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Background: On March 11, 2020, the World Health Organization declared a pandemic caused by the recently emerged severe acute respiratory syndrome coronavirus 2 (SARSCoV-2). This led to increased clinical testing and decentralizing of this testing from provincial health laboratories to regional and private facilities. Leveraging the results from the Canadian Laboratory Response Network's National SARS-CoV-2 Proficiency Test (PT) Program, this study compares multiple commercial and laboratory-developed nucleic acid amplification tests, assessing both sensitivity and specificity across multiple users. Methods: Each panel consisted of six blinded, contrived-clinical samples. Panels were distributed to international, provincial and territorial laboratories and subsequently to partner facilities. Participating laboratories were asked to run these sample through their respective extraction/PCR workflows and submit results to the National Microbiology Laboratory, outlining the nucleic acid extraction platform and nucleic acid amplification test employed, as well as the viral gene target and Ct values or equivalent obtained. Data were compiled for each molecular platform and gene target used. Results: The PT schemes were deployed in May 2020, November 2020 and June 2021, resulting in 683 data sets using 37 different nucleic acid amplification tests. Over the course of three PT schemes, the average score obtained was 99.3% by participants demonstrating consistent testing between laboratories and testing platforms. Conclusion: This study confirmed the rapid and successful implementation of a Canadian PT Program and provided comparative analysis of the various emergency use authorized and laboratory developed tests employed for the detection of SARS-CoV-2 and demonstrated an overall 99.3% test concordance nationwide. [ABSTRACT FROM AUTHOR]

To the Editor: Melioidosis refers to infection caused by the facultative intracellular gram-negative bacterium Burkholderia pseudomallei. The clinical manifestations of melioidosis span a wide spectrum, from asymptomatic exposure or localized [...]

Background: The use of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) systems for bacterial identification has rapidly become a front line tool for diagnostic laboratories, superseding classical microbiological methods that previously triggered the identification of higher risk pathogens. Unknown Risk Group 3 isolates have been misidentified as less pathogenic species due to spectral library availability, content and quality. Consequently, exposure to higher risk pathogens has been reported within Canadian laboratory staff following the implementation of MALDI-TOF MS. This overview aims to communicate the potential risk to laboratory staff of inaccurate identification of security-sensitive biological agents (SSBA) bacteria and to provide suggestions to mitigate.Methods: Cultures were manipulated in a Biosafety Level 3 laboratory, prepared for MALDI-TOF MS analysis via full chemical extraction and analysed on a Bruker Microflex LT instrument. Data were analyzed with Biotyper software; comparing raw spectra against MS profiles in three libraries: Bruker Taxonomy; Bruker Security-Restricted; and National Microbiology Laboratory (NML) SSBA libraries. Four years of Bruker MALDI-TOF MS data acquired in-house were reviewed.Results: In general, the Bruker MS spectral libraries were less successful in identifying the SSBA bacteria. More successful was the NML library. For example, using a high score cut-off (greater than 2.0), the Bruker SR library was unable to identify 52.8% of our Risk Group 3 agents and near neighbours to the species-level with confidence, whereas the custom NML library was unable to identify only 20.3% of the samples.Conclusion: The last four years of data demonstrated both the importance of library selection and the limitations of the various spectral libraries. Enhanced standard operating procedures are advised to reduce laboratory exposure to SSBAs when using MALDI-TOF MS as a front line identification tool. [ABSTRACT FROM AUTHOR]

Botulinum neurotoxins (BoNTs) are extremely potent toxins that can contaminate foods and are a public health concern. Anti-BoNT antibodies have been described that are capable of detecting BoNTs; however there still exists a need for accurate and sensitive detection capabilities for BoNTs. Herein, we describe the characterization of a panel of eight monoclonal antibodies (MAbs) generated to the non-toxic receptor-binding domain of BoNT/A (HC50/A) developed using a high-throughput screening approach. In two independent hybridoma fusions, two groups of four IgG MAbs were developed against recombinant HC50/A. Of these eight, only a single MAb, F90G5-3, bound to the whole BoNT/A protein and was characterized further. The F90G5-3 MAb slightly prolonged time to death in an in vivo mouse bioassay and was mapped by pepscan to a peptide epitope in the N-terminal subdomain of HC50/A (HCN25/A) comprising amino acid residues 985WTLQDTQEIKQRVVF999, an epitope that is highly immunoreactive in humans. Furthermore, we demonstrate that F90G5-3 binds BoNT/A with nanomolar efficiency. Together, our results indicate that F90G5-3 is of potential value as a diagnostic immunoreagent for BoNT/A capture assay development and bio-forensic analysis. [ABSTRACT FROM AUTHOR]

Antibodies against the protective antigen (PA) of Bacillus anthracis play a key role in response to infection by this important pathogen. The aim of this study was to produce and characterize monoclonal antibodies (mAbs) specific for PA and to identify novel neutralizing epitopes. Three murine mAbs with high specificity and nanomolar affinity for B. anthracis recombinant protective antigen (rPA) were produced and characterized. Western immunoblot analysis, coupled with epitope mapping using overlapping synthetic peptides, revealed that these mAbs recognize a linear epitope within domain 2 of rPA. Neutralization assays demonstrate that these mAbs effectively neutralize lethal toxin in vitro. [ABSTRACT FROM AUTHOR]

Monoclonal antibodies (mAbs) have proven to be instrumental in the advancement of research, diagnostic, industrial vaccine, and therapeutic applications. The use of mAbs in laboratory protocols has been growing in an exponential fashion for the last four decades. Described herein are methods for the development of highly specific mAbs through traditional hybridoma fusion. For ultimate success, a series of simultaneously initiated protocols are to be undertaken with careful attention to cell health of both the myeloma fusion partner and immune splenocytes. Coordination and attention to detail will enable a researcher with basic tissue culture skills to generate mAbs from immunized rodents to a variety of antigens (including proteins, carbohydrates, DNA, and haptens) (see Note 1). Furthermore, in vivo and in vitro methods used for antigen sensitization of splenocytes prior to somatic fusion are described herein.

Described herein are methods for the successful screening of monoclonal antibodies (mAbs) of the desired specificities via high-throughput (HTP) homogeneous assay and flow cytometry. We present a combination of screening techniques that allow the scientist to efficiently eliminate nontarget-specific antibody as soon as possible. This compilation of protocols will enable researchers with basic immunology skills to make decisions regarding the design of screening algorithms for the generation of mAbs. Although we have provided an informative overview of both HTP homogeneous assay and flow cytometry, it is imperative for the beginner to acquire fundamental knowledge on how both of these technologies work so as to use these screening strategies effectively.