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1.
Microbiol Spectr ; 9(2): e0068321, 2021 10 31.
Article in English | MEDLINE | ID: covidwho-1476397

ABSTRACT

Antigen-based rapid diagnostics tests (Ag-RDTs) are useful tools for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection. However, misleading demonstrations of the Abbott Panbio coronavirus disease 2019 (COVID-19) Ag-RDT on social media claimed that SARS-CoV-2 antigen could be detected in municipal water and food products. To offer a scientific rebuttal to pandemic misinformation and disinformation, this study explored the impact of using the Panbio SARS-CoV-2 assay with conditions falling outside manufacturer recommendations. Using Panbio, various water and food products, laboratory buffers, and SARS-CoV-2-negative clinical specimens were tested with and without manufacturer buffer. Additional experiments were conducted to assess the role of each Panbio buffer component (tricine, NaCl, pH, and Tween 20) as well as the impact of temperature (4°C, 20°C, and 45°C) and humidity (90%) on assay performance. Direct sample testing (without the kit buffer) resulted in false-positive signals resembling those obtained with SARS-CoV-2 positive controls tested under proper conditions. The likely explanation of these artifacts is nonspecific interactions between the SARS-CoV-2-specific conjugated and capture antibodies, as proteinase K treatment abrogated this phenomenon, and thermal shift assays showed pH-induced conformational changes under conditions promoting artifact formation. Omitting, altering, and reverse engineering the kit buffer all supported the importance of maintaining buffering capacity, ionic strength, and pH for accurate kit function. Interestingly, the Panbio assay could tolerate some extremes of temperature and humidity outside manufacturer claims. Our data support strict adherence to manufacturer instructions to avoid false-positive SARS-CoV-2 Ag-RDT reactions, otherwise resulting in anxiety, overuse of public health resources, and dissemination of misinformation. IMPORTANCE With the Panbio severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen test being deployed in over 120 countries worldwide, understanding conditions required for its ideal performance is critical. Recently on social media, this kit was shown to generate false positives when manufacturer recommendations were not followed. While erroneous results from improper use of a test may not be surprising to some health care professionals, understanding why false positives occur can help reduce the propagation of misinformation and provide a scientific rebuttal for these aberrant findings. This study demonstrated that the kit buffer's pH, ionic strength, and buffering capacity were critical components to ensure proper kit function and avoid generation of false-positive results. Typically, false positives arise from cross-reacting or interfering substances; however, this study demonstrated a mechanism where false positives were generated under conditions favoring nonspecific interactions between the two antibodies designed for SARS-CoV-2 antigen detection. Following the manufacturer instructions is critical for accurate test results.


Subject(s)
Antigens, Viral/analysis , COVID-19 Serological Testing/methods , Drinking Water/virology , Food/virology , SARS-CoV-2/isolation & purification , Buffers , COVID-19/diagnosis , Communication , False Positive Reactions , Humans , SARS-CoV-2/immunology
3.
J Synchrotron Radiat ; 28(Pt 4): 1237-1244, 2021 Jul 01.
Article in English | MEDLINE | ID: covidwho-1334330

ABSTRACT

During the COVID-19 pandemic, synchrotron beamlines were forced to limit user access. Performing routine measurements became a challenge. At the Life Science X-ray Scattering (LiX) beamline, new instrumentation and mail-in protocols have been developed to remove the access barrier to solution scattering measurements. Our efforts took advantage of existing instrumentation and coincided with the larger effort at NSLS-II to support remote measurements. Given the limited staff-user interaction for mail-in measurements, additional software tools have been developed to ensure data quality, to automate the adjustments in data processing, as users would otherwise rely on the experience of the beamline staff, and produce a summary of the initial assessments of the data. This report describes the details of these developments.


Subject(s)
Scattering, Small Angle , Solutions/radiation effects , Synchrotrons/instrumentation , X-Ray Diffraction/instrumentation , Buffers , COVID-19 , Data Collection , Datasets as Topic , Electronic Data Processing , Pandemics , Robotics , SARS-CoV-2 , Software , Specimen Handling , Water
4.
J Immunol Methods ; 496: 113099, 2021 09.
Article in English | MEDLINE | ID: covidwho-1292808

ABSTRACT

Bispecific antibodies (BsAbs) are engineered to simultaneously bind two different antigens, and offer promising clinical outcomes for various diseases. The dual binding properties of BsAbs may enable superior efficacies and/or potencies compared to standard monoclonal antibodies (mAbs) or combination mAb therapies. Characterizing BsAb binding properties is critical during biotherapeutic development, where data is leveraged to predict efficacy and potency, assess critical quality attributes and improve antibody design. Traditional single-target, single-readout approaches (e.g., ELISA) have limited usefulness for interpreting complex bispecific binding, and double the benchwork. To address these deficiencies, we developed and implemented a new dual-target/readout binding assay that accurately dissects the affinities of both BsAb binding domains directly and simultaneously. This new assay uses AlphaPlex® technology, which eliminates traditional ELISA wash steps and can be miniaturized for automated workflows. The optimized BsAb AlphaPlex assay demonstrates 99-107% accuracy within a 50-150% linear range, and detected >50% binding degradation from photo- and thermal stress conditions. To the best of our knowledge, this is the first instance of a dual-target/readout BsAb AlphaPlex assay with GMP-suitable linear range, accuracy, specificity, and stability-indicating properties. As a highly customizable and efficient assay, BsAb AlphaPlex may be applicable to numerous bispecific formats and/or co-formulations against a variety of antigens beyond the clinical therapeutic space.


Subject(s)
Antibodies, Bispecific/immunology , Antibody Specificity , Antigens/immunology , CTLA-4 Antigen/immunology , Immunoassay , Programmed Cell Death 1 Receptor/immunology , Antibodies, Bispecific/metabolism , Antigen-Antibody Complex , Antigens/metabolism , Binding Sites, Antibody , Buffers , CTLA-4 Antigen/metabolism , Enzyme-Linked Immunosorbent Assay , Epitopes , Humans , Hydrogen-Ion Concentration , Kinetics , Predictive Value of Tests , Programmed Cell Death 1 Receptor/metabolism , Protein Binding , Reproducibility of Results
5.
Sci Rep ; 11(1): 9387, 2021 04 30.
Article in English | MEDLINE | ID: covidwho-1209076

ABSTRACT

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), calls for prompt and accurate diagnosis and rapid turnaround time for test results to limit transmission. Here, we evaluated two independent molecular assays, the Biomeme SARS-CoV-2 test, and the Precision Biomonitoring TripleLock SARS-CoV-2 test on a field-deployable point-of-care real-time PCR instrument, Franklin three9, in combination with Biomeme M1 Sample Prep Cartridge Kit for RNA 2.0 (M1) manual extraction system for rapid, specific, and sensitive detection of SARS-COV-2 in cell culture, human, and animal clinical samples. The Biomeme SARS-CoV-2 assay, which simultaneously detects two viral targets, the orf1ab and S genes, and the Precision Biomonitoring TripleLock SARS-CoV-2 assay that targets the 5' untranslated region (5' UTR) and the envelope (E) gene of SARS-CoV-2 were highly sensitive and detected as low as 15 SARS-CoV-2 genome copies per reaction. In addition, the two assays were specific and showed no cross-reactivity with Middle Eastern respiratory syndrome coronavirus (MERS-CoV), infectious bronchitis virus (IBV), porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis (TGE) virus, and other common human respiratory viruses and bacterial pathogens. Also, both assays were highly reproducible across different operators and instruments. When used to test animal samples, both assays equally detected SARS-CoV-2 genetic materials in the swabs from SARS-CoV-2-infected hamsters. The M1 lysis buffer completely inactivated SARS-CoV-2 within 10 min at room temperature enabling safe handling of clinical samples. Collectively, these results show that the Biomeme and Precision Biomonitoring TripleLock SARS-CoV-2 mobile testing platforms could reliably and promptly detect SARS-CoV-2 in both human and animal clinical samples in approximately an hour and can be used in remote areas or health care settings not traditionally serviced by a microbiology laboratory.


Subject(s)
COVID-19/diagnosis , Real-Time Polymerase Chain Reaction/methods , SARS-CoV-2/isolation & purification , Animals , Buffers , Cricetinae , Humans , Mobile Applications , Reagent Kits, Diagnostic , SARS-CoV-2/genetics , Sensitivity and Specificity , Time Factors
6.
PLoS One ; 16(3): e0249149, 2021.
Article in English | MEDLINE | ID: covidwho-1158246

ABSTRACT

Conducting numerous, rapid, and reliable PCR tests for SARS-CoV-2 is essential for our ability to monitor and control the current COVID-19 pandemic. Here, we tested the sensitivity and efficiency of SARS-CoV-2 detection in clinical samples collected directly into a mix of lysis buffer and RNA preservative, thus inactivating the virus immediately after sampling. We tested 79 COVID-19 patients and 20 healthy controls. We collected two samples (nasopharyngeal swabs) from each participant: one swab was inserted into a test tube with Viral Transport Medium (VTM), following the standard guideline used as the recommended method for sample collection; the other swab was inserted into a lysis buffer supplemented with nucleic acid stabilization mix (coined NSLB). We found that RT-qPCR tests of patients were significantly more sensitive with NSLB sampling, reaching detection threshold 2.1±0.6 (Mean±SE) PCR cycles earlier then VTM samples from the same patient. We show that this improvement is most likely since NSLB samples are not diluted in lysis buffer before RNA extraction. Re-extracting RNA from NSLB samples after 72 hours at room temperature did not affect the sensitivity of detection, demonstrating that NSLB allows for long periods of sample preservation without special cooling equipment. We also show that swirling the swab in NSLB and discarding it did not reduce sensitivity compared to retaining the swab in the tube, thus allowing improved automation of COVID-19 tests. Overall, we show that using NSLB instead of VTM can improve the sensitivity, safety, and rapidity of COVID-19 tests at a time most needed.


Subject(s)
Limit of Detection , SARS-CoV-2/isolation & purification , Safety , Specimen Handling/methods , Adult , Buffers , Female , Humans , Male , Pandemics , Polymerase Chain Reaction , SARS-CoV-2/genetics , Time Factors
7.
Lab Med ; 52(1): e15-e16, 2021 Jan 04.
Article in English | MEDLINE | ID: covidwho-933866

ABSTRACT

OBJECTIVE: During the current pandemic, COVID-19 has been detected in patients using real-time reverse transcriptase-polymerase chain reaction (RT-PCR) that confirms the presence of SARS-CoV-2 RNA. The demand for increased testing, particularly for asymptomatic individuals required alternative approaches to single-patient RT-PCR testing, such as pooling. METHODS: This study explored the impact of dilution on the detectability of SARS-CoV-2 in asymptomatic patients using RT-PCR and demonstrated that pooling can be effective in low prevalence populations. RESULTS: The RT-PCR results for the 3:1, 5:1, and 7:1 aliquot samples showed little differences in CT values, confirming detection capability at these dilutions. CONCLUSION: Based on the results of the present study, a pooled approach with up to 5:1 sample aliquots and using the current RT-PCR methodology likely will detect SARS CoV2 RNA among asymptomatic patients.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , RNA, Viral/genetics , Reverse Transcriptase Polymerase Chain Reaction/methods , SARS-CoV-2/genetics , Asymptomatic Diseases , Buffers , COVID-19/virology , Humans , Specimen Handling/methods
8.
Am J Clin Pathol ; 155(2): 280-285, 2021 02 04.
Article in English | MEDLINE | ID: covidwho-872941

ABSTRACT

OBJECTIVES: The increase in the number of patients with coronavirus disease 2019 (COVID-19) has delayed real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR), requiring proper shipping and storage conditions, especially in hot weather. This study aims to assess how some conditions, such as storage period, temperature, media or buffer, and sample types, affect the results of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RT-qPCR. METHODS: SARS-CoV-2-positive specimens were collected from Boramae Medical Center for 2 months (from May to June 2020) and stored in different media or buffers at different temperatures. RESULTS: As a result of examining confirmed patient samples, RT-qPCR results were not significantly affected by 2°C to 8°C storage until after 7 days. When stored at 20°C to 22°C or above 35°C, the results were affected negatively even after 1 day. Higher storage temperatures resulted in a lower probability of detecting viral nucleic acids because of degradation. Samples stored in pH-controlled media or buffer were more stable than those stored in nonbuffer states. CONCLUSIONS: These results emphasize the importance of storage temperature and media or buffer and performing RT-qPCR for SARS-CoV-2 nucleic acid detection as soon as possible after sample collection.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , RNA, Viral/analysis , Real-Time Polymerase Chain Reaction/methods , Reverse Transcriptase Polymerase Chain Reaction/methods , SARS-CoV-2/genetics , Specimen Handling , Buffers , Humans , Temperature
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