title: Development and evaluation of IgY ImmunoCapture PCR ELISA for detection of Staphylococcus aureus enterotoxin A devoid of protein A interference.
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.
authors.affiliation: Department of Microbiology, Defence Food Research Laboratory, Mysore 570011, India.
authors.forename: Prakash
authors.initials: P
authors.lastname: Reddy
authors.affiliation: Department of Microbiology, Defence Food Research Laboratory, Mysore 570011, India.
authors.forename: Shylaja
authors.initials: S
authors.lastname: Ramlal
authors.affiliation: Department of Microbiology, Defence Food Research Laboratory, Mysore 570011, India.
authors.forename: Murali Harishchandra
authors.initials: MH
authors.lastname: Sripathy
authors.affiliation: Department of Microbiology, Defence Food Research Laboratory, Mysore 570011, India. Electronic address: drharshvardhanbatra@gmail.com.
authors.forename: Harsh Vardhan
authors.initials: HV
authors.lastname: Batra
mesh_terms: D000818:Animals; D000918:Antibody Specificity; D003429:Cross Reactions; D004768:Enterotoxins / Q000235:genetics / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000592:standards; D005189:False Positive Reactions; D005506:Food Contamination; D006801:Humans; D007136:Immunoglobulins; D008892:Milk / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000592:standards; D011237:Predictive Value of Tests; D015203:Reproducibility of Results; D013202:Staphylococcal Food Poisoning / Q000175:diagnosis* / Q000276:immunology / Q000382:microbiology; D013205:Staphylococcal Protein A / Q000276:immunology*; D013211:Staphylococcus aureus / Q000235:genetics / Q000276:immunology*
chemical_list: D004768:Enterotoxins; C016512:IgY; D007136:Immunoglobulins; D013205:Staphylococcal Protein A; C026066:enterotoxin A, Staphylococcal
keywords: Enterotoxin A; ImmunoCapture PCR; Immunoglobulin Y; PCR-ELISA; Spiking; Staphylococcal protein A