# Development of Nanobody-Based Sandwich ELISA Resistant to SpA Interference for Sensitive Detection of Staphylococcal Enterotoxin A

## TL;DR

Researchers developed a new, highly sensitive test for detecting a dangerous toxin from Staphylococcus aureus that avoids false positives caused by a bacterial protein.

## Contribution

A novel sandwich ELISA using nanobodies and a monoclonal antibody that effectively eliminates SpA interference for sensitive SEA detection.

## Key findings

- Two nanobodies (SEA-4-20 and SEA-4-31) paired with mAb-4C6 achieved detection limits of 0.135 ng/mL and 0.137 ng/mL for SEA.
- The developed ELISA effectively eliminates false positives caused by S. aureus surface protein A (SpA) interference.
- The method offers a reliable tool for detecting SEA in food, clinical, and environmental samples.

## Abstract

Staphylococcus aureus is a major pathogen responsible for staphylococcal food poisoning (SFP), with its pathogenicity primarily dependent on staphylococcal enterotoxins (SEs). Among these, staphylococcal enterotoxin A (SEA) is a critical risk factor due to its high toxicity, high detection rate (accounting for 80% of SFP cases), strong thermal stability, and resistance to hydrolysis. Traditional SEA immunoassays, such as enzyme-linked immunosorbent assay (ELISA), are prone to false-positive results caused by nonspecific binding interference from S. aureus surface protein A (SpA). In recent years, nanobodies (single-domain heavy-chain antibodies) have emerged as an ideal alternative to address SpA interference owing to their small molecular weight (15 kDa), high affinity, robust stability, and lack of Fc regions. In this study, based on a previously developed highly specific monoclonal antibody against SEA (mAb-4C6), four anti-SEA nanobodies paired with mAb-4C6 were obtained through two-part (four-round) of biopanning from a naive nanobody phage display library. Among these, SEA-4-20 and SEA-4-31 were selected as optimal candidates and paired with mAb-4C6 to construct double-antibody sandwich ELISAs. The detection limits for SEA were 0.135 ng/mL and 0.137 ng/mL, respectively, with effective elimination of SpA interference. This approach provides a reliable tool for rapid and accurate detection of SEA in food, clinical, and environmental samples.