Preparation of labeled staphylococcal enterotoxin A with high specific activity

Abstract

Staphylococcal enterotoxin A (SEA) was labeled by the chloramine-T method with 125I to a specific activity of 68 to 300 /XCi per jig of SEA and with 131I to specific activity of 8 to 218 uCi per jtg of SEA. SEA was partially damaged and aggregated during the labeling and storage. The damage seemed not to be greatly dependent on the specific activity of labeled enterotoxin. Crossed immunoelectrophoresis showed two antigenically active and three inactive components in the ascending part of the labeled enterotoxin peak during fractionation by gel chromatography. During storage at 4°C, the antigenic activity of label decreased faster when labeling had been with 131I than when with 1251. The antigenic activity of labeled SEA was lowered remarkably in the ascending part of the protein peak. Greatest release of radioiodine during storage was in the same part of protein peak. According to these results, the most suitable label for radioimmunoassay is obtained from the descending part of protein peak.

Radioiodination of Enterotoxin from Clostridium perfringens Type A using Chloramine T

“…It has often been reported that the ascending, centre or trailing portion of the elution peak from gel filtration after iodination may show significantly higher immunoreactivity (Hunter 1971;Niskanen & Lindroth 1976). The sub-fractions of the protein peak ( Fig.…”

“…The number of accessible tyrosine residues on the surface of the enterotoxin molecule is not known, but the total number of tyrosine residues has been found to be 18 (Granum & Skjelkvdle 1977). Many proteins are sensitive to chloramine T, and aggregate formation and diminution of the immunoreactivity is usually encountered (Sherman et a f . 1974; Kauffman & Johnson 1975;Niskanen & Lindroth 1976). Cf.…”

Detection of staphylococcal enterotoxins A, B, C, D, and E in foods by radioimnunoassay, using staphyloccal cells containing protein A as immunoadsorbent

“…In any case, the unavailability of the purified enterotoxins, particularly SED, and the small number of laboratories that can provide the necessary facilities for handling radioactive materials will greatly restrict the use of RIA. Labeling of the enterotoxins with 125I or "I has been studied extensively (5,11,16,18,19, 23) because of denaturation problems, particularly with SEA. Our attempts to iodinate the toxins with the lactoperoxidase method of Thorell and Johansson (24) and the modified chloramine-T procedure of Hunter (12) and Greenwood and Hunter (10) failed to give reproducible iodinations with adequate uptake of label.…”

“…P31, p. 259). Chloramine-T (5,15,18,19), lactoperoxidase (23), and gaseous iodine (11) have been used for the labeling of one or more of the enterotoxins with 1251I or 131I with varying degrees of success. The major problem has been in finding a labeling procedure which results in a reasonably stable antigen that retains its specificity for its analogous antibodies.…”