Lab Matters Fall 2023 | Page 91

APHL 2023 POSTER ABSTRACTS wastewater samples were collected and DNA and RNA were isolated using extraction kits specialized for microbiome and inhibitory samples . Extraction efficiency was evaluated by conventional and digital PCR , as well as NGS . In addition , these samples were tested in a hybrid capture based NGS workflow for detection , sequencing , and characterization of AMR gene targets using a variety of sample types . Human stool samples were used as a comparison . Results : Significantly more diverse AMR markers were found in wastewater than in individual fecal samples . Fecal samples showed strong bias towards resistance markers against particular common antibiotics . Very high concordance was found between NGS read number and dPCR copy number , indicating the potential complementary value of these methods . Conclusion : These tools can provide insights into the development and dynamic of AMR , and could thus be a powerful method to combat antibiotic resistance . Identifying the most efficient and consistent workflows will support this effort . Learnings from these workflows can also be extended to further targets or application fields , such as adventitious agents or respiratory pathogens , improving understandings and outcomes for public health .

Presenter : Dylan Barbera , dylan . barbera @ qiagen . com

Optimizing Workflow for Comprehensive Immunological Assessment in Response to Influenza Vaccination and Infection

C . Bryan , U . Shanmugasundaram , S . Sharma , A . Kumar , D . Eddins , M . Mishina , M . Reed , M . Sheth , Y . Wang , J . Pohl , S . Gangappa , S . Sambhara ; US Centers for Disease Control and Prevention

Influenza is a vaccine-preventable respiratory virus infection and is estimated to cause 20 – 60 thousand deaths in the United States . The annual economic burden due to influenza in the US alone is between 6.3 – 25.3 billion dollars . Although annual vaccination is recommended to reduce morbidity , mortality and economic impact , the vaccine effectiveness ranged from 10-60 % in the United States from 2004 – 2021 . Several host factors , including preexisting influenza immunity , age , weight , sex and immune status , can impact vaccine efficacy . In addition , despite being vaccinated , many individuals get infected with influenza and the reasons for these break-through infections are unclear . Here we describe the development and optimization of a workflow to assess the frequency of antigen specific B cells with high-dimensional flowcytometry , B-cell receptor usage , and B- and T- cell transcriptomic signatures in peripheral blood mononuclear cells and hemagglutinin ( HA ) - specific serological repertoire in plasma with liquid chromatography and mass spectrometry to identify the clonotypes of Influenza HAspecific antibodies . The workflow will be utilized to investigate the immunological mechanisms of breakthrough infections of influenzainfected subjects ( break through infection group ) compared to vaccinated and uninfected subjects ( vaccinated control group ).

Presenter : Chena Bryan , chenabryan9 @ hotmail . com

Simultaneous Detection of Respiratory Infectious Diseases using Immunoprecipitation and Liquid Chromatography- Tandem Mass Spectrometry

Y . Song , R . Gibson , S . Samra ; Thermo Fisher Scientific

We are now living in a new era with new normal after the COVID-19 pandemic . Recently , respiratory syncytial virus ( RSV ) has been another concern as it surges among children . In addition to influenza viruses , not only are their symptoms similar at early stages , but they are also both enveloped viruses with several common biological properties , often leading to challenges in accurate identification . Thus , there is a need to develop a faster and more specific analytical tool that can differentiate infectious diseases . This study describes a targeted approach for the simultaneous detection of different respiratory infectious diseases by targeting nucleoprotein ( or nucleocapsid protein , NP ) using immunoprecipitation ( IP ) and liquid chromatography-tandem mass spectrometry ( LC-MS / MS ) because NP is highly conserved and specific for infectious disease virus types among different viral components . Prior to IP , equal amounts of all biotinylated antibodies were pooled together as one antibody panel for this study . The biotinylated antibody panel was added to samples collected via nasopharyngeal swabs in viral transport media ( VTM ). The antigen-antibody complex in VTM was directly subjected to IP using streptavidin magnetic beads . The IP purified samples were then tryptic digested and analyzed by Thermo Scientific™ Vanquish™ MD HPLC system hyphenated to Thermo Scientific™ TSQ Altis MD mass spectrometer . Data acquisition , processing and reporting were performed using TraceFinder™ LDT software 1.0 . Multiple viruses , SARS-CoV-2 , influenza virus A and B types , RSV , and human coronavirus ( HCoV-229E ), were selected to show that this method can distinguish different disease viruses . The workflow was optimized from sample preparation to LC-MS analysis . The protein precipitation and post sample clean-up were eliminated . From IP procedure , two incubation steps for antigen-antibody complex formation and immobilization on the magnetic beads were reduced to 15 minutes each ( originally one hour each ). Trypsin digestion incubation time was optimized to 15 minutes ( previously 90 minutes ). Particularly , the reduction of trypsin digestion time was achieved owing to a generation of much cleaner sample matrix by IP . The entire process was finalized to less than one hour from four hours . LC-MS run time was also optimized to five minutes . A total of 12 peptides were successfully monitored ( 2-3 peptides per disease ) by SRM . Calibration curve was generated with stable isotope-labeled standards ( Thermo Scientific HeavyPeptide AQUA Ultimate ). With criteria of % RSD 0.99 , 0.05 to 1 fmol of peptides on LC column were determined as the LOQs for each peptide with retention time variation ± 0.01 minutes . The method significantly improved the sensitivity and turn-around time , compared to other digestion or peptide enrichment methods .

For Research Use Only – Not For Diagnostic Procedures Presenter : Yvonne Song , yvonne . song @ thermofisher . com