Direct Enzyme-linked Immunosorbent Assay Research Articles

A stable and sensitive enzyme-linked immunosorbent assay (ELISA) for the determination of metsulfuron-methyl residues in foods.

A hapten of metsulfuron-methyl was successfully designed and synthesized from 2-methylester-phenylsulfonamide and succinic anhydride, and the polyclonal antibody against metsulfuron-methyl was prepared by immunization procedure with the hapten-bovine serum albumin conjugate. A stable and sensitive direct competitive enzyme-linked immunosorbent assay (dcELISA) method had been developed under the optimal conditions. The sensitivity (IC50 ) was 37.03± 1.87µg/L, and the detection line (IC15 ) was 1.57± 0.11µg/L. Rice, wheat, oat, flaxseed, milk, and water were chosen to study the recovery test and the recovery rates were 83.11%-117.44% . The matrix effect was eliminated by a simple dilution of the sample extracts. The results from dcELISA were well agreement with the results from HPLC-MS. It was indicated that the developed method had good accuracy and stability. It could be applied for the detection of metsulfuron-methyl residues. It was worth mentioning that the antibody could recognize metsulfuron-methyl and tribenuron-methyl with cross-reactivities of 100% and 49.72%, respectively. In order to understand the cross-reactivity, molecular modeling including molecular alignment and electrostatic potential surfaces were introduced. It was found that the special group of metsulfuron-methyl played an important role, especially on C3 position of the phenyl group. PRACTICAL APPLICATION: A stable, sensitive, and low-cost dc ELISA method had been developed with good accuracy and applied in the determination of metsulfuron-methyl in foods. Molecular simulation was introduced to understand the specificity between the antibody and the analyst. It was a good method to study the cross-reactivity between the antibody and the analyst or analogue.

Does vitamin D affect the association between FTO rs9939609 polymorphism and depression?

ABSTRACT Background & objectives: Depression is a highly prevalent and multifactorial psychological disorder. Fat Mass and Obesity-Associated (FTO) gene and the serum vitamin D level are proposed to be involved in pathophysiology of depression. This study aimed to investigate the interactions between one FTO gene single nucleotide polymorphism, depression, and serum vitamin D level in overweight adults. Methods: One hundred and ninety-seven overweight adults were recruited in this cross-sectional study. FTO genotyping was performed by amplification refractory mutation system polymerase chain reaction (ARMS-PCR). Depression severity was assessed using Beck’s depression inventory (BDI-II). Serum vitamin D levels were measured using a direct competitive enzyme-linked immunosorbent assay (ELISA) method. Results: A-allele carriers had higher Beck’s depression score (P = 0.03). Multivariate regression models showed a positive association between the A-allele of FTO rs9939609 polymorphism and depression. Serum vitamin D level had no effect on the association between FTO genotype and depression. Conclusion: A-allele of FTO rs9939609 polymorphism might be associated with depression independent of serum vitamin D level. Further longitudinal studies are needed to confirm these results.

Survey of Commercial Food Products for Detection of Walnut (Juglans regia) by Two ELISA Methods and Real Time PCR.

Labeling of food allergens in accordance with legal regulations is important to protect the health of allergic consumers. The requirements for detecting allergens in foods involve adequate specificity and sensitivity to identify very small amounts of the target allergens in complex food matrices and processed foods. In this work, one hundred commercial samples were analyzed for walnut detection using three different methods: a sandwich enzyme-linked immunosorbent assay (ELISA) kit based on polyclonal antibodies, a direct ELISA using a recombinant multimeric scFv, and a real time PCR. The most sensitive method was real time PCR followed by sandwich ELISA kit and multimeric scFv ELISA. There was agreement between the three methods for walnut detection in commercial products, except for some heat-treated samples or those that contained pecan. The walnut ELISA kit was less affected by sample processing than was the multimeric scFv ELISA, but there was cross-reactivity with pecan, producing some false positives that must be confirmed by real time PCR. According to the results obtained, 7.0 to 12.6% of samples (depending on the analytical method) contained walnut but did not declare it, confirming there is a risk for allergic consumers. Moreover, there was one sample (3.7%) labelled as containing walnut but that tested negative for this tree nut. Genetic and immunoenzymatic techniques offer complementary approaches to develop a reliable verification for walnut allergen labeling.

Presence of antibodies against SARS-CoV-2 spike protein in bovine whey IgG enriched fraction

Bovine whey IgG enriched fraction contains antibodies against various human bacterial pathogens. It contains antibodies against some viral antigens, including human respiratory syncytial virus and influenza virus. We investigated whether the IgG enriched fraction has cross-reactivity with IgG antibodies against SARS-CoV-2 spike (S) and nucleocapsid (N) proteins. The full-length and partial-length SARS-CoV-2 S, N, a recombinant protein of the receptor binding domain (RBD) and nine peptides covering the receptor binding motif (RBM) of S were prepared. Direct enzyme-linked immunosorbent assays were conducted using these recombinant proteins and peptides as coating antigens and revealed the IgG enriched fraction contained antibodies against partial-length S [amino acids (aa) 177–512, 288–512, 348–578, 387–516 and 408–664], full-length N (aa 1–419) and partial-length N (aa 1–120, 111–220, 1–220 and 210–419), two RBD peptides, covering aa 427–446 and 502–520 of S, and recombinant RBD of S. These results indicate IgG enriched fraction contains antibodies against SARS-CoV-2.

Enzyme and lateral flow monoclonal antibody-based immunoassays to simultaneously determine spirotetramat and spirotetramat-enol in foodstuffs

Spirotetramat is employed worldwide to fight insect pests due to its high efficiency. This chemical is quickly metabolized by plants into spirotetramat-enol, so current regulations establish that both compounds must be determined in foodstuffs for monitoring purposes. Nowadays, immunochemical methods constitute rapid and cost-effective strategies for chemical contaminant analysis at trace levels. However, high-affinity binders and suitable bioconjugates are required. In this study, haptens with opposite functionalisation sites were synthesized in order to generate high-affinity monoclonal antibodies. A direct competitive enzyme-linked immunosorbent assay with an IC50 value for the sum of spirotetramat and spirotetramat-enol of 0.1 μg/L was developed using selected antibodies and a novel heterologous bioconjugate carrying a rationally-designed hapten. Studies with fortified grape, grape juice, and wine samples showed good precision and accuracy values, with limits of quantification well below the maximum residue limits. Excellent correlation of results was observed with a standard reference chromatographic method. As a step forward, a lateral flow immunoassay was developed for onsite screening analysis of spirotetramat in wine. This assay was successfully validated according to Regulation 519/2014/EU for semi-quantitative methods at concentrations in line with the legal levels of spirotetramat and spirotetramat-enol in grapes, with a satisfactory false suspect rate below 2%.

Trypanosoma cruzi infection in Triatoma infestans and high levels of human\u2013vector contact across a rural-to-urban gradient in the Argentine Chaco

BackgroundPeri-urban and urban settings have recently gained more prominence in studies on vector-borne transmission of Trypanosoma cruzi due to sustained rural-to-urban migrations and reports of urban infestations with triatomines. Prompted by the finding of Triatoma infestans across the rural-to-urban gradient in Avia Terai, an endemic municipality of the Argentine Chaco, we assessed selected components of domestic transmission risk in order to determine its variation across the gradient.MethodsA baseline vector survey was conducted between October 2015 and March 2016, following which we used multistage random sampling to select a representative sample of T. infestans at the municipal level. We assessed T. cruzi infection and blood-feeding sources of 561 insects collected from 109 houses using kinetoplast DNA-PCR assays and direct enzyme-linked immunosorbent assays, respectively. We stratified triatomines according to their collection site (domestic or peridomestic), and we further categorized peridomestic sites in ecotopes of low- or high-risk for T. cruzi infection.ResultsThe overall adjusted prevalence of T. cruzi-infected T. infestans was 1.8% (95% confidence interval [CI] 1.3–2.3) and did not differ between peri-urban (1.7%) and rural (2.2%) environments. No infection was detected in bugs captured in the urban setting; rather, infected triatomines were mainly collected in rural and peri-urban domiciles, occurring in 8% of T. infestans-infested houses. The main blood-feeding sources of domestic and peridomestic triatomines across the gradient were humans and chickens, respectively. The proportion of triatomines that had fed on humans did not differ between peri-urban (62.5%) and rural (65.7%) domiciles, peaking in the few domestic triatomines collected in urban houses and decreasing significantly with an increasing proportion of chicken- and dog- or cat-fed bugs. The relative odds ratio (OR) of having a T. cruzi infection was nearly threefold higher in bugs having a blood meal on humans (OR 3.15), dogs (OR 2.80) or cats (OR: 4.02) in a Firth-penalized multiple logistic model.ConclusionsTrypanosoma cruzi transmission was likely occurring both in peri-urban and rural houses of Avia Terai. Widespread infestation in a third of urban blocks combined with high levels of human–triatomine contact in the few infested domiciles implies a threat to urban inhabitants. Vector control strategies and surveillance originally conceived for rural areas should be tailored to peri-urban and urban settings in order to achieve sustainable interruption of domestic transmission in the Chaco region.Graphical

Widespread zoophagy and detection of Plasmodium spp. in Anopheles mosquitoes in southeastern Madagascar

BackgroundMalaria is a top cause of mortality on the island nation of Madagascar, where many rural communities rely on subsistence agriculture and livestock production. Understanding feeding behaviours of Anopheles in this landscape is crucial for optimizing malaria control and prevention strategies. Previous studies in southeastern Madagascar have shown that Anopheles mosquitoes are more frequently captured within 50 m of livestock. However, it remains unknown whether these mosquitoes preferentially feed on livestock. Here, mosquito blood meal sources and Plasmodium sporozoite rates were determined to evaluate patterns of feeding behaviour in Anopheles spp. and malaria transmission in southeastern Madagascar.MethodsAcross a habitat gradient in southeastern Madagascar 7762 female Anopheles spp. mosquitoes were collected. Of the captured mosquitoes, 492 were visibly blood fed and morphologically identifiable, and a direct enzyme-linked immunosorbent assay (ELISA) was used to test for swine, cattle, chicken, human, and dog blood among these specimens. Host species identification was confirmed for multiple blood meals using PCR along with Sanger sequencing. Additionally, 1,607 Anopheles spp. were screened for the presence of Plasmodium falciparum, P. vivax-210, and P. vivax 247 circumsporozoites (cs) by ELISA.ResultsCattle and swine accounted, respectively, for 51% and 41% of all blood meals, with the remaining 8% split between domesticated animals and humans. Of the 1,607 Anopheles spp. screened for Plasmodium falciparum, Plasmodium vivax 210, and Plasmodium vivax 247 cs-protein, 45 tested positive, the most prevalent being P. vivax 247, followed by P. vivax 210 and P. falciparum. Both variants of P. vivax were observed in secondary vectors, including Anopheles squamosus/cydippis, Anopheles coustani, and unknown Anopheles spp. Furthermore, evidence of coinfection of P. falciparum and P. vivax 210 in Anopheles gambiae sensu lato (s.l.) was found.ConclusionsHere, feeding behaviour of Anopheles spp. mosquitoes in southeastern Madagascar was evaluated, in a livestock rich landscape. These findings suggest largely zoophagic feeding behaviors of Anopheles spp., including An. gambiae s.l. and presence of both P. vivax and P. falciparum sporozoites in Anopheles spp. A discordance between P. vivax reports in mosquitoes and humans exists, suggesting high prevalence of P. vivax circulating in vectors in the ecosystem despite low reports of clinical vivax malaria in humans in Madagascar. Vector surveillance of P. vivax may be relevant to malaria control and elimination efforts in Madagascar. At present, the high proportion of livestock blood meals in Madagascar may play a role in buffering (zooprophylaxis) or amplifying (zoopotentiation) the impacts of malaria. With malaria vector control efforts focused on indoor feeding behaviours, complementary approaches, such as endectocide-aided vector control in livestock may be an effective strategy for malaria reduction in Madagascar.

HIGHLY SENSITIVE IMMUNOASSAY OF PENICILLINS IN DAIRY AND MEAT PRODUCTS

Specific immunoreagents have been obtained and a direct enzyme-linked immunosorbent assay with a very high sensitivity to penicillins in standard solutions (0.06 ng/ml) as well as in the samples of milk (0.3 μg/kg) and meat (2.4 μg/kg) has been developed.

Development of a single-chain variable fragment-alkaline phosphatase fusion protein-based direct competitive enzyme-linked immunosorbent assay for furaltadone metabolite in ctenopharyngodon idellus

ABSTRACTTo determine furaltadone metabolites 5-methylmorpholino-3-amino-2-oxazolidinone (AMOZ) residual levels in ctenopharyngodon idellus, direct competitive enzyme-linked immunosorbent assay (dcELISA) was developed based on a single-chain variable fragment (scFv)-alkaline phosphatase (AP) fusion protein. Firstly, VH and VL gene sequences were cloned from hybridoma cell lines secreting monoclonal antibody against AMOZ derivative, and then the productive VH and VL genes were assembled to a scFv gene and engineered to produce scFv-AP fusion protein. The fusion protein was further identified as a bifunctional reagent for immunoassay, then a sensitive one-step dcELISA against AMOZ derivative was developed with a half-maximal inhibitory concentration (IC50) and limit of detection (LOD) of 10.62 and 4.83 ng/mL, respectively. The recovery values of AMOZ from spiked fish samples determined by dcELISA were in 71.31%∼82.88%. The results suggested that the prepared scFv-AP fusion protein in this work can be used for rapid and convenient immunoassays to detect AMOZ residues in fish samples.

Enzyme-linked immunosorbent assay and immunochromatographic strip for rapid detection of atrazine in three medicinal herbal roots

Objectives: An enzyme-linked immunosorbent assay (ELISA) and colloidal gold-based immunochromatographic (ICG) strip assay will be developed for the rapid and high-throughput detection of atrazine (ATZ) in medicinal herbs. Methods: A monoclonal antibody against ATZ was obtained after the immunization of mice, cell fusion, and hybridoma screening, and the antibody was used to develop direct competitive ELISA (dcELISA) and the ICG strip assay. Results: Both dcELISA and ICG strip methods were established, optimized, and validated for the detection of ATZ in Salviae miltiorrhizae radix et rhizome, Astragali radix, and Isatidis radix. dcELISA had a half-maximum inhibition concentration of 10.56 ng/mL (Salviae miltiorrhizae radix et rhizome), 7.6 ng/mL (Astragali radix), and 8.15 ng/mL (Isatidis radix). The limit of detection (LOD) of the ICG strip assay was 12.5 ng/mL (Salviae miltiorrhizae radix et rhizome), 12.5 ng/mL (Astragali radix), and 6.25 ng/mL (Isatidis radix) in different herb matrices. Due to the recognition characteristics of the monoclonal antibody for the pesticides ATZ, propazine, sebuthylazine, and prometryn, the detection results of real samples by the two immunoassays were confirmed by liquid chromatography–tandem mass spectrometry, which proved the accuracy and reliability of the established methods. Conclusion: The proposed dcELISA and ICG strip methods were suitable for the rapid, convenient, and high-throughput detection of ATZ in these medicinal herbs.

Development and Characterization of a Quantitative ELISA to Detect Anti-SARS-CoV-2 Spike Antibodies

A novel clinical assay for the detection and quantitation of antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was adapted from an in-house, research-based enzyme-linked immunosorbent assay (ELISA). Development and validation were performed under regulatory guidelines, and the test obtained emergency use authorization (EUA) from the New York State Department of Health (NYSDOH) and the Food and Drug Administration (FDA). The Mount Sinai COVID-19 antibody assay is an orthogonal, quantitative direct ELISA test which detects antibodies reactive to the receptor binding domain (RBD) and the spike protein of the novel SARS-CoV-2. The assay is performed on 96-well plates coated with either SARS-CoV-2 recombinant RBD or spike proteins. The test is divided into two stages, a qualitative screening assay against RBD and a quantitative assay against the full-length spike protein. The test uses pooled high titer serum as a reference standard. Negative pre-COVID-19 and positive post-COVID-19, PCR-confirmed specimens were incorporated in each ELISA test run, and the assays were performed independently at two different locations. The Mount Sinai COVID-19 serology performed with high sensitivity and specificity, 92.5% (95% CI: 0.785 – 0.980) and 100% (CI: 0.939 – 1.000) respectively. Between-run precision was assessed with a single run repeated over 22 days; and within-run precision was assessed with 10 replicates per day over 22 days. Both were within reported acceptance criteria (CV ≤20%). This population-based study reveals the applicability and reliability of this novel orthogonal COVID-19 serology test for the detection and quantitation of antibodies against SARS-CoV-2, allowing a broad set of clinical applications, including the broad evaluation of SARS-CoV-2 seroprevalence and antibody profiling in different population subsets.Funding: This work was partially supported by the JPB foundation, the Open Philanthropy Project (#2020-215611) and other philanthropic donations.Declaration of Interests: Mount Sinai has licensed serological assays to commercial entities and has filed for patent protection for serological assays. The Icahn School of Medicine at Mount Sinai has filed patent applications relating to the COVID-19 serological assay (“Serology Assay”) and NDV-based SARS-CoV-2 vaccines which list Florian Krammer (“Serology Assay”, vaccines), Daniel Stadlbauer (“Serology Assay”), Damodara Rao Mendu (“Serology Assay”), and Carlos Cordon-Cardo (“Serology Assay”) as co-inventors. The foundational “Serology Assay” intellectual property (IP) was licensed by the Icahn School of Medicine at Mount Sinai to commercial entities including Kantaro Biosciences, a company in which Mount Sinai has a financial interest. Florian Krammer consulted for Merck, Curevac, and Pfizer in the past (before 2020) and is currently consulting for Pfizer, Seqirus, and Avimex. The Krammer laboratory is collaborating with Pfizer on animal models of SARS-CoV-2.Patient Informed Consent Statement: Informed consent was obtained from all individuals included in this study.

Overexpression of Schistosome-specific Antigens for Selection of scFv Antibody from Phage Display Libraries of Water Buffalo for Potential Use in Schistosomiasis Diagnosis

Schistosomiasis is an acute and chronic parasitic disease caused by blood flukes of the genus Schistosoma. The Kato-Katz method is the standard for the diagnosis of intestinal schistosomiasis because of its specificity, simplicity, and low cost. However, Kato-Katz is less useful in light infections and other available detection methods are neither practical nor sensitive for routine or large-scale screening. Thus, improved tests are needed for the assessment of infection. The water buffalo is a natural host and reservoir of Schistosoma but is schistosomiasis-resistant, suggesting a robust immune repertoire, which can be tapped as a source of antibodies for diagnosis of schistosomiasis. Two schistosome antigens had been previously shown to be recognized by a phage display single-chain fragment of variation (scFv) library derived from a schistosome-infected water buffalo. However, specific antibodies have not been isolated using these antigens due to a lack of sufficient amounts and purified forms of the antigens. In this study, the schistosome antigens were cloned and overexpressed in vector with solubility tag, purified by immobilized metal affinity chromatography (IMAC), and used to interrogate the immune scFv library to isolate and identify specific schistosome antibody. An scFv specific to schistosome SAP domain-containing hypothetical protein was isolated but was only 105-bp long although certain regions align with the IgG VL, tetraglycylserine (Gly4Ser) linker, and IgG VH sequences of synthetic scFv constructs. Despite its length, scFv A63 can be potentially designed as a ligand in direct ELISA (enzyme-linked immunosorbent assay) by fusion with enzyme conjugates such as alkaline phosphatase to detect S. japonicum schistosomule and adult worm. The results in this study also suggest the need to construct a bigger and more diverse scFv library and to test a wider range of schistosome antigens, specifically those that will be present on the surface of the schistosome at all stages of S. japonicum life cycle.

Direct competitive ELISA enhanced by dynamic light scattering for the ultrasensitive detection of aflatoxin B1 in corn samples

Compared with absorbance, scattering-based dynamic light scattering (DLS) signal has higher sensitivity because its light-scattering intensity is very sensitive to changes in size, thereby enhancing the sensitivity. Herein, we first developed a DLS-enhanced direct competitive enzyme-linked immunosorbent assay (DLS-dcELISA) for ultrasensitive detection of aflatoxin B1 (AFB1) in corn. By using hydroxyl radical-induced gold nanoparticle (AuNP) aggregation to amplify AuNP scattering signals, the developed DLS-dcELISA exhibited ultrahigh sensitivity for AFB1. The detection limit was 0.12 pg mL−1, which was 153- and 385-fold lower than those obtained using plasmonic and colorimetric dcELISA. In addition, the DLS-dcELISA exhibited excellent selectivity, high accuracy, and strong practicality. Overall, this work presented a simple and universal strategy for improving the sensitivity of traditional ELISA platform only by using the sensitive DLS signals. This technique can replace absorbance-based plasmonic or colored signals as immunoassay signal output for enhanced competitive detection of mycotoxins.

Awareness about ELISA technique among dental students

The enzyme-linked immunosorbent assay (ELISA) can be used to recognize proteins, peptides, antibodies as well as hormones. Often known also as an enzyme immunoassay (EIA), ELISA is used as a diagnostic test in the field of biomedicine and science. This study was conducted to determine the understanding of the ELISA technique among dental students. This survey was performed for assessing the awareness about ELISA technique amongst the dental students. This study was a questionnaire oriented, cross-sectional type of survey comprising 100 dental college students in Chennai. A self-designed questionnaire with 10 questions eliciting the knowledge and awareness about applications of ELISA technique among dental college students. Questionnaires were circulated through an online website survey planet. The questions explored the awareness on ELISA technique diagnostic indications, Direct ELISA, Indirect ELISA, Sandwich ELISA, Competitive ELISA and mechanism of ELISA technique. After the responses were received from 100 participants, data were collected and analysed.67% of the respondents were aware of the ELISA technique .52% were aware of direct ELISA technique. 45%% were aware of the indirect ELISA technique. 42% were aware of the sandwich ELISA technique, 38% were aware of the competitive ELISA technique. 35% were aware of the mechanism of the ELISA technique. The awareness about the ELISA technique in diagnostic medical applications was less among dental students. Increased awareness and educational programs should be initiated to spread knowledge about the ELISA technique among all students and clinicians.

Preparation and Characterization of Two Immunogens and Production of Polyclonal Antibody with High Affinity and Specificity for Darunavir.

Darunavir (DRV) is a potent antiviral drug used for treatment of infections with human immunodeficiency virus (HIV). Effective and safe treatment with DRV requires its therapeutic drug monitoring (TDM) in patient’s plasma during therapy. To support TDM of DRV, a specific antibody with high affinity is required in order to develop a sensitive immunoassay for the accurate determination of DRV in plasma. In this study, two new and different immunogens were prepared and characterized. These immunogens were the DRV conjugates with keyhole limpet hemocyanin (KLH) protein. The first immunogen (DRV-KLH) was prepared by zero-length direct linking of DRV via its aromatic amino group with the tyrosine amino acid residues of KLH by diazotization/coupling reaction. The second immunogen (G-DRV-KLH) was prepared by conjugation of the N-glutaryl derivative of DRV (G-DRV) with KLH. The 5-carbon atoms-spacing G-DRV hapten was synthesized by reaction of DRV via its aromatic amino group with glutaric anhydride. The reaction was monitored by HPLC and the chemical structure of G-DRV was confirmed by mass, 1H-NMR, and 13C-NMR spectroscopic techniques. The hapten (G-DRV) was linked to the KLH protein by water-soluble 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) coupling procedure. The pertinence of the coupling reactions of haptens to protein was confirmed, and the immunogens were characterized by ultraviolet (UV) spectrophotometry. Both DRV-KLH and G-DRV-KLH were used for the immunization of animals and the animal’s antiserum that showed the highest affinity was selected. The collected antiserum (polyclonal antibody) had very high affinity to DRV (IC50 value = 0.2 ng mL−1; defining IC50 as the DRV concentration that can inhibit antibody binding by 50% of its maximum binding) and high specificity to DRV among other drugs used in the combination therapy with DRV. Cumulative results from direct and competitive enzyme-linked immunosorbent assay (ELISA) using this polyclonal antibody proved that the immunogens were highly antigenic and elicited a specific polyclonal antibody. The produced polyclonal antibody is valuable for the development of highly sensitive and selective immunoassays for TDM of DRV.

Enzyme immunoassay systems and a reagent kit for the determination of bacitracin in foods

Two test-systems for a direct and an indirect enzyme-linked immunosorbent assay (ELISA) of peptide antibiotic bacitracin (BC) were developed and studied. For the both systems, polyclonal antibodies were obtained by immunizing rabbits with a conjugate of BC with keyhole limpet hemocyanine synthesized using reaction between the peptide and the high molecular weight protein in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). The product of BC linking to thyroglobulin which was activated with EDC and N-hydroxysulfosuccinimide served as conjugated antigen on a solid phase in the indirect ELISA. For the direct ELISA, the antibodies against BC were immunochemically immobilized onto microplate surface, while the liquid phase contained a conjugate of BC with horseradish peroxidase. This conjugate was obtained by successive reactions of antibiotic amino groups coupling to periodate oxidized carbohydrate chains of enzyme and the reducting of formed Shiff’s base with sodium borohydride. Conjugated antigens binding to anti-BC antibodies provided maximum colorimetric signals of 2.0 and 1.2 optical units for the direct and indirect ELISA, respectively, and depended on BC content in the liquid phase. Antibiotic concentration that caused the inhibition of binding by 50 % was 2.6 ng/ml in the direct ELISA and 10.0 ng/ml in the indirect ELISA. The simple and sensitive direct ELISA system was used as a prototype of the finished reagent kit and a method for measurements with technical-analytical parameters and metrological characteristics allowing the determination of BC residues in a variety of foods including 14 items in a concentration range of 9.0 to 405.0 pg/kg with proper accuracy and precision.

Development of Metal Nanoparticle-immobilized Microplate for High-throughput and Highly Sensitive Fluorescence Analysis.

Enzyme-linked immunosorbent assay (ELISA) is a widespread analytical biochemistry assay. In this work, a direct ELISA method using a metallic nanoparticle (NP)-immobilized 96-well plate was developed for high-throughput, highly sensitive fluorescence analysis. Immobilization of metallic NPs on a 96-well plate effectively amplified fluorescence signals of the assay. The silver (Ag) NP-immobilized plate showed the best fluorescence enhancement effect of all the metal-immobilized plates tested. We used the Ag NP-immobilized plate to detect biomolecules and bacteria and found that both the fluorescence intensity and the limit of detection (LOD) were strongly enhanced by more than 100 times compared with those of the unmodified 96-well plates. Quantitative and qualitative considerations for target bacteria regarding the impact of autofluorescence on detection were successfully obtained for several strains. Our results demonstrate the potential of applying Ag NPs for enhancing the efficiency of direct and indirect ELISA assays.

Total Daily Production and Periodicity of Melatonin Metabolite in Critically Ill Children.

To determine whether total daily 6-sulfatoxymelatonin excretion and diurnal variation of melatonin secretion was maintained during the early phase of PICU admission through examination of the melatonin urinary metabolite, 6-sulfatoxymelatonin. Exploratory prospective, observational study. Twelve-bed medical-surgical PICU of a Children's Hospital. Fifty children 3 months to 18 years old enrolled within 24 hours of PICU admission with access for urinary sampling. None. Urine samples were collected at 4-hour intervals for 24 hours and stored at -80C. 6-sulfatoxymelatonin was determined in duplicate by direct enzyme-linked immunosorbent assay. Patients were heterogeneous for diagnosis, had a mean age of 8.1 years (SD = 6.1 yr), and median (interquartile range) Pediatric Risk of Mortality III of 10 (4-13). Mean (SD) total daily 6-sulfatoxymelatonin production was 30.0 µg (25.6 µg) for the first 24 hours, which did not differ significantly from the means on days 2 (p = 0.56) or 3 (p = 0.29), and was similar to literature controls. Mean 6-sulfatoxymelatonin production for the population fit a periodic function well, with a reliable amplitude of 326 ng/hr and peak excretion from 04:00 to 08:00 (F = 4.4, p = 0.01), even when 6-sulfatoxymelatonin was corrected for body weight (F = 3.4, p = 0.03) and when sedation was included in the model (F = 3.95, p = 0.004). There was no significant correlation between lighting and 6-sulfatoxymelatonin excretion at any time period (R values: 0.11-0.25, p = 0.10-0.94). Mean 6-sulfatoxymelatonin excretion did not fit the model for a periodic function well for the subpopulations studied (sepsis [n = 18, F = 1.1, p = 0.32], respiratory failure requiring deep sedation [n = 10, F = 0.4, p = 0.66], and neurologic injury [n = 7, F = 0.6, p = 0.55]). Total daily and diurnal variation of 6-sulfatoxymelatonin excretion is heterogeneously maintained early in pediatric critical illness. However, this may not hold true for specific diagnostic categories.

Fluorescence Enhancement Effect by Metal Nanoparticles-immobilized Microplate.

In this reported work, we achieved high-throughput, highly sensitive fluorescent analysis using an enzyme-linked immunosorbent assay (ELISA) that employed a metallic nanoparticle (NP)-immobilized 96-well plate. The immobilization of metallic NPs on a 96-well plate effectively amplified fluorescent signals of the assay. The silver (Ag) NP-immobilized plate showed the best fluorescent enhancement effect of all plates immobilized by metal NPs. Our results demonstrate the potential of applying Ag NPs to enhance the efficiency of direct and indirect ELISA by the labeling of targets.

Autoantibody Biomarkers for Basal Ganglia Encephalitis in Sydenham Chorea and Pediatric Autoimmune Neuropsychiatric Disorder Associated With Streptococcal Infections.

Movement, behavioral, and neuropsychiatric disorders in children have been linked to infections and a group of anti-neuronal autoantibodies, implying dopamine receptor-mediated encephalitis within the basal ganglia. The purpose of this study was to determine if anti-neuronal biomarkers, when used as a group, confirmed the acute disease in Sydenham chorea (SC) and pediatric autoimmune neuropsychiatric disorder associated with streptococcal infections (PANDAS). IgG autoantibodies against four neuronal autoantigens (tubulin, lysoganglioside GM1, and dopamine receptors D1 and D2) were detected in SC sera (N=8), sera and/or cerebrospinal fluid (CSF) from two groups of PANDAS cases (N=25 first group and N=35 second group), sera from Tourette's syndrome (N=18), obsessive-compulsive disorder (N=25), attention deficit hyperactivity disorder (N=18), and healthy controls (N=28) by direct enzyme-linked immunosorbent assay (ELISA). IgG specific for neuronal autoantigens was significantly elevated during the acute symptomatic phase, and the activity of calcium/calmodulin-dependent protein kinase II (CaMKII) pathway was significantly elevated in human neuronal cells. Five assays confirmed the disease in SC and in two groups of children with PANDAS. In 35 acute onset PANDAS patients, 32 sera (91.4%) were positive for one or more of the anti-neuronal autoantibodies compared with 9 of 28 healthy controls (32.1%, p<0.0001). Importantly, CSF of 32 (91.4%) PANDAS patients had one or more detectable anti-neuronal autoantibody titers and CaMKII activation. Among healthy control subjects with elevated serum autoantibody titers for individual antigens, none (0%) were positively associated with elevated positive CaMKII activation, which was a striking contrast to the sera of PANDAS subjects, who had 76–89% positive association with elevated individual autoantibody titers and positive CaMKII activity. At 6 months follow-up, symptoms improved for more than 80% of PANDAS subjects, and serum autoantibody titers also significantly decreased. Results reported herein and previously published studies in our laboratory suggest the antibody biomarkers may be a useful adjunct to clinical diagnosis of SC, PANDAS, and related disorders and are the first known group of autoantibodies detecting dopamine receptor-mediated encephalitis in children.