Thiol-reactive compounds prevent nonspecific antibody binding in immunohistochemistry

Area of Science:

• Immunohistochemistry (IHC)
• Molecular Biology
• Biochemistry

Background:

• Nonspecific antibody binding causes confounding background staining in IHC.
• Spontaneous reduction of immunoglobulin disulfide bonds suggests a role for sulfhydryl interactions.
• Understanding these interactions is crucial for improving assay specificity.

Purpose of the Study:

• To test the hypothesis that sulfhydryl interactions mediate nonspecific antibody binding in IHC.
• To identify reagents that can inhibit this background staining.
• To enhance the signal-to-noise ratio in IHC experiments.

Main Methods:

• Coincubation of primary antibodies with various thiophilic reagents (e.g., reduced glutathione (GSH), L-cysteine) in TE buffer.
• Testing oxidized glutathione (GSSG) for comparison.
• Enzyme-linked immunosorbent assay (ELISA) to confirm antibody-GSH binding.
• Tissue preincubation with sulfhydryl-reactive reagents.

Main Results:

• Coincubation with GSH and other thiol-reactive compounds significantly inhibited background staining.
• Oxidized glutathione (GSSG) had no effect on background staining.
• Optimized GSH coincubation improved the IHC signal-to-noise ratio.
• Tissue preincubation with sulfhydryl-reactive reagents reduced background but also target sensitivity.

Conclusions:

• Thiol-reactive compounds effectively prevent nonspecific antibody binding in IHC.
• A mechanism involving interruption of sulfhydryl bonds between antibodies and tissues is proposed.
• Findings provide a molecular basis for improving IHC specificity and have implications for immunoassays, diagnostics, and therapeutics.