APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 1977, p. 824-828 Copyright X 1977 American Society for Microbiology
Vol. 33, No. 4 Printed in U.S.A.
Iodination of Staphylococcal Enterotoxin B by Use of Chloramine-T D. S. ORTH The Procter & Gamble Company, Winton Hill Technical Center, Cincinnati, Ohio 45224
Received for publication 12 October 1976
This report describes the conditions that are necessary for iodination of staphylococcal enterotoxin B (SEB) by use of chloramine-T. Makor Chemical Co. SEB and the two major SEB components, which were prepared by isoelectric focusing of partially purified SEB, were used in these studies. The antigenic activity of the SEB preparations was monitored by radioimmunoassay as the oxidation/reduction (O/R) potential was increased by addition of chloramine-T. The SEB preparations lost antigenic activity rapidly at pH 7.5 and room temperature when suffilcient chloramine-T was added to raise the O/R potential above 250 mV. Iodinated SEB with satisfactory immunoreactivity was prepared by omitting carrier iodide from the iodination reaction mixture and by using at least 1 mg of SEB/ml, steps which made the O/R potential more stable, and by stopping the reaction before the O/R potential exceeded 250 mV. Comparison of the chloramine-T method with a lactoperoxidase/H202 method of iodinating SEB showed the latter to cause a greater loss of immunoreactivity. Strains of Staphylococcus aureus produce several serological types of enterotoxins that cause food poisoning when ingested. Although several methods for detecting staphylococcal enterotoxins have been developed, the use of radioimmunoassay (RIA) offers the advantages of speed and sensitivity. In theory, RIA allows both identification and quantitation of specific serological types of enterotoxin. In attempting to use the solid-phase assay described by Johnson et al. (4) as the basis for an RIA for staphylococcal enterotoxin B (SEB), difficulty was encountered in preparing iodinated SEB (I-SEB) that reacted satisfactorily with homologous antisera. The iodination procedure used by Johnson et al. (4) involved adding several reagents in succession, allowing the iodination reaction to proceed for a few minutes, and stopping the reaction by adding a reducing agent. I produced I-SEB with satisfactory immunoreactivity one time using this method; however, I failed to do so on several repeated tagging attempts. I turned to the standardized chloramine-T method of protein iodination reported by Hunter (3) but encountered problems in my attempts to iodinate SEB by this method. The purpose of this report is to describe these problems and the experiments that were set up to determine the conditions necessary for successful iodination of SEB by use of chloramine-T.
MATERIALS AND METHODS SEB and anti-B. Purified SEB and anti-B were obtained from Makor Chemicals, Ltd., Jerusalem, Israel. Makor SEB stock solution was prepared by adding SEB to 0.15 M phosphate-buffered saline at pH 7.5 (PBS at pH 7.5 = 0.15 M potassium phosphate buffer with 0.85% NaCl). Partially purified SEB, kindly furnished by John A. Troller, was fractionated by isoelectric focusing. The two major protein peaks, which were designated SEB no. 1 and SEB no. 2, caused emesis in kittens (10 jLg of protein), had no hemolysin activity (20 ug of protein/ ml), and had isoionic points of pH 8.3 and 8.1, respectively. Iodination. The first attempts at iodination of SEB were performed by the method of Johnson et al. (4). Later attempts used the standardized chloramine-T procedure of Hunter (3) in a 3- to 5-ml reaction mixture containing carrier iodide (KI). The first iodination of Makor SEB and the purified SEB no. 1 and no. 2 by Hunter's procedure used a 3. 1-ml reaction mixture made up of the following components: 0.3 mg of SEB, 1 ml of 0.15 M PBS, 2 ml of 0.1 M KI in 0.15 M PBS, and 50 or 100 ,uCi of 1251- ion in 100 ,ul of 0.1 N NaOH. The kinetics of 1251- ion incorporation into the SEB were initially determined by the trichloroacetic acid precipitation test described by Hunter. Later, the incorporation of the iodine nuclide was monitored by a rapid thin-layer technique (1). The iodination reactions were stopped by the addition of Na2S2O5 when 50 to 75% of the 1251- ion was bound to the SEB, and this required the addition of sufficient chloramine-T to raise the electromotive force (EMF) to 320 to 400+ mV. 824
VOL. 33, 1977
IODINATION OF STAPHYLOCOCCAL ENTEROTOXIN B
The modified Hunter method that was successful in iodinating SEB preparations is described below. Carrier-free Na'25I (1 mCi in 20 /.l of 0.1 N NaOH) was obtained from New England Nuclear Corp. Before use, 500 ,lp of 0.15 M PBS at pH 7.5 was added to the vial of Na125I. The radioiodide in PBS was added to a 10-ml beaker that contained 1 mg of SEB in 1 ml of PBS, which had been adjusted to an EMF of 10%. The I-SEB prepared by the procedure of Johnson et al. (4) gave (B/T)o ratios
Vol. 33, No. 4 Printed in U.S.A.
Iodination of Staphylococcal Enterotoxin B by Use of Chloramine-T D. S. ORTH The Procter & Gamble Company, Winton Hill Technical Center, Cincinnati, Ohio 45224
Received for publication 12 October 1976
This report describes the conditions that are necessary for iodination of staphylococcal enterotoxin B (SEB) by use of chloramine-T. Makor Chemical Co. SEB and the two major SEB components, which were prepared by isoelectric focusing of partially purified SEB, were used in these studies. The antigenic activity of the SEB preparations was monitored by radioimmunoassay as the oxidation/reduction (O/R) potential was increased by addition of chloramine-T. The SEB preparations lost antigenic activity rapidly at pH 7.5 and room temperature when suffilcient chloramine-T was added to raise the O/R potential above 250 mV. Iodinated SEB with satisfactory immunoreactivity was prepared by omitting carrier iodide from the iodination reaction mixture and by using at least 1 mg of SEB/ml, steps which made the O/R potential more stable, and by stopping the reaction before the O/R potential exceeded 250 mV. Comparison of the chloramine-T method with a lactoperoxidase/H202 method of iodinating SEB showed the latter to cause a greater loss of immunoreactivity. Strains of Staphylococcus aureus produce several serological types of enterotoxins that cause food poisoning when ingested. Although several methods for detecting staphylococcal enterotoxins have been developed, the use of radioimmunoassay (RIA) offers the advantages of speed and sensitivity. In theory, RIA allows both identification and quantitation of specific serological types of enterotoxin. In attempting to use the solid-phase assay described by Johnson et al. (4) as the basis for an RIA for staphylococcal enterotoxin B (SEB), difficulty was encountered in preparing iodinated SEB (I-SEB) that reacted satisfactorily with homologous antisera. The iodination procedure used by Johnson et al. (4) involved adding several reagents in succession, allowing the iodination reaction to proceed for a few minutes, and stopping the reaction by adding a reducing agent. I produced I-SEB with satisfactory immunoreactivity one time using this method; however, I failed to do so on several repeated tagging attempts. I turned to the standardized chloramine-T method of protein iodination reported by Hunter (3) but encountered problems in my attempts to iodinate SEB by this method. The purpose of this report is to describe these problems and the experiments that were set up to determine the conditions necessary for successful iodination of SEB by use of chloramine-T.
MATERIALS AND METHODS SEB and anti-B. Purified SEB and anti-B were obtained from Makor Chemicals, Ltd., Jerusalem, Israel. Makor SEB stock solution was prepared by adding SEB to 0.15 M phosphate-buffered saline at pH 7.5 (PBS at pH 7.5 = 0.15 M potassium phosphate buffer with 0.85% NaCl). Partially purified SEB, kindly furnished by John A. Troller, was fractionated by isoelectric focusing. The two major protein peaks, which were designated SEB no. 1 and SEB no. 2, caused emesis in kittens (10 jLg of protein), had no hemolysin activity (20 ug of protein/ ml), and had isoionic points of pH 8.3 and 8.1, respectively. Iodination. The first attempts at iodination of SEB were performed by the method of Johnson et al. (4). Later attempts used the standardized chloramine-T procedure of Hunter (3) in a 3- to 5-ml reaction mixture containing carrier iodide (KI). The first iodination of Makor SEB and the purified SEB no. 1 and no. 2 by Hunter's procedure used a 3. 1-ml reaction mixture made up of the following components: 0.3 mg of SEB, 1 ml of 0.15 M PBS, 2 ml of 0.1 M KI in 0.15 M PBS, and 50 or 100 ,uCi of 1251- ion in 100 ,ul of 0.1 N NaOH. The kinetics of 1251- ion incorporation into the SEB were initially determined by the trichloroacetic acid precipitation test described by Hunter. Later, the incorporation of the iodine nuclide was monitored by a rapid thin-layer technique (1). The iodination reactions were stopped by the addition of Na2S2O5 when 50 to 75% of the 1251- ion was bound to the SEB, and this required the addition of sufficient chloramine-T to raise the electromotive force (EMF) to 320 to 400+ mV. 824
VOL. 33, 1977
IODINATION OF STAPHYLOCOCCAL ENTEROTOXIN B
The modified Hunter method that was successful in iodinating SEB preparations is described below. Carrier-free Na'25I (1 mCi in 20 /.l of 0.1 N NaOH) was obtained from New England Nuclear Corp. Before use, 500 ,lp of 0.15 M PBS at pH 7.5 was added to the vial of Na125I. The radioiodide in PBS was added to a 10-ml beaker that contained 1 mg of SEB in 1 ml of PBS, which had been adjusted to an EMF of 10%. The I-SEB prepared by the procedure of Johnson et al. (4) gave (B/T)o ratios
