Fluorescent Immunoassay for Determination of Staphylococcal Enterotoxin A in Milk by Immobilized F(ab')(2) Fragment of Anti-enterotoxin A Monoclonal Antibody

Foods with high protein content such as milk, meat and salads have been frequently incriminated in staphylococcal enterotoxins (SЕ) outbreaks. Staphylococcal enterotoxin A (SEA) is most toxic from all 20 SЕ. Potency of SEA requires that the methods of analysis in food must be sensitive enough to measure very low concentrations. A fluorescent immunoassay, sandwich format, for SEA detection in milk was developed, using immobilized F(ab′)2 fragment and anti-SEA monoclonal antibody (mAb) bonded onto the surface of magnetic nanoparticles (MNPs). F(ab′)2 fragment of anti-SEA mAb was obtained by pepsin hydrolysis of mAb. The fragment and antibody were conjugated with fluorescein 5(6)-isothiocyanate (FITC) and formed F(ab')2 fragment-FITC and mAb-FITC conjugates. The obtained conjugates were purified by Sephadex 25 size exclusion chromatography. A fluorescent immunoassay was developed by using immobilized F(ab')2 fragment and anti-SEA mAb and were obtained conjugates F(ab')2 fragment-FITC and mAb-FITC. The analytical parameters of the immunoassay with F(ab′)2 fragment were compared with those obtained from the immunoassay with mAb. When using a immobilized F(ab')2 and fluorescent conjugate F(ab')2 fragment - FITC, the linear range of SEA in buffer was from 0.7 to 300 pg/mL, and in milk from 1.0 to 300 pg/mL. With an immobilized mAb and mAb-FITC conjugate, the linear range of SEA in buffer and in milk was from 50 to 300 pg/mL. It was found that the immunoassay with F(ab')2 fragment allow the measurement of SEA at very low concentrations. The limit detection of SEA in milk samples with F(ab')2 fragment was 0.9 pg/mL and with whole antibody 30 pg/mL.