Lecture 4-Immunoassays PDF

Immunoassays as sensitive analytical methods in protein biochemistry Clinical Neuroscience Dr. rer. nat. Steffen Halbgebauer- Experimental Neurology 13.12.2023  Omics Shiratake et al. 2016...

Immunoassays as sensitive analytical methods in protein biochemistry Clinical Neuroscience Dr. rer. nat. Steffen Halbgebauer- Experimental Neurology 13.12.2023  Omics Shiratake et al. 2016 2 Clinical Neuroscience Immunoassays| Dr. Steffen Halbgebauer| 14.12.2022  Immunoassays vs. Mass spectrometry Immunoassays Mass spectrometry https://www.biomol.com/de/ressourcen/biomol- blog/western-blot-ladekontrollen https://www.bio-techne.com/p/simple- plex/ella-automated-immunoassay- system_600-100 https://ablinc.com/immunology-services/quanterix/ 3 Clinical Neuroscience Immunoassays| Dr. Steffen Halbgebauer| 14.12.2022  Immunoassays vs. Mass spectrometry https://cen.acs.org/articles/93/i20/Mass-Spec-Welcome-Clinical-Labs.html Shiratake et al. 2016 4 Clinical Neuroscience Immunoassays| Dr. Steffen Halbgebauer| 14.12.2022  What are Immunassays Bioanalytical methods for the detection of analytes Macro- or small molecules can be measured Semi-quantitative, quantitative or only presence Recognition via antibodies or antigens Analyte: The molecule detected by the immunoassay Epitope: The binding side of an antibody 5 Clinical Neuroscience Immunoassays| Dr. Steffen Halbgebauer| 14.12.2022  Solutions frequently analyzed in experimental and clinical neurology Body fluids: Blood Plasma (EDTA, Heparin, Citrate) Serum Cerebrospinal fluid (CSF) Urine Saliva Other: Tissue Homogenates Cell culture homogenates Cell culture supernatant 6 Clinical Neuroscience Immunoassays| Dr. Steffen Halbgebauer| 14.12.2022  Immunoassay overview (for this talk) SDS-Page - Staining - Blotting (Western Blot) Isoelectric focusing and 2D Page Classic ELISAs assays -Direct ELISA -Indirect ELISA -Sandwich ELISA -Competitive ELISA Single molecule array (Simoa) ELLA (automated microfluidic system) Novel developments Mesocale Discovery (MSD) Platform (last 10 years) Capillary isoelectric focusing immunoassay Seed aggregation assays (SAA) 7 Clinical Neuroscience Immunoassays| Dr. Steffen Halbgebauer| 14.12.2022  Excursion: Antibody A) by Tokenzero ( CC BY-SA 4.0) via Wikimedia commons B) work has been released into the public https://en.wikipedia.org/wiki/Antibody#/media/File:Antibody.svg domain by its author, Je at uwo at English Wikipedia. C) work has been released into the public domain by its author D) by Tokenzero ( CC BY-SA 4.0) via Wikimedia commons 8 Clinical Neuroscience Immunoassays| Dr. Steffen Halbgebauer| 14.12.2022  SDS-Page Sodium dodecyl sulfate–polyacrylamide gel electrophoresis Ulrich K. Laemmli (developer) Application of current Seperation of molecules mostly between 5 and 250 kDa according to their molecular weight/size https://www.phosphosolutions.com/pages/sds-page-demystified 9 Clinical Neuroscience Immunoassays| Dr. Steffen Halbgebauer| 14.12.2022  SDS-Page https://ruo.mbl.co.jp/bio/e/support/method/sds-page.html 10 Clinical Neuroscience Immunoassays| Dr. Steffen Halbgebauer| 14.12.2022  SDS-Page Wangler, Michael & Bellen, Hugo. (2017). In Vivo Animal Modeling. 10.1016/B978-0-12-803077-6.00012-6. 11 Clinical Neuroscience Immunoassays| Dr. Steffen Halbgebauer| 14.12.2022  SDS-Page SDS-Page result after SDS-Page result protein staining https://proteinark.com/realtime-stain/ Mad-Ali et al. 2019 12 Clinical Neuroscience Immunoassays| Dr. Steffen Halbgebauer| 14.12.2022  Western Blot 13 Clinical Neuroscience Immunoassays| Dr. Steffen Halbgebauer| 14.12.2022  Western Blot Semi Dry blotting https://en.wikipedia.org/wiki/Western_blot#/media/File:Western_blot_transfer.png 14 Clinical Neuroscience Immunoassays| Dr. Steffen Halbgebauer| 14.12.2022  Western Blot Wet blotting https://en.wikipedia.org/wiki/Western_blot#/media/File:Western_blot_transfer.png 15 Clinical Neuroscience Immunoassays| Dr. Steffen Halbgebauer| 14.12.2022  Clinical Example Protein 14-3-3 in CSF of prion disease patients Castellani et al. 2004 Neurology 16 Clinical Neuroscience Immunoassays| Dr. Steffen Halbgebauer| 14.12.2022  Strengths and limitations Strengths Limitations Well established Time consuming Broad applications Denaturation of proteins Cheap Only semi-quantitative Subjective results 17 Clinical Neuroscience Immunoassays| Dr. Steffen Halbgebauer| 14.12.2022  Isoelectric focusing Separation according to the charge of the analytes Pergande et al. 2016 18 Clinical Neuroscience Immunoassays| Dr. Steffen Halbgebauer| 14.12.2022  Clinical Example Oligocloncal Bands Detection of Immunoglobulin Gs in CSF and blood Halbgebauer et al. 2016 19 Clinical Neuroscience Immunoassays| Dr. Steffen Halbgebauer| 14.12.2022  2D-Page https://www.bio-rad.com/de-de/product/readystrip-ipg-strips?ID=56994cbe-ea1b- 48d8-b7e9-bc17726c0d70 Ward et al. 2019 20 Clinical Neuroscience Immunoassays| Dr. Steffen Halbgebauer| 14.12.2022  2D-Page CSF SerpinA1 Isoforms 21 Clinical Neuroscience Immunoassays| Dr. Steffen Halbgebauer| 14.12.2022  ELISA Enzyme linked immunosorbent assay Enzymatische Farbreaktion 4 main types Boguszewska, Karolina & Szewczuk, Michał & Urbaniak, Sandra & Karwowski, Boleslaw. (2019). Review: immunoassays in DNA damage and instability detection. Cellular and Molecular Life Sciences. 76. 10.1007/s00018-019-03239-6. 22 Clinical Neuroscience Immunoassays| Dr. Steffen Halbgebauer| 14.12.2022  Indirect ELISA https://www.leinco.com/indirect-elisa-protocol/ 23 Clinical Neuroscience Immunoassays| Dr. Steffen Halbgebauer| 14.12.2022  Sandwich ELISA 24 Clinical Neuroscience Immunoassays| Dr. Steffen Halbgebauer| 14.12.2022  Sandwich ELISA https://www.leinco.com/sandwich-elisa-protocol/ 25 Clinical Neuroscience Immunoassays| Dr. Steffen Halbgebauer| 14.12.2022  Clinical Examples Abeta 1-42 Total Tau Phospho-Tau … Lim, Ho Jae et al. ‘Comparison of Two Analytical Platforms in Cerebrospinal Fluid Biomarkers for the Classification of Alzheimer’s Disease Spectrum with Amyloid PET Imaging’. 1 Jan. 2020 : 949 – 958. 26 Clinical Neuroscience Immunoassays| Dr. Steffen Halbgebauer| 14.12.2022  Strengths and limitations Strengths Limitations Well established Cross contaminations/reactivity Broad applications Antibody limitation (sandwich) Cheap Needs validation Quantitative Many „bad“ commercial kits Sensitive Native protein detection Easy to use 27 Clinical Neuroscience Immunoassays| Dr. Steffen Halbgebauer| 14.12.2022  Single molecule array (Simoa) Digital ELISA Analog and digital mode Bead based Automated Commercial kits available for certain analytes Option for „homemade assays https://ablinc.com/immunology-services/quanterix/ 28 Clinical Neuroscience Immunoassays| Dr. Steffen Halbgebauer| 14.12.2022  Single molecule array (Simoa) Rissin et al. 2010 29 Clinical Neuroscience Immunoassays| Dr. Steffen Halbgebauer| 14.12.2022  Single molecule array (Simoa) 30 Clinical Neuroscience Immunoassays| Dr. Steffen Halbgebauer| 14.12.2022  Blood pTau in AD (Future) Clinical Examples Neurofilaments in Amyotrophic Lateral sclerosis Karikari et al. 2020 31 Clinical Neuroscience Immunoassays| Dr. Steffen Halbgebauer| 14.12.2022  Strengths and limitations Strengths Limitations Highly sensitive Expensive High throughput Needs some training Fast Sometimes system errors Quantitative Many commercial kits available Option for homemade assays 32 Clinical Neuroscience Immunoassays| Dr. Steffen Halbgebauer| 14.12.2022  Microfluidic Ella platform Microfluidic platform Glass nanoreactors (GNRS) Triplicate measurement Automated Commercial kits available for certain analytes Option for „homemade assays https://www.bio-techne.com/p/simple-plex/ella-automated-immunoassay-system_600-100 33 Clinical Neuroscience Immunoassays| Dr. Steffen Halbgebauer| 14.12.2022  Microfluidic Ella platform Van Manen-Brush et al. 2020 34 Clinical Neuroscience Immunoassays| Dr. Steffen Halbgebauer| 14.12.2022  Microfluidic Ella platform https://www.bio-techne.com/p/simple-plex/ella-automated-immunoassay-system_600-100 35 Clinical Neuroscience Immunoassays| Dr. Steffen Halbgebauer| 14.12.2022 Ella Instrument Video https://www.youtube.com/watch?v=Qe2UyN6 SBjU&t=76s Multiple Sclerosis: clinic, pathophysiology and treatment | Dr. André Huss | Experimental Neurology | 15.01.2020 36  (Future) Clinical Examples Blood measurement of neurofilament light and heavy chain Halbgebauer et al 2022 37 Clinical Neuroscience Immunoassays| Dr. Steffen Halbgebauer| 14.12.2022  Strengths and limitations Strengths Limitations Highly sensitive More expensive than ELISA High throughput Sometimes „channels“ not found Not many neurological commercial Fast kits available Lower throuput for homemade Quantitative assays Easy to use Option for homemade assays 38 Clinical Neuroscience Immunoassays| Dr. Steffen Halbgebauer| 14.12.2022  MSD Platform Electrochemiluminscence technology 39 Clinical Neuroscience Immunoassays| Dr. Steffen Halbgebauer| 14.12.2022  Capillary isoelectric focusing NanoPro1000 or PeggySue Separation according to charge Different pH Gradients Reaction in small glass capillaries (voulme 400nl) Automated 40 Clinical Neuroscience Immunoassays| Dr. Steffen Halbgebauer| 14.12.2022  Capillary isoelectric focusing Sample Loading ~ 400 nl diphosphorylated monophosphorylated nonphosphorylated Separation by Charge UV-light induced immobilization Primary Antibody HRP labeled Secondary Immunodetection Antibody Chemiluminescense 41 Clinical Neuroscience Immunoassays| Dr. Steffen Halbgebauer| 14.12.2022  Capillary isoelectric focusing https://www.bio-techne.com/p/simple-western/nanopro-1000_004-109 42 Clinical Neuroscience Immunoassays| Dr. Steffen Halbgebauer| 14.12.2022  Capillary isoelectric focusing SerpinA1 detection using traditional IEF (left) and CIEF (right) Green: control Blue: Parkinson‘s disease dementia Halbgebauer et al. 2016 43 Clinical Neuroscience Immunoassays| Dr. Steffen Halbgebauer| 14.12.2022  (Future) Clinical Examples Oligoclonal bands using traditional IEF (left) and CIEF (right) 44 Clinical Neuroscience Immunoassays| Dr. Steffen Halbgebauer| 14.12.2022 Halbgebauer et al. 2016  Strengths and limitations Strengths Limitations Isoform readout Expensive High throughput Not so sensitive Different gradients available Semi-quantitative Needs some training 45 Clinical Neuroscience Immunoassays| Dr. Steffen Halbgebauer| 14.12.2022  Seed aggregation assays (SAA) RT-QuIC (Real-time quaking-induced conversion) PMCA (Protein misfolding cyclic amplification) „normally“ folded protein (seed)  mix with misfolded protein from e.g. CSF  amplification of misfolded protein  aggregation  break down of aggregation  further amplification  … In between measuring of aggregates using fluorescence (Thioflavin T) Yes or No answer Examples for Misfolded proteins detected using SAAs Prion Alpha-Synuclein Tau TDP-43 46 Clinical Neuroscience Immunoassays| Dr. Steffen Halbgebauer| 14.12.2022  Seed aggregation assays (SAA) Candelise et al. 2020 47 Clinical Neuroscience Immunoassays| Dr. Steffen Halbgebauer| 14.12.2022  Clinical Examples Detection misfolded prion protein in Prion diseases 48 Clinical Neuroscience Immunoassays| Dr. Steffen Halbgebauer| 14.12.2022  Strengths and limitations Strengths Limitations Highly sensitive Time consuming Opens new possibilties for the detection of highly low abundant Only Yes or No answer (misfolded) proteins Already used in clinics Sometimes subjective results Needs „good“ recombinant protein 49 Clinical Neuroscience Immunoassays| Dr. Steffen Halbgebauer| 14.12.2022  Internships (master thesis) available in our laboratory (Experimental Neurology) Development of biomarker assays for Alzheimer‘s and Parkinson‘s disease using sandwich ELISA (ELLA/Simoa) https://www.bio-techne.com/p/simple-plex/ella-automated-immunoassay-system_600-100 https://ablinc.com/immunology-services/quanterix/ 50 Clinical Neuroscience Immunoassays| Dr. Steffen Halbgebauer| 14.12.2022