Competitive Enzyme Immunoassay Research Articles

Challenges, pitfalls and surprises: development and validation of a monoclonal antibody for enzyme immunoassay of the steroid 1α-hydroxycorticosterone in elasmobranch species

Sharks and rays are popular species used in wildlife ecotourism and aquariums to educate the public on the behavior, ecology and conservation challenges of elasmobranchs. To understand long-term physiological health and welfare under varying social and husbandry conditions, we developed and validated an enzyme immunoassay (EIA) to measure stress/ionoregulatory hormones in managed and semi-free range southern rays (Hypanus americanus). Banked serum and interrenal samples from 27 female rays managed at Disney’s The Seas with Nemo and Friends® and Castaway Cay were used to evaluate measurement of 1α-hydroxycorticosterone (1αOHB) relative to corticosterone (B). Although commercial EIAs are available for B, those tested exhibit only low relative cross-reactivity to 1αOHB (3–5%). To improve measurement of 1αOHB, we developed a monoclonal antibody using a synthesized 1αOHB-derivative for evaluation using high-performance liquid chromatography (HPLC) and EIA. Relative displacements of cross-reactant compounds showed that the antibody had good sensitivity for the target antigen 1αOHB, and low sensitivity to related steroids (desoxycorticosterone and B), but greater sensitivity to 11-dehydrocorticosterone. Tests of competitive vs. noncompetitive EIA formats, reagent titration, and incubation times of the antibody and conjugate were used to optimize sensitivity, repeatability and precision of measured 1αOHB in standards and samples (4 ng/ml, 90% binding). Tests of sample pre-treatment (pH adjustment) and extraction with varying solvent polarity were used to optimize measurement of 1αOHB in <1 ml (serum) or 1 g (interrenal) samples. HPLC analysis revealed the 1αOHB EIA to be superior for measurement of 1αOHB compared to use of a B EIA with or without HPLC fractioning. Results may prove useful for extrapolation to guide best practices for 1αOHB measurement in other elasmobranch species. Improved measurement of stress/ionoregulatory hormones in sharks and rays will be important for many aspects of collection, transport, medical treatment in aquaria and conservation management of these charismatic and ecologically important species.

A conventional chemical reaction for use in an unconventional assay: A colorimetric immunoassay for aflatoxin B1 by using enzyme-responsive just-in-time generation of a MnO2 based nanocatalyst.

The authors describe a colorimetric immunoassay for the model nalyte aflatoxin B1 (AFB1). It is based on the just-in-time generation of an MnO2 nanocatalyst. Unlike previously developed immunoassay, the chromogenic reaction relies on the just-in-time formation of an oxidase mimic without the aid of the substrate. Potassium permanganate (KMnO4) is converted into manganese dioxide (MnO2) which acts as an oxidase mimic that catalyzes the oxidation 3,3',5,5'-tetramethylbenzidine (TMB) by oxygen to give a blue colored product. In the presence of ascorbic acid (AA), KMnO4 is reduced to Mn(II) ions. This results in a decrease in the amount of MnO2 nanocatalyst. Hence, the oxidation of TMB does not take place. By adding ascorbate oxidase, AA is converted into dehydroascorbic acid which cannot reduce KMnO4. Based on these observations, a colorimetric competitive enzyme immunoassay was developed where ascorbate oxidase and gold nanoparticle-labeled antibody against AFB1 and magnetic beads carrying bovine serum albumin conjugated to AFB1 are used for the determination of AFB1. In presence of AFB1, it will compete with the BSA-conjugated AFB1 (on the magnetic beads) for the labeled antibody against AFB1 on the gold nanoparticles. This makes the amount of ascorbate oxidase/anti-AFB1 antibody-labeled gold nanoparticles, which conjugated on magnetic beads, reduce, and resulted in an increase of ascorbic acid. Under optimal conditions, the absorbance (measured at 652nm) decreases with increasing AFB1 concentrations in the range from 0.1 to 100ng mL-1, with a 0.1ng mL-1 detection limit (at the 3Sblank level). The accuracy of the assay was validated by analyzing spiked peanut samples. The results matched well with those obtained with a commercial ELISA kit. Conceivably, the method is not limited to aflatoxins but has a wide scope in that it may be applied to many other analytes for which respective antibodies are available. Graphical abstract Schematic illustration of ascorbate oxidase (AOx)-mediated potassium permanganate (KMnO4)-responsive ascorbic acid (AA) for visual colorimetric immunoassay of aflatoxin B1 (AFB1) by coupling with hydrolytic reaction of AOx toward AA and the KMnO4-Mn(II)-TMB system [note: 3,3',5,5'-tetramethylbenzidine: TMB].

Protein Ingredients Control in Gluten Free Products Using SDS-PAGE, Developed Competitive Enzyme Immunoassays and Commercial ELISA Kits

Some protein ingredients declared in the label of gluten free products are allergenic proteins (milk, soy and egg). The proper identification of these proteins in food products is important for consumers who have food allergies. The aim of this study was to control the presence of protein ingredients declared in the label of gluten free products. Samples were analyzed with sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), using an extractive solution of total proteins (Tris-ClH buffer 0.0625 M with 3% sodium dodecylsulfate and 2% 2-mercaptoethanol; pH: 6.8) and a selective solvent for the extraction of caseins (isopropanol 55° + 2-mercaptoethanol / ISO + ME). Developed Competitive enzyme immunoassays were used for the detection / quantification of milk, soy and egg proteins in products elaborated with rice flour. Specific polyclonal rabbit antiserums against milk, soy and egg proteins were used as primary antibodies in these competitive enzyme immunoassays. Commercial ELISA kits from Neogen, R-Biopharm and Romer were used to verify the results. In some samples undeclared allergens were detected. Correct allergens labeling is very important for the safety of allergic consumers. In conclusion, it is possible to identify all the proteins ingredients in these gluten-free foods studied, using a combination of electrophoretic methods and immunochemical methods.

Osteogenic Effect of Inducible Nitric Oxide Synthase (iNOS)-Loaded Mineralized Nanoparticles on Embryonic Stem Cells

Background/Aims: This study investigated the effect of inducible nitric oxide synthase-loaded mineralized nanoparticles (iNOS-MNPs) on the osteogenic differentiation of mouse embryonic stem cells (ESCs). Methods: We prepared iNOS-MNPs using an anionic block copolymer template-mediated calcium carbonate (CaCO₃) mineralization process in the presence of iNOS. iNOS-MNPs were spherical and had a narrow size distribution. iNOS was stably loaded within MNPs without denaturation. In order to confirm the successful introduction of iNOS-MNPs into the cytosol of ESCs, intracellular levels of nitric oxide (NO) was determined with a fluorometric analysis. A NO effector molecule, cyclic guanosine 3’,5’ monophosphate (cGMP) was also quantified with a competitive enzyme immunoassay. Cell viability in response to iNOS-MNP treatment was determined using the cell counting kit-8 (CCK-8) assay. Alkaline phosphatase (ALP) activity assay, intracellular calcium quantification assay, and Alizarin red S staining for matrix mineralization were performed to investigate osteogenic differentiation of ESCs. The protein levels of Runt-related transcription factor 2 (RUNX2), osteocalcin (OCN), and osterix (OSX) as osteogenic-related factors were also assessed by immunofluorescence staining and Western blot analysis. The complex pathways associated with iNOS-MNP-derived osteogenic differentiation of ESCs were evaluated by network-based analysis. Results: Cells with iNOS-MNPs displayed a significant increase in NO and cGMP concentration compared with the control group. When cells were exposed to iNOS-MNPs, there were no adverse effects on cell viability. Importantly, iNOS-MNP uptake promoted the osteogenic differentiation of ESCs. Using transcriptome profiling, we obtained 1,836 differentially-induced genes and performed functional enrichment analysis with ClueGO and KEGG. These analyses identified significantly enriched and interconnected molecular pathways such as protein kinase activity, estrogen receptor activity, bone morphogenetic protein (BMP) receptor binding, ligand-gated ion channel activity, and phosphatidylinositol 3-phosphate binding. Conclusion: These findings suggest that iNOS-MNPs can induce osteogenic differentiation in ESCs by integrating complex signaling pathways.

Development of a chemiluminescent enzyme immunoassay to detect cadmium in cereal samples

ABSTRACT In this study, we develop a sensitive indirect competitive chemiluminescent enzyme immunoassay (ic-CLEIA) to detect cadmium (Cd) in cereals. A luminol-hydrogen peroxide chemiluminescence system catalysed by horseradish peroxidase (HRP) was used as the signal detecting system. The method showed a half-maximal inhibition concentration (IC50) of 0.5 ng/mL and a limit of determination (LOD) of 0.06 ng/mL. The assay indicated an effective linear range from 0.16 to 1.56 ng/mL. There were no obvious cross-reactivities with other metal ions, including Hg2+, Pb2+, As2+, Ag+, Fe3+, Cu2+, Cr2+, Au3+, Ca2+, Mg2+, Mn2+ and Zn2+. Recovery rates ranged from 79.2% to 120.0% for rice, maize, wheat, brown rice and glutinous rice samples. Twenty-five practical cereal samples were analysed, and the comparative analysis of the results showed a strong correlation (r = 0.984) between the CLEIA and the GFAAS methods. The results indicated that the ic-CLEIA developed was acceptable to screen for cadmium in cereal samples.

A Rapid Screening Method for the Tremorgenic Indole-Diterpene Alkaloid Mycotoxin Paxilline in Beer

The tremorgenic paxilline (PAX), an indole-diterpene alkaloid mycotoxin, was recently detected as a natural contaminant of ergot of barley and rye. To check the possibility of a transfer of this mycotoxin into beer, a rapid and sensitive immunochemical method for the analysis of PAX in commercial bottled beer was developed. A straightforward sample preparation procedure could be established, including degassing, pH adjustment, optional filtering, and finally a 1:5 dilution with a methanolic phosphate-buffered saline solution. Analysis of PAX was performed by a competitive indirect enzyme immunoassay (EIA). The detection limit at a cut-off value of 80% B/B0 of the EIA standard curve was evaluated by analysis of spiked beer. PAX at levels of 3 μg/L and 5 μg/L yielded 69% and 100% positive results, therefore the detection limit in beer was at 5 μg/L. Recoveries of PAX at levels of 5–20 μg/L were 88–97%, coefficients of variation were 17–22%. With these characteristics, the EIA was considered to be a suitable screening method for PAX in beer. A survey of bottled beer from the German market was conducted which included 38 samples of domestic and international brands, the latter containing various flavoring ingredients. All samples were clearly negative for PAX, which indicates that this toxin is not a relevant contaminant in beer.

Study on the antigenicity of metallofullerenol: antibody production, characterization, and its enzyme immunoassay application.

With their intriguing structures and properties, metallofullerenols have attracted considerable attention in biological and medical applications. Due to the increasing biomedical interest, effective detection methods are important to monitor and control metallofullerenols. However, the detection of metallofullerenols becomes very difficult after polyhydroxylated modification due to the lack of detectable features. Antibody-based immunoassay methods have been important tools for detection and will better meet the needs of analysis of metallofullerenols. Thus, the antigenicity of metallofullerenol has been studied for the first time. In this study, no immune response was detected when metallofullerenol Gd@C82(OH)x was used as immunogen. However, the polyclonal antibody against metallofullerenol was produced using metallofullerenol-KLH (keyhole limpet hemocyanin) as immunogen, indicating that metallofullerenol can act as hapten. The specificity of the obtained antibody was investigated. It has been found that the hydroxyl groups on the surface of the carbon cage, the encapsulated metal, and the size and shape of the carbon cage did not affect the recognition specificity of the antibody. Based on the obtained antibody, an indirect competitive enzyme immunoassay was developed for the determination of metallofullerenol with detection limits of 18ng/mL in PBS. This enzyme immunoassay method was successfully used to detect metallofullerenol in serum. This work can provide an innovative way to determine metallofullerenols. Graphical abstract The polyclonal antibody against metallofullerenol was produced using metallofullerenol-KLH (keyhole limpet hemocyanin) as immunogen. Based on the obtained antibody, a competitive enzyme immunoassay was developed for the determination of metallofullerenol.

Evaluation of ecdysteroid antisera for a competitive enzyme immunoassay and extraction procedures for the measurement of mosquito ecdysteroids

Ecdysteroid hormones regulate several aspects of insect development and reproduction. The predominant ecdysteroids produced by insects including mosquitoes are ecdysone (E) and 20-hydroxyecdysone (20E). The ability to measure E and 20E titers is essential for many studies, but few sensitive, low cost options are currently available for doing so. To address this deficiency, we developed a new enzyme-linked immunoassay (EIA). In the first part of the study, we compared the affinity of two new antisera named EAB25 and EAB27 to other available ecdysteroid antisera. EAB25 had a 27-fold higher affinity for 20E than E, while EAB27 had a four-fold higher affinity for 20E. In the second part of the study, EIA protocols were developed for analyzing E and 20E produced by the mosquito Aedes aegypti. Results indicated that pelts from fourth instar larvae and ovaries from blood-fed, adult females produced E and 20E. Methanol extraction in the presence of magnesium from whole body samples altered antibody recognition of E and 20E by EIA. However, extraction with 1-butanol and two organic/water phase separations eliminated this problem and improved assay performance. We conclude the new antisera used in the EIA provide a low-cost, flexible, and sensitive method for measuring E and 20E in insects.

Chitotriosidase, a marker of innate immunity, is elevated in patients with primary breast cancer.

Cancer progression has been associated with altered immune cell function and activation. Neopterin, which is secreted by interferon-γ stimulated macrophages, exhibits an association with multiple cancer types and metastatic disease. Chitotriosidase, which is secreted by chronically activated macrophages and granulocyte-macrophage colony-stimulating factor stimulated neutrophils has not been studied in the setting of cancer. The goal of this discovery study was to screen chitotriosidase for diagnostic capacity in detecting cancer and compare its operating characteristics with those of neopterin. Serum from subjects with breast (n= 66) or prostate (n= 70) cancer, and from 204 subjects free of malignant disease were studied. Chitotriosidase was measured by enzyme activity assay, while neopterin was measured by a competitive enzyme immunoassay. Statistical analyses included group comparisons by Mann Whitney U test, diagnostic capacity by receiver operating characteristics (ROC) curve analysis and biomarker associations with physiologic and clinical measures by Spearman correlation. Chitotriosidase activity was significantly higher in both cancer types compared with gender matched controls, though only in breast cancer was the diagnostic capacity significant (area under the ROC curve of 0.97 ± 0.01). In contrast, neopterin was significantly elevated in prostate cancer and exhibited discriminatory capacity (area under the ROC curve of 0.76 ± 0.05). Age, BMI, % body fat and metastasis were variables that correlated with neopterin, but not chitotriosidase levels. The operating characteristics of serum chitotriosidase were different from neopterin and further analysis of chitotriosidase as a biomarker for breast cancer is warranted.

Differences in Neuropeptide Y Secretion Between Intracerebral Hemorrhage and Aneurysmal Subarachnoid Hemorrhage

Neuropeptide Y (NPY) is one of the most potent endogenous vasoconstrictors, and its contribution to the multifactorial cascade of cerebral vasospasm due to nontraumatic subarachnoid hemorrhage (SAH) is not yet fully understood. This experimental study compared the hemorrhage-specific course of NPY secretion into cerebrospinal fluid (CSF) and into plasma between 2 groups: patients with SAH and patients with basal ganglia hemorrhage (BGH) or cerebellar hemorrhage (CH) over the first 10 days after hemorrhage. Seventy-nine patients were prospectively included: SAH patients (n=66) (historic population) and intracerebral hemorrhage patients (n=13). All patients received an external ventricular drain within 24 hours of the onset of bleeding. CSF and plasma were drawn daily from day 1 to day 10. The levels of NPY were determined by means of competitive enzyme immunoassay. The CSF samples of 29 patients (historic population) who had undergone spinal anesthesia due to orthopedic surgery served as the control group. NPY levels in CSF were significantly higher in the 2 hemorrhage groups than in the control group. However, the 2 hemorrhage groups showed significant differences in NPY levels in CSF (SAH mean, 0.842 ng/mL vs. BGH/CH mean, 0.250 ng/mL; P<0.001) as well as in the course of NPY secretion into CSF over the 10-day period. NPY levels in plasma did not differ significantly among SAH, BGH/CH, and controls. Our findings support the hypothesis that excessive release of NPY into CSF but not into plasma is specific to aneurysmal SAH in the acute period of 10 days after hemorrhage. In BGH/CH, CSF levels of NPY were also increased, but the range was much lower.

Malachite green residue in farmed fish in north-west part of Iran

ABSTRACTThe occurrence of malachite green (MG) residue was determined in 177 farmed fish samples (i.e., 85 carp, Cyprinus carpio, and 92 rainbow trout, Oncorhynchus mykiss) purchased from north-west part of Iran from 2014 to 2015. Competitive enzyme immunoassay method validated according to the criteria of Commission Decision 2002/657/EC was used to determine the MG residue. The mean recoveries were in the range of 79.1–86.2% and the limit of detection of the method was 0.30 µg/kg. The value of detection capability for MG in fish muscle was less than 1 µg/kg, which was below the minimum required performance limit of 2 µg/kg. MG was detected in 108 fish samples, corresponding to 61.0% of the total examined samples, ranging between 0.35 and 7.12 µg/kg. Most of the contaminated samples (28.8%) contained MG at the level of 0.3–1 µg/kg. Altogether, 18 out of 177 samples contained residue of MG higher than 2 µg/kg. The results indicated that the usage of MG in Iranian fish farms is frequent. It is alarming and could be a potential hazard for public health.

Magnetic nanoparticles-phenanthrene conjugates (MNPs-Phe) probe based competitive chemiluminescence enzyme immunoassay (MNPs-icCLEIA) for phenanthrene and other homologous polycyclic aromatic hydrocarbons

In this study, a magnetic nanoparticle-phenanthrene hybrid conjugates (MNPs-Phe) probe was synthesized and an indirect competitive chemiluminescence enzyme immunoassay (MNPs-icCLEIA) was developed for rapid and sensitive detection of phenanthrene (Phe) and its homolog in water samples. The developed MNPs-icCLEIA was based on competitive reaction between Phe in samples and the MNPs-Phe probe. In the presence of Phe, the monoclonal antibody (McAb) would be competitive captured by MNPs-Phe probe and Phe in samples. Then MNPs-Phe-McAb complexes would be separated by a magnetic field. After the reaction of horseradish peroxidase conjugated goat anti-mice IgG (HRP-IgG) with MNPs-Phe-McAb complexes, chemiluminescent substrate was added and chemiluminescence was produced. The linear range of the assay for Phe was from 1.85 to 71.51ngmL−1 and the limit of detection (LOD) was 0.85ngmL−1. Compared with the traditional enzyme linked immunosorbent assay (ELISA), the sensitivity of the developed MNPs-icCLEIA was about 3 times higher and more than 90min was saved to fulfill the procedure of developed method while the linear range was nearly the same. The recoveries of polycyclic aromatic hydrocarbons (PAHs) from three different water samples were 97.2%–103.4%, 95.8%–104.8% and 96.9%–103.5% respectively, which had a good correlation (R2=0.9986) with those from ELISA. The results indicated that the developed MNPs-icCLEIA is sensitive, rapid and reliable for the detection of Phe and other homologous PAHs in environmental water.

An efficient direct competitive nano-ELISA for residual BSA determination in vaccines.

A simple, fast, and highly sensitive direct competitive enzyme-linked immunosorbent assay (ELISA) based on bovine serum albumin (BSA) antigen labeled amine-terminated silicon dioxide (SiO2-NH-BSA) nanoparticles was developed to determine residual BSA in vaccines. As nano-ELISA using nanomaterials with a very high surface-to-volume ratio has emerged as a promising strategy, SiO2-NH-BSA nanoparticles were prepared in this study by the coupling of BSA to SiO2 nanoparticles modified with amidogen, followed by the quantification of BSA via a direct competitive binding of BSA-antigen-labeled SiO2 nanoparticles to anti-BSA antibody conjugated with horseradish peroxidase. The validation study showed that the linear range of this method was from 1 to 90ng/mL (r = 0.998) and the limit of detection was 0.67ng/mL. The intra-assay and interassay coefficients of variation were less than 10% at three concentrations (10, 40, and 70ng/mL), and the recovery was 92.4%, indicating good specificity. As a proof of principle, this new method was applied in the analysis of residual BSA in five different vaccines. Bland-Altman plots revealed that there was no significant difference in the accuracy and precision between our new method and the most commonly used sandwich ELISA. From the results taken together, the new method developed in this study is more sensitive and facile with lower cost and thus demonstrated potential to be applied in the quality control of biological products. Graphical Abstract Illustration of the procedures of the direct competitive enzyme immunoassay.

A PRELIMINARY STUDY ABOUT GLUTEN LEVELS IN SARDINIAN CRAFT BEERS

The aim of this study was to determine the gluten content of a representative sampling of Sardinian craft beers. Twenty-five craft beers produced by seven Sardinian microbreweries were analyzed. All beers were produced without micro-filtration, pasteurization or preservative additions provided by Italian law. The competitive enzyme immunoassay (Ridascreen competitive ELISA kit) was used for gluten quantification. The levels of gluten found ranged from 39 mg/L in a Pilsner beer to 2400 mg/L in a Weizen. No gluten-free beer was found and eight (32%) of the twenty-five beers analyzed contained less than 100 mg/L of gluten and could be labeled as “very low gluten” according to the Commission Regulation (EC) No 41/2009. Furthermore a significant positive correlation between gluten content and foam stability was found.

Antisecretory Factor (AF) egg-yolk peptides reflects the intake of AF-activating feed in hens

Summary Objective The aim of the present investigation was to determine the level of the active form of the endogenous protein Antisecretory Factor (actAF), and also the active form of AF immunoreactive molecules in the affinity purified egg yolk. Design Determination of AF in affinity purified egg yolk by means of in vivo and in vitro methods. Setting Sahlgrenska University Hospital and Chalmers University of Technology, both units located in Gothenburg, Sweden. The farm housing the egg-laying hens is situated some 10 miles west to Stockholm. Samples Egg yolk collected weekly from hens subjected to AF-stimulating feed for up to 18 weeks. Methods and main outcome measures The methods used were: a/ The in vivo rat ligated jejunal loop assay. b/ Two variations of ELISA, i.e. 1.An indirect ELISA using a polyclonal antibody against AF-16 antibodies. 2.A competitive enzyme immunoassay for detection of the peptide AF-16 and also related immunoreactive molecules. c/ Matrix-Assisted Laser Desorption Ionization Mass Spectrometry, (MALDI-MS). The numeric variables registered represents: a/ The rat jejunal ligated loop assay demonstrates the influence of AF on the in vivo secretory response (mg/cm) to cholera toxin challenge. The ligated loop is some 12–15 cm long and placed on the mid part of jejunum, and the cholera toxin induced secretion is registered after a 5 h long challenge period. b/ The indirect ELISA method demonstrates the relative concentration of immunogenic AF peptides/AF-16 peptides and also related immunoreactive compounds by means of absorbance values, while the values of the competitive immunoassay represent the concentration of AF-16 peptides including similar immunoreactive peptides in ng/ml. c/ The MALDI-MS method provide information about the concentration of the AF-16 peptide down to nanogram per ml. levels after mass spectrometry analysis of the sample. Results All methods revealed similar results by demonstrating a continuous increase over time in the collected egg yolk samples. Thus, low AF activity was registered in egg yolk collected in the period of 1–10 weeks of AF-stimulated feeding, significantly higher AF values was registered in yolk collected between 10 and 15 weeks of feeding, while maximal AF concentration was determined after 15 weeks of feeding. Thus, in the period between 15 and 18 weeks of stimulated AF-feeding, no further increase of the endogenous AF activity could be registered despite continuous AF-stimulated feeding. Conclusion During the period of AF-stimulated feeding of the egg laying hens the registration of AF concentration in the affinity purified egg yolk samples must be continuously registered over time. The various methods used for determination of AF concentration in the affinity purified egg yolk might all serve as tools in order to achieve the optimal concentration of active AF. Together, these methods will provide information about the optimal AF concentration in the final product consisting of spray dried egg yolk (Salovum®) used for disease treatment.

Homocysteine Metabolism and Hematological Parameters in Early Stage of Phenytoin Treated Epileptic Children.

Long-term antiepileptic drug (AED) therapy has been associated with metabolic consequences that lead to an increase in risk of atherosclerosis in patients with epilepsy. Earlier published studies showed conflicting results about the levels of hematological parameters, serum homocysteine, folate, and vitamin B12, in epileptics treated with phenytoin monotherapy. Therefore, we evaluated homocysteine metabolism and hematological parameters in early stage of phenytoin treated epileptic children. A total of 64 newly diagnosed epileptic children with mean age 10.09 ± 2.56 years were enrolled at the start of study. However, after 3 months follow up, the final total sample size was only 50 epileptic children. Fourteen children dropped out of study due to poor follow up. Serum homocysteine levels were measured by enzyme immunoassay method. Serum folate and vitamin B12 levels were estimated by Competitive Chemiluminescent Enzyme Immunoassay method. Hematological parameters were analysed by an automated hematology analyzer (Cell counter), Sysmex XT-1800i, using commercially available reagents. In our study the anthropometric and hematological parameters did not show any significant difference after phenytoin monotherapy as compared to before therapy in epileptic children. The serum homocysteine level in epileptic children was found to be significantly increased after phenytoin (PHT) monotherapy as compared to before therapy. Moreover, a highly significant decrease was observed in the serum folate and vitamin B12 levels after phenytoin monotherapy as compared to before therapy in epileptic children. Phenytoin monotherapy may cause a significant increase in the levels of serum homocysteine and a significant decrease in the serum folate and vitamin B12 levels in children with epilepsy, and the significant changes in above mentioned parameters occur early in the course of treatment. This could be responsible for a higher prevalence of cardiovascular incidents in epileptic children taking phenytoin monotherapy. Therefore, it may be useful to do early screening and treatment of increased serum homocysteine levels in epileptic children under phenytoin monotherapy to prevent atherosclerosis and its complications. Hematological parameters should also be strictly monitored regularly in individuals administered with PHT monotherapy. If there are persistent alterations, the administration of the drugs should be discontinued.

Development of a Competitive Enzyme Immunoassay Technique for the Detection of Soy Traces in Meat Products

The aim of this work was to develop a competitive enzyme immunoassay technique, to detect the presence of traces of soy in meat products. Specific rabbit polyclonal antiserum against soy protein was used as primary antibody. The optimal antigen concentration to be immobilized on the plate and the concentration of primary antibody to be used in competition was determined. The calibration curve was fitted using increasing concentrations of an extract of soy product. The soy product was extracted with Tris-HCl buffer 0.0625M with 3% sodium dodecylsulfate and 2% mercaptoethanol. The working range used in the enzyme immunoassay to detect soy was 9-280ppm SP with adequate linearity (R2: 0.9880). All validation parameters studied were appropriate. Commercial samples of meat products were analyzed with this enzyme immunoassays and a commercial ELISA kit. Significant differences were observed in the quantitative results obtained with both methods; nevertheless the developed enzyme immunoassay could be used as screening method.

Comparison of two competitive enzyme immunoassay kits for quantification of plasma Urotensin-II in rats

ABSTRACTChanges in Urotensin-II (U-II) concentration, a potent vasoconstrictor peptide, have been detected in various pathologies, but it has been impossible to define a normality range. We aimed to analyze the concordance and interchangeability between two enzyme immunoassay methods developed by Phoenix Pharmaceuticals, Inc. to measure U-II plasma concentration in rats: ELISA and fluorescent EIA. Assays resulted positively correlated (r = 0.850; p < 0.01). There was a significant difference between assays values (p < 0.001). The analysis of agreement (Bland and Altman plot) stated that the mean of the differences was 2.055 (SD ± 0.588). Hence, we concluded that the two U-II assays were correlated but not interchangeable.

Effects of Intratesticular Injection of 70% Glycerin on Stallions

The removal of endogenous germ cells of recipient stallions is a key step to produce donor germ cell-derived sperm using the germ cell transplantation technique. Six Thoroughbred stallions were divided into a treatment (n = 3) and a control group (n = 3), and 70% glycerin (1, 2, 3-trihydroxypropane, 40 mL per testis) or phosphate-buffered saline, respectively, was locally injected into testes. General semen evaluation, libido, and testicular volume were performed weekly from 3 weeks before to 10 weeks after treatment. The number of round germ cells in the ejaculate was counted using a hemocytometer. The hematoxylin and eosin staining was performed on the cross sections of testicular tissue obtained 11th week of treatment. Plasma testosterone levels in blood collected weekly were measured using a colorimetric competitive enzyme immunoassay kit. The sperm number was significantly lower than that of the control group at 5 and 10 weeks after glycerin injection. No differences in the status of spermatogenesis in the cross sections of seminiferous tubules and testicular volume were found between the two groups. The 70% glycerin-treated stallions had reduced total and progressively motile sperm and exhibited a significantly higher population of round germ cells in the ejaculate. Testosterone levels, testicular volumes, and libido of stallions were not significantly different between the groups. In conclusion, although intratesticular injection of 70% glycerin may have caused disassociation of some germ cells in the seminiferous tubules for several weeks, it did not significantly ablate germ cells in the tubules at 11 week in stallions.

Abstract 2401: RD3-loss orchestrates GC-signaling independent evolution of progressive neuroblastoma

Abstract Recently we recognized the transcriptional/translational loss of Retinal degeneration Protein 3 (RD3) in high-risk progressive neuroblastoma (HR-NB) and defined its novel tumor evolution stabilization function. Studies have shown that RD3 competitively binds to guanylate cyclases (GCs) and maintains GC expression, stability, as well as restores GC localization. Accordingly, we investigated the role of GCs, if any, in RD3-loss associated evolution of HR-NB. Utilizing an in vivo mouse model of spontaneous HR-NB coupled with ex vivo approaches using metastatic site derived aggressive cells (MSDACs), we examined the transcriptional and translational expression of GCs and validated with high-content confocal immunofluorescence. Further, utilizing a cGMP competitive enzyme immunoassay, we investigated both intracellular and soluble GC activity. Whole genome microarray analysis revealed a differential activation of GC signaling transcriptional machinery in three different clones of MSDACs (compared to parental SH-SY5Y cells) ex vivo. Immunoblotting identified a consistent translation of GCs (GC-1 and GC-2) in a manifold of metastatic tumors (compared to non-metastatic primary xenografts) in vivo and in MSDACs ex vivo. Confocal immunofluorescence analysis validated the near-identical expression of GC1 and GC2 both in SH-SY5Y cells and MSDACs. cGMP immunoassay recognized indistinguishable intracellular and soluble GC activity in clones of differentiated/undifferentiated MSDACs as opposed to the clones of SH-SY5Y. For the first time, the results of this study define the transcription and translation blueprint of GCs in HR-NB and, further imply that the RD3-loss may dictate GCs-signaling independent alternate molecular flow-through in the evolution of HR-NB. Acknowledgements: Stephenson Cancer Center, NIH COBRE 1P20GM103639-01, Broken Arrow Lady Elks Auxiliary, and Oklahoma TSET. Citation Format: Singh Meghna, Wolansky Rachel, Terence S. Herman, Natarajan Aravindan. RD3-loss orchestrates GC-signaling independent evolution of progressive neuroblastoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2401.