Table 1 The pros and cons of different immunoassay technologies

Size-CNIA

Nanogram sample loading, good reproducibility, quantitative data, medium throughput for multi-sample or multi-target analysis, straight forward assay transfer from conventional Western blot, automated operation, easy protocol standardization

Limited matrix options for high resolution analysis on small and big MW proteins

Charge-CNIA

Nanogram sample loading, quantitative data, good reproducibility, medium throughput for multi-sample or multi-target analysis, distinguish and detect isoform variations or PTMs with pan-reactive antibody, automated operation, easy protocol standardization

Often need to test multiple antibodies for a target, limited ampholyte options for low and high pI protein analysis, peak identity determination is challenging

Conventional Western blot

Has been top choice for studying protein isoform variations, post-translational modifications

Microgram sample loading, poor data quantitation and reproducibility, low throughput, tedious manual processing steps, challenge to transfer assay from discovery research for clinical application

IHC

Provide information about protein localization within the cell, most used immunoassay for clinical tissue sample analysis

Poor data quantitation and reproducibility, data interpretation is subjective, difficult in protocol standardization

ELISA

Has been gold standard for protein concentration measurement, quantitative data, good reproducibility

Microgram sample loading, need isoform specific antibody, stringent antibody evaluation for specificity, relative long assay development

Multiplexed bead assays

Multiplex, high throughput, small sample consumption, automation

Potential cross-reactivity issues, stringent antibody evaluation for specificity, long assay development process, expensive reagents and consumables

Antibody array

Multiplex, high throughput, small sample consumption, automation

Potential cross-reactivity issues, stringent antibody evaluation for specificity, long assay development process

RPPA

Multiplex, high throughput, ng sample loading, automation

Stringent antibody evaluation for specificity, long assay development process, high cost when perform low sample number multi-target analysis