Antibody Arrays: Methods and Protocols

Preface
Contents
Contributors
Chapter 1: Sandwich-Based Antibody Arrays for Protein Detection
	1 Introduction
	2 Materials
	3 Methods
		3.1 Slide, Sample, and Standard Preparation
		3.2 Blocking and Incubation
		3.3 Fluorescence Detection and Image Analysis
	4 Notes
	References
Chapter 2: Well-Based Antibody Arrays
	1 Introduction
		1.1 The Elephant in the Room-Antibody Sourcing
		1.2 Cross-Reactivity
		1.3 Printing Aspects
		1.4 Preparation of Antibodies for Printing
	2 Materials
		2.1 Immobilization by Physical Adsorption
		2.2 Colorimetric Immunoassay Development
		2.3 Immobilization by Covalent Attachment
			2.3.1 Activated Polypropylene Plate
			2.3.2 Epoxy-Activated Glass Surface
		2.4 Immobilization by Molecular Bridging
		2.5 Fluorescent Multiplex ELISA/Fluorescent Immunoassay (FIA)
	3 Methods
		3.1 Physical Adsorption
			3.1.1 Printing of the Capture Antibody Array Plate
			3.1.2 Colorimetric Immunoassay Development
			3.1.3 Imaging and Data Analysis
			3.1.4 ImageJ Analysis of Well-Based Antibody Arrays
		3.2 Covalent Coupling
			3.2.1 Covalent Cross-Linking of Antibodies to Activated Polypropylene
			3.2.2 Covalent Attachment of Antibodies onto Epoxy Glass Surfaces
		3.3 Molecular Bridging
			3.3.1 Preparation of Oligonucleotide-BSA Conjugates
			3.3.2 Immobilization of the Oligo-BSA-Antibody onto Polystyrene Plates
		3.4 Multiplex Immunoassays
			3.4.1 Oligo-BSA Antibody Array Fluorescent Multiplex ELISA
			3.4.2 Oligo-BSA Antibody Array FIA
	4 Notes
	References
Chapter 3: One-Step Antibody Arrays
	1 Introduction
	2 Materials
	3 Methods
		3.1 Preparation of the Antibody Array Glass Chip
		3.2 Sample Processing
		3.3 Imaging and Data Analysis
	4 Notes
	References
Chapter 4: Global Detection of Proteins by Label-Based Antibody Array
	1 Introduction
	2 Materials
		2.1 Array Printing
		2.2 Sample Preparation and Storage
		2.3 Sample Detection
	3 Methods
		3.1 Array Printing
		3.2 Sample Preparation
		3.3 Sample Detection
		3.4 Fluorescence Detection (Fig. 5)
	4 Notes
	References
Chapter 5: Surface Plasmon Resonance Imaging-Mass Spectrometry Coupling on Antibody Array Biochip: Multiplex Monitoring of Bio...
	1 Introduction
	2 Materials
		2.1 Chemical Reagents
		2.2 Proteins (Receptors and Ligands)
		2.3 Solvents, Buffer, and Solutions for SPRi Experiment
		2.4 Equipment
		2.5 Instrument
			2.5.1 SPR
			2.5.2 MALDI-TOF Mass Spectrometry
	3 Methods
		3.1 Gold Surface Cleaning
		3.2 PEG Self-Assembled Monolayer
		3.3 PEG Activation
		3.4 Antibody Grafting
		3.5 Biochip - SPRi Prism Assembly
		3.6 Passivation of the Biochip Surface
		3.7 SPRi Experiment
		3.8 MALDI-TOF MS Experiment
	4 Notes
	References
Chapter 6: Multiplexed Protein Biomarker Detection with Microfluidic Electrochemical Immunoarrays
	1 Introduction
	2 Materials
		2.1 Preparation of Immunoassays
		2.2 Microfluidic System (Fig. 1)
		2.3 Antigens and Conjugates
		2.4 Detection Chamber
	3 Methods
		3.1 Preparation of Modified Arrays
		3.2 Multiplexed Detection
	4 Notes
	References
Chapter 7: Cytometry Multiplex Bead Antibody Array
	1 Introduction
	2 Materials
	3 Methods
		3.1 Preparation of Sample and Reagents
		3.2 Preparation of Protein Standard Dilutions
		3.3 Array Procedure
		3.4 Flow Cytometer Setup and Data Acquisition
		3.5 Data Analysis (Fig. 5)
	4 Notes
	References
Chapter 8: Antibody Arrays: Barcode Technology
	1 Introduction
	2 Materials
		2.1 Preparation of Polydimethylsiloxane (PDMS) Chip for Barcode Flow Patterns
		2.2 Construction and Validation of One/Two-Dimensional DNA Barcode Array
		2.3 Conversion of Two-Dimensional DNA Barcode Array into an Antibody Array
	3 Methods
		3.1 Preparation of PDMS Chip for Barcode Flow Patterns
		3.2 Construction and Validation of One-Dimensional DNA Barcode Array
		3.3 Construction and Validation of Two-Dimensional DNA Barcode Array
		3.4 Conversion of Two-Dimensional DNA Barcode Array into an Antibody Array
	4 Notes
	References
Chapter 9: Reverse Phase Protein Arrays
	1 Introduction
	2 Materials
		2.1 Specimen Preparation
		2.2 Cell Culture
		2.3 RPPA Construction
			2.3.1 RPPA Printing
			2.3.2 RPPA Total Protein Test Print
		2.4 RPPA Immunostaining
		2.5 RPPA Colorimetric Image Acquisition
	3 Methods
		3.1 Specimen Preparation
		3.2 Cell Culture
			3.2.1 Adherent Cell Lines
			3.2.2 Suspension Cell Lines
		3.3 RPPA Construction
			3.3.1 RPPA Printing
			3.3.2 RPPA Total Protein Test Print
		3.4 RPPA Immunostaining
		3.5 RPPA Colorimetric Image Acquisition
	4 Notes
	References
Chapter 10: Enhanced Protein Profiling Arrays for High-Throughput Quantitative Measurement of Cytokine Expression
	1 Introduction
	2 Materials
	3 Methods (Fig. 1)
	4 Notes
	References
Chapter 11: Methods for Membranes and Other Absorbent Surfaces
	1 Introduction
	2 Materials
	3 Methods
	4 Notes
	References
Chapter 12: Precise Chip-to-Chip Reagent Transfer for Cross-Reactivity-Free Multiplex Sandwich Immunoassays
	1 Introduction
	2 Materials
		2.1 Buffers and Solutions
		2.2 Material and Specialized Equipment
	3 Methods
		3.1 Sample Preparation and Incubation
		3.2 SNAP: Precisely Deliver the Detection Antibodies to Their Cognate Spots
		3.3 Pre-wash  SNAP
		3.4 Measuring the Assay Signals
	4 Notes
	References
Chapter 13: Antibody Printing Technologies
	1 Introduction
	2 Contact Printing
		2.1 Pin Printing
		2.2 Microstamp Printing
		2.3 Stencil Lithography
		2.4 Microfluidic Printing
	3 Noncontact Printing
		3.1 Inkjet Printing
	4 Lithography
		4.1 Photolithography
		4.2 Electron Beam Patterning
	5 AFM Nanoscale Patterning Techniques
		5.1 Dip-Pen Lithography
		5.2 Nanoshaving
	6 Summary
	References
Chapter 14: Self-Assembling Peptide Hydrogels for 3D Microarrays
	1 Introduction
	2 Materials
		2.1 Peptide Synthesis and Purification: Resin Loading
		2.2 Peptide Coupling
		2.3 Cleavage from the Resin
		2.4 Work-Up, RP-HPLC Analysis, and Purification
		2.5 Microarrays
	3 Methods
		3.1 Peptide Synthesis
		3.2 Hydrogel Formation
		3.3 Microarrays: General Procedure
		3.4 3D Immunoassay  Test
	4 Notes
	References
Chapter 15: Surface Modification of Glass Slides with Aminosilanes for Microarray Use
	1 Introduction
	2 Materials
	3 Methods
	4 Notes
	References
Chapter 16: Dried Blood-Based Protein Profiling Using Antibody Arrays
	1 Introduction
	2 Materials
		2.1 Blood Collection and Preparation
		2.2 Antibody Array Analysis
	3 Methods
		3.1 DBS Collection
		3.2 DBS Preparation
		3.3 Antibody Array Analysis
	4 Notes
	References
Chapter 17: Profiling Glycoproteins on Functionalized Reverse Phase Protein Array
	1 Introduction
	2 Materials
	3 Methods
		3.1 Synthesis of Hydroxylamine Functionalized PAMAM
		3.2 Periodate Oxidation of Samples
			3.2.1 Standard Glycoprotein
			3.2.2 Plasma
			3.2.3 Extracellular Vesicles of Plasma
			3.2.4 Urine
		3.3 polyGPA Analysis
	4 Notes
	References
Chapter 18: Determining Protein Phosphorylation Status Using Antibody Arrays and Phos-Tag Biotin
	1 Introduction
	2 Materials (See Note 1)
		2.1 Lysate Samples
		2.2 Reactions on Arrays
		2.3 Phosphate-Affinity Probing with Phos-Tag
		2.4 Enhanced Chemiluminescence Detection Reagent and Equipment
	3 Methods
		3.1 Reactions on Arrays
		3.2 Phosphate-Affinity Probing with Phos-Tag and Detecting Phosphoproteins
	4 Notes
	References
Chapter 19: Analyzing Signaling Pathways Using Antibody Arrays
	1 Introduction
	2 Materials
	3 Methods
		3.1 Sample Preparation (See Note 1)
		3.2 Array Assay
		3.3 Data Extraction and Analysis
	4 Notes
	References
Chapter 20: Using Antibody Arrays for Biomarker Discovery
	1 Introduction
	2 Materials
		2.1 Antibody Pair Preparation
		2.2 Printing of Antibody Microarrays
		2.3 Clinical Sample Preparation
		2.4 Antibody Array Performance
	3 Methods
		3.1 Preparation of Antibody Pairs
		3.2 Printing of Antibody Arrays
		3.3 Exclusion of Cross-Reactivity
		3.4 Sample Detection
		3.5 Data Analysis
	4 Notes
	References
Chapter 21: Immune Cell Isolation from Murine Intestine for Antibody Array Analysis
	Abbreviations
	1 Introduction
	2 Materials
		2.1 Equipment
		2.2 Reagents and Solutions
		2.3 Antibody and Staining Reagents
		2.4 Disposable Reagents
	3 Methods
		3.1 Dissection and Preparation of Small and Large Intestines
		3.2 Removal of the Epithelial Layer
		3.3 Digestion of Intestinal Tissue
		3.4 Isolation of Immune Cells
		3.5 FACS Sorting of Immune Cell Populations
		3.6 Optional: MACS Separation
	4 Notes
	References
Chapter 22: Database Development for Antibody Arrays
	1 Introduction
	2 Materials
	3 Methods
		3.1 Design the Database Framework
		3.2 Construct the Database in Alibaba Cloud
		3.3 Fill in Composition of Database Structure
		3.4 Construct the Analysis Tools
	4 Notes
	References
Chapter 23: Data Analysis for Antibody Arrays
	1 Introduction
	2 Data Preprocessing Transformation
	3 Differential Expression Analyses
		3.1 Fold Change
		3.2 Hypothesis Testing
		3.3 Linear Model
		3.4 Analysis of Variance
	4 Classification Analysis
		4.1 Unsupervised Classification
		4.2 Supervised Classification
	5 Biological Annotation Analyses
	References
Chapter 24: Statistical Analysis Options for Antibody Array Data
	1 Introduction
	2 A Brief Introduction of R
	3 Packages Outside the R Base Module
	4 Analysis of Antibody Array Data with R
		4.1 Data Cleanup and Transformation
		4.2 Visualization of Data Profile: Heatmap of Antibody Array Data
		4.3 Principal Component Analysis and Visualization
		4.4 Differential Expression Analysis
			4.4.1 Differential Expression Analysis by Target with Multiple Comparison Correction
			4.4.2 Differential Expression Analysis Based on Permutation
			4.4.3 Differential Expression Analysis Basing on Variance Estimated with Empirical Bayes Method
		4.5 Co-expression Network Analysis
		4.6 Functional/Knowledge Database-Based Enrichment Analysis
		4.7 Statistical Modeling
	5 Conclusion
	References
Index