Direct competitive enzyme-linked immunosorbent assay for the determination of the highly polar short-chain sulfophenyl carboxylates

A direct enzyme-linked immunosorbent assay for the detection of the short-chain sulfophenylcarboxylic acids (SPCs), the main metabolites of the linear alkylbenzenesulfonates, is reported. Six SPCs (2C3, 2C4, 3C4, 2C5, 3C5, 3C6), differing in the length of the alkyl chain (between C3 and C6) and in the position of the phenylsulfonic group versus the carboxylic group, have been synthesized. Antibodies have been raised against a mixture of the corresponding horseshoe crab hemocyanin conjugates prepared by coupling the carboxylic acid to the lysine amino acid residues. The immunoassay As115/3C4-HRP achieves an IC50 value of 23 nM (6.67 microg L(-1)) and a detection limit of 0.85 nM (0.24 microg L(-1)), using as standard analyte an equimolar mixture of the six SPCs. The immunoassay has found to work better in media with low or moderate ionic strength (4-30 mS cm(-1)). The decrease in the detectability produced by the potential formation of SPC salts with divalent cations such as Ca2+ can be prevented by lowering the pH of the assay medium below the pKa value of the SPC carboxylic group and using a buffer chelating with properties such as citrate buffer. The assay can be considered specific for short-chain SPCs since congeners with longer alkyl chains and other pollutants containing sulfonic groups in their structure do not interfere significantly in the assay. Preliminary experiments addressed to evaluate the potential application of this assay to environmental water samples demonstrate the usefulness of the assay.     

Quantitative enzyme-linked immunosorbent assay for determination of polychlorinated biphenyls in environmental soil and sediment samples

An enzyme-linked immunosorbent assay (ELISA) for the quantitative determination of Aroclors 1242, 1248, 1254, and 1260 in soil and sediments was developed and its performance compared with that of gas chromatography (GC). The detection limits for Aroclors 1242 and 1248 in soil are 10.5 and 9 ng/g, respectively. The assay linear dynamic range is 50-1333 ng/g. Cross-reactivity of the assay with 37 structurally related potential cocontaminants in environmental soil samples was examined; none of the chlorinated anisoles, benzenes, or phenols exhibited >3% cross-reactivity, with <0.1% cross-reactivity being the norm. Soil spike recoveries of 107% and 104% were obtained for Aroclors 1242 and 1248, respectively, for a spike level of 5 mg/kg, with corresponding relative standard deviations of 14% and 17%. One hundred forty-eight environmental soil, sediment, and paper pulp samples, obtained from two EPA listed Superfund sites, were analyzed by ELISA and standard GC methods. Samples were extracted for ELISA analysis by shaking with methanol. Additional extractions of the same samples were performed either with supercritical carbon dioxide or by Soxhlet extraction with methanol. ELISA results for both the supercritical fluid and the Soxhlet extracts were in close agreement with the GC results, while the ELISA results for the methanol shake extracts were not. The data for the environmental samples demonstrated the capability of the ELISA to provide accurate results and reinforced the dependence of any detection method, including ELISA, on appropriate extraction procedures.     

Design of a compact disk-like microfluidic platform for enzyme-linked immunosorbent assay

This paper presents an integrated microfluidic device on a compact disk (CD) that performs an enzyme-linked immunosorbent assay (ELISA) for rat IgG from a hybridoma cell culture. Centrifugal and capillary forces were used to control the flow sequence of different solutions involved in the ELISA process. The microfluidic device was fabricated on a plastic CD. Each step of the ELISA process was carried out automatically by controlling the rotation speed of the CD. The work on analysis of rat IgG from hybridoma culture showed that the microchip-based ELISA has the same detection range as the conventional method on the 96-well microtiter plate but has advantages such as less reagent consumption and shorter assay time over the conventional method.     

Development of an enzyme-linked immunosorbent assay for the determination of the linear alkylbenzene sulfonates and long-chain sulfophenyl carboxylates using antibodies generated by pseudoheterologous immunization

ELISA methods have been developed for screening contamination of water resources by linear alkyl benzene sulfonates (LAS) or the most immediate degradation products, the long chain sulfophenyl carboxylates, SPCs. The assay uses antibodies raised through pseudoheterologous immunization strategies using an equimolar mixture of two immunogens (SFA-KLH and 13C(13)-SPC-KLH) prepared by coupling N-(4-alkylphenyl)sulfonyl-3-aminopropanoic acid (SFA) and p-(1-carboxy-13-tridecyl)phenylsulfonic acid (13C(13)-SPC) to keyhole limpet hemocyanin (KLH). The immunizing haptens have been designed to address recognition versus two different epitopes of the molecule. The SFA hapten maximizes recognition of the alkyl moiety while preserving the complexity of the different alkyl chains present in the LAS technical mixture. The 13C(13)-SPC hapten addresses recognition of the common and highly antigenic phenylsulfonic group. The antisera raised using this strategy have been shown to be superior to those obtained through homologous immunization procedures using a single substance. By using an indirect ELISA format, LAS and long-chain SPCs can be detected down to 1.8 and 0.2 microg L(-1), respectively. Coefficients of variation of 6 and 12% within and between assays, respectively, demonstrate immunoassay reproducibility. The assay can be used in media with a wide range of pH and ionic strength values. Preliminary experiments performed to assess matrix effects have demonstrated the potential applicability of the method as a screening tool to assess contamination by these types of surfactants in natural water samples.     

Preparation of antibodies for the designer steroid tetrahydrogestrinone and development of an enzyme-linked immunosorbent assay for human urine analysis

A highly sensitive enzyme-linked immunosorbent assay (ELISA) has been developed for tetrahydrogestrinone (THG), the new designer anabolic steroid responsible for the well-known Balco scandal announced in year 2003. Antibodies have been raised against 18a-homo-pregna-4,9,11-trien-17beta-ol-3-carboxymethyl oxime coupled to horseshoe crab hemocyanin. The hapten has been synthesized from gestrinone by controlled reduction of the triple bond of the ethinyl group at position C-17, without affecting the double bonds of the steroidal rings, followed by reaction of the keto group at C-3 with (carboxymethoxy)amine hemihydrochloride to form the oxime bond. The antisera obtained has been used in combination with 18a-homo-pregna-4,9,11-trien-17beta-ol-20-yn-3-carboxymethyl oxime, a hapten derivative of gestrinone, coupled to bovine serum albumin to establish a competitive ELISA. Under the conditions used, THG can be detected in buffer with a limit of detection (LOD) of 0.045 +/- 0.015 microg L(-1) (N = 9). The assay is very selective since other steroids assessed are not recognized. Preliminary experiments performed with human urine samples demonstrate that the assay can be applied to the analysis of these samples after a simple sample treatment method reaching a LOD of 0.25 +/- 0.14 microg L(-1). Accuracy is very good as demonstrated by the excellent correlation obtained when analyzing blind spiked urine samples (slope 0.93, R2 = 0.992).     

Validation of accuracy of enzyme-linked immunosorbent assay in hybridoma screening and proposal of an improved screening method

The 96-well plate format of enzyme-linked immunosorbent assay (ELISA) is the de facto standard in screening hybridomas for active antibody. Despite its widespread use, there have been few or no systematic attempts to validate its accuracy and answer the fundamental question, is it finding all the positives? We report here on a comparison between ELISA and a semiautomated flow-based kinetic exclusion assay (KinExA), both used in screening the same hybridoma cell line. Our finding is that ELISA is both overreporting (false positives) and underreporting (false negatives) compared to the KinExA system. The large number of hybridoma cells (e.g., cultured in six 96-well plates) that must be checked is daunting in considering any method other than ELISA for routine screening. To overcome this, we devised a sampling strategy in which wells are combined in a specified pattern, allowing a significant reduction in the total number of measurements required.     

Enzyme-linked immunosorbent assay compared with gas chromatography/mass spectrometry for the determination of triazine herbicides in water

An enzyme-linked immunosorbent assay (ELISA) was compared to a gas chromatography/mass spectrometry (GC/MS) procedure for the analysis of triazine herbicides and their metabolites in surface water and groundwater. Apparent recoveries from natural water and spiked water by both methods were comparable at 0.2-2 micrograms/L. Solid-phase extraction (SPE) was examined also, and recoveries were determined for a suite of triazine herbicides. A significant correlation was obtained between the ELISA and GC/MS method for natural water samples that were extracted by SPE. Because ELISA was developed with an atrazine-like compound as the hapten with conjugation at the 2-position, it was selective for triazines that contained both ethyl and isopropyl side chains. Concentrations for 50% inhibition (IC50) were as follows: atrazine, 0.4 microgram/L; ametryne, 0.45 microgram/L; prometryn and propazine, 0.5 microgram/L; prometon, 0.7 microgram/L; simazine and terbutryn, 2.5 micrograms/L; hydroxyatrazine, 28 micrograms/L; deethylatrazine and deisopropylatrazine, 30 micrograms/L; cyanazine, 40 micrograms/L; didealkylatrazine had no response. The combination of screening analysis by ELISA, which requires no sample preparation and works on 160 microL of sample, and confirmation by GC/MS was designed for rapid, inexpensive analysis of triazine herbicides in water.     

Development of a monoclonal antibody against ochratoxin A and its application in enzyme-linked immunosorbent assay and gold nanoparticle immunochromatographic strip

A monoclonal antibody (mAb) specific to ochratoxin A (OTA) was produced from a stable hybridoma cell line, 9C9H9, generated by the fusion of P3/NS1/1-AG4-1 myeloma cells with spleen cells isolated from a BALB/c mouse immunized with OTA-keyhole limpet hemocyanin. The 9C9H9 mAb belongs to the immunoglobulin G1 (kappa chain) isotype. A competitive direct enzyme-linked immunosorbent assay (cdELISA) and a competitive indirect ELISA were established for antibody characterization. The concentrations causing 50% inhibition of binding of OTA-horseradish peroxidase to the antibody by OTA, OTB, and OTC were found to be 0.32, 0.17, and 0.28 ng/mL, respectively, in the cdELISA. A sensitive and rapid mAb-based gold nanoparticle immunochromatographic strip was also developed using this mAb. This strip has a detection limit of 5 ng/mL for OTA and can be completed in 10 min. Analysis of OTA in coffee samples revealed that data obtained from immunochromatographic strip were in a good agreement with those obtained from cdELISA. The mAb-based cdELISA and immunochromatographic strip assay established in this study were sensitive and accurate for rapid screening of OTA in coffee samples.     

Design and synthesis of immunoconjugates and development of competition inhibition enzyme-linked immunosorbent assay (CIEIA) for the detection of O-isopropyl methylphosphonofluoridate (sarin): an organophosphorous toxicant

Three haptens of the organophosphorus (OP) toxicant ‘sarin’ having different spacer arm were designed and synthesized. Haptens were conjugated with BSA (bovine serum albumin) and ovalbumin (OVA) for raising antibody and coating antigen. High antibody titer with higher specificity was obtained from 4-(4-(isopropoxy(methyl)phosphoryloxy)phenylamino)-4-oxobutanoic acid (hapten B) having reasonable long spacer arm. For the standard curve, an IC₅₀ (inhibitory concentration) of free antigen was found to be 0.415 μg mL⁻¹ on the basis of indirect competitive ELISA. The study revealed that heterology in competition inhibition enzyme immunoassay (CIEIA) produced remarkable improvement in the sensitivity and specificity of the assay. Under the optimized conditions, the quantitative working range was found to be 0.19-1.56 μg mL⁻¹ with a limit of detection (LOD) of 0.05 μg mL⁻¹. The antibodies showed negligible cross reactivity (CR) with other OP toxicants and pesticides, which makes the assay suitable for the selective detection of sarin.     

A gas chromatographic separation for the h and C stable isotope ratio determination of coal compounds

A new, completely automated gas chromatography technique has been developed to separate the different gaseous compounds produced during underground coal gasification for their (13)C/(12)C and D/H isotope ratio measurements. The technique was designed for separation and collection of H(2), CO, CO(2), H(2)O, H(2)S, CH(4), and heavier hydrocarbons. These gaseous compounds are perfectly separated by the gas-phase chromatograph and quantitatively sent to seven combustion and collection lines. H(2), CO, CH(4), and heavier hydrocarbons are quantitatively oxidized to CO(2) and/or H(2)O. The isotopic analyses are performed by the sealed-tube method. The zinc method is used for reduction of both water and H(2)S to hydrogen for D/H analysis. Including all preparation steps, the reproducibility of isotope abundance values, for a quantity higher than or equal to 0.1 mL of individual components in a mixture (5 mL of gases being initially injected in the gas chromatograph), is ±0.1‰ for δ(13)C(PDB) and ±6‰ for δD(SMOW).