Biomedical subjects
Publications and source records attributed to Rosa Puchades.
The potential of using compact discs as high throughput screening platforms for DNA microarraying is discussed and applied to discriminate genetic variations of Plum pox virus.
Highly sensitive enzyme-linked immunoassays for chlorpyrifos, one of the most applied insecticides, are presented. Several haptens were synthesized for immunoreagent production and ELISA development. The best immunoassays obtained are based on an indirect coated-plate immunoassay format. Two assays were optimized; one shows a limit of detection of 0.3 ng L(-1), an I50 of 271 ng L(-1), and a dynamic range between 4 and 16 474 ng L(-1). The other one has a limit of detection of 0.07 ng L(-1), an I50 of 7 ng L(-1), and a dynamic range between 0.4 and 302 ng L(-1). The assays were used to quantify chlorpyrifos in olive oil using a simple and rapid sample extraction procedure. The good recoveries achieved in both assays (109% mean value) and the agreement with values given by the GC reference method (110% mean value) indicate their potential for either screening or laboratory quantification.
A fully automated immunosensor for the herbicide glyphosate has been developed on the basis of the immunocomplex capture assay protocol. The sensor carries out on-line analyte derivatization prior to the assay and uses a selective anti-glyphosate serum, a glyphosate peroxidase enzyme tracer, and fluorescent detection. Under optimal conditions, the detection limit achieved is 0.021 microg/L with an analysis rate of 25 min per assay, autonomy of more than 48 h, and sensor reusability >500 analytical cycles. The immunosensor is able to discriminate structurally related molecules, such as aminomethylphosphonic acid, the main metabolite of glyphosate, and other related herbicides, such as glufosinate and glyphosine. Interferences from naturally occurring species (anions, cations, and humic substances) and their elimination were also studied. The immunosensor has been successfully applied to water and soil sample analysis, with good recoveries at levels lower than 1 microg/L. Results obtained with the immunosensor correlate well with data from a magnetic particle ELISA and LC/LC/MS chromatographic method.
Malathion is an organophosphorus insecticide commonly used in crops and indoor applications. Negative effects of malathion on human health and ecosystems are of growing concern. In this work, novel malathion haptens are synthesized to develop an ELISA screening method. The immunoassay is based on a conjugate-coated format and shows a limit of detection of 0.11 ng/mL, an IC50 of 1.58 ng/mL, a dynamic range between 0.23 and 10.94 ng/ mL, and a cross-reactivity of <2% with structurally related compounds. The developed ELISA has been used to quantify malathion in groundwater and surface water samples. The good recoveries achieved and the agreement with those given by the GC-MS reference method indicate the potential of the assay for environmental monitoring of malathion in natural waters without purification or preconcentration steps. The effect of dissolved organic matter (humic acids) on the ELISA was evaluated, resulting in the conclusion that the immunoassay can be successfully performed in surface water samples with a humic acid content up to 10 mg/L, without sample pretreatment. Samples with a high humic acid content can be analyzed through ELISA after solid-phase extraction, eluting with acetone or acetonitrile.
Vitellogenin (VTG) is a protein produced in the liver of oviparous animals in response to oestrogens. Abnormal production of VTG by males, therefore, is used as a biological indicator of exposure to xeno-oestrogens. In this study, a sandwich-ELISA for measuring VTG in Liza aurata (golden grey mullet) was developed and validated. Plasma VTG was purified from 17beta-oestradiol-injected immature individuals of mullet, by size-exclusion and ion-exchange chromatography. Polyclonal antibodies against VTG were raised in rabbits. A sensitive immunoassay was developed for measurement of vitellogenin in L. aurata serum, reaching a quantification limit of 0.01 microg mL(-1) and a dynamic range from 0.02 to 2 microg mL(-1). The assay is specific, because high levels (>100 microg mL(-1)) of carp (Cyprinus carpio), goldfish (Carassius auratus), tilapia (Oreochromis niloticus), tench (Tinca tinca), rainbow trout (Oncorhynchus mykiss), European eel (Anguilla anguilla) and frog (Rana perezi) purified VTG, give negligible responses. The assay was used to analyse plasma samples from wild mullet.
Novel procedures for fenthion hapten synthesis are described following three different strategies. The first one attaches the spacer arm to the oxygen atom of the aromatic fenthion ring. The second one binds it through the thiophosphate moiety and the third strategy consists on the attachment of the spacer arm to the sulfur atom of the molecule. A total of nine fenthion haptens have been synthesized and used for immunoreagent production (protein conjugates and polyclonal antibodies). The developed conjugate-coated format ELISA exhibited a detection limit of 0.03 ng/ml, an IC50 of 0.05 ng/ml and a dynamic range between 0.03 and 1 ng/ml. There was little or no cross-reactivity to similar tested compounds. The ELISA was used to determine fenthion residues in white wine samples without any purification or preconcentration steps. Recoveries ranged between 81% and 113%.
A highly sensitive and specific enzyme-linked immunosorbent assay has been developed for detection of sulfathiazole (STZ, 4-amino- N-thiazol-2-yl-benzenesulfonamide). A set of haptens was synthesized in order to produce polyclonal antibodies against sulfonamides. Two ELISA formats (antibody-coated and conjugate-coated) were also investigated using all the serum/coating conjugate combinations that showed specific recognition. The developed ELISA succeeded in detection of STZ at concentrations as low as 0.03 ng mL(-1) over a measurable range of 0.12-6.71 ng mL(-1). Selectivity studies have demonstrated that other sulfonamides do not interfere significantly (<10%) with analysis of STZ by this immunochemical technique. Analysis of spiked bee honey samples by the developed ELISA method showed recoveries were good. The selectivity and sensitivity (IC(50)=1.6 ng mL(-1)) make it a suitable screening method for determination of low levels of STZ in food samples.
Diazinon is a broad organophosphate insecticide used in agricultural and other treatments, resulting in widespread water contamination. The development of easy-to-use screening immunoanalytical methods is an interesting tool to study environmental pollution impact. Two novel strategies for diazinon hapten synthesis are addressed. One of them attaches the spacer arm to the oxygen atom of the diazinon aromatic ring. The other one retains the diazinon basic structure linking the spacer to an aromatic carbon. A total of eight diazinon haptens were synthesized, demonstrating that they are suitable for immunoreagent (protein conjugates and polyclonal antibodies) production. The optimized ELISA is based on conjugate-coated format and had a detection limit of 0.40 microg/L, showing little or no cross-reactivity to similar tested compounds. The immunoassays were used as a tool to quantify diazinon in natural waters. Results are in agreement with those given by GC-MS reference method. Mean recoveries ranging between 99% and 105% confirm the potential of our approach to determine diazinon in samples without purification or preconcentration steps, being applied as a screening method for field monitoring of diazinon in river waters.
A sensitive, simple and reliable method has been developed for the determination of atrazine in extra virgin olive oil. The analytical procedure involves direct extraction of the target analyte from oil matrix with methanol and a freezing clean-up step (-80 degrees C) followed by plate or sensor immunoassay determination. A detection limit of 0.7 ng/mL, with a dynamic range from 1.0 to 10.4 ng/mL, was reached. The method was highly selective for atrazine and propazine, showing little or no cross-reactivity to other similar compounds. The excellent recoveries obtained (mean value 91.3%) confirm the potential of this approach to detect atrazine in olive oil for application as screening and complementary method in pesticide regulatory and food safety programs. The proposed method correlates well with the reference gas chromatography (GC-MS) technique.
A highly sensitive and specific indirect enzyme-linked immunosorbent assay is described for Silvex, 2-(2,4,5 trichlorophenoxy)propionic acid, (2,4,5-TP). One specific feature of the immunoassay is the use of simple chemical activation of chlorophenoxy acids to prepare both the immunizing and coating conjugates. The assay is based on the use of polyclonal antibodies raised against 2,4,5-TP, and a peroxidase-labeled secondary antibody for colorimetric detection. The effect of different chemical conditions (pH, and salt and detergent concentration) on immunoassay performance has been studied. Under the best conditions the least detectable dose and the sensitivity (IC(50)) for 2,4,5-TP were 0.05 micro g L(-1) and 0.80 micro g L(-1), respectively. The optimized immunoassay was also highly specific, showing little (6.9% for 2,4,5-T) or no cross-reactivity with other similar herbicides. The assay was used to determine 2,4,5-TP in water and soils. The excellent recoveries obtained (mean values ranging between 89% and 104%) make this immunoassay a suitable screening method for either environmental monitoring or laboratory quantification of 2,4,5-TP.
The use of a rapid and specific (cross-reactivity<4%) enzyme-linked immunosorbent assay (ELISA) for the determination of alachlor residues in water and vegetable samples is addressed. The analytical method consists of a fast extraction procedure followed by an optimised ELISA. The detection limit was 0.44 microg l(-1), with a linear range from 0.89 to 143.2 microg l(-1). For alachlor extraction from water samples, different solid-phase cartridges (C, Ph, C8 and C2) were assayed using MeOH as eluent. Extracts were diluted (1:4) with distilled water before ELISA. This procedure gave recoveries close to 100% with RSDs<14%. For vegetable samples, alachlor was extracted directly with MeOH and the extracts diluted 1:40 (v/v) with saline buffer prior to ELISA. The results obtained by the proposed procedure correlate well with the reference method (multiresidue extraction-GC-MS) for vegetable samples (r>0.85).
Selected physico-chemical and chemical characteristics of 13 coconut coir dust (mesocarp pithy tissue plus short-length fibres) samples from Asia, America and Africa were evaluated as peat alternatives. All properties studied differed significantly between and within sources, and from the control Sphagnum peat. pH of coir dust was slightly acidic, whereas salinity varied dramatically between 39 and 597 mS m(-1) in the saturated media extract. The cation exchange capacity and carbon/nitrogen (C/N) ratio ranged from 31.7 to 95.4 cmol(c) kg(-1) and from 75 to 186, respectively. Most carbon was found as lignin and cellulose. The concentrations of available nitrogen, calcium, magnesium and micro-elements were low, while those of phosphorus and potassium were remarkably high (0.28-2.81 mol m(-3) and 2.97-52.66 mol m(-3) for P and K, respectively). Saline ion concentrations, especially chloride and sodium, were also high.
The development of an immunoassay for screening contamination by chlorophenols is presented. Two haptens were synthesized and conjugated to immunizing proteins to raise rabbit polyclonal antibodies. The antibody-coated format (direct) gave better sensitivity than the conjugate-coated format (indirect) if 2,4,6-trichlorophenol is used as target analyte. The measurement range was 86.4 microg l(-1) to 0.7 microg l(-1), with an average I50 of 7.8 microg l(-1) and a detection limit of 0.2 microg l(-1). The assay detects the presence of trichloropyridinol and other chlorophenols such as di-, tetra- and pentachlorophenols constituting thus a suitable tool for the early warning of the presence of such family contaminants. The optimized method permits the detection of the most important chlorophenols in a fast and reproducible way using no more than one antibody and a single assay. The results achieved with water samples spiked with different chlorophenols fit with a multiple linear regression model when expressing the total concentration of chlorophenols as equivalent of 2,4,6-trichlorophenol (P < 0.01), demonstrating the usefulness of the assay as a screening tool to detect contamination by chlorophenols.