Comparative Inhibitory Effects of Antigen and Antibody in the Staphylococcal Enterotoxin Solid-Phase Radioimmunoassay System

A solid-phase radioimmunoassay employing 125I-labeled enterotoxins and polystyrene tubes coated with specific antibody has been developed for assaying the relative concentrations of antibodies to staphylococcal enterotoxins A and B. Competitive binding occurs between tube-bound antibody and free antibody for binding sites on 125I-labeled enterotoxin. The sensitivity of the system is affected by the amount of antibody on the walls of the tubes, the concentration of 125I-labeled enterotoxin added to the system, and probably by the relative binding affinities of the bound and unbound antibodies. Antibody, 0.01 to 0.07 mug/ml, inhibited the uptake of 125I-labeled enterotoxin by 20%. Both the antibody and antigen solid-phase radioimmunoassay inhibition systems can be appropriately represented by either of the following two models: Loge (Y/1 - Y) = alpha0 + alpha1 LogeX and LogeY = beta0 + beta1 LogeX, where Y is bound activity, X is antigen or antibody concentration for inhibition, and alpha0, alpha1, beta0, and beta1 are regression coefficients. Estimates from the first model were slightly more precise for the antibody system, whereas the reverse was true for the antigen system.     

Staphylococcal Enterotoxin C: Solid-Phase Radioimmunoassay

A solid-phase radioimmunoassay test employing 125I-labeled enterotoxin C and polystyrene tubes coated with specific antibody was used for the detection and quantitation of entertoxin C in condensed milk, cheddar cheese, custard, and ham salad. The assay was sensitive to 1 to 10 ng of toxin per g of food; nonspecific inhibitions were 16% or less.     

Modified Radioimmunoassay Determination for Staphylococcal Enterotoxin B in Foods

The sensitivity of solid-phase radioimmunoassay for the measurement of staphylococcal enterotoxin B (SEB) in foods was decreased by food constituents that react with rabbit anti-SEB with an equivalency of over 2 ng/ml. This activity was minimized by a conditioning step for anti-SEB and by removal of interfering compounds in the sample by extraction. The assay was a sequential solid-phase radioimmunoassay technique in which polystyrene test tubes were initially incubated with antisera and then with bovine serum albumin. The tubes were then conditioned with either a centrifuged aqueous cheese extract or, equally effective, reconstituted nonfat dry milk for 16 h at 4°C. Samples of milk or heat-treated and CHCl₃-extracted cheese or chicken salad slurries were incubated in the assay tubes for 6 h at 37°C. The samples were replaced by ¹²⁵I-labeled SEB and incubated for a further 2 to 4 h before the contents were removed and the tubes were washed and counted. A buffer solution containing known concentrations of toxin served as standards for assaying SEB in the food extracts. The entire assay can be accomplished within 24 h with a sensitivity of 1 ng/ml in milk and in the cheese extract or 1.3 ng/ml in the chicken salad extract.     

Staphylococcal Enterotoxins A and B: Solid-Phase Radioimmunoassay in Food

An immunoassay employing (125)I labeled enterotoxins A and B and polystyrene tubes coated with specific antibodies was used for detection and quantitation of enterotoxin in food. Ham salad, cheddar cheese, custard, condensed milk, and salami were studied. Enterotoxin was successfully determined in all the foods by simple extraction procedures. The assay was sensitive to 1 to 10 ng of toxin per g of food; nonspecific inhibitions were 15% or less.     

Interaction of Escherichia coli heat-stable enterotoxin B with rat intestinal epithelial cells and membrane lipids

The binding of 125I-labeled Escherichia coli heat-stable enterotoxin B to rat intestinal epithelial cells was unsaturable and nonspecific, at concentrations well above that required to mediate biological events. Following its interaction with intestinal cells, approximately 50-80% of heat-stable enterotoxin B remained stably associated with the cells, implying that it was partitioned into the membrane and/or internalized by the cell. The toxin bound with different affinities to lipids isolated from intestinal epithelial cells, phospholipids, glycolipids, neutral lipids and to model membrane vesicles containing negatively charged lipids. These results indicate that heat-stable enterotoxin B utilizes the membrane bilayer, rather than a surface protein or glycoprotein in modulating toxin-induced enterotoxicity.     

Paw oedema test for detection of Salmonella enterotoxin : modification and standardization

Paw oedema test (POT) was standardized with modifications for the detection of Salmonella enterotoxin. Instead of measuring the weight of the inoculated paws after amputating the limbs at 48 hr post-inoculation, percent relative thickness of the order of 121 +/- 3.8% at 24-48 hr was found to be a better index. This test yielded parallel results to rabbit ligated ileal loop (RLIL) technique. The test was positive with enterotoxic crude cell lysates (CL) and cell free-culture-supernatants (CFCS) of S. newport and S. typhimurium, partially purified and purified enterotoxin of S. newport and purified cholera toxin. The test was found to be specific in that non-enterotoxic CFCS did not cause significant increase in the thickness. Minimum detection level of purified S. newport enterotoxin was estimated to be as low as 20 micrograms. Thus, the modified POT was considered to be an effective and economical bioassay model for the detection of Salmonella enterotoxin.     

Thermal Inactivation of Staphylococcal Enterotoxin B in Veronal Buffer

The times and temperatures required to inactivate staphylococcal enterotoxin B were studied by use of the double-gel-diffusion technique to assay enterotoxin. Enterotoxin B (99 +% pure) was suspended in 0.04 M Veronal buffer, dispensed into borosilicate vials, and the vials were sealed and heated in an oil bath. An amount of 30 mug/ml of this toxin was reduced to less than 0.7 mug/ml in 103.0, 87.1, 70.5, 57.2, 39.1, 27.6, 16.4, and 12.0 min, respectively, at temperatures of 96, 99, 101.7, 104.4, 110, 115.6, 121, and 126.7 C. The end point for enterotoxin inactivation by gel diffusion was identical to that by intravenous injection of cats. Limited studies with crude enterotoxin B showed that the crude preparation was slightly more thermostable. The respective D values of crude and purified enterotoxin B were 64.5 and 52.3, 40.5 and 34.4, 29.7 and 23.5, 18.8 and 16.6, and 11.4 and 9.9 min at temperatures of 99, 104.4, 110, 115.6, and 121 C. The z value for purified enterotoxin B was 32.4 C. The experimental activation energy was 20,700 cal/g mole, standard enthalpy of activation at 120 C was 19,900 cal/g mole, standard entropy of activation at 120 C was -21.4 cal/g mole K, and the standard free energy of activation at 120 C was 28,200 cal/g mole.     

Use of glycosyltransferases to restore pertussis toxin receptor activity to asialoagalactofetuin

Fetuin derivatives with enzymatically altered oligosaccharide units were tested for their ability to inhibit pertussis toxin-mediated agglutination of goose erythrocytes and the binding of 125I-labeled fetuin to pertussis toxin-coated polystyrene tubes. Fetuin oligosaccharides were sequentially degraded by treatment with: neuraminidase (asialofetuin) followed by beta-galactosidase (asialoagalactofetuin) and, lastly, with beta-N-acetylhexosaminidase (asialoagalacto-a[N-acetylglucosamino]fetuin). Asialofetuin retained only 19 and 53% of the inhibitory activity of native fetuin in the hemagglutination and 125I-fetuin binding assays, respectively. Asialoagalactofetuin showed no further reduction of inhibition in the hemagglutination system and, instead, resulted in partial recovery of inhibition in the 125I-fetuin-pertussis toxin binding assay. Asialoagalacto-a[N-acetylhexosamino]fetuin showed a further decrease in ability to inhibit pertussis toxin binding in both assays. The inhibitory activity of asialoagalactofetuin could be restored to that of native fetuin by adding back D-galactose with UDP-Gal:D-glucosyl-1,4-beta-galactosyltransferase, followed by the addition of terminal sialic acid residues with CMP-N-acetylneuraminic acid:beta-D-galactosyl-1,4-N-acetyl-beta-D-glucosamine-alpha-2,6-N- acetylneuraminyltransferase. The data suggested that a requirement for pertussis toxin binding to fetuin may be the presence of acetamido-containing sugar groups in the nonreducing terminal position of fetuin's oligosaccharides.     

Specificity and cross-reactivity of staphylococcal enterotoxin A monoclonal antibodies with enterotoxins B, C1, D, and E.

The cross-reactivity of monoclonal antibodies produced against staphylococcal enterotoxin A with purified and crude enterotoxins B, C1, D, and E and the specificity of such reactions were evaluated by the indirect enzyme-linked immunosorbent assay and immunoblotting of Western blots (from sodium dodecyl sulfate-polyacrylamide gel electrophoresis) followed by autoradiography. Purified and crude enterotoxins B were also tested with polyclonal antibodies. Specificity of reactivity was demonstrated by immunoblotting of crude enterotoxin A, crude enterotoxin A treated with trypsin, crude enterotoxin E, and also with crude A, B, C1, and D that were pretreated with Sepharose-4B-linked normal rabbit immunoglobulin G to remove protein A. A band corresponding to each staphylococcal enterotoxin was seen with monoclonal antibodies under all conditions tested and also with crude and purified enterotoxin B with two different (rabbit and goat) polyclonal antisera.     

Specificity and cross-reactivity of staphylococcal enterotoxin A monoclonal antibodies with enterotoxins B, C1, D, and E

The cross-reactivity of monoclonal antibodies produced against staphylococcal enterotoxin A with purified and crude enterotoxins B, C1, D, and E and the specificity of such reactions were evaluated by the indirect enzyme-linked immunosorbent assay and immunoblotting of Western blots (from sodium dodecyl sulfate-polyacrylamide gel electrophoresis) followed by autoradiography. Purified and crude enterotoxins B were also tested with polyclonal antibodies. Specificity of reactivity was demonstrated by immunoblotting of crude enterotoxin A, crude enterotoxin A treated with trypsin, crude enterotoxin E, and also with crude A, B, C1, and D that were pretreated with Sepharose-4B-linked normal rabbit immunoglobulin G to remove protein A. A band corresponding to each staphylococcal enterotoxin was seen with monoclonal antibodies under all conditions tested and also with crude and purified enterotoxin B with two different (rabbit and goat) polyclonal antisera.     

Resistance of Free-Living Nematodes to Staphylococcal Enterotoxin

The usefulness of free-living nematodes for assaying staphylococcal enterotoxin was evaluated with a 98% pure enterotoxin B on five different nematodes. Included in the evaluation was an enterotoxin B in a crude culture filtrate. The filtrate of a culture of nonenterotoxigenic strains of Staphylococcus aureus, the uninoculated respective broth media, and distilled water were used as controls. The purified enterotoxin was found to exert no toxic effects at dosages ranging from 10 to 1,000 μg/ml for as long as 24 hr. Utilization of the toxin-protein by these nematodes was evidenced by their propagation after exposure times longer than 24 hr. The crude filtrate, containing 28 μg of enterotoxin per ml, was detrimental to nematodes to the same degree as the nontoxic filtrate and the uninoculated broths, in that they all caused irritation to external genitalia, motility changes, and death after comparable exposure times. This is in agreement with earlier observations that standard bacteriological fluid media, or broths containing over 1% protein hydrolysate or 1 to 2% salts, exert toxic effects on free-living nematodes.     

Staphylococcal Enterotoxin D

Purification of enterotoxin D from Staphylococcus aureus 494 was attempted by utilizing the following techniques: Concentration of bacterial culture supernatant with polyethylene glycol 20000, CM-cellulose column chromatography, Sephadex G-100 gel-filtration, chromatography on a DEAE cellulose column with concave gradient elution and gel-filtration with Sephadex G-50. The final preparation obtained gave a single precipitin line with anti-crude enterotoxin D serum and no precipitation with anti-enterotoxin A, B or C when it was tested by Ouchterlony's technique. It was capable of producing 100% emesis in monkeys at about 1.25 μg of protein per kg body weight. Antiserum prepared by injecting rabbits with the purified preparation gave a single precipitin line with concentrated crude toxin, and it also gave a single arc when tested in immunoelectrophoresis with concentrated crude toxin. A type-specific neutralization effect in monkeys was obtained. Thus, enterotoxin D has been identified with a purified preparation.     

A novel method for the detection of receptors and membrane proteins by scintillation proximity radioassay

A rapid and convenient binding assay for receptors and membrane proteins has been developed. It is based on the binding of 125I-labeled ligands to membrane proteins adsorbed to polyvinyltoluene plastic scintillation microspheres. Membranes or isolated membrane proteins adsorb to the beads upon mixing, and addition of 125I-labeled ligand induces photon emission which is proportional to the amount of added receptor or membrane protein. The interaction of acetylcholine receptor with 125I-labeled alpha-bungarotoxin and antigens with 125I-labeled antibodies or protein A were used as models to test the system. As little as 1 ng of acetylcholine receptor is detected by the assay and a linear relationship with receptor concentration is observed up to 50 ng of receptor per 250 microliter reaction medium. The effects of detergents, salts, soluble proteins, and neutral membranes were studied. Inclusion of bovine serum albumin up to 1 mg/ml, sodium chloride up to 0.5 M, and membranes up to 10 micrograms/ml cause little or no effect on the assay. Detergents at 10-fold below their critical micelle concentrations had little or no effect on the assay. The pharmacological effects of agonists such as acetylcholine were conveniently studied by following the displacement of the 125I-labeled ligand. Similarly, the amount of toxin in crude snake venom can be assayed by measuring competition with the labeled toxin. Only a few seconds are required to perform each binding assay.     

Tetrodotoxin-insensitive sodium channels. Binding of polypeptide neurotoxins in primary cultures of rat muscle cells

The binding of 125I-labeled derivatives of scorpion toxin and sea anemone toxin to tetrodotoxin-insensitive sodium channels in cultured rat muscle cells has been studied. Specific binding of 125I-labeled scorpion toxin and 125I-labeled sea anemone toxin was each blocked by either native scorpion toxin or native sea anemone toxin. K0.5 for block of binding by several polypeptide toxins was closely correlated with K0.5 for enhancement of sodium channel activation in rat muscle cells. These results directly demonstrate binding of sea anemone toxin and scorpion toxin to a common receptor site on the sodium channel. Binding of both 125I-labeled toxin derivatives is enhanced by the alkaloids aconitine and batrachotoxin due to a decrease in KD for polypeptide toxin. Enhancement of polypeptide toxin binding by aconitine and batrachotoxin is precisely correlated with persistent activation of sodium channels by the alkaloid toxins consistent with the conclusion that there is allosteric coupling between receptor sites for alkaloid and polypeptide toxins on the sodium channel. The binding of both 125I-labeled scorpion toxin and 125I-labeled sea anemone toxin is reduced by depolarization due to a voltage-dependent increase in KD. Scorpion toxin binding is more voltage-sensitive than sea anemone toxin binding. Our results directly demonstrate voltage-dependent binding of both scorpion toxin and sea anemone toxin to a common receptor site on the sodium channel and introduce the 125I-labeled polypeptide toxin derivatives as specific binding probes of tetrodotoxin-insensitive sodium channels in cultured muscle cells.     

Detection of staphylococcal enterotoxins by enzyme-linked immunosorbent assays and radioimmunoassays: comparison of monoclonal and polyclonal antibody systems.

Murine monoclonal antibodies reactive with at least one of the serological types of staphylococcal enterotoxin were examined for use in assay systems for the detection of enterotoxin at the level of 1.0 ng of enterotoxin per ml. An antibody sandwich enzyme-linked immunosorbent assay was devised for each toxin type by identifying an effective antibody pair. One antibody (the coating antibody) was coated onto a polystyrene plate and removed the enterotoxin from the test solution; the second antibody (the probing antibody) was conjugated to horseradish peroxidase and detected the captured toxin. Enterotoxins A and E could be detected in the same system by the use of cross-reacting monoclonal antibodies. All subtypes of enterotoxin C could be detected in one assay system. Two effective systems were described for each of types B and D. Each of these systems, when compared with the homologous enterotoxin-specific polyclonal rabbit antibody systems, was found to compare favorably. The monoclonal enzyme-linked immunosorbent assay systems for the detection of enterotoxins A and C2 were examined for a variety of food extracts; no abnormal interference could be detected from these extracts. The monoclonal antibody systems were also compared with the homologous enterotoxin-specific polyclonal serum for the detection of enterotoxin by the competitive radioimmunoassay (RIA). Single monoclonal antibodies generally did not perform as well in the RIA as did the homologous toxin-specific polyclonal serum. However, pools of monoclonal antibodies were prepared that approached the sensitivity and precision of the polyclonal system for the detection of each toxin by the RIA.     

Detection of staphylococcal enterotoxins by enzyme-linked immunosorbent assays and radioimmunoassays: comparison of monoclonal and polyclonal antibody systems

Murine monoclonal antibodies reactive with at least one of the serological types of staphylococcal enterotoxin were examined for use in assay systems for the detection of enterotoxin at the level of 1.0 ng of enterotoxin per ml. An antibody sandwich enzyme-linked immunosorbent assay was devised for each toxin type by identifying an effective antibody pair. One antibody (the coating antibody) was coated onto a polystyrene plate and removed the enterotoxin from the test solution; the second antibody (the probing antibody) was conjugated to horseradish peroxidase and detected the captured toxin. Enterotoxins A and E could be detected in the same system by the use of cross-reacting monoclonal antibodies. All subtypes of enterotoxin C could be detected in one assay system. Two effective systems were described for each of types B and D. Each of these systems, when compared with the homologous enterotoxin-specific polyclonal rabbit antibody systems, was found to compare favorably. The monoclonal enzyme-linked immunosorbent assay systems for the detection of enterotoxins A and C2 were examined for a variety of food extracts; no abnormal interference could be detected from these extracts. The monoclonal antibody systems were also compared with the homologous enterotoxin-specific polyclonal serum for the detection of enterotoxin by the competitive radioimmunoassay (RIA). Single monoclonal antibodies generally did not perform as well in the RIA as did the homologous toxin-specific polyclonal serum. However, pools of monoclonal antibodies were prepared that approached the sensitivity and precision of the polyclonal system for the detection of each toxin by the RIA.     

Subcellular and regional distribution of 125I-labeled alpha-bungarotoxin binding in rat brain and its relationship to acetylcholinesterase and choline acetyltransferase.

1. The subcellular distribution of binding sites for 125I-labeled alpha-bungarotoxin was studied in rat cerebral cortex. Primary fractions showing higher specific activity than homogenate were P2 (crude mitochondria and nerve endings) and P3-P2 was subfractionated on a Ficoll gradient with the P2B (nerve ending) subfraction exhibiting the greatest recovery (65%) and enrichment of toxin binding. Toxin binding showed a distribution similar to that of acetylcholinesterase, choline acetyltransferase, and sodium and potassium ion-activated ATPase. 2. P2B and P3 were subfractionated on five-step discontinuous sucrose gradients. The highest specific activity of toxin binding and acetylcholinesterase was associated with fractions of relatively low buoyant density, while choline acetyltransferase activity was associated with fractions of higher density. 3. Toxin binding, acetylcholinesterase, and choline acetyltransferase activities were relatively high in olfactory lobes, cerebral cortex, thalamic region, caudate nucleus, and brain stem; intermediate in hippocampus; low in cerebellum. 4. The relationship of toxin binding to the putative acetylcholine receptor in brain is discussed.     

Evaluation of ELISA and GM1-ELISA for detection of Salmonella enterotoxin.

The ELISA and GM1-ELISA, by using antiserum to purified Salmonella enterotoxin (SE), were standardized and carried out to screen salmonellae isolated from foods of animal origin for enterotoxigenicity. Of the 101 strains of Salmonella belonging to 15 different serogroups tested, 76 (75.24%) strains from 13 serogroups were found enterotoxigenic. ELISA correlated well with rabbit ligated ileal loop (RLIL) test for the detection of enterotoxin producing salmonellae with 24 test strains. ELISA also yielded positive reaction with 7 of 13 RLIL negative strains. GM1-ELISA could not be carried out as none of the 101 cell free culture supernatants (CFCS) were able to bind with GM1-ganglioside. ELISA and GM1-ELISA were also standardized with antiserum to cholera toxin for the detection of salmonellae producing cholera related enterotoxin. None of the 101 strains was found to produce cholera related enterotoxin. ELISA could detect as low as 15 ng/100 microliters of purified SE and 10 ng/100 microliters of cholera toxin when tested with their homologous antisera.     

Binding of enterotoxin from Clostridium perfringens type A to liver cells in vivo and in vitro. The enterotoxin causes membrane leakage

Enterotoxin from Clostridium perfringens was shown to retain its biological activity after labelling with 125I. When injected intravenously into mice and rats, most of the radioactivity in the organs was present in the form of intact toxin. Studies of the tissue distribution of labelled enterotoxin showed the largest amounts in the liver, where the activity reached a maximum 10--15 min after administration. The highest concentration per g tissue was found in liver and kidneys. The radioactivity was excreted in the urine as a mixture of intact labelled toxin and low molecular weight degradation products. In vitro studies with purified parenchymal liver cells showed rapid release of lactate dehydrogenase (LDH) during treatment with enterotoxin, thus indicating severe membrane damage.     

Binding specificity of the combining site of cholera toxin for human erythrocytes.

The reactivity of cholera toxin (CT) with blood-group determinant(s) on human erythrocytes was studied by competitive binding assays. 125I-labeled CT was found to bind more efficiently to pronase- and neuraminidase-treated human type A, B, and O erythrocytes than their untreated ones. The binding of 125I-labeled CT to neuraminidase-treated human type B erythrocytes was effectively inhibited by ganglioside GM1, but not by porcine gastric mucin with both A and H determinants (hog A + H), blood group specific lectins, and other substances at the highest concentrations used. Ganglioside GM1 was at least 10(5) times more potent than other inhibitors. These findings strongly suggest that the predominant binding substance for CT on human erythrocytes is not the blood-group determinant(s) but ganglioside GM1.