Multiplex Immunoassay for Biomarker Profiling of Whole Blood Cell Lysates and Supernatants and Pathogen Response in Neat Whole Blood Cultures
Replicating in vivo conditions is essential for understanding immune responses and measuring immune biomarkers in blood. Sampling immune biomarkers in plasma or serum often fails to detect disease-relevant signals, possibly because these markers are sequestered in immune cells or extracellular vesic...
Saved in:
| Main Authors: | , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2025-05-01
|
| Series: | Methods and Protocols |
| Subjects: | |
| Online Access: | https://www.mdpi.com/2409-9279/8/3/46 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1839653197059194880 |
|---|---|
| author | Irina Balan Alejandro G. Lopez A. Leslie Morrow |
| author_facet | Irina Balan Alejandro G. Lopez A. Leslie Morrow |
| author_sort | Irina Balan |
| collection | DOAJ |
| description | Replicating in vivo conditions is essential for understanding immune responses and measuring immune biomarkers in blood. Sampling immune biomarkers in plasma or serum often fails to detect disease-relevant signals, possibly because these markers are sequestered in immune cells or extracellular vesicles. Furthermore, traditional whole blood cultures using external media may not accurately mimic the physiological environment of blood cells. To address these limitations, we developed a strategy using whole blood cell lysates and supernatants to optimize biomarker detection. Additionally, we employed neat whole blood culture methods, preserving the natural cellular and biochemical environment to assess sensitivity to immune modulators, such as lipopolysaccharide (LPS). This cost-effective approach minimizes variability and contamination risks. By utilizing Luminex multiplex immunoassays, we profiled immune biomarkers with higher sensitivity and efficiency than traditional ELISAs. Blood samples from individuals with high alcohol consumption validated our method by assessing biomarker levels before and after LPS stimulation, providing insights into intracellular responses and inflammatory pathways. This method enhances our understanding of inflammatory processes in blood cells, demonstrating the advantages of cell lysates, supernatants, and advanced multiplex assays in immunological research. |
| format | Article |
| id | doaj-art-9097d154d9a24e69b2553b1e7e7fd8ef |
| institution | Matheson Library |
| issn | 2409-9279 |
| language | English |
| publishDate | 2025-05-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Methods and Protocols |
| spelling | doaj-art-9097d154d9a24e69b2553b1e7e7fd8ef2025-06-25T14:13:58ZengMDPI AGMethods and Protocols2409-92792025-05-01834610.3390/mps8030046Multiplex Immunoassay for Biomarker Profiling of Whole Blood Cell Lysates and Supernatants and Pathogen Response in Neat Whole Blood CulturesIrina Balan0Alejandro G. Lopez1A. Leslie Morrow2Bowles Center for Alcohol Studies, School of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USABowles Center for Alcohol Studies, School of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USABowles Center for Alcohol Studies, School of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USAReplicating in vivo conditions is essential for understanding immune responses and measuring immune biomarkers in blood. Sampling immune biomarkers in plasma or serum often fails to detect disease-relevant signals, possibly because these markers are sequestered in immune cells or extracellular vesicles. Furthermore, traditional whole blood cultures using external media may not accurately mimic the physiological environment of blood cells. To address these limitations, we developed a strategy using whole blood cell lysates and supernatants to optimize biomarker detection. Additionally, we employed neat whole blood culture methods, preserving the natural cellular and biochemical environment to assess sensitivity to immune modulators, such as lipopolysaccharide (LPS). This cost-effective approach minimizes variability and contamination risks. By utilizing Luminex multiplex immunoassays, we profiled immune biomarkers with higher sensitivity and efficiency than traditional ELISAs. Blood samples from individuals with high alcohol consumption validated our method by assessing biomarker levels before and after LPS stimulation, providing insights into intracellular responses and inflammatory pathways. This method enhances our understanding of inflammatory processes in blood cells, demonstrating the advantages of cell lysates, supernatants, and advanced multiplex assays in immunological research.https://www.mdpi.com/2409-9279/8/3/46biomarker profilingcytokineshuman whole blood cell culturelipopolysaccharideinflammatory pathways |
| spellingShingle | Irina Balan Alejandro G. Lopez A. Leslie Morrow Multiplex Immunoassay for Biomarker Profiling of Whole Blood Cell Lysates and Supernatants and Pathogen Response in Neat Whole Blood Cultures Methods and Protocols biomarker profiling cytokines human whole blood cell culture lipopolysaccharide inflammatory pathways |
| title | Multiplex Immunoassay for Biomarker Profiling of Whole Blood Cell Lysates and Supernatants and Pathogen Response in Neat Whole Blood Cultures |
| title_full | Multiplex Immunoassay for Biomarker Profiling of Whole Blood Cell Lysates and Supernatants and Pathogen Response in Neat Whole Blood Cultures |
| title_fullStr | Multiplex Immunoassay for Biomarker Profiling of Whole Blood Cell Lysates and Supernatants and Pathogen Response in Neat Whole Blood Cultures |
| title_full_unstemmed | Multiplex Immunoassay for Biomarker Profiling of Whole Blood Cell Lysates and Supernatants and Pathogen Response in Neat Whole Blood Cultures |
| title_short | Multiplex Immunoassay for Biomarker Profiling of Whole Blood Cell Lysates and Supernatants and Pathogen Response in Neat Whole Blood Cultures |
| title_sort | multiplex immunoassay for biomarker profiling of whole blood cell lysates and supernatants and pathogen response in neat whole blood cultures |
| topic | biomarker profiling cytokines human whole blood cell culture lipopolysaccharide inflammatory pathways |
| url | https://www.mdpi.com/2409-9279/8/3/46 |
| work_keys_str_mv | AT irinabalan multipleximmunoassayforbiomarkerprofilingofwholebloodcelllysatesandsupernatantsandpathogenresponseinneatwholebloodcultures AT alejandroglopez multipleximmunoassayforbiomarkerprofilingofwholebloodcelllysatesandsupernatantsandpathogenresponseinneatwholebloodcultures AT alesliemorrow multipleximmunoassayforbiomarkerprofilingofwholebloodcelllysatesandsupernatantsandpathogenresponseinneatwholebloodcultures |