Neeraja Venkateswaran - Academia.edu (original) (raw)

Papers by Neeraja Venkateswaran

The Journal of Immunology

Arboviruses cause a serious public health problem in tropical and subtropical countries and are e... more Arboviruses cause a serious public health problem in tropical and subtropical countries and are emerging as a threat in western hemisphere also. Rapid and accurate detection of these infections can help in improving patient care and disease control. The cross-reactivity of immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies to ZIKV with dengue virus (DENV), West Nile virus (WNV), yellow fever virus (YFV) and some other related flaviviruses poses a significant challenge for an accurate detection of ZIKV infection in serological assay such as IgM capture ELISA. To overcome these challenges we developed a multiplex serological assay that will simultaneously detect antibody response to ZIKV, other related flaviviruses and Chikungunya virus (CHIKV). Various recombinant arboviral antigens were coupled to optically encoded microspheres from Luminex Corporation. This fluorescent microsphere immunoassay was used to differentiate between recent, past, and/or co-infections that may oc...

Kesmas: Jurnal Kesehatan Masyarakat Nasional

The SARS-CoV-2 transmission dynamics in low- and middle-income countries remain poorly understood... more The SARS-CoV-2 transmission dynamics in low- and middle-income countries remain poorly understood. This study aimed to estimate the SARS-CoV-2 antibodies seroprevalence in Jakarta, Indonesia, and to increase knowledge of SARS-CoV-2 transmission in urban settings. A population-based serosurvey among individuals aged one year or older was conducted in Jakarta. Employing a multistage sampling design, samples were stratified by district, slum, and non-slum residency, sex, and age group. Blood samples were tested for IgG against three different SARS-CoV-2 antigens. Seroprevalence was estimated after applying sample weights and adjusting for cluster characteristics. In March 2021, this study collected 4,919 respondents. The weighted estimate of seroprevalence was 44.5% (95% CI = 42.5-46.5). Seroprevalence was highest among adults aged 30-49 years, with higher seroprevalence in women and the overweight/obese group. Respondents residing in slum areas were 1.3-fold more likely to be seroposi...

Health Security

We conducted a comprehensive, multiphase laboratory evaluation of InBios Active Melioidosis Detec... more We conducted a comprehensive, multiphase laboratory evaluation of InBios Active Melioidosis Detect (AMD) rapid test, a lateral flow immunoassay designed to detect capsular polysaccharides produced by Burkholderia mallei or Burkholderia pseudomallei, used in conjunction with the Omni Array Reader (OAR) for the rapid detection of B mallei or B pseudomallei in environmental (nonclinical) samples at 2 sites. The limit of detection, using reference strains B mallei strain ATCC 23344 and B pseudomallei strain ATCC 11668, was determined to be 10 3 to 10 4 CFU/mL. In different phases of the evaluation, inclusivity strains that included geographically diverse strains of B mallei (N = 13) and B pseudomallei (N = 22), geographically diverse phylogenetic near neighbor strains (N = 66), environmental background strains (N = 64), white powder samples (N = 26), and environmental filter extracts (N = 1 pooled sample from 10 filter extracts) were also tested. A total of 1,753 tests were performed, which included positive and negative controls. Visual and OAR results showed that the AMD test detected 92.3% of B mallei and 95.5% of B pseudomallei strains. Of the 66 near-neighbor strains tested, cross-reactivity was observed with only B stabilis 2008724195 and B thailandensis 2003015869. Overall, the specificity and sensitivity were 98.8% and 98.7%, respectively. The results of this evaluation support the use of the AMD test as a rapid, qualitative assay for the presumptive detection of B mallei and B pseudomallei in suspicious environmental samples such as white powders and aerosol samples by first responders and laboratory personnel.

Health Security

We conducted a comprehensive, multiphase laboratory evaluation of the InBios Active Melioidosis D... more We conducted a comprehensive, multiphase laboratory evaluation of the InBios Active Melioidosis Detect (AMD) rapid test, a lateral flow immunoassay designed to detect capsular polysaccharides produced by Burkholderia mallei or Burkholderia pseudomallei, used in conjunction with the Omni Array Reader for the rapid identification of culture isolates of B mallei or B pseudomallei to support clinical diagnosis for response and triage during a mass casualty event, such as a biological attack. The study was conducted at 2 sites to assess the performance of the AMD test. The sensitivity, specificity, and reproducibility of the assay was determined using 5 replicates of 35 inclusivity strains and 64 clinical background strains. A total of 520 tests were performed, which included both positive and negative controls. Results obtained visually and with the Omni Array Reader showed a sensitivity of 92.3% for B mallei and 95.6% for B pseudomallei; no cross-reactivity was observed with any of the 64 clinical background organisms. The results from this study indicate that the AMD test for the presumptive identification of B mallei and B pseudomallei isolates to support clinical diagnosis is highly robust, specific, and sensitive. This evaluation supports the use of this test as a rapid, qualitative assay for the presumptive identification of B mallei and B pseudomallei in a clinical setting.

Emerging Infectious Diseases

SSRN Electronic Journal, 2021

PLOS ONE, 2020

The increasing prevalence of individuals with multiple food allergies and the need to distinguish... more The increasing prevalence of individuals with multiple food allergies and the need to distinguish between foods containing homologous, cross-reactive proteins have made the use of single-analyte antibody-based methods (e.g., ELISAs) sometimes insufficient. These issues have resulted in the need to conduct multiple analyses and sometimes employ orthogonal methods like mass spectrometry or DNA-based methods for confirmatory purposes. The xMAP Food Allergen Detection Assay (xMAP FADA) was developed to solve this problem while also providing increased throughput and a modular design suitable for adapting to changes in analytical needs. The use of built-in redundancy provides the xMAP FADA with built-in confirmatory analytical capability by including complementary antibody bead sets and secondary analytical end points (e.g., ratio analysis and multi-antibody profiling). A measure of a method's utility is its performance when employed by analysts of varying expertise in multiple laboratory environments. To gauge this aspect, a multi-laboratory validation (MLV) was conducted with 11 participants of different levels of proficiency. The MLV entailed the analysis of incurred food samples in four problematic food matrices, meat sausage, orange juice, baked muffins, and dark chocolate. Except for a couple of instances, involving two confirmatory components in the analysis of baked muffins, the allergenic foods were detected by all participants at concentrations in the analytical samples comparable to � 10 μg/g in the original food sample. In addition, despite high levels of inter-lab variance in the absolute intensities of the responses, the intralaboratory reproducibility was sufficient to support analyses based on the calibration standards and direct comparison controls (DCCs) analyzed alongside the samples. In contrast, ratio analyses displayed inter-laboratory %CV (RSD R) values < 20%; presumably because the ratios are based on inherent properties of the antigenic elements. The

Analytical Chemistry, 2019

Single-domain antibodies (sdAb), recombinantly produced variable heavy domains derived from the u... more Single-domain antibodies (sdAb), recombinantly produced variable heavy domains derived from the unconventional heavy chain antibodies found in camelids, provide stable, well-expressed binding elements with excellent affinity that can be tailored for specific applications through protein engineering. Complex matrices, such as plasma and serum, can dramatically reduce assay sensitivity. Thus, to achieve highly sensitive detection in complex matrices a highly efficient assay is essential. We produced sdAb as genetically linked dimers, and trimers, each including SpyTag at their C-terminus. The constructs were immobilized onto dyed magnetic microspheres to which SpyCatcher had been coupled and characterized in terms of their performance as capture reagents in sandwich assays. Initial tests on the ability of oriented monomer, dimer, and trimer captures to improve detection versus unoriented constructs in an assay for staphylococcal enterotoxin B spiked into buffer showed the oriented dimer format provided the best sensitivity while offering robust protein production. Thus, this format was utilized to improve a sdAb-based assay for the detection of dengue virus (DENV) nonstructural protein 1 (NS1) in serum. Detection of NS1 from each of the four DENV serotypes spiked into 50% normal human serum was increased by at least a factor of 5 when using the oriented dimer capture. We then demonstrated the potential of using the oriented dimer capture to improve detection of NS1 in clinical samples. This general method should enhance the utility of sdAb incorporated into any diagnostic assay, including those for high consequence pathogens.

Prevention, Early Detection, and Interception, 2019

Many cancers overexpress FASN which catalyzes the final synthetic step of de novo fatty acid synt... more Many cancers overexpress FASN which catalyzes the final synthetic step of de novo fatty acid synthesis pathway. FASN has been considered as a potential drug target for several decades now. Many publications provide evidence of the elevated FASN with aggressive tumors, but still, there are very few inhibitors of FASN that have successfully been utilized as therapeutics in the clinical arena. An accurate measurement of FASN in association with tumor-specific markers in solid tumors and serum may help to propel forward the therapeutic significance of FASN inhibitors. With this objective in mind, we are currently working on the development of a multiplex assay for detection and quantitation of FASN and Cripto-1 in serum samples. We have optimized a magnetic fluorescent bead based multiplex sandwich immunoassay for detection and quantitation of FASN and Cripto-1 (CR-1) in human serum and plasma samples. We selected Cripto-1 to pair with FASN because it is an EGF-like domain carrying growth factor associated with a number of different types of human carcinomas. We use two different monoclonal antibodies for FASN and one for CR-1 as capture antibodies covalently coupled to magnetic fluorescent beads and a mixture of two biotin-labeled detection antibodies in this assay. The streptavidin-phycoerythrin (SAPE) conjugate is used to report the positive capture of a target in this assay. We have also included various internal controls in this assay to monitor the assay performance and assure quality of the reagents used in each well. The dynamic range of this assay was determined to be 100pg/mL to 100 ng/mL using recombinant FASN and Cripto-1 from commercial sources. The R2 values were 0.98 and above for each of the analytes and recovery of each standard were between 90 % and 125%. We spiked recombinant FASN and Cripto-1 in normal human serum and plasma at different concentrations and spike recovery was between 70 % and 130 %. We further evaluated this assay with some commercially obtained clinical samples. This presentation includes detailed results of this study. Citation Format: Neeraja Venkateswaran, William M. Nelson. A novel multiplex assay for detection and quantitation of fatty acid synthase (FASN) in serum or plasma samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-248.

Journal of immunological methods, 2018

Advances in high-throughput proteomic approaches have provided substantial momentum to novel dise... more Advances in high-throughput proteomic approaches have provided substantial momentum to novel disease-biomarker discovery research and have augmented the quality of clinical studies. Applications based on multiplexed microsphere suspension array technology are making strong in-roads into the clinical diagnostic/prognostic practice. Conventional proteomic approaches are designed to discover a broad set of proteins that are associated with a specific medical condition. In comparison, multiplex microsphere immunoassays use quantitative measurements of selected set(s) of specific/particular molecular markers such as cytokines, chemokines, pathway signaling or disease-specific markers for detection, metabolic disorders, cancer, and infectious agents causing human, plant and animal diseases. This article provides a foundation to the multiplexed microsphere suspension array technology, with an emphasis on the improvements in the technology, data analysis approaches, and applications to tran...

DESCRIPTION Multiplex PCR assay for detection of BoNT A and BoNT B on T-COR 8

Introduction: Dengue virus (DENV) is a mosquito borne human virus causing both asymptomatic and s... more Introduction: Dengue virus (DENV) is a mosquito borne human virus causing both asymptomatic and severe infection in tropical and subtropical regions of the world. A rapid detection of the virus is critical to patient management and for surveillance of disease. A dry format quantitative reverse transcriptase polymerase chain reaction assay was developed for field use and systematically evaluated previously using Cepheid SmartCycler platform for rapid detection of DENV1. In the current study we optimized this assay for use with T-COR 8™ integrated system that includes a portable thermocycler, simple sample collection and processing devices to detect DENV in whole blood samples directly. The objective of the study was to develop an assay suitable for not only the laboratory settings but also for point-of-care settings, low resource settings and austere environments. Methods: The assay specificity was previously tested by using a panel of related flaviviruses. Eighty one confirmed dengu...

Botulinum neurotoxins (BoNT) are some of the most lethal bacterial toxins that are usually identi... more Botulinum neurotoxins (BoNT) are some of the most lethal bacterial toxins that are usually identified in form of botulism through clinical manifestations and diagnosis that is subsequently confirmed by various laboratory tests. Several new methods are being developed and validated for the identification of BoNT in foods, environmental or clinical samples which combine immunoassays and PCR based assays that improve the sensitivity of detection and reduce the time required. In addition, these methods are user-friendly, with minimal training requirements, and are field deployable. This presentation describes the use of a novel field-portable real time PCR thermal cycler (Tetracore T-COR 8™) for the rapid detection of BoNT A. The T-COR 8 is an eight well field portable, battery operated, four/five color instrument that weighs less than 10 pounds, and the system incorporates room temperature stable dry RT-PCR reagents. The T-COR 8 allows for remote access control and data analysis via Et...

ABSTRACT Introduction: Bacillus anthracis (Ba) spores can survive harsh conditions of temperature... more ABSTRACT Introduction: Bacillus anthracis (Ba) spores can survive harsh conditions of temperature, humidity and ultraviolet radiation and are the predominant form in the environment. On entry into host, it germinates to vegetative cell form and becomes infective to cause anthrax disease. Inhalational, cutaneous and intestinal anthrax are found in humans and many animals. This study describes development of differential detection of Ba spores and vegetative cells using panels of monoclonal antibodies and polyclonal antibodies in a multiplex assay format. Methods: A multiplex sandwich immunoassay was designed using fluorescent microspheres coupled with various capture antibodies. Three mouse monoclonal antibodies that react with high specificity with EA1 protein, which is well known as a major S-layer component of vegetative cells and is tightly bound to spore surfaces even after extensive washes, and a spore specific monoclonal antibody directed against glycosyl hydrolase were used a...

DESCRIPTION Detection of DENV and CHIKV using T-COR 8

PLoS ONE, 2013

Serodiagnosis of arthropod-borne viruses (arboviruses) at the Division of Vector-Borne Diseases, ... more Serodiagnosis of arthropod-borne viruses (arboviruses) at the Division of Vector-Borne Diseases, CDC, employs a combination of individual enzyme-linked immunosorbent assays and microsphere immunoassays (MIAs) to test for IgM and IgG, followed by confirmatory plaque-reduction neutralization tests. Based upon the geographic origin of a sample, it may be tested concurrently for multiple arboviruses, which can be a cumbersome task. The advent of multiplexing represents an opportunity to streamline these types of assays; however, because serologic crossreactivity of the arboviral antigens often confounds results, it is of interest to employ data analysis methods that address this issue. Here, we constructed 13-virus multiplexed IgM and IgG MIAs that included internal and external controls, based upon the Luminex platform. Results from samples tested using these methods were analyzed using 8 different statistical schemes to identify the best way to classify the data. Geographic batteries were also devised to serve as a more practical diagnostic format, and further samples were tested using the abbreviated multiplexes. Comparative error rates for the classification schemes identified a specific boosting method based on logistic regression "Logitboost" as the classification method of choice. When the data from all samples tested were combined into one set, error rates from the multiplex IgM and IgG MIAs were <5% for all geographic batteries. This work represents both the most comprehensive, validated multiplexing method for arboviruses to date, and also the most systematic attempt to determine the most useful classification method for use with these types of serologic tests.

Acta microbiologica Hungarica, 1992

A modification that simplifies the spot hybridization technique is described for using biotinylat... more A modification that simplifies the spot hybridization technique is described for using biotinylated DNA probes. Plasmid EWD299 having LT gene insert, labelled with biotin either by nick translation or using photobiotin was used as DNA probe for the specific detection of enterotoxigenic Escherichia coli. A simple protocol has been described for easy lysis of test samples by boiling in distilled water followed by detergent treatment and was found to be as efficient as the lysis using lysozyme and protease. Three different solid supports namely DEAE-cellulose paper, nitrocellulose paper and nylon membrane were also compared for their suitability in this spot hybridization test. Nitrocellulose paper was found to give better colour signal with the photobiotinylated DNA probe.

Biochemistry and molecular biology international, 1994

The degradation of bacteriophage lambda (lambda) deoxyribonucleic acid (DNA) by interaction with ... more The degradation of bacteriophage lambda (lambda) deoxyribonucleic acid (DNA) by interaction with 0.1, 0.5 and 1 mM concentrations of sulfur mustard (SM) was investigated using agarose gel electrophoresis. Alkaline agarose gel electrophoresis also revealed single strand breaks at 0.5 and 0.1 mM concentrations of SM. The presence of magnesium ions in the reaction mixture prevented DNA degradation. It is proposed that the degradation of lambda DNA by its interaction with an excess of SM may be caused by the breakage of phosphodiester backbone of DNA via the formation of an intermediate phosphotriester bond.

Defence Science Journal, 1991

Single-stranded nucleic acid molecules can interact under certain conditions with other nucleic a... more Single-stranded nucleic acid molecules can interact under certain conditions with other nucleic acids in the regions of base complementarity to form double-stranded hybrid molecules. From measurement of kinetics of the hybridisation anti the extent of pybrid formation, information on nucleotide sequence heterogeneity of nucleic acid fractions, sequence properties of purified classes of molccules or the degree of homology between different samples of nucleic acids can be obtained. A specific deoxyribonucleic acid or ribonucleic acid sequence with an identifiable marker can be used as a nucleic acid probe. This paper reviews the use of nucleic acid probes for classifying the microorganisms and their detection in clinical or environmcntal samples.

The Journal of Immunology

Arboviruses cause a serious public health problem in tropical and subtropical countries and are e... more Arboviruses cause a serious public health problem in tropical and subtropical countries and are emerging as a threat in western hemisphere also. Rapid and accurate detection of these infections can help in improving patient care and disease control. The cross-reactivity of immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies to ZIKV with dengue virus (DENV), West Nile virus (WNV), yellow fever virus (YFV) and some other related flaviviruses poses a significant challenge for an accurate detection of ZIKV infection in serological assay such as IgM capture ELISA. To overcome these challenges we developed a multiplex serological assay that will simultaneously detect antibody response to ZIKV, other related flaviviruses and Chikungunya virus (CHIKV). Various recombinant arboviral antigens were coupled to optically encoded microspheres from Luminex Corporation. This fluorescent microsphere immunoassay was used to differentiate between recent, past, and/or co-infections that may oc...

Kesmas: Jurnal Kesehatan Masyarakat Nasional

The SARS-CoV-2 transmission dynamics in low- and middle-income countries remain poorly understood... more The SARS-CoV-2 transmission dynamics in low- and middle-income countries remain poorly understood. This study aimed to estimate the SARS-CoV-2 antibodies seroprevalence in Jakarta, Indonesia, and to increase knowledge of SARS-CoV-2 transmission in urban settings. A population-based serosurvey among individuals aged one year or older was conducted in Jakarta. Employing a multistage sampling design, samples were stratified by district, slum, and non-slum residency, sex, and age group. Blood samples were tested for IgG against three different SARS-CoV-2 antigens. Seroprevalence was estimated after applying sample weights and adjusting for cluster characteristics. In March 2021, this study collected 4,919 respondents. The weighted estimate of seroprevalence was 44.5% (95% CI = 42.5-46.5). Seroprevalence was highest among adults aged 30-49 years, with higher seroprevalence in women and the overweight/obese group. Respondents residing in slum areas were 1.3-fold more likely to be seroposi...

Health Security

We conducted a comprehensive, multiphase laboratory evaluation of InBios Active Melioidosis Detec... more We conducted a comprehensive, multiphase laboratory evaluation of InBios Active Melioidosis Detect (AMD) rapid test, a lateral flow immunoassay designed to detect capsular polysaccharides produced by Burkholderia mallei or Burkholderia pseudomallei, used in conjunction with the Omni Array Reader (OAR) for the rapid detection of B mallei or B pseudomallei in environmental (nonclinical) samples at 2 sites. The limit of detection, using reference strains B mallei strain ATCC 23344 and B pseudomallei strain ATCC 11668, was determined to be 10 3 to 10 4 CFU/mL. In different phases of the evaluation, inclusivity strains that included geographically diverse strains of B mallei (N = 13) and B pseudomallei (N = 22), geographically diverse phylogenetic near neighbor strains (N = 66), environmental background strains (N = 64), white powder samples (N = 26), and environmental filter extracts (N = 1 pooled sample from 10 filter extracts) were also tested. A total of 1,753 tests were performed, which included positive and negative controls. Visual and OAR results showed that the AMD test detected 92.3% of B mallei and 95.5% of B pseudomallei strains. Of the 66 near-neighbor strains tested, cross-reactivity was observed with only B stabilis 2008724195 and B thailandensis 2003015869. Overall, the specificity and sensitivity were 98.8% and 98.7%, respectively. The results of this evaluation support the use of the AMD test as a rapid, qualitative assay for the presumptive detection of B mallei and B pseudomallei in suspicious environmental samples such as white powders and aerosol samples by first responders and laboratory personnel.

Health Security

We conducted a comprehensive, multiphase laboratory evaluation of the InBios Active Melioidosis D... more We conducted a comprehensive, multiphase laboratory evaluation of the InBios Active Melioidosis Detect (AMD) rapid test, a lateral flow immunoassay designed to detect capsular polysaccharides produced by Burkholderia mallei or Burkholderia pseudomallei, used in conjunction with the Omni Array Reader for the rapid identification of culture isolates of B mallei or B pseudomallei to support clinical diagnosis for response and triage during a mass casualty event, such as a biological attack. The study was conducted at 2 sites to assess the performance of the AMD test. The sensitivity, specificity, and reproducibility of the assay was determined using 5 replicates of 35 inclusivity strains and 64 clinical background strains. A total of 520 tests were performed, which included both positive and negative controls. Results obtained visually and with the Omni Array Reader showed a sensitivity of 92.3% for B mallei and 95.6% for B pseudomallei; no cross-reactivity was observed with any of the 64 clinical background organisms. The results from this study indicate that the AMD test for the presumptive identification of B mallei and B pseudomallei isolates to support clinical diagnosis is highly robust, specific, and sensitive. This evaluation supports the use of this test as a rapid, qualitative assay for the presumptive identification of B mallei and B pseudomallei in a clinical setting.

Emerging Infectious Diseases

SSRN Electronic Journal, 2021

PLOS ONE, 2020

The increasing prevalence of individuals with multiple food allergies and the need to distinguish... more The increasing prevalence of individuals with multiple food allergies and the need to distinguish between foods containing homologous, cross-reactive proteins have made the use of single-analyte antibody-based methods (e.g., ELISAs) sometimes insufficient. These issues have resulted in the need to conduct multiple analyses and sometimes employ orthogonal methods like mass spectrometry or DNA-based methods for confirmatory purposes. The xMAP Food Allergen Detection Assay (xMAP FADA) was developed to solve this problem while also providing increased throughput and a modular design suitable for adapting to changes in analytical needs. The use of built-in redundancy provides the xMAP FADA with built-in confirmatory analytical capability by including complementary antibody bead sets and secondary analytical end points (e.g., ratio analysis and multi-antibody profiling). A measure of a method's utility is its performance when employed by analysts of varying expertise in multiple laboratory environments. To gauge this aspect, a multi-laboratory validation (MLV) was conducted with 11 participants of different levels of proficiency. The MLV entailed the analysis of incurred food samples in four problematic food matrices, meat sausage, orange juice, baked muffins, and dark chocolate. Except for a couple of instances, involving two confirmatory components in the analysis of baked muffins, the allergenic foods were detected by all participants at concentrations in the analytical samples comparable to � 10 μg/g in the original food sample. In addition, despite high levels of inter-lab variance in the absolute intensities of the responses, the intralaboratory reproducibility was sufficient to support analyses based on the calibration standards and direct comparison controls (DCCs) analyzed alongside the samples. In contrast, ratio analyses displayed inter-laboratory %CV (RSD R) values < 20%; presumably because the ratios are based on inherent properties of the antigenic elements. The

Analytical Chemistry, 2019

Single-domain antibodies (sdAb), recombinantly produced variable heavy domains derived from the u... more Single-domain antibodies (sdAb), recombinantly produced variable heavy domains derived from the unconventional heavy chain antibodies found in camelids, provide stable, well-expressed binding elements with excellent affinity that can be tailored for specific applications through protein engineering. Complex matrices, such as plasma and serum, can dramatically reduce assay sensitivity. Thus, to achieve highly sensitive detection in complex matrices a highly efficient assay is essential. We produced sdAb as genetically linked dimers, and trimers, each including SpyTag at their C-terminus. The constructs were immobilized onto dyed magnetic microspheres to which SpyCatcher had been coupled and characterized in terms of their performance as capture reagents in sandwich assays. Initial tests on the ability of oriented monomer, dimer, and trimer captures to improve detection versus unoriented constructs in an assay for staphylococcal enterotoxin B spiked into buffer showed the oriented dimer format provided the best sensitivity while offering robust protein production. Thus, this format was utilized to improve a sdAb-based assay for the detection of dengue virus (DENV) nonstructural protein 1 (NS1) in serum. Detection of NS1 from each of the four DENV serotypes spiked into 50% normal human serum was increased by at least a factor of 5 when using the oriented dimer capture. We then demonstrated the potential of using the oriented dimer capture to improve detection of NS1 in clinical samples. This general method should enhance the utility of sdAb incorporated into any diagnostic assay, including those for high consequence pathogens.

Prevention, Early Detection, and Interception, 2019

Many cancers overexpress FASN which catalyzes the final synthetic step of de novo fatty acid synt... more Many cancers overexpress FASN which catalyzes the final synthetic step of de novo fatty acid synthesis pathway. FASN has been considered as a potential drug target for several decades now. Many publications provide evidence of the elevated FASN with aggressive tumors, but still, there are very few inhibitors of FASN that have successfully been utilized as therapeutics in the clinical arena. An accurate measurement of FASN in association with tumor-specific markers in solid tumors and serum may help to propel forward the therapeutic significance of FASN inhibitors. With this objective in mind, we are currently working on the development of a multiplex assay for detection and quantitation of FASN and Cripto-1 in serum samples. We have optimized a magnetic fluorescent bead based multiplex sandwich immunoassay for detection and quantitation of FASN and Cripto-1 (CR-1) in human serum and plasma samples. We selected Cripto-1 to pair with FASN because it is an EGF-like domain carrying growth factor associated with a number of different types of human carcinomas. We use two different monoclonal antibodies for FASN and one for CR-1 as capture antibodies covalently coupled to magnetic fluorescent beads and a mixture of two biotin-labeled detection antibodies in this assay. The streptavidin-phycoerythrin (SAPE) conjugate is used to report the positive capture of a target in this assay. We have also included various internal controls in this assay to monitor the assay performance and assure quality of the reagents used in each well. The dynamic range of this assay was determined to be 100pg/mL to 100 ng/mL using recombinant FASN and Cripto-1 from commercial sources. The R2 values were 0.98 and above for each of the analytes and recovery of each standard were between 90 % and 125%. We spiked recombinant FASN and Cripto-1 in normal human serum and plasma at different concentrations and spike recovery was between 70 % and 130 %. We further evaluated this assay with some commercially obtained clinical samples. This presentation includes detailed results of this study. Citation Format: Neeraja Venkateswaran, William M. Nelson. A novel multiplex assay for detection and quantitation of fatty acid synthase (FASN) in serum or plasma samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-248.

Journal of immunological methods, 2018

Advances in high-throughput proteomic approaches have provided substantial momentum to novel dise... more Advances in high-throughput proteomic approaches have provided substantial momentum to novel disease-biomarker discovery research and have augmented the quality of clinical studies. Applications based on multiplexed microsphere suspension array technology are making strong in-roads into the clinical diagnostic/prognostic practice. Conventional proteomic approaches are designed to discover a broad set of proteins that are associated with a specific medical condition. In comparison, multiplex microsphere immunoassays use quantitative measurements of selected set(s) of specific/particular molecular markers such as cytokines, chemokines, pathway signaling or disease-specific markers for detection, metabolic disorders, cancer, and infectious agents causing human, plant and animal diseases. This article provides a foundation to the multiplexed microsphere suspension array technology, with an emphasis on the improvements in the technology, data analysis approaches, and applications to tran...

DESCRIPTION Multiplex PCR assay for detection of BoNT A and BoNT B on T-COR 8

Introduction: Dengue virus (DENV) is a mosquito borne human virus causing both asymptomatic and s... more Introduction: Dengue virus (DENV) is a mosquito borne human virus causing both asymptomatic and severe infection in tropical and subtropical regions of the world. A rapid detection of the virus is critical to patient management and for surveillance of disease. A dry format quantitative reverse transcriptase polymerase chain reaction assay was developed for field use and systematically evaluated previously using Cepheid SmartCycler platform for rapid detection of DENV1. In the current study we optimized this assay for use with T-COR 8™ integrated system that includes a portable thermocycler, simple sample collection and processing devices to detect DENV in whole blood samples directly. The objective of the study was to develop an assay suitable for not only the laboratory settings but also for point-of-care settings, low resource settings and austere environments. Methods: The assay specificity was previously tested by using a panel of related flaviviruses. Eighty one confirmed dengu...

Botulinum neurotoxins (BoNT) are some of the most lethal bacterial toxins that are usually identi... more Botulinum neurotoxins (BoNT) are some of the most lethal bacterial toxins that are usually identified in form of botulism through clinical manifestations and diagnosis that is subsequently confirmed by various laboratory tests. Several new methods are being developed and validated for the identification of BoNT in foods, environmental or clinical samples which combine immunoassays and PCR based assays that improve the sensitivity of detection and reduce the time required. In addition, these methods are user-friendly, with minimal training requirements, and are field deployable. This presentation describes the use of a novel field-portable real time PCR thermal cycler (Tetracore T-COR 8™) for the rapid detection of BoNT A. The T-COR 8 is an eight well field portable, battery operated, four/five color instrument that weighs less than 10 pounds, and the system incorporates room temperature stable dry RT-PCR reagents. The T-COR 8 allows for remote access control and data analysis via Et...

ABSTRACT Introduction: Bacillus anthracis (Ba) spores can survive harsh conditions of temperature... more ABSTRACT Introduction: Bacillus anthracis (Ba) spores can survive harsh conditions of temperature, humidity and ultraviolet radiation and are the predominant form in the environment. On entry into host, it germinates to vegetative cell form and becomes infective to cause anthrax disease. Inhalational, cutaneous and intestinal anthrax are found in humans and many animals. This study describes development of differential detection of Ba spores and vegetative cells using panels of monoclonal antibodies and polyclonal antibodies in a multiplex assay format. Methods: A multiplex sandwich immunoassay was designed using fluorescent microspheres coupled with various capture antibodies. Three mouse monoclonal antibodies that react with high specificity with EA1 protein, which is well known as a major S-layer component of vegetative cells and is tightly bound to spore surfaces even after extensive washes, and a spore specific monoclonal antibody directed against glycosyl hydrolase were used a...

DESCRIPTION Detection of DENV and CHIKV using T-COR 8

PLoS ONE, 2013

Serodiagnosis of arthropod-borne viruses (arboviruses) at the Division of Vector-Borne Diseases, ... more Serodiagnosis of arthropod-borne viruses (arboviruses) at the Division of Vector-Borne Diseases, CDC, employs a combination of individual enzyme-linked immunosorbent assays and microsphere immunoassays (MIAs) to test for IgM and IgG, followed by confirmatory plaque-reduction neutralization tests. Based upon the geographic origin of a sample, it may be tested concurrently for multiple arboviruses, which can be a cumbersome task. The advent of multiplexing represents an opportunity to streamline these types of assays; however, because serologic crossreactivity of the arboviral antigens often confounds results, it is of interest to employ data analysis methods that address this issue. Here, we constructed 13-virus multiplexed IgM and IgG MIAs that included internal and external controls, based upon the Luminex platform. Results from samples tested using these methods were analyzed using 8 different statistical schemes to identify the best way to classify the data. Geographic batteries were also devised to serve as a more practical diagnostic format, and further samples were tested using the abbreviated multiplexes. Comparative error rates for the classification schemes identified a specific boosting method based on logistic regression "Logitboost" as the classification method of choice. When the data from all samples tested were combined into one set, error rates from the multiplex IgM and IgG MIAs were <5% for all geographic batteries. This work represents both the most comprehensive, validated multiplexing method for arboviruses to date, and also the most systematic attempt to determine the most useful classification method for use with these types of serologic tests.

Acta microbiologica Hungarica, 1992

A modification that simplifies the spot hybridization technique is described for using biotinylat... more A modification that simplifies the spot hybridization technique is described for using biotinylated DNA probes. Plasmid EWD299 having LT gene insert, labelled with biotin either by nick translation or using photobiotin was used as DNA probe for the specific detection of enterotoxigenic Escherichia coli. A simple protocol has been described for easy lysis of test samples by boiling in distilled water followed by detergent treatment and was found to be as efficient as the lysis using lysozyme and protease. Three different solid supports namely DEAE-cellulose paper, nitrocellulose paper and nylon membrane were also compared for their suitability in this spot hybridization test. Nitrocellulose paper was found to give better colour signal with the photobiotinylated DNA probe.

Biochemistry and molecular biology international, 1994

The degradation of bacteriophage lambda (lambda) deoxyribonucleic acid (DNA) by interaction with ... more The degradation of bacteriophage lambda (lambda) deoxyribonucleic acid (DNA) by interaction with 0.1, 0.5 and 1 mM concentrations of sulfur mustard (SM) was investigated using agarose gel electrophoresis. Alkaline agarose gel electrophoresis also revealed single strand breaks at 0.5 and 0.1 mM concentrations of SM. The presence of magnesium ions in the reaction mixture prevented DNA degradation. It is proposed that the degradation of lambda DNA by its interaction with an excess of SM may be caused by the breakage of phosphodiester backbone of DNA via the formation of an intermediate phosphotriester bond.

Defence Science Journal, 1991

Single-stranded nucleic acid molecules can interact under certain conditions with other nucleic a... more Single-stranded nucleic acid molecules can interact under certain conditions with other nucleic acids in the regions of base complementarity to form double-stranded hybrid molecules. From measurement of kinetics of the hybridisation anti the extent of pybrid formation, information on nucleotide sequence heterogeneity of nucleic acid fractions, sequence properties of purified classes of molccules or the degree of homology between different samples of nucleic acids can be obtained. A specific deoxyribonucleic acid or ribonucleic acid sequence with an identifiable marker can be used as a nucleic acid probe. This paper reviews the use of nucleic acid probes for classifying the microorganisms and their detection in clinical or environmcntal samples.