Slot Blot Assay as Method for Testing Sensitivity and Specificity of Antibodies

Lectin-like oxidized LDL receptor-1 (Lox-1) was recently reported as a marker for myeloid-derived suppressor cells (MDSCs) in the blood circulation of patients with lung disease and COVID-19-induced acute respiratory distress syndrome (ARDS). In previous studies, however, there were numerous inconsistencies in the expression pattern of Lox-1, with a variety of commercially available antibodies. This study utilized a slot blot approach to identify antibodies with a specific and high affinity binding to the extracellular and intracellular domains of Lox-1 (ECD and ICD, respectively). In the slot blot assay, protein samples are drawn under vacuum onto PVDF-F membrane then subjected to immunodetection with a horseradish peroxidase-based detection method. Recombinant Lox-1 ECD protein (BioTechne), recombinant Lox-1 ECD with RIPA and Laemmli, and ICD peptide (Biomatik) were used at 3 different concentrations as positive controls, and BSA was included as a negative control. HL60 cells, which were found by our laboratory to express negligible levels of Lox-1, and HEK293 cells expressing abundant levels of full-length Lox-1 protein were used to determine specificity of the antibodies for endogenous Lox-1 protein. Five antibodies previously used in research were tested using these negative (BSA, HL60 extract) and positive controls (recombinant protein, HEK293-Lox-1+ extract, primary antibody). Overall, this slot blot assay shows promise as a quick, qualitative way to determine antibody specificity and sensitivity. More refinement is required to ensure that all samples are properly placed onto the membrane.

Hello, my name is Ewen Crunkhorn, and my research project was on validating the slot blot assay as a method for testing sensitivity and specificity of commercial antibodies.

The lab I did my experiment with was studying Lox-1 expression in Covid-19 immune response, specifically in immune suppressive cells. However, during experimentation different antibodies showed conflicting molecular weights, and upon further literature review, these molecular weights were supported in studies that used a specific antibody, but were still in conflict across antibodies. As such, we sought a way to show the specificity and sensitivity of an antibody to Lox-1. The slot blot was identified as a potential avenue for validation.

We chose a variety of factors as follows. Two versions of the extracellular domain, or ECD (one denatured to show potential conformational bindings) and one version of the intracellular domain, or ICD, at 3 concentrations to test for sensitivity. Bovine Serum Albumin (BSA) , a bovine protein, and cell extracts shown to have negligible amounts of Lox-1 were used as negative controls, while the antibody itself and cell extracts shown to contain high levels of full length Lox-1 were used as positive controls.

Here we have the ICD antibody results. The first antibody is from ThermoFisher, and only a very faint band of the antibody positive control is seen, so it would have to be retested at higher concentrations. The second is Abcam, and shows even binding to the cell extracts, but only faint binding to the ICD. This would also have to be retested as the positive control band is not visible, but does suggest that the antibody is more sensitive to other protein species. Finally the Biorbyt antibody showed very high recognition of the ICD peptide and positive control. It also bound to both cell extracts, though much fainter.

Here we have the two ECD antibodies. The Biotechne antibody shows only faint binding at the highest non-denatured ECD concentration, which suggest a conformational binding to Lox-1. The Sigma antibody only shows binding the negative control cell extracts, suggesting that it recognizes a species other than Lox-1. Both would need to be retested as the positive control bands are not visible.

In conclusion, the slot blot shows promise as a method to validate commercial antibodies for laboratory use, but needs refinement for publication data.