Simple solutions to false-positive staphylococcal enterotoxin assays with seafood tested with an enzyme-linked immunosorbent assay kit (TECRA)

Abstract

The TECRA kit, a commercial staphylococcal enterotoxin visual immunoassay kit, is an enzyme-linked immunosorbent assay system which utilizes polyvalent antisera against staphylococcal enterotoxin types A to E. The test is simple and rapid to perform (4 h) and has therefore been widely used for screening purposes. In this study, the TECRA kit produced a number of false-positive reactions with seafood; 25% of 218 samples of seven types of seafood gave false-positive results, particularly shellfish such as mussels (85%), clams (32%), oysters (23%), winkles (20%), and squid (13%). Some nonshellfish samples also gave false-positive results with the TECRA kit (smelt [20%] and trout [10%]). The substance contributing to the false-positive results differed from true staphylococcal enterotoxins in that it was: (i) heat labile, being completely inactivated by heating for 3 min at 70°C, compared with 5% inactivation of true staphylococcal enterotoxins by the same heat treatment, (ii) in a selective reaction with normal rabbit or calf serum (nonspecific reactions were completely abolished by these sera, whereas staphylococcal enterotoxins were not affected), and (iii) incapable of binding to a copper-chelate Sepharose gel (all of the substance remained in the unbound wash fraction, whereas staphylococcal enterotoxins were quantitatively bound to the gel). The false-positive reactions occurring with seafood were not associated with substances produced by microorganisms, since the bacterial isolates from the samples did not give positive results with the TECRA kit. The problems of nonspecific reactions occurring with the TECRA kit could be resolved by any of the following procedures: (i) metal chelate affinity chromatography (2 h) with copper chelate Sepharose, (ii) treatment with rabbit or calf normal serum (0.1 volume of serum added to food extract and held at room temperature for 2 min), or (iii) heat treatment at 70°C for 3 min. The recovery of staphylococcal enterotoxin following any of these procedures ranged from 90 to 95%.