A BIOSENSOR METHOD FOR A COMPETITIVE IMMUNOASSAY DETECTION OF STAPHYLOCOCCAL ENTEROTOXIN B (SEB) IN MILK²

Abstract

A sensitive and more rapid biosensor method for the detection of staphylococcal enterotoxins (SE) is needed by the food industry. Staphylococcal enterotoxin B (SEB) is highly heat resistant and is a potential bioterrorism agent. Our research objective was to develop a competitive immunoassay using a surface plasmon resonance (SPR) biosensor for the detection of SEB below 1 ng/mL [part per billion (ppb)] in fresh fluid milk. The assay consisted of SEB immobilization on the sensor surface. An anti‐SEB was allowed to bind with the SEB in samples off line prior to the biosensor analysis. The excess and unbound anti‐SEB was then captured by SEB sensor. The assay conditions were optimized to detect SEB in HEPES buffer and in whole milk. An analysis of milk samples spiked with 0.312–50 ppb. SEB consisted of heating the samples at 95C followed by rapid cooling and centrifugation at 2961 × g to separate the skim fraction. Aliquots of the skim fraction containing SEB were allowed to bind with anti‐SEB for 30 or 60 min. The SEB and anti‐SEB complex were separated from the free anti‐SEB by centrifugation, and the supernatants were injected over the sensor. SEB was detectable in buffer at 0.78–50 ppb and in spiked whole and skim milk from 0.312–25 ppb. The biosensor analysis including the sensor regeneration was 15 min per sample in a fully automated system. The competitive assay format resulted in higher detection sensitivity and greater sample throughput than the SPR biosensor sandwich assay. The competitive assay will be utilized for the detection of SEB in various foods and will be optimized for the detection of other staphylococcal toxins in foods.

A Biosensor Method for Detection of Staphylococcal Enterotoxin a in Raw Whole Egg*

“…The displacements shown in Table 1 are lower than those reported for the analysis of SEB in milk (Medina 2005), where the spiked SEB was detected from 0.312–25 ppb. Presumably, there are two reasons for these differences.…”

“…The centrifuged samples were analyzed with the Biacore system without decanting the supernatant from the pellet. We have reported that in the analysis of SEB in ham extracts (Medina 2003) and in milk samples (Medina 2005), SEB and anti‐SEB IgG remained in the supernatant after centrifugation at 2961 × g. Figure 3 shows the dose response curves in five trials when 0, 1–40 ppb SEB spiked in whole egg supernatant and in homogenized egg was plotted against RU of excess and unbound anti‐SEA. In trials I–III, 10 µg anti‐SEA was added to a 1‐g sample.…”

Toxin immunosensors and sensor arrays for food quality control

“…Antibodies bound to toxin molecules from the sample are washed away and the mass increase on the surface measured. A LOD of 312 pg/ mL of SEB in fresh whole milk and skimmed milk could be achieved [110]. SEA was verifiable in a similar approach in spiked raw egg supernatants with an LOD of 1 ng/mL and a dynamic range of 1-40 ng/mL within 15-min of assay time [111].…”

A Review of the Methods for Detection of Staphylococcus aureus Enterotoxins

“…Another sandwich immunoassay using SPR was also used for the determination of 2.5 ppb SEB-spiked ham tissue [ 90 ]. Moreover, a competitive pattern of SPR biosensor for detecting SEA and SEB in whole egg was established with the concentration range of 1–40 ng/mL SEA [ 91 ] and the LOD of SEB was lower than 1 ng/mL in fresh fluid milk [ 92 ]. SPR immunoassay combined mass spectrometry was established to detect SEB in spiked milk and mushrooms, and achieved a detection limit of 1 ng/mL [ 93 , 94 ].…”