Genetic variability and population structure of the Montezuma quail (Cyrtonyx montezumae) in the northern limit of its distribution.

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 (A = 2.41 ± 0.27). The global population genetic differentiation index RST = 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.

High Fundamental Frequency (HFF) Monolithic Quartz Crystal Microbalance with Dissipation Array for the Simultaneous Detection of Pesticides and Antibiotics in Complex Food.

As in the case of the food industry in general, there is a global concern about safety and quality in complex food matrices, such as honey, which is driving the demand for fast, sensitive and affordable analytical techniques across the honey-packaging industry. Although excellent techniques such as liquid chromatography-tandem mass spectrometry (LC-MS/MS) are available, these are located in centralized laboratories and are still lacking in speed, simplicity and cost-effectiveness. Here, a new approach is presented where a competitive immunoassay is combined with a novel High Fundamental Frequency Quartz Crystal Microbalance with Dissipation (HFF-QCMD) array biosensor for the simultaneous detection of antibiotics and pesticides in honey. Concretely, thiabendazole and sulfathiazole residues were monitored in spiked honey samples. Results revealed that HFF-QCMD arrays provide a complementary and reliable tool to LC-MS/MS for the analysis of contaminants in these kinds of complex matrices, while avoiding elaborate sample pre-treatment. The good sensitivity achieved (I50 values in the 70-720 µg/kg range) and the short analysis time (60 min for 24 individual assays), together with the ability for multiple analyte detection (24 sensor array) and its cost-effectiveness, pave the way for the implementation of a fast on-line, in situ routine control of potentially hazardous chemical residues in honey.

Comparative Analysis of Chromatin-Delivered Biomarkers in the Monitoring of Sepsis and Septic Shock: A Pilot Study.

Sepsis management remains one of the most important challenges in modern clinical practice. Rapid progression from sepsis to septic shock is practically unpredictable, hence the critical need for sepsis biomarkers that can help clinicians in the management of patients to reduce the probability of a fatal outcome. Circulating nucleoproteins released during the inflammatory response to infection, including neutrophil extracellular traps, nucleosomes, and histones, and nuclear proteins like HMGB1, have been proposed as markers of disease progression since they are related to inflammation, oxidative stress, endothelial damage, and impairment of the coagulation response, among other pathological features. The aim of this work was to evaluate the actual potential for decision making/outcome prediction of the most commonly proposed chromatin-related biomarkers (i.e., nucleosomes, citrullinated H3, and HMGB1). To do this, we compared different ELISA measuring methods for quantifying plasma nucleoproteins in a cohort of critically ill patients diagnosed with sepsis or septic shock compared to nonseptic patients admitted to the intensive care unit (ICU), as well as to healthy subjects. Our results show that all studied biomarkers can be used to monitor sepsis progression, although they vary in their effectiveness to separate sepsis and septic shock patients. Our data suggest that HMGB1/citrullinated H3 determination in plasma is potentially the most promising clinical tool for the monitoring and stratification of septic patients.

Real-time monitoring of fenitrothion in water samples using a silicon nanophotonic biosensor.

Due to the large quantities of pesticides extensively used and their impact on the environment and human health, a prompt and reliable sensing technique could constitute an excellent tool for in-situ monitoring. With this aim, we have applied a highly sensitive photonic biosensor based on a bimodal waveguide interferometer (BiMW) for the rapid, label-free, and specific quantification of fenitrothion (FN) directly in tap water samples. After an optimization protocol, the biosensor achieved a limit of detection (LOD) of 0.29 ng mL-1 (1.05 nM) and a half-maximal inhibitory concentration (IC50) of 1.71 ng mL-1 (6.09 nM) using a competitive immunoassay and employing diluted tap water. Moreover, the biosensor was successfully employed to determine FN concentration in blind tap water samples obtaining excellent recovery percentages with a time-to-result of only 20 min without any sample pre-treatment. The features of the biosensor suggest its potential application for real time, fast and sensitive screening of FN in water samples as an analytical tool for the monitoring of the water quality.

Detection of DDT and carbaryl pesticides in honey by means of immunosensors based on high fundamental frequency quartz crystal microbalance (HFF-QCM).

BACKGROUND: In recent years there has been a concern about the presence of pesticides in honey because residues of DDT and carbaryl were found in honey samples. Traditional techniques, such as chromatography, reach the required limits of detection (LOD) but are not suitable for in situ implementation in the honey-packaging industry due to their high cost and the need for highly qualified staff for routine operation. Biosensors offer simplicity, low cost, and easy handling for analytical purposes in food applications. RESULTS: Piezoelectric immunosensors based on high fundamental frequency quartz crystal microbalance (HFF-QCM) have been developed for the detection of carbaryl and DDT in honey. Biorecognition was based on competitive immunoassays in the conjugate-coated format, using monoclonal antibodies as specific immunoreagents. The assay LODs attained by the HFF-QCM immunosensors were 0.05 µg L-1 for carbaryl and 0.24 µg L-1 for DDT, reaching a similar level of detectability to that of the usual reference techniques. The practical LODs in honey samples were 8 µg kg-1 for carbaryl and 24 µg kg-1 for DDT. The immunosensors' analytical performance allow the detection of these pesticides in honey at EU regulatory levels with good accuracy (recovery percentages ranging from 94% to 130% within the working range of each pesticide standard curve) and precision (coefficients of variation in the 9-36% range). CONCLUSION: The proposed immunosensor is a promising analytical tool that could be implemented for quality control in the honey packaging industry, to simplify and to reduce the cost of the routine pesticide analysis in this appreciated natural food. © 2020 Society of Chemical Industry.

Survey on Drosophila suzukii Natural Short-Term Dispersal Capacities Using the Mark-Release-Recapture Technique.

Spotted wing drosophila, Drosophila suzukii Matsumura (Diptera: Drosophilidae), has become a key pest for soft fruits and cherries in Europe in less than a decade since the first outbreak in 2007. Although this pest's passive dispersal ability has been observed over more than 1400 km in 1 year, active spread has not yet been extensively studied. A mark-release-recapture (MRR) method based on protein-marked flies was employed to determine the flight capacity of D. suzukii. Sterile marked flies were released and recaptured in a trap grid at increasing distances from 10 to 250 m from the releasing point to study flight distance during periods ranging from 3 h to 1 week. MRR experiments were replicated in the presence and absence of host fruits to study how they could affect dispersal behavior. The dispersal capacity of the Mediterranean fruit fly, Ceratitis capitata Wiedemann (Diptera: Tephritidae) was also studied under the same conditions. The results showed a low dispersal ability for D. suzukii, with a daily flight distance below 100 m with no predominant wind. The implications on natural dispersion and control methods based on attractants are discussed.

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.

A High Fundamental Frequency (HFF)-based QCM Immunosensor for Tuberculosis Detection.

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.

High-frequency phase shift measurement greatly enhances the sensitivity of QCM immunosensors.

In spite of being widely used for in liquid biosensing applications, sensitivity improvement of conventional (5-20MHz) quartz crystal microbalance (QCM) sensors remains an unsolved challenging task. With the help of a new electronic characterization approach based on phase change measurements at a constant fixed frequency, a highly sensitive and versatile high fundamental frequency (HFF) QCM immunosensor has successfully been developed and tested for its use in pesticide (carbaryl and thiabendazole) analysis. The analytical performance of several immunosensors was compared in competitive immunoassays taking carbaryl insecticide as the model analyte. The highest sensitivity was exhibited by the 100MHz HFF-QCM carbaryl immunosensor. When results were compared with those reported for 9MHz QCM, analytical parameters clearly showed an improvement of one order of magnitude for sensitivity (estimated as the I50 value) and two orders of magnitude for the limit of detection (LOD): 30µgl(-1) vs 0.66µgL(-1)I50 value and 11µgL(-1) vs 0.14µgL(-1) LOD, for 9 and 100MHz, respectively. For the fungicide thiabendazole, I50 value was roughly the same as that previously reported for SPR under the same biochemical conditions, whereas LOD improved by a factor of 2. The analytical performance achieved by high frequency QCM immunosensors surpassed those of conventional QCM and SPR, closely approaching the most sensitive ELISAs. The developed 100MHz QCM immunosensor strongly improves sensitivity in biosensing, and therefore can be considered as a very promising new analytical tool for in liquid applications where highly sensitive detection is required.

Love wave immunosensor for the detection of carbaryl pesticide.

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.

Bisphenol A determination in baby bottles by chemiluminescence enzyme-linked immunosorbent assay, lateral flow immunoassay and liquid chromatography tandem mass spectrometry.

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.

Development and application of recombinant antibody-based immunoassays to tetraconazole residue analysis in fruit juices.

Tetraconazole is currently used as a fungicide in fruit and vegetables. The aim of this work was the development of immunochemical techniques based on recombinant antibodies for the screening of tetraconazole residues in fruit juices. Recombinant antibodies were produced from a hybridoma cell line secreting a monoclonal antibody specific for tetraconazole and from lymphocytes of mice hyperimmunised with tetraconazole haptens conjugated to bovine serum albumin. From these antibodies, enzyme-linked immunosorbent assays in the conjugate-coated format were developed, which were able to detect tetraconazole standards down to 1ng/mL. From recovery studies with spiked samples, these immunoassays determined tetraconazole in orange and apple juices with acceptable reproducibility (coefficients of variation below 25%) and recoveries (ranging from 78% to 145%) for a screening technique. The analytical performance of RAb-based immunoassays was fairly similar to that of the MAb-based immunoassays. Due to their simplicity and high sample throughput, the developed recombinant-based immunoassays can be valuable analytical tools for the screening of tetraconazole residues in fruit juices at regulatory levels.

Indirect competitive immunoassay for the detection of fungicide Thiabendazole in whole orange samples by Surface Plasmon Resonance.

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.

Development of a chemiluminescence-based quantitative lateral flow immunoassay for on-field detection of 2,4,6-trinitrotoluene.

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).

Validation of a phase-mass characterization concept and interface for acoustic biosensors.

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.

Development of monoclonal antibody-based immunoassays for the analysis of bisphenol A in canned vegetables.

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.

Development of monoclonal immunoassays for the determination of triazole fungicides in fruit juices.

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.

Application of a monoclonal-based immunoassay for the determination of imazalil in fruit juices.

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.

Performance of two monoclonal immunoassays in mixtures of cross-reacting dithiophosphorus pesticides.

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.

Rapid detection and counting of viable beer-spoilage lactic acid bacteria using a monoclonal chemiluminescence enzyme immunoassay and a CCD camera.

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.