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J Immunol Methods. 2014 Jun;408:114-22. doi: 10.1016/j.jim.2014.05.012. Epub 2014 Jun 3.

Development and evaluation of IgY ImmunoCapture PCR ELISA for detection of Staphylococcus aureus enterotoxin A devoid of protein A interference.

Author information

1
Department of Microbiology, Defence Food Research Laboratory, Mysore 570011, India.
2
Department of Microbiology, Defence Food Research Laboratory, Mysore 570011, India. Electronic address: drharshvardhanbatra@gmail.com.

Abstract

In the present study, a sensitive and specific IgY mediated ImmunoCapture-PCR-ELISA (IC-PCR-ELISA) was developed for the detection of staphylococcal enterotoxin A (SEA) from culture supernatants and suspected contaminated samples. Due to the virtue of avian immunoglobulins (IgY) to have the least affinity towards staphylococcal protein A (SpA) responsible for false positives, we employed anti-SEA IgY for capture of SEA toxin and revealed with SEA specific rabbit antibodies conjugated to a 524bp DNA marker. Biotin-11-dUTP was incorporated during PCR amplification and post PCR analysis was performed by PCR-ELISA. Unlike IgG immunocapture, IgY mediated immunocapture of SEA was free from false positives due to protein A. The developed assay was specific to SEA except for minor cross reactivity with staphylococcal enterotoxin E (SEE). Several raw milk samples were evaluated for the presence of SEA with and without enrichment. Three samples were found to be positive for SEA after enrichment for 8h. Though IC-PCR-ELISA for SEA showed 100% correlation with PCR analysis for sea gene, the assay was unique in terms of sensitivity of detecting ~10pg/ml of SEA toxin from spiked milk samples. Result of IC-PCR-ELISA was further confirmed by conventional methods of isolation and characterization. The presented method can be very useful for rapid analysis of milk samples for SEA contamination and can be further extended for detection of multiple SE's in different wells of same PCR plate using common DNA substrate.

KEYWORDS:

Enterotoxin A; ImmunoCapture PCR; Immunoglobulin Y; PCR-ELISA; Spiking; Staphylococcal protein A

PMID:
24941881
DOI:
10.1016/j.jim.2014.05.012
[Indexed for MEDLINE]

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