Presence of IgE antibodies to staphylococcal exotoxins on the skin of patients with atopic dermatitis. Evidence for a new group of allergens.

In the current study, we investigated whether Staphylococcus aureus grown from affected skin of atopic dermatitis (AD) patients secreted identifiable toxins that could act as allergens to induce IgE-mediated basophil histamine release. The secreted toxins of S. aureus grown from AD patients were identified by ELISA using antibodies specific for staphylococcal enterotoxin (SE) exfoliative toxin (ET), or toxic shock syndrome toxin (TSST-1). S. aureus isolates from 24 of 42 AD patients secreted identifiable toxins with SEA, SEB, and TSST accounting for 92% of the isolates. 32 of 56 AD sera (57%) tested contained significant levels of IgE primarily to SEA, SEB, and/or TSST. In contrast, although SEA, SEB, or TSST secreting S. aureus could be recovered from the skin of psoriasis patients, their sera did not contain IgE antitoxins. Freshly isolated basophils from 10 AD patients released 5-59% of total histamine in response to SEA, SEB, or TSST-1 but only with toxins to which patients had specific IgE. Basophils from eight other AD patients and six normal controls who had no IgE antitoxin failed to demonstrate toxin-induced basophil histamine release. Stripped basophils sensitized with three AD sera containing IgE to toxin released 15-41% of total basophil histamine only when exposed to the relevant toxin, but not to other toxins. Sensitization of basophils with AD sera lacking IgE antitoxin did not result in release of histamine to any of the toxins tested. These data indicate that a subset of patients with AD mount an IgE response to SEs that can be grown from their skin. These toxins may exacerbate AD by activating mast cells, basophils, and/or other Fc epsilon-receptor bearing cells armed with the relevant IgE antitoxin.     

NVC-422 Inactivates Staphylococcus aureus Toxins

Bacterial pathogens have specific virulence factors (e.g., toxins) that contribute significantly to the virulence and infectivity of microorganisms within the human hosts. Virulence factors are molecules expressed by pathogens that enable colonization, immunoevasion, and immunosuppression, obtaining nutrients from the host or gaining entry into host cells. They can cause pathogenesis by inhibiting or stimulating certain host functions. For example, in systemic Staphylococcus aureus infections, virulence factors such as toxic shock syndrome toxin 1 (TSST-1), staphylococcal enterotoxin A (SEA), and staphylococcal enterotoxin B (SEB) cause sepsis or toxic shock by uncontrolled stimulation of T lymphocytes and by triggering a cytokine storm. In vitro, these superantigens stimulate the proliferation of human peripheral blood mononuclear cells (PBMC) and the release of many cytokines. NVC-422 (N,N-dichloro-2,2-dimethyltaurine) is a broad-spectrum, fast-acting topical anti-infective agent against microbial pathogens, including antibiotic-resistant microbes. Using mass spectrometry, we demonstrate here that NVC-422 oxidizes methionine residues of TSST-1, SEA, SEB, and exfoliative toxin A (ETA). Exposure of virulence factors to 0.1% NVC-422 for 1 h prevented TSST-1-, SEA-, SEB-, and ETA-induced cell proliferation and cytokine release. Moreover, NVC-422 also delayed and reduced the protein A- and clumping factor-associated agglutination of S. aureus cultures. These results show that, in addition to its well-described direct microbicidal activity, NVC-422 can inactivate S. aureus virulence factors through rapid oxidation of methionines.     

Genotypic identification of staphylococcal enterotoxins and toxic shock syndrome toxin 1 by the enzymatic detection of polymerase chain reaction-amplified DNA

The enzymatic detection of a polymerase chain reaction product (ED-PCR), a new detection method of PCR-amplified DNA, was evaluated for the identification of staphylococcal enterotoxin (SE) and toxic shock syndrome toxin 1 (TSST-1) genes. A total of 61Staphylococcus aureus strains, including reference strains and strains isolated from clinical specimens and food poisoning outbreaks, were examined by ED-PCR and by reverse passive latex agglutination (RPLA) phenotypic identification. There was 100% agreement between the genotypic and phenotypic identification of SEA, SEB, SEC, SEE strains and TSST-1. In the case of SED, however, 4 strains were positive by ED-PCR and negative by RPLA. ED-PCR offers an accurate alternative to traditional immunoassays or conventional PCR using electrophoresis for the detection of SE and TSST-1 production yielding results that are more precise than with older techniques.     

金黄色葡萄球菌肠毒素SEA、SEB、SEC、SED序列多态性及进化分析

目的对不同来源金黄色葡萄球菌肠毒素SEA、SEB、SEC和SED的序列多态性及进化进行分析,为进一步了解不同亚型的毒素表达差异及食品风险评估提供理论依据。方法选择我国部分地区不同来源经典肠毒素SEA、SEB、SEC和SED阳性的菌株共90株,采用PCR方法扩增其序列并测序。 并与GenBank下载的经典肠毒素亚型代表序列进行比对分析,用MEGA 7软件构建系统发生树。结果SEA氨基酸序列存在3个突变位点,主要位于N端和C端;有3个亚型,SEA阳性菌株以SEA1亚型为主。 SEB氨基酸序列在两端的突变较中间活跃,存在18个突变位点;有11个亚型,SEB阳性菌株主要为SEB1和SEB2亚型。 SEC氨基酸序列突变位点有24个,分布在整个序列;共8个亚型,SEC阳性菌株以SEC3亚型为主。 SED氨基酸序列相对保守,共7个亚型,SED阳性菌株以SED1为主。 本研究还发现2个新亚型,分别命名为SEB9和SEC5,均来自患者分离菌株。结论SEA和SED的氨基酸序列较为保守,SEB和SEC的氨基酸突变位点较多。4种肠毒素的氨基酸序列进化树均分为2个分支。 本研究选取的菌株以SEA1、SEB1、SEB2、SEC3和SED1亚型为主。     

Antibodies to peptidoglycan in the sera from population surveys.

A recently described latex agglutination test was used to determine peptidoglycan antibody titers in sera from healthy human subjects and in sera from patients with a history of streptococcal infections or rheumatoid arthritis. Using latex particles coated with group A streptococcal peptidoglycan 32.8% of the sera from 961 healthy donors reacted positively with titers ranging from 1 : 5 to 1 : 320. Peptidoglycan antibodies were more frequently present in the younger population (45.1% in the 20-29 years old) and considerably decreased in advanced age (15.7% in the 70 years or older). Sera from 82 patients with elevated ASO-titers showed detectable peptidoglycan antibody levels in 40.2%; a statistically significant correlation between ASO and peptidoglycan antibody titers could not be substantiated. Sera from 25 patients with rheumatoid arthritis gave a high incidence (56-92%) of positive results. However there was evidence that this may be due to the action of rheumatoid factor present in such sera.     

Transcytosis of Staphylococcal Superantigen Toxins

Staphylococcus aureus produces a set of proteins (e.g., staphylococcal enterotoxin A [SEA], SEB, toxic shock syndrome toxin 1 [TSST-1]) which act both as superantigens (SAgs) and toxins. Although their mode of action as SAgs is well understood, little is known about how they enter the body via the intestine and cause food poisoning. To examine this problem we used an in vitro culture system to study the capacity of class II MHC-negative human intestinal epithelial cells (Caco-2) to transcytose several staphylococcal toxins. We found that Caco-2 cells are capable of dosedependent, facilitated transcytosis of SEB and TSST-1, but not SEA. We extended these studies in vivo in mice by showing that ingested SEB appears in the blood more efficiently than SEA. Our data suggest that these toxins can cross the epithelium in an immunologically intact form. These results may have important implications for the pathogenesis of food poisoning. S taphylococcus aureus produces a set of exotoxins including staphylococcal enterotoxins (SEs)¹ and toxic shock syndrome toxin 1 (TSST-1) which cause food poisoning and toxic shock syndrome in human beings and other species (1–4). These toxins are intermediate molecular weight proteins (22–30 kD) that also act as superantigens (SAgs; 5–7), due to their ability to bind to MHC class II molecules on APCs, and stimulate all T cells bearing particular Vβs on their T cell receptors (5–11). For example, the mouse in SEA stimulates T cells bearing Vβs 1, 3, 11, 17, and the closely related SEB and SECs stimulate T cells bearing Vβ7 and members of the Vβ8 family (12). TSST-1 stimulates nearly all T cells bearing Vβ15 in the mouse or Vβ2 in human (13). Thus, an individual toxin can potentially activate a very high proportion of T cells (5–30%). The toxic shock associated with these toxins has been attributed to the flood of cytokines (TNF, IL-2, etc.) released during this massive T cell stimulation. For example, in mice SEB can cause a shock-like syndrome resulting in rapid weight loss and death (14). Nude mice or mice lacking T cells bearing the relevant Vβ elements are resistant to the toxic effects of SEB (14). A role for T cells in generation of shock is also suggested by the greatly elevated percentage of T cells bearing Vβ2 in patients with TSST-1–mediated toxic shock syndrome (13, 15).On the other hand, in toxin-mediated food poisoning, the symptoms of which include diarrhea and vomiting within about 4–6 h of ingesting contaminated food, pathogenesis is not so well understood. It is possible that these toxins stimulate intestinal lymphocytes to release cytokines which induce the symptoms by local inflammation. Alternatively, they may have an additional functional domain which can interact directly with receptors on the cells lining the intestine to cause the symptoms. Distinguishing between these possibilities has been difficult due to the lack of a rodent model for toxin-mediated food poisoning.To shed some light on the problem, we performed experiments to see if these toxins can cross the intestinal epithelium, since to stimulate T cells, ingested toxin must escape the intestine in an intact form that is capable of interacting with the host immune system. We performed two types of experiments. In the first we directly examined transepithelial transcytosis of toxins in vitro using a well-characterized human enterocytic cell line, Caco-2 (16, 17). In the second we used T cell stimulation/deletion assays to detect toxins in the blood of mice after they had been fed toxins. Our results show that these toxins cross epithelial membranes, in some cases by a facilitated mechanism. While our results do not rule out a direct effect of these toxins on intestinal epithelium, they show that the toxins can gain rapid access to the immune system after ingestion supporting a role for T cells in the pathogenesis of food poisoning.     

Positive direct and indirect antiglobulin tests associated with oxaliplatin can be due to drug antibody and/or drug-induced nonimmunologic protein adsorption

BACKGROUND: Two patients were suspected of having immune hemolytic anemia (IHA) due to oxaliplatin. A related drug, cisplatin, is known to cause nonimmunologic protein adsorption (NIPA). Studies were performed to determine the presence of oxaliplatin-dependent antibodies in addition to oxaliplatin-induced NIPA.
STUDY DESIGN AND METHODS: Sera and eluates from the two patients were tested against red blood cells (RBCs) treated with oxaliplatin, cisplatin, and carboplatin (another platinum drug). Sera were also tested against untreated RBCs in the presence of the same drugs. Testing with pooled normal sera and anti-human albumin was used to demonstrate the presence of NIPA. Oxaliplatin-treated RBCs sensitized with the patients' sera and pooled normal sera were tested by a monocyte monolayer assay (MMA) to determine potential clinical significance.
RESULTS: Both patients had high-titer antibodies to oxaliplatin in their sera that reacted with oxaliplatin-treated RBCs and with untreated RBCs in the presence of oxaliplatin. RBCs treated with oxaliplatin, cisplatin, and carboplatin all demonstrated NIPA (pooled normal sera and anti-human albumin were reactive to low titers). NIPA was also detected in tests with untreated RBCs in the presence of oxaliplatin and cisplatin. Lower-titer reactivity of both patients' sera with cisplatin may have been due to NIPA and/or cross-reactivity of anti-oxaliplatin with cisplatin. MMAs were weakly positive due to NIPA and more strongly positive due to oxaliplatin antibodies.
CONCLUSION: Two patients with IHA were demonstrated to have oxaliplatin-dependent antibodies. Oxaliplatin was also shown to cause NIPA. The drug-dependent antibody and/or the drug-induced NIPA could have contributed to the patients' hemolytic anemia.     

Serologic responses to toxic shock syndrome (TSS) toxin-1 in menstrual and nonmenstrual TSS

Toxic shock syndrome toxin-1 (TSST-1) is implicated as the major exotoxin associated with menstrual toxic shock syndrome. The role of TSST-1 in nonmenstrual TSS is less certain. We examined serum IgG responses to TSST-1 in 16 nonmenstrual (9 female, 7 male) and 14 menstrual TSS patients, and in 87 women and 66 men as age-matched healthy controls, using an enzyme-linked immunosorbent assay (ELISA). Relative ELISA titers were expressed as percent activity of a mid level serum standard tested concurrently. Based on 95% confidence estimates for predicting a negative titer (20.6 +/- 8.2%) using sera in which TSST-1 specific IgG was demonstrated to be absent by western blot, 24% of control women and 9% of control men lacked TSST-1 specific IgG in the random survey (p less than 0.05, Fisher's exact test). Relative titers in acute sera of menstrual TSS women (26.2 +/- 5.2%, mean +/- S.E.), but not nonmenstrual TSS women (71.8 +/- 18.6%), were significantly lower than those of control women (78.9 +/- 7.3%, p less than 0.01, Mann-Whitney test). Acute titers from male TSS patients (37.0 +/- 15.6%) were also significantly lower than those in control men (114.6 +/- 11.0% (p less than 0.05). Antibody titers from menstrual TSS women and TSS men remained low during convalescence. Nevertheless, seroconversion to TSST-1 was demonstrated by western blot in 7 of 10 patients in whom TSST-1 positive S. aureus was isolated, but in neither of two patients without toxigenic S. aureus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Development of antibodies to OEP, exotoxin A and exoenzymes of Pseudomonas aeruginosa in man

Antibody titers to OEP, exotoxin A and exoenzymes (protease and elastase) in normal sera of 256 specimens of cord blood, children and adults were measured by passive hemagglutination test. Serum antibody to exotoxin A was detected in cord blood samples. The level of mean antibody titers dropped during the first year of age, then rose and reached a plateau at the age of 2 to 5 years and dropped thereafter. Mean antibody titers to OEP by age groups were similar to those of exotoxin A. Antibodies to exoenzymes were not detectable initially, but the level rose during the second year of age and reached a plateau during childhood. Positive antibody titers (1:20) to exotoxin A and OEP were found in all sera belonging to some age groups between 11 to 30 years. The rate of acquisition of antibodies to exoenzymes was low. As for the antibodies to exotoxin A, the disappearance of detectable antibody by treatment with 2-mercaptoethanol was observed during the age of 1 to 4 years. Initial pseudomonas colonization may be common and asymptomatic in infants and young children.     

Acid pH-induced changes in the immunoreactivity of specific antigen and antibody in circulating immune complexes from tuberculosis sera.

The effect of acid pH treatment of circulating immune complexes (CIC) derived from tuberculosis sera was studied on the relative titers of specific antibody (CIC Ab) and mycobacterial antigen (CIC Ag) in the complexes. While the specific antibody titers increased, the titers of CIC Ag declined as a result of acid pH treatment of CIC, both changes being highly significant statistically. Direct exposure of TB sera to pH 2.8 also resulted in significant enhancements in the titers of antibodies directed against Mycobacterium tuberculosis (M.tb.). Competition experiments indicated that the acid pH-treated antibodies retained their antigen specificity. TB sera treated with pH 2.8 for 30 min and then neutralized back to pH 7.4 retained the enhanced antibody reactivity even after 7 days of storage at 4 degrees C. Our results indicate that acid pH treatment induces higher antigen binding ability in anti-M.tb. antibodies, present in free or complexed form in TB sera. These changes appear to be irreversible. Dissociation of CIC and analyzing the titers of released antibody and antigen components offered no advantage with respect to the immunodiagnosis of tuberculosis, as compared to the levels of undissociated CIC components or the serum antibody.     

Staphylococcal superantigens and toxins are detectable in the serum of adult burn patients

Bacterial infection in burn patients is still a devastating contributor to morbidity and mortality. Little is known regarding the presence of staphylococcal toxins in the burn-injured patient. The aim of this study was to characterize the prevalence of several of these toxins and their relationship to clinical metrics and mortality in burn patients. Levels of exotoxins staphylococcal enterotoxin A (SEA), staphylococcal enterotoxin B, toxic shock syndrome toxin 1 (TSST-1), and α-hemolysin were assayed from the serum of 207 adult burn patients aged 16–92 years. Clinical, demographic, and microbiological data from these patients were then compared to toxin levels. Staphylococcal exotoxins α-hemolysin and SEA were present in 45% and 25% of the population, respectively. Bacterial cultures concomitantly showed a high prevalence of Staphylococcus aureus in 48% of patients, of which 59% were methicillin resistant. Several metrics may be predictive of high toxin concentrations of α-hemolysin and TSST-1 and SEA including burn size, length of stay, and bacteremia. Mortality associations indicated that burn size, bacteremia, age, and the presence of α-hemolysin and SEA may be predictors of mortality. A high prevalence of staphylococcal toxin α-hemolysin and superantigens TSST-1 and SEA can be found in the circulation of the adult burn population. The presence of these toxins may contribute to the morbidity and mortality of the burn patient.     

金黄色葡萄球菌肠毒素的检测及结果分析

目的了解引起临床感染的金黄色葡萄球菌(SA)携带肠毒素(SE)的情况。方法对从临床标本中分离出的260株SA,用反向间接血凝试验检测其肠毒素,耐甲氧西林金黄色葡萄球菌(MRSA)检测按2005年美国临床实验室标准化委员会(NCCLS)推荐的检测头孢西丁方法进行。结果260株SA中有172株携带肠毒素,占66.2%,主要为SEA型10.8%(28/260)、SEB型14.6%(38/260)、SEC型10.0%(26/260),同时携带二种或二种以上肠毒素的占23.8%(62/260);其中MRSA134株,全部携带肠毒素,且以SEA、SEB、SEC为主,分别为19.4%、16.4%、13.4%;甲氧西林敏感金黄色葡萄球菌(MSSA)126株,携带肠毒素率为30.0%(38/126),以SEB为主,占9.5%。结论临床上SA分离株大部分都携带肠毒素,且以SEA、SEB、SEC为主,MRSA的带毒率高于MSSA。     

Dissociation of the stimulatory activities of staphylococcal enterotoxins for T cells and monocytes

The staphylococcal enterotoxins (SEs) are homologous proteins related in their capacity for stimulating both T cells and monocytes. To assess the importance of conserved structure and sequence to functional activity, the role of the disulfide loop and adjacent sequence in these toxins was evaluated. Contrary to previous reports, we demonstrate here that the disulfide loop was required for the mitogenic activity of SEA and SEB. While T cell-stimulatory activity was compromised, reduced and alkylated SEs retained major histocompatibility complex class II-binding and monocyte-stimulatory activities, suggesting that their inability to induce T cell proliferation was due to failure to interact with T cell receptor (TCR) rather than with class II molecules. Reduction and alkylation did not affect the far-ultraviolet circular dichroic spectrum of SEA, suggesting that the loss of mitogenic activity was not associated with significant changes in secondary structure. The disulfide linkage imparts considerable stability to these toxins as peptide cleavages within the loop of SEB were not associated with detectable loss of function, although cleavage in the conserved sequence outside the loop of SEA resulted in loss of mitogenic activity. This report thus establishes a functional role for a conserved element in SEs, the disulfide loop, and further indicates that their class II- and TCR-binding activities can be dissociated.     

V3-specific neutralizing antibodies in sera from HIV-1 gp160-immunized volunteers block virus fusion and act synergistically with human monoclonal antibody to the conformation-dependent CD4 binding site of gp120. NIH-NIAID AIDS Vaccine Clinical Trials Network.

Sera from 11 volunteers immunized with a recombinant HIV-1 gp160-expressing vaccinia virus (HIVAC-1e; Oncogen/Bristol-Myers Squibb, Seattle, WA) and boosted with baculovirus-derived rgp160 (VaxSyn; MicroGeneSys, Inc., Meriden, CT) were evaluated for functional serum antibodies and their epitopes. Sera obtained prior to boosting had undetectable HIV-1-specific IgG and neutralizing activity, and did not block HIV-1 from binding or fusing to CD4+ MT-2 cells. 14 d after boosting, sera from each volunteer contained HIV-1-specific IgG titers of 1:40 to 1:1,280. Five of these sera also contained neutralizing antibodies, where most or all neutralizing activity was blocked by a synthetic peptide corresponding to amino acids 307-330 of the V3 loop of gp120, indicating that neutralizing antibodies were mostly V3 loop-specific. All sera obtained after boosting contained HIV-1 binding/fusion-inhibition antibodies, and a significant portion of their activity was blocked by the V3 loop peptide, a result consistent with the presence of antibodies against the region of the V3 loop that participates in fusion. Three sera with V3 loop-specific neutralizing and fusion-inhibition antibodies were studied further. In competitive antibody binding experiments, antibodies reactive with the conformation-dependent, CD4 binding site of gp120 were undetectable in each serum. When evaluated in combination with a monoclonal antibody to the CD4 binding site of gp120, two sera demonstrated synergism in neutralizing assays, and all three sera demonstrated synergism in binding/fusion-inhibition assays, further indicating that the functional antibodies were primarily V3 loop-specific. The synergism also suggests that a vaccine that elicits strong serum antibody responses to both regions of gp120 may improve the potential for inducing protective immunity.     

Evaluating porcine RBC and platelet alpha-galactosyl expression

BACKGROUND: Naturally occurring human xenoreactive antibodies bind and agglutinate porcine RBCs. STUDY DESIGN AND METHODS: To determine if xenoantigen expression on RBCs of individual pigs of different breeds and blood groups is variable, and if it correlates with platelet (PLT) expression, we measured adsorption of affinity-purified antibodies to alpha-galactosyl (alphaGal) by RBCs or PLTs from 22 pigs representing four breeds. Hemagglutination of RBCs from these pigs was also performed with pools of human group OAB, A, B, and AB sera, as well as with anti-alphaGal-depleted pooled OAB human serum. RESULTS: There was significant variation in alphaGal expression on RBCs and PLTs among pigs. PLT alphaGal expression did not correlate with RBC alphaGal. RBCs from all pigs were agglutinated by pooled group O, AB, A, or B sera, whereas titers were reduced by 87 percent with anti-alphaGal-depleted serum and by 82 percent with AB sera from two volunteers. Agglutination titers were higher against RBCs from the five highest RBC alphaGal expressers compared with those from the five lowest RBC alphaGal expressers (92 +/- 12 vs. >160, p < 0.05, where 160 was the maximum dilution tested). CONCLUSION: Hemagglutination is a feasible alternative for rapid identification of pigs with RBCs carrying less alphaGal.     

Anti-skeletal muscle antibodies in the sera from myasthenic patients with thymoma: identification of anti-myosin, actomyosin, actin, and alpha-actinin antibodies by a solid-phase radioimmunoassay and a western blotting analysis

We recently reported a strong association between the occurrence of anti-skeletal muscle (SM) antibodies and the presence of thymoma in patients with myasthenia gravis (MG). To further examine the immunoreactivity of MG sera against human muscle antigens, we developed a solid-phase radioimmunoassay (RIA) using purified muscle antigens and a Western blotting analysis in MG sera with high titers of anti-SM antibodies. Our results showed that MG patients with thymoma (thymoma group) had markedly high titers of anti-myosin and anti-actomyosin antibodies than those without thymoma (non-thymoma group). Furthermore, a close correlation was found between titers of anti-SM, anti-myosin and anti-actomyosin antibodies. The antibody titers against actin, alpha-actinin and tropomyosin were all low and did not correlate with titers of anti-SM antibodies. But, significant levels of these three antibodies were found in the thymoma group. By a Western blotting analysis, immunoreactivity of sera from the thymoma group appeared to be predominantly directed against myosin, actin and alpha-actinin.     

Immunization of Humans with Polyribophosphate, the Capsular Antigen of Hemophilus influenzae, Type b

In human volunteers, single injections of purified polyribophosphate elicited antibodies detectable by passive hemagglutination and by serum bactericidal and opsonizing activities against viable Hemophilus influenzae, type b. All three activities rose by 2 wk to maximal levels, at which they remained for at least 6 months. Doses of 1 mug elicited antibody responses in nearly all recipients; higher doses of the antigen, however, produced larger increases in titer. Booster doses of 1 mug given at 6 months did not further increase the antibody titers. A tuberculin-like response was often observed at the site of injections given intradermally.     

Identification of Staphylococcus aureus enterotoxins A and B genes by PCR-ELISA

We developed PCR-enzyme linked immunosorbent (ELISA) assays to detect Staphylococcus aureus enterotoxins A and B genes. The assays use internal biotin-labelled oligonucleotides as capture probes for immobilizing and subsequently detecting target sequences on microtiter plates. The detection limits of the PCR-ELISAs were approximately 250 gene copies, versus 2500 gene copies by agarose gel analysis. The sensitivity of the assays, as determined from a reference panel of 46 coded samples that included DNA purified from 31 different bacterial species and strains, SEA and SEB plasmid controls, and no-template controls was 100%. No cross-reactivity was observed with DNA from non-staphylococcal species. Using 27 clinical isolates of S. aureus, the SEA PCR-ELISA identified the enterotoxin A (sea) gene in 26 samples, and the SEB PCR-ELISA identified the enterotoxin B (seb) gene in all 27 samples. Compared with conventional antigen capture ELISAs for SEA and SEB toxins, the PCR-ELISAs showed overall superior detection limits. The sensitivity and specificity levels of the SEA PCR-ELISA and the SEA toxin ELISA were comparable within their respective detection thresholds, but the sensitivity and specificity of the SEB PCR-ELISA was much greater than that of SEB toxin ELISA.     

Evaluation of antibodies to the Epstein-Barr virus immediate early gene product ZEBRA by a new enzyme-linked immunosorbent assay.

For the serodiagnosis of Epstein-Barr virus (EBV) infections, we have developed a new enzyme-linked immunosorbent assay (ELISA) for antibodies to the ZEBRA product of EBV immediate early gene BZLF1. ZEBRA protein fused with glutathione-S-transferase (GST) was expressed in Escherichia coli and purified by affinity chromatography with glutathione-Sepharose 4B. An ELISA sandwich capture system was constructed with the GST-ZEBRA immobilized on plastic microtiter plates which had been coated with a mouse monoclonal antibody to GST. ZEBRA-IgG antibodies in patients' sera with chronic active EBV infection (CAEBV) and infectious mononucleosis (IM) had, respectively, very high and high titers. Anti-ZEBRA antibodies were also detected at low titers in sera of some healthy controls. ZEBRA-IgM antibodies were detected in sera of patients with IM and CAEBV but not in sera of healthy controls. In sera of patients with CAEBV, the titers of IgG antibodies to ZEBRA correlated with the antibody titers to early antigens obtained with an immunofluorescence assay, but not to EBV nuclear antigens. This ELISA is a useful diagnostic and prognostic test for EBV infection.     

Neutralizing antibodies against AAV serotypes 1, 2, 6, and 9 in sera of commonly used animal models

Adeno-associated virus (AAV)-based vectors are promising gene delivery vehicles for human gene transfer. One significant obstacle to AAV-based gene therapy is the high prevalence of neutralizing antibodies in humans. Until now, it was thought that, except for nonhuman primates, pre-existing neutralizing antibodies are not a problem in small or large animal models for gene therapy. Here, we demonstrate that sera of several animal models of cardiovascular diseases harbor pre-existing antibodies against the cardiotropic AAV serotypes AAV1, AAV6, and AAV9 and against AAV2. The neutralizing antibody titers vary widely both between species and between serotypes. Of all species tested, rats displayed the lowest levels of neutralizing antibodies. Surprisingly, naive mice obtained directly from commercial vendors harbored neutralizing antibodies. Of the large animal models tested, the neutralization of AAV6 transduction by dog sera was especially pronounced. Sera of sheep and rabbits showed modest neutralization of AAV transduction whereas porcine sera strongly inhibited transduction by all AAV serotypes and displayed the largest variation between individual animals. Importantly, neutralizing antibody titers as low as 1/4 completely prevented in vivo transduction by AAV9 in rats. Our results suggest that prescreening of animals for neutralizing antibodies will be important for future gene transfer experiments in these animal models.