Tixagevimab Plus Cilgavimab Does Not Affect the Interpretation of Electrophoretic and Free Light Chain Assays.

OBJECTIVES: There is concern that the anti-severe acute respiratory syndrome coronavirus 2 therapeutic monoclonal antibodies, used as preexposure prophylaxis in patients with multiple myeloma, may appear as a detectable monoclonal protein by electrophoretic methods, resulting in misinterpretation or inability to measure therapeutic responses in some patients. In this pilot study, we characterize the effect of tixagevimab plus cilgavimab (Evusheld; T + C) on interpretation of serum protein electrophoresis (SPE), immunofixation electrophoresis (IFE), and serum free light chain (sFLC) assays. METHODS: We performed spiking experiments with T + C at serum maximum concentration following a 300-mg dose (1× Cmax) and at 10 times the concentration of Cmax (10× Cmax) with pooled serum samples. SPE and IFE technical procedures were performed on the SPIFE 3000, and sFLC and immunoglobulin G1 (IgG1) subtype quantitation was performed on the Optilite. RESULTS: T + C-associated interference was not visible as an M-spike in normogammaglobulinemic pooled samples. Hypogammaglobulemic pooled samples at 10× Cmax demonstrated an M-spike in SPE and immunoglobulin Gκ pattern in IFE. No increases were noted in the results of sFLC or IgG1 levels. CONCLUSIONS: This study indicates that T + C at pharmacologic Cmax is unlikely to interfere with SPE, IFE, sFLC, or IgG1 analyses when spiked into patient serum samples, but further evaluation of recently injected patients may be warranted.

Identification of SARS-CoV-2 Main Protease (Mpro) Cleavage Sites Using Two-Dimensional Electrophoresis and In Silico Cleavage Site Prediction.

The main protease (Mpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays a crucial role in its life cycle. The Mpro-mediated limited proteolysis of the viral polyproteins is necessary for the replication of the virus, and cleavage of the host proteins of the infected cells may also contribute to viral pathogenesis, such as evading the immune responses or triggering cell toxicity. Therefore, the identification of host substrates of the viral protease is of special interest. To identify cleavage sites in cellular substrates of SARS-CoV-2 Mpro, we determined changes in the HEK293T cellular proteome upon expression of the Mpro using two-dimensional gel electrophoresis. The candidate cellular substrates of Mpro were identified by mass spectrometry, and then potential cleavage sites were predicted in silico using NetCorona 1.0 and 3CLP web servers. The existence of the predicted cleavage sites was investigated by in vitro cleavage reactions using recombinant protein substrates containing the candidate target sequences, followed by the determination of cleavage positions using mass spectrometry. Unknown and previously described SARS-CoV-2 Mpro cleavage sites and cellular substrates were also identified. Identification of target sequences is important to understand the specificity of the enzyme, as well as aiding the improvement and development of computational methods for cleavage site prediction.

Recent advances in microchip electrophoresis for analysis of pathogenic bacteria and viruses.

Life-threatening diseases, such as hepatitis B, pneumonia, tuberculosis, and COVID-19, are widespread due to pathogenic bacteria and viruses. Therefore, the development of highly sensitive, rapid, portable, cost-effective, and selective methods for the analysis of such microorganisms is a great challenge. Microchip electrophoresis (ME) has been widely used in recent years for the analysis of bacterial and viral pathogens in biological and environmental samples owing to its portability, simplicity, cost-effectiveness, and rapid analysis. However, microbial enrichment and purification are critical steps for accurate and sensitive analysis of pathogenic bacteria and viruses in complex matrices. Therefore, we first discussed the advances in the sample preparation technologies associated with the accurate analysis of such microorganisms, especially the on-chip microfluidic-based sample preparations such as dielectrophoresis and microfluidic membrane filtration. Thereafter, we focused on the recent advances in the lab-on-a-chip electrophoretic analysis of pathogenic bacteria and viruses in different complex matrices. As the microbial analysis is mainly based on the analysis of nucleic acid of the microorganism, the integration of nucleic acid-based amplification techniques such as polymerase chain reaction (PCR), quantitative PCR, and multiplex PCR with ME will result in an accurate and sensitive analysis of microbial pathogens. Such analyses are very important for the point-of-care diagnosis of various infectious diseases.

Production and characterization of lentivirus vector-based SARS-CoV-2 pseudoviruses with dual reporters: Evaluation of anti-SARS-CoV-2 viral effect of Korean red ginseng.

Background: Pseudotyped virus systems that incorporate viral proteins have been widely employed for the rapid determination of the effectiveness and neutralizing activity of drug and vaccine candidates in biosafety level 2 facilities. We report an efficient method for producing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pseudovirus with dual luciferase and fluorescent protein reporters. Moreover, using the established method, we also aimed to investigate whether Korean red ginseng (KRG), a valuable Korean herbal medicine, can attenuate infectivity of the pseudotyped virus. Methods: A pseudovirus of SARS-CoV-2 (SARS-2pv) was constructed and efficiently produced using lentivirus vector systems available in the public domain by the introduction of critical mutations in the cytoplasmic tail of the spike protein. KRG extract was dose-dependently treated to Calu-3 cells during SARS2-pv treatment to evaluate the protective activity against SARS-CoV-2. Results: The use of Calu-3 cells or the expression of angiotensin-converting enzyme 2 (ACE2) in HEK293T cells enabled SARS-2pv infection of host cells. Coexpression of transmembrane protease serine subtype 2 (TMPRSS2), which is the activator of spike protein, with ACE2 dramatically elevated luciferase activity, confirming the importance of the TMPRSS2-mediated pathway during SARS-CoV-2 entry. Our pseudovirus assay also revealed that KRG elicited resistance to SARS-CoV-2 infection in lung cells, suggesting its beneficial health effect. Conclusion: The method demonstrated the production of SARS-2pv for the analysis of vaccine or drug candidates. When KRG was assessed by the method, it protected host cells from coronavirus infection. Further studies will be followed for demonstrating this potential benefit.

Present status of microfluidic PCR chip in nucleic acid detection and future perspective

Polymerase chain reaction (PCR) amplifies specific fragment of DNA molecules and has been extensively applied in fields of pathogens and gene mutation detection, food safety and clinical diagnosis which on the other hand, holds the drawbacks of large size instrument, high heat dissipation etc. It has been demonstrated that microfluidics technique coupling with PCR reaction exhibits characteristics of integration, automatization, miniaturization, and portability. Meanwhile, various designed fabrication of microchip could contribute to diverse applications. In this review, we summarized major works about a variety of microfluidic chips equipped with several kinds of PCR techniques (PCR, RT-PCR, mPCR, dPCR) and detection methods like fluorescence, electrochemistry, and electrophoresis detection. The development and application of PCR-based microfluidic chip in pathogen and gene mutation detection, diseases prevention and diagnosis, DNA hybridization and low-volume sample treatment were also discussed. Copyright © 2022 Elsevier B.V.

Tixagevimab Plus Cilgavimab Does Not Affect the Interpretation of Electrophoretic and Free Light Chain Assays.

OBJECTIVES: There is concern that the anti-severe acute respiratory syndrome coronavirus 2 therapeutic monoclonal antibodies, used as preexposure prophylaxis in patients with multiple myeloma, may appear as a detectable monoclonal protein by electrophoretic methods, resulting in misinterpretation or inability to measure therapeutic responses in some patients. In this pilot study, we characterize the effect of tixagevimab plus cilgavimab (Evusheld; T + C) on interpretation of serum protein electrophoresis (SPE), immunofixation electrophoresis (IFE), and serum free light chain (sFLC) assays. METHODS: We performed spiking experiments with T + C at serum maximum concentration following a 300-mg dose (1× Cmax) and at 10 times the concentration of Cmax (10× Cmax) with pooled serum samples. SPE and IFE technical procedures were performed on the SPIFE 3000, and sFLC and immunoglobulin G1 (IgG1) subtype quantitation was performed on the Optilite. RESULTS: T + C-associated interference was not visible as an M-spike in normogammaglobulinemic pooled samples. Hypogammaglobulemic pooled samples at 10× Cmax demonstrated an M-spike in SPE and immunoglobulin Gκ pattern in IFE. No increases were noted in the results of sFLC or IgG1 levels. CONCLUSIONS: This study indicates that T + C at pharmacologic Cmax is unlikely to interfere with SPE, IFE, sFLC, or IgG1 analyses when spiked into patient serum samples, but further evaluation of recently injected patients may be warranted.

Direct SARS-CoV-2 Detection System Utilizing Simple-to-Use Capillary Gel Electrophoresis Sample-to-Result.

We present a Direct SARS-CoV-2 Detection System that achieves sample-to-results in less than two hours in three simple steps. The Detection System includes Direct one-step Reverse Transcription PCR (RT-PCR) reagents (Qexp-MDx kit), a portable thermal cycler (Qamp-mini) with a pre-programmed chip and a simple-to-use Capillary Gel Electrophoresis system (Qsep Series Bio-Fragment Analyzer) with high fluorescence detection sensitivity to solve the problems associated with traditional real-time PCR (qPCR) systems which produces inaccurate test results with high false negative and false positive rates. The proposed simple-to-use detection platform can provide high detection sensitivity (identify less than 20 copies), fast results (less than 120 minutes) and cost-effective results which should be suitable for decentralized testing application of COVID-19.