An improved slot blot assay for protein quantification in serum samples

Enzyme-linked immunosorbent assay (ELISA) is the current preferred method for analyzing serum samples; however, it is resource-intensive, and serum samples have classically posed challenges for traditional immunoblot-based assays due to interference from serum proteins. This may be attributed to the non-specific binding between secondary antibodies and immunoglobulin G (IgGs) in the serum.

To establish an improved slot blot assay protocol that eliminates non-specific staining for an effective analysis of proteins in serum samples.

We explored the utility of Protein G-agarose beads for depleting serum IgGs prior to slot blot analysis, comparing 1-2 rounds of IgG clearance with no clearance. Serum samples were subsequently analyzed for the relative abundance of four target proteins using cleared serum and slot blotting. Densitometric analysis of immunodetected protein bands was performed and normalized to total protein loading, as determined by Ponceau S red staining of membranes. Slot blot results were compared to ELISA-detected levels.

Optimal results were achieved with two rounds of serum incubation with Protein G-agarose beads to deplete IgGs prior to slot blotting. This method eliminated non-specific signals in membranes probed with secondary antibody alone and enabled the specific detection of serum proteins with primary antibodies. Spearman's rank correlations showed moderate to strong correlations between slot blots and ELISAs for each protein examined.

The addition of Protein G-agarose-mediated IgG depletion permits the reliable use of slot blots for the analysis of serum proteins.