Monoclonal Antibodies for the Mycotoxins Deoxynivalenol and 3-Acetyl-Deoxynivalenol

The mycotoxin deoxynivalenol (DON) is produced by the mold Fusarium graminearum and is found worldwide on cereal grains, in particular wheat and maize. Each year this compound, also known as 'vomitoxin' causes substantial losses to agricultural productivity. Three monoclonal antibodies were developed following the immunization of mice with a conjugate of DON and ovalbumin. One of these antibodies, produced by clone #22, was selected for the development of a competitive direct ELISA (CD-ELISA). This format consists of competition between a DON horseradish peroxidase conjugate (DON-HRP) and free DON for antibody attached to microwell plates. Color development in the assay was inhibited 50% (IC₅₀) by 18 ng DON/ml in phosphate-buffered saline (PBS). The antibody from this clone showed strong cross-reactivity to 3-acetyl deoxynivalenol (3-Ac-DON), with an IC₅₀ of 2.9 ng ml^-1 Cross-reactivity to 19 other trichothecene mycotoxins was low. The CD-ELISA was applied to wheat spiked with DON over the range 0.01-10 μg/g and extracted with a 10-fold excess of PBS. The midpoint for color development in the assay using this extraction was 0.27 μg DON/g wheat. Recoveries over the range 0.05-5 μg/g averaged 88.7% with a coefficient of variation of 10.9%. This assay is sufficiently sensitive and rapid to permit the screening of DON in wheat below the US Food and Drug Administration advisory level of 1 ppm in human food.     

Quantification of κ-casein in milk by an optical immunosensor

κ-casein (κ-CN) plays an important role in the stability and coagulation properties of milk. An immunoassay to quantify κ-casein in milk, using an optical biosensor based on surface plasmon resonance (SPR) measurement, has been developed. The assay consists of a two-step sandwich strategy, with two anti-κ-casein antibodies directed against each extremity of the casein to quantify only native κ-casein. The analysis time per sample was less than ten minutes. The antibody-coated surface could be used for more than 150 determinations. The detection limit was established at 0.45 μg.ml^-1 and the intra- and inter-assay variation coefficients were 4.28% and 6.8%, respectively. The method was applied to raw milk in order to quantify intact κ-casein, with no pretreatment of the sample. It also allowed the monitoring of κ-CN concentration during milk coagulation after addition of rennet.     

Androgen dependence in hamsters: overdose, tolerance, and potential opioidergic mechanisms

Anabolic steroids are drugs of abuse. However, the potential for steroid reward and addiction remains largely unexplored. This study used i.c.v. testosterone self-administration and controlled infusions of testosterone or vehicle in hamsters to explore central mechanisms of androgen overdose. Forty-two hamsters used nose-pokes to self-administer 1 μg/μl testosterone i.c.v. 4 h/day in an operant chamber. During 1–56 days of androgen self-administration, 10 (24%) hamsters died. Deaths correlated with peak daily intake of testosterone. Of the hamsters that self-administered a peak intake of <20 μg/day, there was 100% survival (10/10). Survival decreased to 86% (19/22) when daily testosterone intake peaked at 20–60 μg/day. Only 30% (three of 10) survived when daily testosterone intake exceeded 60 μg/day. Deaths are not due to volume or vehicle because i.c.v. infusions of 80 μl vehicle had no effect. Testosterone overdose resembles opiate intoxication. When male hamsters received infusions of 40 μg testosterone, locomotion (25.1±18.8 grid-crossings/10 min), respiration (72.7±5.4 breaths/min) and body temperature (33.5±0.4 °C) were significantly reduced, compared with males receiving vehicle infusions (186.1±8.1 crossings/10 min, 117.6±1.0 breaths/min, 35.9±0.1 °C, P<0.05). However, males developed tolerance to continued daily testosterone infusion. After 15 days, locomotion (170.2±6.3 crossings), respiration (118.4±1.3 breaths/min), and body temperature (35.3±0.3 °C) in testosterone-infused males were equivalent to that in vehicle controls (P>0.05). The depressive effects of testosterone infusion are blocked by the opioid antagonist, naltrexone. With naltrexone pre-treatment (10 mg/kg s.c.), locomotion (183.7±1.8 crossings/10 min), respiration (116.9±0.3 breaths/min), and body temperature (36.1±0.4 °C) during testosterone infusion were equivalent to vehicle controls. Likewise, naltrexone prevents the reinforcing effects of i.c.v. testosterone self-administration. These results indicate that testosterone at high doses causes central autonomic depression, which may be a factor in deaths during self-administration. As well, the depressive effects of large quantities of testosterone may be mediated, at least in part, by an opioidergic mechanism.     

Trichothecene Mycotoxins Depress the Mononuclear-Phagocytic System of Young Turkeys

Macrophage cells isolated from the abdominal cavity of 21-day-old turkeys after a single injection of Sephadex suspension were used to quantitate the effects of direct in vitro exposure to deoxynivalenol (DON), 3-acetyldeoxynivalenol (3ac-DON), scirpentriol (STO), or 15-acetylscirpenol (15-MAS). Macrophage monolayers were established on glass surfaces and cells were exposed to graded levels of individual mycotoxins for 1 hour: DON, 20 -640 μ9/μ1 of culture; 3ac-DON, STO, 15-MAS, 20 -1280 μg/μ1 of culture. All four mycotoxins caused dose-related effects. A concentration of 50 μg/ml DON caused a significant decrease in macrophage adherence, phagocytosis of opsonized SRBC, and number of opsonized SRBC per macrophage; at 200 μg/ml, phagocytosis of unopsonized SRBC was decreased. There were also increasing percentages of damaged macrophages with increasing DON doses as indicated by morphological alterations. Linear decreases in macrophage viability on exposure to 3-acDON and STO were observed. Moreover, STO and 15-MAS decreased macrophage adherence to glass and 3-acDON, STO, and 15-MAS induced macrophage morphological alterations. This study suggests that trichothecene mycotoxins may be immunosuppressive by affecting viability, adherence and phagocytic potential of mononuclear phagocytic cells of young turkeys.     

Application of a high-performance liquid chromatographic assay for the neuromuscular blocker gallamine to analysis of rat plasma, muscle and microdialysate samples

A reliable high-performance liquid chromatographic method has been validated for determination of gallamine in rat plasma, muscle tissue and microdialysate samples. A C18 reversed-phase column with mobile phase of methanol and water containing 12.5 mM tetrabutyl ammonium (TBA) hydrogen sulphate (22:78, v/v) was used. The flow-rate was 1 ml/min with UV detection at 229 nm. Sample preparation involved protein precipitation with acetonitrile for plasma and muscle tissue homogenate samples. Microdialysate samples were injected into the HPLC system without any sample preparation. Intra-day and inter-day accuracy and precision of the assay were <13%. The limit of quantification was 1 microg/ml for plasma, 1.6 microg/g for muscle tissue and 0.5 microg/ml for microdialysate samples. The assay was applied successfully to analysis of samples obtained from a pharmacokinetic study in rats using the microdialysis technique.     

A competitive direct enzyme-linked immunosorbent assay for the rapid detection of deoxynivalenol: development and application in agricultural products and feedstuff

A competitive direct enzyme-linked immunosorbent assay (cd-ELISA) was developed for the rapid detection of deoxynivalenol (DON) in food and feedstuff. Polyclonal antibodies against DON were generated by immunizing rabbits with 3-HS-DON-BSA conjugates. In this assay, the sensitivity (measured as IC₅₀) and limit of detection (measured as IC₁₅) were 0.03 and 0.003?mg?kg^?1, respectively. A total of eight sample types (barley, wheat, oat, maize, rice, flour, milk and feedstuff) were chosen to evaluate the cd-ELISA assay performance. The sample could be directly detected after extraction and dilution with double-distilled water. The limit of detection in the samples was in the range of 0.15–0.48?mg?kg^?1. In the end, the spike and recovery results in both cd-ELISA and high-performance liquid chromatography (HPLC) were compared for assay validation. The recovery results ranged between 70% and 100%. A good correlation (R²?=?0.9613) between ELISA and HPLC was obtained.     

Detection of weak ligand interactions of leukocyte Ig-like receptor B1 by fluorescence correlation spectroscopy

Fluorescence correlation spectroscopy (FCS) can directly and quickly detect the translational diffusion of individual fluorescence-labeled molecules in solutions. Although FCS analyses for protein–protein interactions have been performed, the very weak interactions generally observed in cell–cell recognition of the immune system have not been examined in detail. Here, we report the FCS analysis for low-affinity and fast-kinetic binding (K_d greater than μM range) of the human inhibitory immune cell surface receptor, leukocyte immunoglobulin-like receptor B1 (LILRB1), to its ligands, MHC (major histocompatibility complex) class I molecules (MHCIs) by using the single-molecule FCS detection system which requires only a small amount of sample. Since the random labeling technique for LILRB1 disturbed the MHCI binding, we performed site-specific labeling of LILRB1 by introducing a cysteine residue at the C-terminus, which could be covalently attached with the fluorescence reagent, Alexa647. This technique can be applied to other type I membrane receptors. The low-affinity binding of LILRB1-Alexa647 to MHCIs (HLA-Cw4, and -G1) was detected by FCS, even though non-labeled MHCIs were only twice as big as the labeled LILRB1. Their dissociation constants (7.5 μM (HLA-Cw4) and 5.7 μM (HLA-G1)) could be determined and were consistent with surface plasmon resonance (SPR) data. These results indicate that the single-molecule FCS detection system is capable of analyzing the binding characteristics of immune cell surface receptors even in difficult cases such as (1) small amount of protein samples, (2) small difference in molecular weight and (3) weak affinity. Therefore, it is a powerful tool for characterization and high throughput inhibitor screening of a wide variety of cell–cell recognition receptors involved in immunologically relevant events.     

Immunogenicity of immunoliposomes

Multilamellar immunoliposomes were prepared from dipalmitoylphosphatidylcholine (DPPC), cholesterol (CH), sphingomyelin (SPH) and biotinylated dipalmitoylphosphatidylethanolamine (PEB) in the molar ratio of 1:1:1:0.1 with surface linked avidin-biotinylated sheep (anti-mouse IgG) IgG (AV-sIgG_B) or GK1.5 monoclonal rat (anti-mouse L3T4 antigen) IgG (AV-GK1.5_B). The ability of these immunoliposomes to induce antibody responses against AV, sIgG or GK1.5 was determined. GK1.5_B and sIgG_B elicited a low-level antibody response (5–10 μg/ml serum) after i.v. immunization and boosting. Liposomes (1 μmol) containing GK1.5_B or sIgG_B were more effective than free GK1.5_B or sIgG_B in eliciting antibodies (20–30 and 100–120 μg/ml serum, respectively). Liposomal AV mixed with either sIgG or GK1.5 gave antibody levels comparable to immunization with free GK1.5_B or sIgG_B. Liposomes with surface AV-sIgG_B or AV-GK1.5_B elicited antibodies against AV and high levels against GK1.5 or sIgG. Immunoliposomes possessing surface AV-sIgG_B or AV-GK1.5_B were eliminated from the circulation of normal mice relatively slowly (T_1/2 15.5 and 30 min): in contrast, liposomal AV-sIgG_B or AV-GK1.5_B was rapidly eliminated from the circulation of immunized mice (T_1/2 4.5 and 4.0 min). These results demonstrate that liposomes with surface IgG (immunoliposomes) are immunogenic, and that repeated administration elicits anti-IgG antibodies that result in a significant reduction in blood circulation residence times.     

Determination of Carbaryl in Vegetables Using an Immunosensor Working in Organic Media

Sensitive and rapid analysis of carbaryl using a flow immunosensor is described. An azlactone polymeric gel with covalently bound Protein A/G was used as immunofiltration support. The immunosensor was able to work at high concentrations of organic solvents (e.g. up to 50% MeOH) using a competitive enzyme-immunoassay protocol, with an analysis rate of a complete assay cycle in 22 min. Good sensitivity (I₅₀ = 9.05 μgl^-1) was achieved in organic media and the reusability of the sensor was at least 300 cycles without loss of performance. The sensor was applied to the analysis of carbaryl extracted by both multiresidue method and methanol in fresh and processed vegetable samples. The analytical results were compared with those obtained by ELISA and high-performance liquid chromatography (HPLC) methods. A good agreement was achieved by both immunological methods (r = 0.98) and those obtained by HPLC technique. The results indicated the suitability of the immunosensor for carbaryl analysis in vegetable samples due to its reproducibility, rapidity and compatibility with organic solvents.     

Real time assessment of surface interactions with a titanium passivation layer by surface plasmon resonance

Due to the high corrosion resistance and strength to density ratio titanium is widely used in industry, and also in a gamut of medical applications. Here we report for the first time on our development of a titanium passivation layer sensor that makes use of surface plasmon resonance (SPR). The deposited titanium metal layer on the sensor was passivated in air, similarly to titanium medical devices. Our "Ti-SPR sensor" enables analysis of biomolecule interactions with the passivated surface of titanium in real time. As a proof of concept, corrosion of a titanium passivation layer exposed to acid was monitored in real time. The Ti-SPR sensor can also accurately measure the time-dependence of protein adsorption onto the titanium passivation layer at sub-nanogram per square millimeter accuracy. Besides such SPR analyses, SPR imaging (SPRI) enables real time assessment of chemical surface processes that occur simultaneously at "multiple independent spots" on the Ti-SPR sensor, such as acid corrosion or adhesion of cells. Our Ti-SPR sensor will therefore be very useful to study titanium corrosion phenomena and biomolecular titanium-surface interactions with application in a broad range of industrial and biomedical fields.     

Standardization of a Siddha Formulation Amukkara Curanam by HPTLC

Amukkara curanam, a Siddha formulation, currently used in all types of gastric disorders, rheumatic pain, insomnia and sexual insufficiency, was investigated for the estimation of the marker compounds, withaferine A and piperine contents in a prepared standard formulation and a commercial formulation by using HPTLC method of analysis. The two formulations were subjected to methanol, ethyl acetate and chloroform extractions by using Soxhhlet apparatus The chromatogram was developed using chloroform: methanol (8.5:1.5 v/v) and toluene: ethyl acetate (7:3 v/v) as mobile phases for the estimation of withferine A and piperine respectively. The detection and quantification were performed at a wavelength of 220 nm for withaferine A and 254 nm for piperine. The linear regression analysis of calibration plots of withferine A and piperine exhibited linear relationship in the range of 5 – 15 µg and 50 – 150 ng respectively, while the % recovery was found to be 94.52% w/w of withaferine A and 98.73%w/w of piperine, thus proving the accuracy and precision of the analysis. Methanol and ethyl acetate were found to be the suitable solvents for the extraction of withaferin A and piperine respectively. The withaferine A content in standard formulation was found to be much higher in all the three extracts than that of the commercial sample. However, the piperine content in all the three extracts of standard formulation was slightly lower than the respective extracts of commercial formulation. The proposed HPTLC method was found to be rapid, simple and accurate for quantitative estimation of withferine A and piperine in different formulation extracts.     

Rapid quantification of indinavir in human plasma by high-performance liquid chromatography with ultraviolet detection

A rapid, sensitive and specific high-performance liquid chromatography (HPLC) procedure for the quantification of indinavir, a potent human immunodeficiency virus (HIV) protease inhibitor, in human plasma is described. Following C18 solid-phase extraction, indinavir was chromatographed on a reversed-phase C8 column using a simple binary mobile phase of phosphate buffer-acetonitrile (60:40, v/v). UV detection at 210 nm led to an adequate sensitivity without interference from endogenous matrix components. The limit of quantification was 25 ng/ml with a 0.1 ml plasma sample. The standard curve was linear across the range from 25 to 2500 ng/ml with an average recovery of 91.4%. The mean relative standard deviations for concentrations within the standard curve ranged between 1.4 and 9.7%. Quality control standards gave satisfactory intra- and inter-assay precision (R.S.D. from 3.5 to 15.8%) and accuracy within 15% of the nominal concentration. Sample handling experiments, including HIV heat inactivation, demonstrated analyte stability under expected handling processes. The assay is suitable for the analysis of samples from adult and pediatric patients infected with HIV.     

Development and validation of a biosensor-based immunoassay for progesterone in bovine milk

We have developed a rapid automated immunoassay, using the BIACORE surface plasmon resonance (SPR) biosensor, to measure progesterone in bovine milk. The assay was designed as an inhibition assay with progesterone covalently immobilised to the carboxymethyl dextran matrix of a CM5 sensor chip. A fixed amount of monoclonal anti-progesterone antibody 39C5H7 was mixed 9:1 with the sample and the amount of free antibody was then determined using biomolecular interaction analysis (BIA) by injection of the mixture over the immobilised progesterone sensor surface. The assay was designed to cover the concentration range 0.5 to 50 ng/ml. The limit of detection (LOD) was 3.56 ng/ml. Reproducibility of the assay was very good with both intra-assay and inter-assay coefficients of variation <5%. As results become available within minutes of injection and the procedure involves fully automated instrumentation, we believe that this BIA assay for progesterone in milk could be used in-line in the milking parlour and, thus, provide an important tool for reproductive management of dairy cattle to detect heat and predict pregnancy.     

Routine and sensitive method for determination of nicorandil in human plasma developed for liquid chromatography with ultraviolet and mass spectrometric detection

A rapid and sensitive method using HPLC has been developed for the quantification of nicorandil (SG-75) in human plasma samples for routine bioequivalence studies. The sample preparation needs two liquid-liquid extractions, first with CH3Cl and HClO4 as denaturation reagent and second with addition of ethyl acetate and Na2CO3(aq). Detection wavelength was 256 nm. The obtained correlation coefficient for weighted linear curve in the range from 5.0 to 300 ng/ml was higher than 0.9950. The limit of quantitation (LOQ) was established at 5.0 ng/ml. The HPLC separation was accomplished on Nucleosil Phenyl (5 microm) stainless steel column within 7 min. The mixture of 0.01 M ammonium acetate buffer (pH 6.2) and acetonitrile 10:3 (v/v) was used as the mobile phase. The same separation method was examined on HPLC-MS system. Using this system, the LOQ was established at 1.0 ng/ml and the linearity was obtained in the range from 1.0 to 150 ng/ml.     

Determination of anti-HBsAg IgM monoclonal antibodies in cell culture media by perfusion immunoassay

A rapid, specific, perfusion immunoassay for active anti-HBsAg monoclonal IgM is described. The immunoassay requires less than 3.5 min per sample. The precision was found to be 3.6% at an IgM concentration of 17 μg/ml. A detection limit of 1 μg/ml IgM in culture media was determined. Assay results were found to correlate very well with standard size exclusion chromatography and radial immunodiffusion techniques. This perfusion immunoassay was demonstrated to be useful for determining anti-HBsAg IgM in complex matrices such as cell culture media. The utility of the immunoassay for monitoring production of anti-HBsAg IgM in a perfusion bioreactor is demonstrated.     

Molecularly imprinted based surface plasmon resonance nanosensors for microalbumin detection

Human serum albumin (HSA) is a major blood plasma protein also found in urine where its existence may be a marker of some types of liver or kidney dysfunction. Herein, we fabricated a novel surface plasmon resonance (SPR) nanosensor for selective, sensitive, and label-free microalbumin detection both in aqueous and urine sample solutions. First, HSA-imprinted nanoparticles were synthesized, which consist of ethylene glycol dimethacrylate and N-methacryloyl-L-leucine methyl ester as a cross-linker and functional monomer. The nanoparticles were characterized by zeta-size and scanning electron microscope analyses and were dropped onto the SPR chip surface to make HSA sensitive nanosensor. Characterization studies of HSA-imprinted SPR chip were carried out by atomic force microscopy, Fourier-transform infrared spectroscopy, contact angle, and ellipsometer. The limit of detection and limit of quantification values of HSA-imprinted SPR nanosensor were calculated as 0.7?pM and 1.9?pM for the concentration range of 0.15–500?nM. Selectivity studies of HSA-imprinted SPR nanosensor were achieved with hemoglobin and transferrin proteins which were chosen as competitor molecules. HSA-imprinted SPR nanosensor was displayed highly selective and sensitive to HSA.     

The production of polyclonal and monoclonal antibodies in mice using novel immunization methods

Two novel immunization methods (intrasplenic and intra-inguinal lymph node) have been developed for the production of polyclonal and monoclonal antibodies in mice. Freund's complete adjuvant and antigen were mixed in the ratio of 1 : 2 (v/v). Various concentrations of human serum albumin (HSA) were used as antigen. No primary immune response was induced with 0.1 μg of HSA in either of the methods studied. Intrasplenic immunization resulted in the strongest primary immune responses using all other doses of HSA. The primary immune response induced by intrasplenic immunization with 0.5 μg of HSA was higher than any response induced by subcutaneous immunization with various doses of HSA. Inguinal lymph node immunization was less effective than intrasplenic immunization but better than subcutaneous immunization with 1–50 μg of HSA. Comparisons were also made of the efficacy of different adjuvants when inducing primary immune responses with 1 μg of HSA. Freund's complete adjuvant resulted in a much stronger response than Freund's incomplete adjuvant and alum. Both intrasplenic and inguinal lymph node immunization using 1–5 μg of HSA were able to induce strong primary immune responses. Secondary immunization with either method or intravenous injection 3 days before fusion resulted in a higher frequency of specific monoclonal antibodies.     

Effect of wheat (Triticum aestivum L.) resistance,Fusarium graminearum DNA content,strain potential toxin production,and disease severity on deoxynivalenol content

Six wheat cultivars with varied resistance to Gibberella zeae (Anamorph, Fusarium graminearum Schwabe) were inoculated with six monoconidial strains of G. zeae to investigate the effect of wheat resistance to Fusarium head blight on deoxynivalenol (DON) contents. Samples were selected from grains from each plot, and heavily infected kernels and sound (uninfected) kernels prepared at 10% and 20% Fusarium‐diseased kernels (FDK). The proportions of scabbed spikelets (PSS) in the field, total DON (containing DON, 3‐acetyl‐deoxynivalenol, and 15‐acetyl‐deoxynivalenol), and F. graminearum DNA (Tri5 DNA) in the samples were quantified in 2006 and 2007. PSS exhibited significant variability among the six wheat cultivars. Potential DON production also had significant differences among the six strains. DON toxin concentrations and F. graminearum DNA (Tri5 DNA) showed no significant differences among the six wheat cultivars following inoculation with similar F. graminearum strains at similar FDK levels and at similar disease severity after culture in similar conditions. DON content in grains of the tested wheat cultivars varied with inoculation strain and FDK level, but not with the resistance level of the cultivars to F. graminearum.     

Development of a Biosensor-based Immunoassay for Screening of Chloramphenicol Residues in Milk

Biosensor immunoassays were developed recently for antibiotics with an established maximum residue limit (MRL). In this study, according to the regulatory banning of chloramphenicol (CAP) use for food producing animals, the main objectives were: the specificity of the biosensor assay and the lowest detection limit possible. The assay was based on the inhibition of the binding of polyclonal antibodies against CAP to immobilized CAP on a sensor chip by CAP in solution. The response varied inversely with the antibiotic concentration in the sample. Two different antibodies and two immobilization protocols were tested. As in ELISA tests the antibody influenced the assay performances. Moreover, we showed that particular care should be concentrated on the immobilization step because it is a critical point in the assay development. Three different protocols were developed in milk. The best assay was obtained with antibody 1 in milk on the CAP base surface because of its very low detection limit (0.1 μg l^-1) and the decreased consumption of antibody (four times less than on the CAP surface). This assay is rapid (3 min/run), sensitive, and specific for CAP and CAP glucuronide. It could be integrated in a multi-residue screening test and applied to other matrices (bile, urine, meat).     

Hydrogel-microsphere-enhanced surface plasmon resonance for the detection of a K-ras point mutation employing peptide nucleic acid

Highly-sensitive detection of a K-ras point mutation in codon 12, frequently found in pancreatic cancer, based on DNA-carrying hydrogel microspheres as a response enhancer for surface plasmon resonance (SPR), is described. Acrylamide-based microspheres with carboxyl groups were conjugated with DNA probes. Use of the DNA-carrying microsphere in the sandwich method, that is, binding of the microspheres with target DNAs at the sensor surface, enhanced the SPR response as a combined result of increased dielectric constant by the DNA-carrying microspheres. Microspheres lead to response enhancement, as shown by a 100-fold increase in sensitivity compared to that of non-amplified DNA target hybridization. In addition, the advantage of peptide nucleic acid (PNA) in the detection of a K-ras point mutation at the sensor surface by increasing temperature and flow rate is discussed. Results illustrate that the sandwich method through DNA-carrying microspheres for a SPR sensor is a promising approach for ultrasensitive DNA detection.