ELISA: Methods and Protocols

Dedication
Preface
Contents
Contributors
Chapter 1: Enzyme-Linked Immunosorbent Assay: Types and Applications
	1 Introduction
	2 Historical Background of ELISA
	3 General ELISA Procedure
		3.1 Direct ELISA
		3.2 Indirect ELISA
		3.3 Sandwich ELISA
		3.4 Competitive ELISA
	4 Applications of ELISA and Clinical Significance
	References
Chapter 2: ELISA Essentials: Surfaces, Antibodies, Enzymes, and Substrates
	1 Introduction
	2 Immunosorbent
	3 Antibodies
	4 Enzymes
		4.1 Horseradish Peroxidase
		4.2 Alkaline Phosphatase
	5 Streptavidin-Biotin Systems
	6 The Hook Effect
	7 The Importance of Buffers
		7.1 Coating Buffer
		7.2 Wash Buffer
		7.3 Blocking Buffer
		7.4 Diluents
			7.4.1 Antibody Diluent
			7.4.2 Matrix Diluent
			7.4.3 Checking for Matrix Effects
	References
Chapter 3: Antibody Immobilization
	1 Introduction
		1.1 Surface Chemistries
		1.2 Coupling Strategy
	2 Materials
		2.1 Passive Adsorption of Antibody to Polystyrene Plate
		2.2 Immobilization of Biotinylated Antibody to Streptavidin-Coated Plate
		2.3 Protein a Plate Capture of Monoclonal Antibody
	3 Methods
		3.1 Passive Adsorption of Antibody
		3.2 Immobilization of Biotinylated Antibody
		3.3 Protein a Capture of Monoclonal Antibody
	4 Notes
	References
Chapter 4: Screening for Antibody Specificity and Sensitivity with ELISA
	1 Introduction
	2 Materials
	3 Methods
		3.1 Determine Test Conditions and Prepare Ag-Coated Plate
		3.2 Wash and Block Plate
		3.3 Incubate with Ab
		3.4 Incubate with HRP Conjugate
		3.5 Incubate with TMB Substrate
		3.6 Add Stop Solution and Read Plate
		3.7 Process and Review Data
	4 Notes
	References
Chapter 5: Effective Blocking and Stabilizing Methods Using Synthetic Polymer on ELISA
	1 Introduction
	2 Materials
		2.1 Blocking Method for Enzyme-Labeled Antibody on Microplate
		2.2 Blocking Method for Enzyme-Labeled Antibody on Magnetic Beads
		2.3 Sandwich ELISA
		2.4 Stabilization of Enzyme Conjugate
		2.5 Application of BIOLIPIDURE for Different Immunoassay-Like Lateral Flow Test
			2.5.1 Application for Coronavirus (SARS-CoV-2) IgM/IgG Test Kit
			2.5.2 Application for Substances of Coronavirus (SARS-CoV-2) IgG Test Kit
	3 Methods
		3.1 Blocking Method for Enzyme-Labeled Antibody on Microplate
		3.2 Blocking Method for Enzyme-Labeled Antibody on Magnetic Beads
		3.3 Sandwich ELISA
		3.4 Stabilization of Enzyme Conjugate
		3.5 Application of BIOLIPIDURE for Different Immunoassay-Like Lateral Flow Test
			3.5.1 Application for Coronavirus (SARS-CoV-2) IgM/IgG Test Kit
			3.5.2 Application for Substances of Coronavirus (SARS-CoV-2) IgG Test Kit
	4 Notes
	References
Chapter 6: Gas Plasma Surface Modification for Biological Assays
	1 Introduction
	2 Materials
	3 Methods
		3.1 Plasma Process Design
		3.2 Process Development
		3.3 Plasma Treatment Under Partial Pressure
		3.4 Plasma Treatment Using Atmospheric Plasma System
		3.5 Evaluation of Plasma-Treated Surface
	4 Notes
	References
Chapter 7: Interference in ELISA
	1 Introduction
	2 Interference or Not?
	3 Fixing the Problem
	References
Chapter 8: Determination of Proinflammatory and Antiinflammatory Cytokines by ELISA Technique
	1 Introduction
	2 Materials
		2.1 Determination of IL-6 by ELISA
		2.2 Determination of VEGF-R1 (sFlt-1) by ELISA
	3 Methods
		3.1 Determination of IL-6 by ELISA
		3.2 Determination of VEGF-R1 (sFlt-1) by ELISA
	4 Notes
	References
Chapter 9: Gyrolab Immunoassays: Miniaturization, Automation, and Integration into a Rapid Workflow
	1 Introduction
	2 Overview of Gyrolab Technology
		2.1 Gyrolab Bioaffy CDs
		2.2 Gyrolab Systems
		2.3 Gyrolab System Buffers
		2.4 Assay Buffers
		2.5 96-Well Plate
		2.6 Gyrolab Assay Development Workflow
			2.6.1 Selection of Gyrolab Method
			2.6.2 Design Run
			2.6.3 Capture and Detection Reagents
			2.6.4 Improved Assay Performance with Bioaffy 1000HC
			2.6.5 Titration of Reagents
			2.6.6 Prepare Samples
			2.6.7 Prepare a Run
			2.6.8 Data Analysis
	3 Case Studies
		3.1 Materials
			3.1.1 Pembrolizumab (Keytruda) PK Assay
			3.1.2 IL-2 Biomarker Assay
		3.2 Gyrolab Methods
		3.3 Pharmacokinetics Analysis: Pembrolizumab
			3.3.1 Background
			3.3.2 Results of Method Development
		3.4 Biomarker Analysis: Interleukin 2 (IL-2)
			3.4.1 Background
			3.4.2 Assay Performance
	4 Conclusions
	References
Untitled
Chapter 10: Assessment of Immunologically Potent Carbohydrates
	1 Introduction
	2 Materials
	3 Methods
		3.1 Antigen Coating and Blocking
		3.2 Primary Antibody Reaction
		3.3 Secondary Antibody Reaction
		3.4 Substrate Preparation and Development
		3.5 Examples of Application
			3.5.1 Indirect ELISA
			3.5.2 ELISA Competition Assays
			3.5.3 ELISA ``Sandwich´´ Assays (Capture ELISA)
	4 Notes
	References
Chapter 11: Lateral Flow Microarray-Based ELISA for Cytokines
	1 Introduction
		1.1 Rapid Tests
		1.2 Lateral Flow Microarray
	2 Materials
		2.1 Immobilization of Anti-Cytokine Capture Antibody
		2.2 Construction of LFM-Cytokine Cassettes (See Note 5)
		2.3 LFM-Cytokine Rapid Test
		2.4 Signal Development and Analysis (See Note 9)
	3 Methods
		3.1 Immobilization of Anti-cytokine Capture Antibody to Nitrocellulose Substrate
			3.1.1 Construction of the Nitrocellulose Print Card
			3.1.2 Printing
			3.1.3 Storage
		3.2 Construction of the Lateral Flow Cassette
		3.3 Lateral Flow Microarray Cytokine Rapid Test
			3.3.1 Performing the LFM-Cytokine Rapid Test
			3.3.2 Signal Development and Analysis
	4 Notes
	References
Chapter 12: A Unique Multiplex ELISA to Profile Growth Factors and Cytokines in Cerebrospinal Fluid
	1 Introduction
	2 Materials
	3 Methods
		3.1 Multiplex ELISA Protocol for Standards and Samples
		3.2 Results
	4 Notes
	References
Chapter 13: Well-Based Multiplex Food Allergen Colorimetric ELISA
	1 Introduction
	2 Materials
		2.1 Extraction of Allergens
		2.2 Printing of Allergens
		2.3 Colorimetric ELISA
	3 Methods
		3.1 Extraction of Allergens
		3.2 Printing of Allergens
		3.3 Colorimetric ELISA
	4 Notes
	References
Chapter 14: Mycotoxin Quantification by Competitive ELISA
	1 Introduction
		1.1 Mycotoxin Quantification with Competitive ELISA
	2 Materials
		2.1 Extraction
		2.2 Evaporation
		2.3 Colorimetric ELISA Development
	3 Methods
		3.1 Extraction
		3.2 Evaporation
		3.3 Colorimetric ELISA Development
	4 Notes
	References
Chapter 15: Lumit: A Homogeneous Bioluminescent Immunoassay for Detecting Diverse Analytes and Intracellular Protein Targets
	1 Introduction
	2 Materials
		2.1 Labeling of Antibodies with NanoBiT Subunits
		2.2 Detection of Cytokine Release from Cells in Culture
		2.3 Detection of Phosphorylated ERK1 (p-Thr202) in Cell Lysate
		2.4 Detection of SARS-CoV2 Spike RBD: Human ACE2 Receptor Protein-Protein Interaction
		2.5 Equipment
	3 Methods
		3.1 Labeling of Antibodies with SmBiT and LgBiT
			3.1.1 Chemical Labeling of Antibodies with HaloTag Ligand
			3.1.2 Conjugating HaloTag-SmBiT and HaloTag-LgBiT to Antibody-HaloTag Ligand
			3.1.3 Removing Unreacted HaloTag-BiTs Using Magne-HaloTag Beads
		3.2 Lumit Cytokine Immunoassay
			3.2.1 Selection and Labeling of Candidate Primary Antibodies
			3.2.2 Rapid Screening of Candidate Antibody Pairs
			3.2.3 Lumit Cytokine Immunoassay Optimization
			3.2.4 Calibration Curve and Cell-Based Detection of IL-4
		3.3 Lumit Phospho-ERK1 (Thr202) Immunoassay
			3.3.1 Detection of Phosphorylated ERK1 in MCF-7 Cells
			3.3.2 Testing Effect of a MEK Kinase Inhibitor on Phosphorylation of ERK1
			3.3.3 Lumit Immunoassay Cellular System Protocol
		3.4 Lumit SARS-CoV2 Spike RBD: hACE2 Immunoassay
			3.4.1 General Protocol of Lumit SARS-CoV 2 Spike RBD: hACE2 PPI Immunoassay
			3.4.2 Detection of Neutralizing Antibody Interference with RBD: ACE2 PPI
			3.4.3 Evaluation of Patient-Derived Samples for RBD: ACE2 PPI Neutralizing Effect
			3.4.4 Lumit PPI Data Analysis
	4 Notes
	References
Chapter 16: ELISA-Based Biosensors
	1 Introduction
	2 Integration of ELISA with Microfluidics
	3 Signal Enhancements: Enzymatic Amplification
		3.1 Enzyme-Labeled Fluorescence (ELF)
		3.2 Tyramide Signal Amplification (TSA)
	4 Digital ELISA
		4.1 dELISA
		4.2 ddELISA
		4.3 TSA-dELISA
		4.4 Pre-equilibrium Digital ELISA (PEdELISA)
	5 Other Immunosensor Platforms
		5.1 Temperature-Responsive Liposome-Linked Immunosorbent Assay (TLip-ELISA)
		5.2 Electrochemical Signal
	References
Index