Immunocapture strategies in translational proteomics

Figures & data

Figure 1. Schematic representation of immunocapture strategies discussed in this review. AP-MS: Affinity purification mass spectrometry for interaction studies; FPPA: Forward phase protein array; IC-MS: Immunocapture coupled to mass spectrometry (DDA or DIA); IC-SRM: Immunocapture coupled to SRM; IHC: Immunohistochemistry; RPPA: Reverse phase protein array; SBA: Suspension bead arrays; SIA: Sandwich immunoassay.

Table 1. Main analytical features of antibody capture technologies.

	SIA	TMA-IHC^†	FPPA	RPPA	SBA	IC-MS	IC-SRM
Specificity	High	Low	Low	Low	Low	High	High
Sensitivity	pg/ml	Semiquantitative^‡	ng/ml	ng/ml	ng-pg/ml	ng-pg/ml	ng-pg/ml
Throughput (samples)	~96	~230	~12	~500	~384	~5 (same time of an ELISA)	~40
Multiplex (affinity reagents)	Low	Low	High	Low	High	High	High
Automation	High	Low	Low	Low	High	High	High
Turnaround time	Low	Low	High	High	High	High	High
Sample type	Fluid	Tissue	Tissue	Fluid/tissue	Fluid/tissue	Fluid/tissue	Fluid/tissue
Sample required	1–100 µl		50 µg	0.1 µl/10 ng	0.1 µl/50 µg	10–1000 µl/1–50 mg	10–1000 µl/1–50 mg
Applications	Clinical	Clinical	Discovery/clinical	Discovery/clinical	Discovery	Validation	Validation

^†High-throughput TMA-IHC.

^‡For TMA-IHC, it is not possible to provide an absolute concentration, but continuous values of protein expression in tissues are obtained by methods such as Automated Quantitative Analysis (AQUA).[8] For this reason, the method has been defined semi-quantitative.

FPPA: Forward phase protein array; IC-MS: Immunocapture coupled to mass spectrometry (shotgun); IC-SRM: Immunocapture coupled to mass spectrometry (selected reaction monitoring); IHC: Immunohistochemistry; RPPA: Reverse phase protein array; SIA: Sandwich immunoassay; SBA: Suspension bead arrays; TMA-IHC: Tissue microarray immunohistochemistry.

Table 2. Analytical features of current and emerging immunoassay systems.

	Affinity reagents	Surface	Quantification	Multiplex (affinity reagents)	Sample volume	Dynamic range (logs)	Reference
ELISA	Abs	Microplates	Fluorescence or Chemiluminescence	Limited	>25 µl	2–3	[54]
Mesoscale	Abs	Microplates	Electrochemiluminescence	Limited	>25 µl	2–5	[63]
Singulex-Erenna®	Abs	Magnetic microparticles	Fluorescence (Ab-biotinylated)	No	10–100 µl	3–4	[64]
Bead based Sandwich assay	Abs	Magnetic microparticles	Fluorescence (Ab-biotinylated)	Scalable	50–100 µl	3–4	[65]
Bead based Antibody array	Abs or antigens	Magnetic microparticles	Fluorescence (Streptavidin-labeled)	Scalable	3 µl	3–4	[52]
Olink-Proximity Ligation Assays	Abs	In solution	PCR amplification	Scalable	1 µl	2–5	[53]
Quanterix-Simoa ®	Abs	Magnetic beads	Fluorescence (Streptavidin-labeled)	Scalable	150 µl	2–4	[66]
Somalogic- SOMAmer®	Modified Aptamers	Magnetic beads	DNA microarrays or qPCR	Scalable	150 µl	7–8	[55]
Simplex	Abs	Glass nanoreactors	Fluorescence (Ab-biotinylated)	Limited	<25	3–4	[67]
Immunosensors	Abs or aptamers	Nanomaterials	Chemiluminescence or label-free	Scalable	10 µl	3–7	[60]

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