvents were optimized to obtain the best performance of the ELISA with chemiluminescent end-point detection. The influence of different buffers on LFIA performance was also evaluated. Both methods showed good repeatability (mean CV value around 13%) and sensitivity. Reproducibility tests for CL-ELISA gave a mean CV value of about 25%. The IC50 and Limit of Detection (LOD) values of CL-ELISA were 0.2 and 0.02 ng mL(-1), respectively. The LOD of LFIA was 0.1 μg mL(-1). A LC-MS/MS method was also optimized. The separation was performed in a C18 column with a triple-quadrupole mass spectrometer with electrospray ionisation interface. The method showed a good linearity in the range 2 to 500 ng mL(-1), with a regression coefficient of 0.998. In the simulating solutions the detection and quantification limits, calculated by the signal to noise level of 3 (S/N = 3), were 5.8 ng mL(-1) and 17.4 ng mL(-1), respectively. This limit of quantification was about 3 and 35 times lower than the permitted limits set by the official method CEN/TS 13130-13 (0.05 μg mL(-1)) and by the Directive 2004/19/EC (0.6 μg mL(-1)), respectively. The methods were applied to determine BPA release from baby bottles, performing repeated procedures according to EU and national regulations. The results demonstrated that no BPA migration from the tested plastic materials occurred with only one exception. The migrated amount, above the regulatory limits, was detected by all the mentioned assays.

A highly sensitive and specific SPR-based competitive immunoassay for the detection of Thiabendazole (TBZ) has been developed. An indirect format where a TBZ-protein conjugate is immobilized onto gold surfaces has been selected. Under the optimal conditions, a LOD of 0.67 nM (0.13 μg L(-1)) and an IC(50) of 3.2 nM (0.64 μg L(-1)) have been achieved which are comparable to the values obtained by conventional ELISA. Analysis of real samples has been attempted by first evaluating the influence of complex matrix samples coming from whole oranges and secondly measuring samples containing TBZ previously evaluated by chromatographic methods. A methanolic extraction procedure followed by a simple dilution in assay buffer has proven to be sufficient to measure orange samples using the developed immunoassay with an excellent recovery percentage. The sensitivity and the feasibility of measuring whole orange samples demonstrate the effectiveness and robustness of the SPR biosensor, which can be useful for the determination of TBZ in food at concentrations below the Maximum Residue Levels (MRLs) established by the European legislation.

Simple, rapid and highly sensitive assays, possibly allowing on-site analysis, are required in the security and forensic fields or to obtain early signs of environmental pollution. Several bioanalytical methods and biosensors based on portable devices have been developed for this purpose. Among them, Lateral Flow ImmunoAssays (LFIAs) offer the advantages of rapidity and ease of use and, thanks to the high specificity of antigen-antibody binding, allow greatly simplifying and reducing sample pre-analytical treatments. However, LFIAs usually employ colloidal gold or latex beads as labels and they rely on the formation of colored bands visible by the naked eye. With this assay format, only qualitative or semi-quantitative information can be obtained and low sensitivity is achieved. Recently, the use of enzyme-catalyzed chemiluminescence detection in LFIA has been proposed to overcome these problems. In this work, we describe the development of a quantitative CL-LFIA assay for the detection of 2,4,6-trinitrotoluene (TNT) in real samples. Thanks to the use of a portable imaging device for CL signal measurement based on a thermoelectrically cooled CCD camera, the analysis could be performed directly on-field. A limit of detection of 0.2 μg mL(-1) TNT was obtained, which is five times lower than that obtained with a previously described colloidal gold-based LFIA developed employing the same immunoreagents. The dynamic range of the assay extended up to 5 μg mL(-1) TNT and recoveries ranging from 97% to 111% were obtained in the analysis of real samples (post blast residues obtained from controlled explosion)., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The aim of this work was the development of monoclonal antibodies (MAbs) and highly sensitive immunoassays (ELISAs) to bisphenol A (BPA), a well-known endocrine disruptor able to migrate from the internal coating of cans to food contained inside, particularly vegetables. To produce MAbs to BPA, four synthetic compounds were conjugated to proteins and used as immunizing haptens in mice. By applying hybridoma technology, several MAbs were produced and selected. These antibodies were characterized in the conjugate-coated and in the antibody-coated formats, using both homologous and heterologous conjugates. Three indirect ELISA based on the MAbs showing the highest affinity to BPA were selected. The limit of detection of the most sensitive ELISA was 0.22 nM (0.05 ng/mL), with an I₅₀ value of around 1 nM (0.23 ng/mL). An homologous ELISA based on the MAb BPAB-11 was applied to the simple, direct determination of BPA in the liquid portion of canned artichoke, peas, and sweet corn. Only sample dilution in an appropriate saline buffer was required to minimize matrix effects and to enter the ELISA working range. Recovery and precision of the method were evaluated by spiking the liquid portion of these cans with BPA at 20, 50, and 100 ng/mL. Coefficients of variation were below 20% in most cases. With regard to recovery, the analytical data obtained were also acceptable. This immunoassay has therefore proved its potential as a new tool for the rapid, sensitive and accurate determination of BPA in canned food.

Acoustic wave resonator techniques are widely used in in-liquid biochemical applications. The main challenges remaining are the improvement of sensitivity and limit of detection, as well as multianalysis capabilities and reliability. The sensitivity improvement issue has been addressed by increasing the sensor frequency, using different techniques such as high fundamental frequency quartz crystal microbalances (QCMs), surface generated acoustic waves (SGAWs) and film bulk acoustic resonators (FBARs). However, this sensitivity improvement has not been completely matched in terms of limit of detection. The decrease on frequency stability due to the increase of the phase noise, particularly in oscillators, has made it impossible to increase the resolution. A new concept of sensor characterization at constant frequency has been recently proposed based on the phase/mass sensitivity equation: Δφ/Δm ≈ -1/m(L), where m(L) is the liquid mass perturbed by the resonator. The validation of the new concept is presented in this article. An immunosensor application for the detection of a low molecular weight pollutant, the insecticide carbaryl, has been chosen as a validation model.

Enzyme-linked immunosorbent assays (ELISAs) based on monoclonal antibodies for the detection of triazole fungicides have been developed. With this aim, hapten-protein conjugates, containing the common triazole and chlorinated aromatic moieties, were prepared. From mice immunized with these conjugates, several monoclonal antibodies (MAbs) with the ability to sensitively bind several triazoles with different specificity were obtained. Both analyte- and class-specific ELISAs were developed. The hexaconazole-specific immunoassay can determine this fungicide with a limit of detection of 0.3 mug/L in standard buffer. The so-called triazole-specific immunoassay allowed for the detection of tetraconazole, penconazole, cyproconazole, and myclobutanil, with limits of detection in the 0.1-0.7 mug/L range. These immunoassays were applied to the determination of triazoles in spiked fruit juices. Samples were adequately diluted to minimize the matrix effects. Coefficients of variation were below 30%, and recoveries ranged from 62 to 135%. Therefore, the developed immunoassays can determine triazole fungicides in fruit juices down to the maximum residue limits currently legislated, without any sample treatment other than dilution.

A statistical approach for the analysis of complex samples by immunoassay is proposed in this article. Two enzyme-linked immunosorbent assays (ELISAs), one of them in the conjugate-coated format and the other in the antibody-coated format, were evaluated for their suitability to the analysis of mixtures of three organodithiophosphorus pesticides: azinphos-methyl, azinphos-ethyl and phosmet. It was found that the apparent affinity of the antibody to each analyte changed in the presence of a cross-reacting compound in the antibody-coated ELISA format, but not when the conjugate-coated ELISA format was used. The assays were thereafter applied to the analysis of mixtures of the three recognized pesticides. With the conjugate-coated ELISA format, accurate and precise determinations of mixtures could be performed if an azinphos-methyl standard curve was employed, with recoveries between 71% and 130% and with coefficients of variation lower than 12.7%. Neither accurate nor precise measurements could be accomplished with the enzyme immunoassay using the antibody-coated ELISA format, independently of the standard curve used. It is thought that the study presented here will have applicability in a variety of cases where the analytical goal is semiquantitative screening based on the total quantity of an unknown mixture of related compounds.

A monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) was developed for the quantification of imazalil [(RS)-1-(beta-allyloxy-2,4-dichlorophenylethyl)imidazole] in apple, tomato and orange juice samples. From an imazalil hapten, which mimics the analyte structure, several monoclonal antibodies were obtained. An ELISA in the conjugate-coated format was developed and optimized using the antibody showing the highest sensitivity. For standards, the detection limit of the ELISA was 0.2 nM (0.06 ng ml(-1)), with an I(50) value of 1.6 nM (0.5 ng ml(-1)). The study of the influence of matrices on assay reliability indicated that the ELISA could determine imazalil in fruit juices at the low ng ml(-1) level simply by diluting the sample, without any clean-up or concentration step. Recovery and precision of the method were evaluated by spiking juice samples with imazalil in the 10-500 ng ml(-1) range. The mean recovery from fruit juices was 97% and the mean coefficient of variation was approximately 20%. In addition to being precise and accurate, the method has proved to be simple and sensitive, with a quantification limit well below the maximum residue limits for imazalil in these matrices.

A chemiluminescence enzyme immunoassay carried out with a monoclonal antibody (MAb) and a charge-coupled device (CCD) camera was developed for rapid enumeration of viable beer-spoilage lactic acid bacteria. LA-4 MAb, which recognizes a broad spectrum of lactic acid bacteria isolated from several breweries across Spain, was produced and characterized. Test samples were filtered through polycarbonate membranes, and the membranes with retained bacteria were incubated at 31 degrees C for 2 days. They were then subjected to a two-step chemiluminescence enzyme immunoassay with MAb LA-4, and light-emitting points were detected and counted with a CCD camera. Eighteen out of 19 beer-spoilage lactic acid bacteria analysed produced luminous spots that could be enumerated. Results provided by the immunochemiluminescence assay correlated very well with those obtained by visual plate counting within a range of 3-100 CFU/100 ml. Correlation coefficients were 0.994 for four strains in sterile saline solution and 0.984 for 14 strains in artificially contaminated beer. The excellent agreement suggests that luminous spots detected within 2 days of culture are produced only by viable cells.

Nanocolloidal gold particles were prepared and labeled to an anti-carbofuran monoclonal antibody (Mab). This conjugate was dispensed on the conjugated pad of a porous glass fiber. Ovalbumin (OVA)-carbofuran and goat anti-mouse IgG were dispensed on the nitrocellulose (NC) membrane and served as the test line and control line, respectively. The carbofuran-containing sample migrated to the NC membrane and reacted with the anti-carbofuran Mab labeled with the colloidal gold. The mixture diffused along the membrane and passed through the OVA-carbofuran in the test line via capillary action. The more analyte present in the sample, the more effectively it will compete with the carbofuran immobilized on the test line for binding to the limited amount of antibody labeled with colloidal gold. An adequate amount of carbofuran could prevent attachment of the colored conjugate to the test line. The presence or absence of a colored band on the test line could indicate a negative or positive result, respectively. When measured to the water sample spiked with carbofuran, this was obtained at or above 0.25 mg/L of carbofuran. The major advantages of the one-step strip test are that the detection time needed was <10 min and all of the reagents are included in the test device.

Organochlorine pesticides still generate public health concerns because of their unresolved health impact and their persistence in living beings, which is demanding appropriate analytical techniques for their monitoring. In this study, an enzyme-linked immunosorbent assay based on monoclonal antibodies (MAbs) for the detection of an important group of organochlorine pesticides, the cyclodiene group, has been developed. With this aim, several hapten-protein conjugates, characterized by exposure of the common hexachlorinated bicyclic (norbornene) moiety and differing in the linking structure to the carrier protein, were prepared. From mice immunized with these conjugates, several MAbs with the ability to sensitively bind the majority of cyclodienes were obtained. Among them CCD2.2 MAb displaying the broadest recognition to cyclodiene compounds (endosulfan, dieldrin, endrin, chlordane, heptachlor, aldrin, toxaphene: I(50) values in the 6-25 nM range) was selected for the assay. Interestingly, this MAb showed certain stereospecificity toward other polychlorinated cycloalkanes because the gamma-isomer of hexachlorocyclohexane (lindane) was also very well recognized (I(50) value of 22 nM). This immunoassay is potentially a very valuable analytical tool for the rapid and sensitive determination of cyclodiene insecticides and related compounds, which in turn may contribute to the understanding of the biological activities and of the overall environmental impact of these persistent organic pollutants.

Two enzyme-linked immunosorbent assays (ELISA) with chemiluminescent (CL) detection for the insecticide DDT and the group of DDT-related compounds have been optimized and characterized. Both conjugate-coated ELISAs are based on monoclonal antibodies (MAbs) of different specificity and homologous protein conjugates. Effects of several physicochemical factors (ionic strength, pH, Tween-20 and Bovine serum albumin (BSA) concentrations) and solvents (methanol, ethanol, acetone and N,N'-dimethylformamide) on the performance of the assays were studied and optimized. For the DDT-selective assay, the sensitivity, estimated as the I(50) value, was 0.6 microg/l, with a linear working range between 0.1 and 2 microg/l and a limit of detection of 0.06 microg/l. For the DDT group-selective assay, the sensitivity was 0.2 microg/l, with a linear working range between 0.07 and 1 microg/l and a limit of detection of 0.04 microg/l. CL-ELISAs were four times more sensitive compared to colorimetric ELISAs. Finally, both immunoassays were applied to the detection of DDT and group of DDT-related compounds in spiked real water, soil and food samples.

We report the use of a novel technique for detection of the organochlorine insecticide compound dichlorodiphenyltrichloroethane (DDT) by measuring the nanometer-scale bending of a microcantilever produced by differential surface stress. A synthetic hapten of the pesticide conjugated with bovine serum albumin (BSA) was covalently immobilised on the gold-coated side of the cantilever by using thiol self assembled monolayers. The immobilisation process is characterised by monitoring the cantilever deflection in real-time. Then specific detection is achieved by exposing the cantilever to a solution of a specific monoclonal antibody to the DDT hapten derivative. The specific binding of the antibodies on the cantilever sensitised side is measured with nanomolar sensitivity. Direct detection is proved by performing competitive assays, in which the cantilever is exposed to a mixed solution of the monoclonal antibody and DDT. The future prospects and limitations to be overcome for the application of nanomechanical sensors for pesticide detection are discussed.

Azinphos-methyl (AM), O,O-dimethyl S-[(4-oxo-1,2,3-benzotriazin-3(4H)-yl)methyl] phosphorodithioate, is a dithiophosphorous insecticide extensively used for the control of fruit culture pests. In this work the ELIFA system, initially developed and marketed to substitute conventional ELISA methods, was used for the screening of supports and immunoreagents in the development of a flow immunosensor to AM. The objective was to find the optimal antibody concentration, support quantity and enzymatic tracer concentration to develop a sensitive and reusable immunosensor. The influence of chitosan as protein stabilizing agent was also investigated. We observed that, on the basis of immunosorbent characterization, chitosan-modified silica with immobilized LIB-MFH14 monoclonal antibody (MAb) showed the best sensitivity, with a I(50) value of 6 nM AM. All of the immobilized MAbs either in alkylaminated or chitosan-modified silica showed I(50) values between 10 and 36 nM. Regarding the regeneration capability, the best desorption agent tested was 0.1 M glycine/HCl, pH 2.0, performing in most cases a 100% desorption after just one wash and maintaining the antibody activity even after 20 cycles of regeneration. The chitosan-modified silica seemed to be the best support for this purpose.