Profiling of Metabolomic Changes in Plasma and Urine of Pigs Caused by Illegal Administration of Testosterone Esters.

The use of anabolic steroid hormones as growth promoters in feed for farm animals has been banned in the European Union since 1988 on the basis of Council Directive 96/22/EC. However, there is still ongoing monitoring and reporting of positive findings of these banned substances in EU countries. The aim of this work was to investigate the efficacy and discriminatory ability of metabolic fingerprinting after the administration of 17ß-testosterone esters to pigs. Plasma and urine samples were chromatographically separated on a Hypersil Gold C18 column. High resolution mass spectrometry metabolomic fingerprints were analysed on a hybrid mass spectrometer Q-Exactive. Three independent multivariate statistical methods, namely principal component analysis, clustre analysis, and orthogonal partial least squares discriminant analysis showed significant differences between the treated and control groups of pigs even 14 days after the administration of the hormonal drug. Plasma samples were also analysed by a conventional quantitative analysis using liquid chromatography with tandem mass spectrometry and a pharmacokinetic curve was constructed based on the results. In this case, no testosterone residue was detected 14 days after the administration. The results clearly showed that a metabolomics approach can be a useful and effective tool for the detection and monitoring of banned anabolic steroids used illegally in pig fattening.

Immunoaffinity chromatography combined with tandem mass spectrometry: A new tool for the selective capture and analysis of brassinosteroid plant hormones.

Brassinosteroids (BRs) are plant-specific steroid hormones that play essential roles in the regulation of many important physiological processes in plant life. Their extremely low concentrations (~pmoles/g FW) in plant tissue and huge differences in polarity of individual members within the BR family hamper their detection and quantification. To address this problem, an immunoaffinity sorbent with broad specificity and high capacity for different BR metabolites containing a monoclonal antibody (mAb) against a BR spacer (20S)-2α,3α-dihydroxy-7-oxa-7α-homo-5α-pregnane-6-one-20 carboxylic acid (BR4812) was used for the rapid and highly selective isolation of endogenous BRs containing a 2α,3α-diol in ring A from minute plant samples. This enrichment procedure was successfully applied as a sample preparation method prior to quantitative analysis of BRs in real plant tissues by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Use of immunoaffinity chromatography (IAC) increased the sensitivity of the UHPLC-MS/MS analysis owing to improvements in the BR signal-to-noise ratio (S/N) and matrix factor (MF). Although MF values of BRs analyzed in classical samples ranged from 8.9% to 47.4%, MF values for the IAC purified samples reached 44.5-96.6%. Thus, the developed IAC-UHPLC-MS/MS approach was shown to be a simple, robust, effective and extremely fast procedure requiring minute amounts of plant samples suitable for the quantitative profiling of many BR metabolites, helping to overcome the major problems associated with their determination in very complex plant matrices.

2,4-D and IAA Amino Acid Conjugates Show Distinct Metabolism in Arabidopsis.

The herbicide 2,4-D exhibits an auxinic activity and therefore can be used as a synthetic and traceable analog to study auxin-related responses. Here we identified that not only exogenous 2,4-D but also its amide-linked metabolite 2,4-D-Glu displayed an inhibitory effect on plant growth via the TIR1/AFB auxin-mediated signaling pathway. To further investigate 2,4-D metabolite conversion, identity and activity, we have developed a novel purification procedure based on the combination of ion exchange and immuno-specific sorbents combined with a sensitive liquid chromatography-mass spectrometry method. In 2,4-D treated samples, 2,4-D-Glu and 2,4-D-Asp were detected at 100-fold lower concentrations compared to 2,4-D levels, showing that 2,4-D can be metabolized in the plant. Moreover, 2,4-D-Asp and 2,4-D-Glu were identified as reversible forms of 2,4-D homeostasis that can be converted to free 2,4-D. This work paves the way to new studies of auxin action in plant development.

Determination of alkylresorcinols and their metabolites in biological samples by gas chromatography-mass spectrometry.

High throughput GC-MS methods for quantification of alkylresorcinols (AR), biomarkers of whole grain wheat and rye intake, in plasma and adipose tissue and their metabolites in urine were developed and optimised. Alkylresorcinols in plasma (200µL) and adipose tissues (10-50mg) were extracted with diethyl ether, whereas main AR metabolites such as DHBA and DHPPA and newly identified metabolites in urine (50µL) were extracted with ethyl acetate after enzymatic deconjugation. All extracts were purified on OASIS-MAX solid phase extraction cartridges. Plasma and adipose tissue sample extracts were then derivatised with trifluoroacetic anhydride and reconstituted in undecane, whereas AR metabolites in urine samples were derivatised with BSTFA+TMCS (99:1, v/v, 100µL). Prepared samples were quantified by GC-MS (EI-SIM). Analysis of all compounds in the different matrices showed good selectivity, sensitivity, linearity, precision (<15% within and between batches), adequate recovery (75-108%), and short total run time (10-12min). The methods developed are applicable to large-scale sample sets such as epidemiological studies.

Detection of biomarkers for inflammatory diseases by an electrochemical immunoassay: the case of neopterin.

An electrochemical immunoassay for neopterin was developed using recently produced specific antibodies immobilized to protein A-coated magnetic beads in combination with differential pulse voltammetry and screen-printed array of electrodes. Neopterin-alkaline phosphatase conjugate was used as label in a competitive assay format. Multiplexed analysis of neopterin was demonstrated by replacing the traditional ELISA with electrochemical detection and the traditional plastic wells with screen-printed array of electrodes. The optimized electrochemical method, based on polyclonal antibodies, reached a limit of detection of 0.008 ng/mL with an average RSD %=10. Serum samples collected from patients with sepsis, healthy volunteers and other patients without a confirmed clinical diagnosis were also analyzed. The obtained results, compared with those of a commercial ELISA kit, had a significant correlation, showing the possibility to distinguish among the serum samples from ill or healthy subjects.

Analytical evaluation of a high-throughput enzyme-linked immunosorbent assay for acrylamide determination in fried foods.

The analytical performance and evaluation of a kit-based ELISA for the determination of acrylamide in fried potato and corn chip samples are described. The sample homogenate is subjected to clean-up using SPE, followed by analyte derivatization and ELISA detection. Accuracy, precision and linearity of the ELISA procedure have been validated using spiked samples. Analytical recovery ranged from 91.8% to 96.0% with coefficients of variation below 15%. Good linearity over a wide range of dilution and minimal assay drift was observed within a microtiter plate. IC50 value of the calibration curve was 110 ng/mL, with the limit of detection about 5 ng/mL and dynamic range from 10 to 1000 ng/mL. The high specificity of the ELISA was demonstrated by cross-reactivity study using 11 potential cross-reactants. A good correlation between the results obtained from the ELISA and GC-MS within the concentration range 120-1500 µg/kg was found in the chip samples (r=0.992, n=120). The data demonstrate that the evaluated and validated ELISA has a potential utility in a quick, simple and reliable acrylamide screening analysis for the medium- and large-sized food companies, as well as for residue laboratories and the food industry dealing with improving the chemical safety of foods available to the consumer.

Multiplex immunoassay for persistent organic pollutants in tilapia: comparison of imaging- and flow cytometry-based platforms using spectrally encoded paramagnetic microspheres.

Recent developments in spectrally encoded microspheres (SEMs)-based technologies provide high multiplexing possibilities. Most SEMs-based assays require a flow cytometer with sophisticated fluidics and optics. A new imaging super-paramagnetic SEMs-based alternative platform transports SEMs with considerably less fluid volume into a measuring chamber. Once there SEMs are held in a monolayer by a magnet. Light-emitting diodes (LEDs) are focused on the chamber to illuminate the SEMs - instead of lasers and they are imaged by a charge-coupled device (CCD) detector, offering a more compact sized, transportable and affordable system. The feasibility of utilising this system to develop a 3-plex SEMs-based imaging immunoassay (IMIA) for the screening of persistent organic pollutants (POPs) was studied. Moreover the performance characteristics of 3-plex IMIA were critically compared with the conventional 3-plex flow cytometric immunoassay (FCIA). Both SEM technologies have potential for the multiplex analysis of polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs) and polycyclic aromatic hydrocarbons (PAHs) in buffer and fish extract with insignificant differences in assay sensitivities. Furthermore, we developed a faster and simpler, modified QuEChERS-like generic POPs extraction from tilapia fillet using sodium hydrogen carbonate as one of the salt additives and dispersive solid-phase extraction (dSPE) as a clean-up. Finally, a preliminary in-house validation using 40 different blank and spiked tilapia fillet samples was performed in both systems and the results obtained were critically compared. The lower-cost imaging SEMs-based system performed similarly to the original flow cytometer and, in combination with the new quicker QuEChERS-like extraction, it has high potential for future rapid screening of POPs in several other sample matrices such as other fish species, vegetable refined oils and environmental samples.

POCIS sampling in combination with ELISA: screening of sulfonamide residues in surface and waste waters.

Sulfonamide antibiotics coming from both human and veterinary medicine are among the most common emerging pollutants in freshwater. The present paper shows the successful application of passive sampling using POCIS in combination with an immunochemical ELISA technique and HPLC/MS/MS analysis to study the distribution of sulfonamides in streams around small towns in the Czech Republic, as well as around a major agglomeration of the city of Brno, including its waste water treatment plant (WWTP). Results indicated the presence of sulfonamides at most studied sites with concentrations ranging from <20 up to 736 ng of sulfamethoxazole equivalents per POCIS. Very high levels were detected in both the influent and effluent of the Brno WWTP with maxima > 8000 ng SMX per POCIS. All samplers collected down-stream of the studied towns and WWTPs clearly showed an increase in sulfonamide drug residues. Higher concentrations were determined in rivers at the city of Brno agglomeration. In agreement with other available studies, these findings indicate low efficiency of conventional WWTPs to eliminate polar pharmaceuticals such as sulfonamides. Good performance and correlation with the LC/MS results, as well as ease of use, indicate good potential for the immunochemical ELISA technique to become the screening tool for sulfonamide determination in surface waters including passive samplers.

Multiplex screening of persistent organic pollutants in fish using spectrally encoded microspheres.

Persistent organic pollutants (POPs) are environmental and food-related contaminants of global public health concern and known to be carcinogenic and endocrine disruptors. Their monitoring is essential, and an easy-to-use, rapid, and affordable multianalyte screening method with simplified sample preparation can be a valuable tool prior to instrumental analysis. For this purpose, a flow cytometric immunoassay (FCIA), based on a spectrally encoded microbeads technology, was developed for the multiplex detection of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), and polybrominated diphenyl ethers (BDEs) in buffer and fish extracts. The sensitivities of the assays in the three-plex FCIA format were similar to the individual FCIAs for the marker compounds benzo[a]pyrene (BaP), 3,3',4,4'-tetrachlorobiphenyl (PCB77), and 2,2',4,4'-tetrabromodiphenyl ether (BDE47) in buffer with IC(50) values of 0.4, 20, and 2 µg L(-1), respectively. Apart from the three markers, we could detect at least 14 other POPs. Extracts of fish with different fat content, prepared with a simplified extraction and cleanup procedure, had an insignificant influence on the overall three-plex FCIA performance, with the exception of some impact on the PAHs detection. The performance of the three-plex FCIA, in combination with the simple extraction procedure, is adequate for regulatory control in accordance with the required limits.

Determination of atrazine in surface waters by combination of POCIS passive sampling and ELISA detection.

Polar organic compound integrative samplers (POCIS) in combination with instrumental techniques such as LC-MS-MS were previously used to monitor environmental pollutants but the performance of alternative immunochemical methods such as ELISA (enzyme-linked immunosorbent assay) has been explored less. In the present study, POCIS technology was applied to surface water sampling in the Czech Republic, and ELISA was used as a detection technique for the herbicide atrazine. In the first study, 28 samples from streams around small municipal waste water treatment plants (WWTPs) were collected using two different devices (POCISpest and POCISpharm) over the course of 21 days. Elevated atrazine concentrations (up to 25 ng per POCIS) were found in samples down-stream of WWTPs. This observation was also confirmed in another two year study (4 sampling periods) investigating 7 river sites around a major city of Brno as well as the inlet and outlet of the city's WWTP. High atrazine levels were systematically determined at the outlet from the WWTPs (120-605 ng per POCIS). A decreasing trend in the atrazine concentrations in rivers around the city of Brno has been observed, with the highest levels observed within the first sampling period in spring 2007 (100-600 ng per POCIS, with an extreme value of 2760 ng per POCIS). Results of the atrazine ELISA were closely correlated with LC-MS/MS, which confirmed good applicability of ELISA as a cost-effective screening tool.

Pushing antibody-based labeling systems to higher sensitivity by linker-assisted affinity enhancement.

The sensitivity of antibody/hapten-based labeling systems is limited by the natural affinity ceiling of immunoglobulins. Breaking this limit by antibody engineering is difficult. We thus attempted a different approach and investigated if the so-called bridge effect, a corecognition of the linker present between hapten and carrier protein during antibody generation, can be utilized to improve the affinity of such labeling systems. The well-known haptens 2,4-dinitrophenol (2,4-DNP) and 2,4-dichlorophenoxyacetic acid (2,4-D) were equipped with various linkers, and the resulting affinity change of their cognate antibodies was analyzed by ELISA. Anti-2,4-DNP antibodies exhibited the best affinity to their hapten when it was combined with aminobutanoic acid or aminohexanoic acid. The affinity of anti-2,4-D antibodies could be enhanced even further with longer aliphatic spacers connected to the hapten. The affinity toward aminoundecanoic acid-2,4-D derivatives, for instance, was improved about 100-fold compared to 2,4-D alone and yielded detection limits as low as 100 amoles of analyte. As the effect occurred for all antibodies and haptens tested, it may be sensible to implement the bridge effect in future antibody/hapten-labeling systems in order to achieve the highest sensitivity possible.

Production and analytical characterization of neopterin immunoreagents for biosensor developments.

Neopterin is a valuable biomarker of cellular immunity associated with various pathological situations such as viral and bacterial infections, autoimmune, cardiovascular, neurodegenerative and malignant disorders. To produce specific antibodies against neopterin for a rapid multi-biomarker-based diagnosis, a novel hapten derivative was synthesized and attached to carrier proteins. The thoroughly characterized conjugates were used for immunization of BALB/c mice and rabbits. The produced monoclonal antibody reached in both direct and indirect enzyme-linked immunosorbent assay (ELISA) format LoD of 0.18 and 0.45 µg L(-1), respectively, and was a superior immunoreagent for further biosensor developments with regard to assay sensitivity and material availability. The best polyclonal antibody was somewhat more sensitive in direct ELISA with LoD of 0.05 µg L(-1). The optimized ELISA method was evaluated with blood samples collected from patients with renal insufficiency, patients with sepsis, patients without confirmed clinical diagnosis, and healthy volunteers. In plasma samples, neopterin concentrations ranging from 3.2 to 103 µg L(-1) could be determined with the monoclonal ELISA whereas twofold lower results were obtained with the polyclonal ELISA. A satisfactory correlation of results was found between the polyclonal ELISA and IBL Neopterin ELISA kit within the concentration range 0.5-16 µg L(-1) (R = 0.874; n = 40), and slightly lower correlation was found for monoclonal-based ELISA (R = 0.819; n = 40). These data show that the generated antibodies may be used as functional analytical reagents for the integration into multianalyte biochip detection systems.

Biolabeling with 2,4-dichlorophenoxyacetic acid derivatives: the 2,4-D tag.

Many bioanalytic and diagnostic procedures rely on labels with which the molecule of interest can be tracked in or discriminated from accompanying like substances. Herein, we describe a new labeling and detection system based on derivatives of 2,4-dichlorophenoxyacetic acid (2,4-D) and anti-2,4-D antibodies. The 2,4-D system is highly sensitive with a K(D) of 7 x 10(-11) M for the hapten-antibody pair, can be used on a large variety of biomolecules such as proteins, peptides, carbohydrates, and nucleic acids, is not hampered by endogenous backgrounds because 2,4-D is a xenobiotic, and is robust because 2,4-D is a very stable compound that withstands the conditions of most reactions usually performed on biomolecules. With this unique blend of properties, the 2,4-D system compares favorably with its rivals digoxigenin (DIG)/anti-DIG and biotin/(strept)avidin and provides an interesting and powerful tool in biomolecular labeling.

Rapid profiling of peptide stability in proteolytic environments.

The notorious degradation susceptibility of peptides is a major obstacle to their use as medicinal drugs. Assays with which the stability of peptides in complex proteolytic environments can be determined are thus indispensable for peptide drug development. Herein, we describe a new peptide proteolysis assay that meets that demand. It unites the high-throughput capacity of heterogeneous with the well-defined kinetic characteristics of homogeneous assay formats and operates on the cleavage-caused loss of a detection handle. We have confirmed the assay's accuracy with well-defined model interactions and proved its high versatility and robustness with a representative application where we determined the half-lives of 375 different peptides in a crude intestinal protease preparation. With this reliable, reproducible, and efficient assay the enzyme kinetics of any peptide-protease interaction is accessible even for complex protease solutions.

ELISA for sulfonamides and its application for screening in water contamination.

Two enzyme-linked immunosorbent assays (ELISAs) were tested for their suitability for detecting sulfonamides in wastewater from various stages in wastewater treatment plants (WWTPs), the river into which the wastewater is discharged, and two swine-rearing facilities. The sulfamethoxazole ELISA cross-reacts with several compounds, achieving detection limits of <0.04 microg/L for sulfamethoxazole (SMX), sulfamethoxypyridine, sulfachloropyridine, and sulfamethoxine, whereas the sulfamethazine (SMZ) ELISA is more compound specific, with a detection limit of <0.03 microg/L. Samples from various stages of wastewater purifications gave 0.6-3.1 microg/L by SMX-ELISA, whereas river samples were approximately 10-fold lower, ranging from below detection to 0.09 microg/L. Swine wastewater samples analyzed by the SMX-ELISA were either at or near detectable limits from one facility, whereas the other facility had concentrations of approximately 0.5 microg/L, although LC-MS/MS did not confirm the presence of SMX. Sulfamethazine ELISA detected no SMZ in either WWTP or river samples. In contrast, wastewater samples from swine facilities analyzed by SMZ-ELISA were found to contain approximately 30 microg/L [piglet (50-100 lb) wastewater] and approximately 7 microg/L (market-weight hog wastewater). Sulfamethazine ELISA analyses of wastewater from another swine facility found concentrations to be near or below detection limits. A solid phase extraction method was used to isolate and concentrate sulfonamides from water samples prior to LC-MS/MS multiresidue confirmatory analysis. The recoveries at 1 microg/L fortification ranged from 42 +/- 4% for SMZ to 88 +/- 4% for SMX ( n = 6). The ELISA results in the WWTPs were confirmed by LC-MS/MS, as sulfonamide multiresidue confirmatory analysis identified SMX, sulfapyridine, and sulfasalazine to be present in the wastewater. Sulfamethazine presence at one swine-rearing facility was also confirmed by LC-MS/MS, demonstrating the usefulness of the ELISA technique as a rapid and high-throughput screening method.

In-house validation of an ELISA method for screening of semicarbazide in eggs.

An enzyme-linked immunosorbent assay (ELISA) method is described for the semi-quantitative determination of semicarbazide (SEM), the marker residue for the banned nitrofuran drug, nitrofurazone, in chicken eggs. The sample homogenate is subjected to acid hydrolysis and derivatisation with o-nitrobenzaldehyde, followed by ethyl acetate/hexane extraction and detection by ELISA. The ELISA procedure has been validated using 0.3, 1.0 and 3 microg kg(-1) of SEM in fortified samples. Detection capability (CC(ss)) was based on the acceptance of 5% false compliant results for a given concentration level according to Commission Decision 2002/657/EC and was determined to be 0.3 microg kg(-1) with a respective limit of detection of 0.13 microg kg(-1). A validated LC-MS/MS method was used for the analysis of incurred egg samples and the results compared with ELISA. A good correlation between the results obtained from ELISA and LC-MS/MS within the concentration range 0.12-20.3 microg kg(-1) was observed in samples collected from chickens fed with a medicated ration of nitrofurazone (r = 0.992, n = 14). Validated ELISA enabled reliable monitoring of SEM levels in eggs collected from incurred chickens over a 90-day period.

Broad-specificity immunoassays for sulfonamide detection: immunochemical strategy for generic antibodies and competitors.

Development of antibodies with broad specificity recognition for sulfonamide drugs was found to be surprisingly difficult when conventional immunochemical strategies were applied to hapten design. To improve the cross-reactivity pattern of antibodies for the family of sulfonamide drugs, a novel strategy based on the single-ring (fragment-derived) hapten moieties with different spacer substituent lengths was employed for the preparation of immunogens, coating conjugates, and enzyme competitors. The rabbit antibodies raised against a common (one-ring) p-aminobenzenesulfonamide hapten moiety (attached to a carrier protein through the N-1 position) in combination with a homologous hapten-peroxidase tracer allowed the detection of 15 sulfonamide species at the maximum residue limit level using direct ELISA. The two-ring 6-(4-aminobenzensulfonylamino)hexanoic hapten mimics, previously reported in the literature as a weak generic antigen, generated surprisingly superior immune responses in rabbits. The antibodies raised against this two-ring hapten were capable of detecting at least 19 and 17 sulfonamides in a direct ELISA system at the regulatory level with sensitivities corresponding to 20 and 50% binding inhibition, respectively. A negligible cross-reaction with N4 metabolites makes it possible to measure responses of parent sulfonamides in the presence of their metabolized forms. In skimmed milk, the highest limit of detection (LOD) for sulfacetamide defined as 20% inhibition was 65.2 microg x L(-1) (IC20 value), whereas the additional 18 sulfonamides tested exhibited LODs in the range of 0.2-36.8 microg x L(-1). This sensitivity allows simple multisulfonamide tests to be established for use in the laboratory or on site.

Biacore biosensor immunoassay for 4-nonylphenols: assay optimization and applicability for shellfish analysis.

A rapid Biacore biosensor immunoassay of 4-nonylphenols was developed. Two types of antibodies were used in the study: polyclonal antibodies with high cross-reactivity towards technical 4-nonylphenol and a monoclonal antibody very specific to 4-n-nonylphenol. 9-(p-Hydroxyphenyl)nonanoic acid was immobilized onto surface of a sensor chip. The best assay sensitivity was achieved using a flow rate of 50 microl min(-1) and injection time of 2 min. For the assay incorporating monoclonal antibodies a limit of detection 2 ng ml(-1) for 4-n-nonylphenol was achieved. With polyclonal antibodies one order lower sensitivity was observed for 4-nonylphenols. High background level of calibration curve for technical 4-nonylphenol was decreased by using IgG fraction of polyclonal antibodies in combination with lower amount of immobilised 9-(p-hydroxyphenyl)nonanoic acid. Sensitivity of the assay was improved by using a chip with a new derivative on a surface-N-aminobutyl [2-(4-hydroxyphenyl)ethylamine] (limit of detection--5 ng ml(-1)). Applicability of the developed assays to ecological monitoring was checked in experiments using shellfish samples. 4-n-Nonylphenol from spiked samples was extracted into hexane followed by clean-up on NH2 SPE columns. Calibration curves generated for cockles, mussels and oyster samples were identical (limit of detection about 10 ng g(-1)) whereas for scallop samples a slight decrease (about 5-10%) of absolute response was observed. In the assay using the monoclonal antibody specific to 4-n-nonylphenol 31 shellfish samples were found to be negative. Results obtained with polyclonal antibodies indicated that two scallop samples contained a quantity of 4-nonylphenols. The developed biosensor assay could be applied for shellfish analysis as a preliminary screening method.

Development of direct ELISA for the determination of 4-nonylphenol and octylphenol.

Development of direct competitive enzyme-linked immunoadsorbent assays (ELISAs) based on polyclonal and monoclonal antibodies raised against 4-n-alkylphenol hapten mimics is described. A strong tendency to recognize 4-nonylphenol (NP) and 4-octylphenol (OP) as a total analyte amount was indicated by cross-reactivity pattern established for two polyclonal antibodies. These antibodies were employed for development of class-selective assays exhibiting IC(50) values around 40 microg.L(-1) for technical 4-NP. Specificity of the monoclonal antibody 4H6 and additional two polyclonal antibodies allowed sensitive detection of linear long-chain forms of 4-n-alkylphenols (4-n-AP). The assays incorporating these antibodies offer a potential for detecting the minor fraction of NP/OP isomer spectrum having IC(50) = 11.5 microg.L(-1) for 4-n-NP. No cross-reactivity interference was indicated for linear alkylbenzene sulfonates and phenolic compounds. To interpret the measured data in terms of analytical equivalents, a reliable relationship between the assay responses and AP content of contaminated samples should be verified and validated.

Identification of monoclonal antibodies against 2,4-D herbicide by ELISA and DNA sequencing.

Seven hybridoma clones, E2/G2, E2/B5, E4/C2, G5/E10, F6/C10, B5/C3, and B7, produced within one fusion experiment in 1991 and the clone E4/C2 originated from 1995 were characterized by sequencing of genes coding for variable domains of the antibodies against 2,4-D herbicide. Amino acid sequences of selected antibodies, deduced from DNA analysis, were confirmed by mass spectrometry. Surprisingly, nucleotide sequence analysis of the clones E2/G2 and E2/B5, producing the most sensitive antibodies, proved to have sequence homology of their variable domains, although the IC(50) values determined for these antibodies 9 years prior to the DNA analysis were 2.0 and 8.2 ng/mL, respectively. The same findings arose from the comparison of the immunochemical to DNA data established for G5/E10, F6/C10, and B5/C3 clones producing antibodies with IC(50) values in the range of 26.3-43.1 ng /mL. The clone E4/C2, originating from the later fusion experiment, did not share nucleotide homology with any of the examined clones. Data obtained by ELISA, immunosensor, and DNA analysis within a 9 year period are discussed with respect to hybridoma stability, methodic artifacts, measurement reliability, and other possible factors influencing the result interpretation.