Immunoblot assay for serodiagnosis of Helicobacter pylori infections.

An immunoblot assay for the serological diagnosis of Helicobacter pylori infection was evaluated. Serum samples from patients whose gastric biopsy specimens were known to be positive or negative for H. pylori on culture were used to establish interpretive criteria for the immunoblot assay. A panel of sera from patients with diseases other than H. pylori infection and sera from healthy blood donors were included to validate these criteria. All sera were initially assessed in an enzyme immunoassay (Ge-EIA), based on acid glycine-extracted cell surface proteins of H. pylori NCTC 11637. The same antigen extract was used in the immunoblot assay. In addition, the Ge-EIA and the immunoblot assay were compared with a commercially available EIA (Seradyn, Color Vue Pylori). Bands of 110/120 kDa and/or two of five low-molecular-mass proteins (26, 29, 30, 31, and 33 kDa, in any combination) showed a strong correlation with the H. pylori culture-positive patients (97.5%) compared to the correlation obtained with the EIA results (Ge-EIA, 87.5%; Seradyn EIA, 92.5%), and the antibody responses to these proteins were considered specific reactions. In 37 of 40 serum samples from culture-negative patients and also in sera from patients with other disorders, a moderate antibody reactivity to the medium-size proteins (43 to 66 kDa) was observed, and these were considered not valuable for a specific immunoblot assay. Among sera from culture-positive patients, 39 of 40 serum samples were defined to be immunoblot positive, and from among sera from culture-negative patients, 3 of 40 serum samples were defined to be immunoblot positive. The use of sera from patients with negative cultures for H. pylori as negative controls may decrease the sensitivity due to sampling error and false-negative culture results. Immunoblot assay-positive results were detected among 10% of sera from patients with other diseases, whereas they were detected among 42.5% of sera by the Ge-EIA and 47.5% of sera by the Seradyn-EIA. The higher number of EIA-positive sera in this group reflects a possible cross-reactivity (false-positive EIA result). Of the blood donors, representing asymptomatic but possibly colonized subjects, 24% were immunoblot positive. In conclusion, our data indicate that immunoblotting is more sensitive as well as more specific than EIA. Moreover, it permits detection of antibody responses to specific antigens, e.g., the cytotoxin-associated CagA protein, which may have pathological implications.     

Serum total tryptase levels are increased in patients with active chronic urticaria

Background We have demonstrated previously mast cell histamine release upon incubation with chronic urticaria (CU) sera, presumably by degranulation. Objective To explore total and mature tryptase in order to assess whether any increase in total tryptase levels is due in part to mast cell degranulation or to mast cell burden. We also wanted to explore differences between the autoimmune groups called idiopathic (serum unable to activate basophils), and to correlate total and mature tryptase levels with different urticaria features. Methods We measured total and mature tryptase serum levels in 81 CU patients, 16 atopic donors and 21 healthy control sera. We assessed autoimmunity by measuring the CD63 expression in normal basophil donors upon incubation with CU sera. Results We found significantly higher levels of total tryptase in the sera of CU patients (6.6 ±4.1 μg/L) than in sera from healthy non‐atopic subjects (4.4 ±2.8 μg/L) and from atopic subjects (4.5 ±1.7 μg/L). Mature tryptase levels were undetectable (<1 ng/mL). Total tryptase levels in the autoimmune urticaria group were significantly higher (9.8 ±5.4 μg/L) than the idiopathic urticaria group (4.4 ±2.2 μg/L). A significant difference in total tryptase was found between symptomatic patients (7.3 ±4.1 μg/L) compared with asymptomatic ones (5.7 ±4.1 μg/L) at the time of venesection. No difference was found in mature tryptase levels either. Conclusion Total elevated tryptase levels are not accompanied by an elevated mature tryptase levels, as might be expected if the serum levels reflected mast cell degranulation. Cite this as: M. Ferrer, J. M. Nuñez‐Córdoba, E. Luquin, C. E. Grattan, J. M. De la Borbolla, M. L. Sanz, L. B. Schwartz, Clinical & Experimental Allergy, 2010 (40) 1760–1766.     

Levels of Schistosoma mansoni Circulating Antigen in Chronic Hepatitis C Patients with Different Stages of Liver Fibrosis

The goal of this study was to determine the levels of S. mansoni antigen in different liver fibrosis stages with chronic hepatitis C (CHC) Egyptian patients. A total of 174 CHC patients showing HCV-NS4 antigen and HCV- RNA in their sera were included. S. mansoni antigen was detected in serum using Western blot and ELISA. The levels of interferon-γ (IFN- γ) were determined using ELISA. The 50 kDa S. mansoni antigen discriminated patients infected with S. mansoni from healthy individuals with 0.93 area under curve (AUC), 92% sensitivity, and 97% specificity. The level of S. mansoni antigen (μg/ml) was significantly (P < 0.0001) increased with the progression of liver fibrosis stages (26.9 ± 17.5 in F1, 42.1 ± 25.2 in F2, 49.8 ± 30.3 in F3 and 62.2 ± 26.3 μg/mL in F4 liver cirrhosis), 26.9 ± 17.59 in significant fibrosis (F2–F4); 51.2 ± 27.9 in advanced fibrosis (F3–F4). A significant correlation (r = 0.506; P < 0.0001) was shown between the levels of the S. mansoni antigen and the HCV-NS4 antigen. In conclusion, the presence of S. mansoni antigen in different liver fibrosis stages of CHC patients confirming that concomitant schistosome infection aggravates liver disease.     

Diagnosis of Babesiosis Using an Immunoblot Serologic Test

Although the current indirect immunofluorescent assay (IFA) diagnostic antibody test for human babesiosis is sensitive and specific, an immunoblot antibody test may be easier to standardize and to perform. Our objective, therefore, was to determine the efficacy of and develop interpretive criteria for an immunoblot antibody test for diagnosing acute human babesiosis using a Babesia microti whole-cell lysate as the antigen. We compared the reactivity of sera to a B. microti immunoblot assay in 24 human subjects experiencing symptoms and expressing laboratory evidence of babesiosis, 28 subjects who experienced Lyme disease, 12 subjects who experienced human granulocytic ehrlichiosis, and 51 subjects who reported no history of any of these diseases and whose sera did not react against B. microti antigen in an IFA test. Immunoblot strips were impregnated with proteins derived from the GI strain of B. microti that had been electrophoresed in an acrylamide sodium dodecyl sulfate gel, followed by electroblotting onto nitrocellulose membranes. The sera of all subjects who experienced babesiosis reacted against the B. microti antigen in the IFA and against at least one of nine immunoblot protein bands specific to B. microti. In contrast, none of the sera from people who appeared not to have experienced this infection reacted against the B. microti antigen in the IFA (compared to 4% in the immunoblot assay). When two reactive bands were considered as definitive, immunoblot test sensitivity was 96%, while specificity was 99% and predictive positivity and predictive negativity were 96 and 99%, respectively. Our B. microti immunoblot procedure shows promise as a sensitive, specific, and reproducible assay for routine clinical diagnosis of acute babesiosis.     

Polyclonal B Cell Activation and Antigen-Driven Antibody Response as Mechanisms of Autoantibody Production Insle

We studied 16 patients affected by autoimmune hemolytic anaemia (AIHA), both idiopathic and associated with other diseases (B and T lymphoma, B hepatitis, gastric carcinoma, systemic lupus erythematosus) or a-methyldopa therapy, in order to value T- and B-cell activation. We determined the count of T- and B-cell subsets in peripheral blood, the proliferative response of peripheral blood lymphocytes (PBL) to phytohe-magglutinin (PHA) and to pokeweed mitogen (PWM), the percentage of CD25 ⁺ cells in culture and interleukin (1L)-lα, IL-2, IL-4, tumor necrosis factor (TNF)α and soluble IL-2 receptor (sIL-2R) levels in sera and in culture. Except for an increase in CD4 ⁺ and CD8 ⁺ T cell number in a case of AIHA associated with a T lymphoma and an increase in the percentage of CD5 ⁺ and PCAl⁺ B cells in two cases of AIHA associated with B lymphoma and with SLE, no further data showed a relationship with the disease possibly associated with AIHA, so both idiopathic and secondary AIHA cases were analyzed together. CD4 ⁺ T cells were reduced in number in 9 cases, while CD8 ⁺ T cells were reduced in 6 cases. The percentage of CD5 ⁺ B cells was increased in 5 cases. The percentage of PCAl⁺ cells was increased in all cases (mean ± sd: 18 ± 22 vs 0,2 ± 1 in controls). The average PBL proliferative response to PHA was reduced (S.I. 71 ± 55 vs 138 ± 45 in controls) as well as that to PWM (S.I. 27 ± 21 vs 75 ± 24 in controls), despite IL-2 high levels, in all cases, in both sera (meanfsd: 648 ± 351 pg/ml vs 16 ± 4 pg/ml in controls) and culture supernatants (meanksd: 1045 f 677 pg/ml vs 195 ± 51 pg/ml in controls). In PHA stimulated cultures the percentage of CD25⁺ cells was reduced (meanasd: 37 ± 18 vs 63 ± 14 in controls), sIL-2R levels were like controls in 7 cases. In sera sIL-2R levels were increased in all cases (meanfsd 1256 ± 465 U/ml vs 256 ± 114 U/ml in controls), IL-lα was increased in all cases too, while IL-4 levels were increased only in 7 cases. Linear regression analysis generally showed a low relationship between S.I. and IL-2, IL-4 and sIL-2R levels in supernatants of PHA stimulated culture as well as between S.I. and the percentage of CD25 ⁺ cells. Taken together these data suggest a state of B- and T-cell hyperactivation in AIHA. The low PBL proliferative response in vim, explained in previous studies as a temporary functional exhaustion, might be itself a sign of the complete lymphocyte activation occurring in vivo in AIHA.     

Human schistosomiasis haematobium: effective diagnosis of active infection using a pair of monoclonal antibodies against soluble egg antigen

The present study was designed to prepare monoclonal antibodies (MAbs) against Schistosoma haematobium soluble egg antigen (SEA) with immunodiagnostic potential for urinary schistosomiasis. From a panel of MAbs, a pair of IgG1 MAbs (2D/11C and 10B/2C) specific for S. haematobium SEA was selected. Both MAbs recognized one band with a 42-kDa molecular weight by western blots. The pair of MAbs was employed in sandwich ELISA for the detection of circulating schistosome antigen (CSA), one as antigen-capturing antibody and the other as peroxidase-conjugated antigen-detecting antibody. The lower detection limit of the assay was 1 ng/ml of S. haematobium SEA. The assay was performed on sera of 65 S. haematobium-infected patients, 25 patients infected with other parasites (Fasciola hepatica, Echinococcus granulosus), and 20 noninfected individuals. CSA was demonstrated in 89% of the S. haematobium-infected group. However, CSA was negative in the sera of healthy individuals and patients infected with other parasites, giving an overall specificity of 100% for the CSA assay. A positive correlation (r=0.37, p<0.01) was detected between the number of S. haematobium eggs excreted in 10 ml urine and the CSA level detected in the sera of S. haematobium-infected patients. Our data show that the use of anti-S. haematobium MAbs for the detection of CSA provides a sensitive and specific method for the immunodiagnosis of active S. haematobium-infected patients. Moreover, CSA assay using this anti-S. haematobium MAb/ELISA system was proven to correlate with intensity of infection and hence morbidity assessment.     

Prevention and identification of Salmonella Enteritidis infection via novel diagnostic stained antigens and polyclonal antibodies

Analysis of Salmonella Enteritidis with specific antisera and stained antigen helps to reduce the identification time of the infection and offers clinical and epidemiological advantages both for poultry health and food industry for the control of salmonellosis. In this study, S. Enteritidis O and H specific antisera were obtained from rabbits immunised subcutaneously using formalin-treated whole bacterial cells as antigens. S. Enteritidis specific stained O and H antigens were prepared and tested for their diagnostic usefulness. In order to eliminate cross reactivity, Salmonella Adeyo was used as an antigen for the production of H specific polyclonal antibody and stained H antigen. Enzyme-linked immunosorbent assay results of antisera were obtained, ranging from 273,300±0.041–312,200±0.028 A492 nm. Stained O and H antigen preparations were achieved with a stronger binding degree to the hyperimmune rabbit sera and without being shown cross-reactivity between O and H antigens.     

Prediction of total body water and fatness from anthropometry: Importance of skinfold measurements

Prediction equations for total body water (TBW) generally use weight and height as predictors, but their ability may be limited because they implicitly assume a constancy of TBW among individuals of similar body size. The objective of this study was to evaluate the relative importance of anthropometric dimensions in predicting TBW and body composition. TBW determined by doubly labeled water (DLW) dilution techniques was used as the frame of reference in 23 healthy Aymara subjects, 4–65 years, in a rural community of the Bolivian Andes. Predictive performances of anthropometric variables for TBW were examined with multiple regression analyses. The generated equations were tested for cross-validity, using published data for U.S. adults. The resulting errors were compared with those of the published prediction equations of Mellits and Cheek (M&C) and Durnin and Womersley (D&W). The simplified prediction equation using weight and the triceps skinfold (Eq-A1:R² = 0.989, SEE = 1.041 L) and that using weight and the triceps and subscapular skinfolds (Eq-A2: R² = 0.990, SEE = 1.020 L) had better R² and smaller SEE than those using any combination of variables, weight, height, age, and sex. In the cross-validation sample, Eq-A1 and Eq-A2 demonstrated higher precision than the D&W and M&C equations. Evaluated by the method of Bland and Altman (mean difference ± 2SD), prediction errors for fat mass and fat percent were 0.2 ± 2.8 kg and 0.4 ± 5.2% in Eq-A2, 1.1 ± 3.5 kg, and 1.8 ± 6.1% in Eq-A1, −2.4 ± 3.6 kg and −3.4 ± 5.1% in D&W, and −2.3 ± 7.6 kg and −2.6 ± 10.3% in M&C. Significant underestimation of fat mass and sex differences in the biases were observed with D&W and M&C (P < 0.05), but not with Eq-A2. By including skinfold measurements, a single prediction equation for TBW was valid for males and females across different population samples. © 1996 Wiley-Liss, Inc.     

Detection of IgE antibody against Candida albicans enolase and its crossreactivity to Saccharomyces cerevisiae enolase

Candida albicans 46 kDa protein, a glycolytic enolase enzyme, is an important allergen of the yeast. The purpose of the study was to detect circulating IgE and IgG antibodies against C. albicans enolase (CAE). We isolated CAE using sequential DEAE Sephacel and Pl 1 column chromatography from spheroptasts of C. albicans, and delected IgE and IgG antibody against CAE by immunoblotting. Crossreactivity of enolose of C. albicans and Saccharomyces cerevisiae was also examined by immunoblotting and immunoblot inhibition test. Among 54 sera with positive IgE RAST to C. albicans, IgE antibody against CAE was detected in 20 sera (37%) and IgG antibody in 27 sera (50%). The allergenic potency of CAE was confirmed using a skin-prick test in three patients. Simultaneous IgE binding to S. cerevisiae enolase was only observed in four out of 20 sera reacting to CAE. Pre-treatment of sera with CAE completely inhibited IgE binding to S. cerevisiae enolase. Whereas the latter only partially inhibited IgE binding to CAE. These results suggest that CAE shares some crossreacting epitopes with S. cerevisiae enolase, representing minor components of CAE but dominant segments of S. cerevisiae enolase.     

Allergenic crossreactivity between Lepidoglyphus destructor and Blomia tropicalis

Background In general, the non-pyroglyphid mites Lepidoglyphus destructor and Blomia tropicalis show a different geographical distribution. Allergic sensitization to both species have been demonstrated in several investigations. However, whether this reflects crossreactivity or dual sensitization is so far not known. Objective The aim of the study was to investigate the allergenicity and allergenic crossreactivity of L destructor and B. tropicalis using sera from Sweden and Brazil. Objective Allergens in extracts of L. destructor and B. tropicalis were identified with SDS-PAGE and immunoblotting and the crossreactivity was studied by an immunoblot inhibition method. In addition to mite extracts, a recombinant major allergen of L destructor, Lep d 2, was used. Results The extract prepared from L. destructor contained 21 IgE-binding components when using the Swedish or the Brazilian sera. A 15 kDa allergen was recognized by 85% of the Swedish sera and 78% of the Brazilian. The B. tropicalis extract exposed 23 IgE-binding components when the Brazilian sera were used and 19 when the Swedish sera were used. A total of 83% of the Brazilian sera and 80% of the Swedish sera identified a 14.5 kDa allergen. The IgE response of the Swedish serum pool to 10 B. tropicalis allergens was inhibited by L. destructor extract. Likewise, the response of the Brazilian serum pool to four different L. destructor allergens was inhibited by B. tropicalis extract. The recombinant Lep d 2 allergen inhibited 33% of the IgE binding of the Swedish serum pool to the 14.5 kDa allergen in the B. tropicalis extract. Conclusion crossreactivity with several proteins from L. destructor and B. tropicalis was demonstrated. The results suggest that a B. tropicalis 14.5 kDa allergen is antigenically crossreactive with recombinant L. destructor allergen Lep d 2.     

Use of Lymphocyte Platelet Binding Assay for Detecting a Preimplantation Factor: A Quantitative Assay

PROBLEM: To evaluate the ability of the lymphocyte/platelet binding assay to identify a preimplantation factor (PIF). METHOD: Percentages of binding of lymphocytes by platelets in the presence of sera from 30 known pregnant and 30 nonpregnant individuals were compared using a novel lymphocyte/platelet binding assay. The assay is performed using a combination of a heat inactivated sera with donor O+ lymphocytes, activated complement and an antibody against CD₂(T11, Ortho Pharmaceuticals). RESULTS: In nonpregnant females (23.6 ± 6.5%) and males (17.7 ± 4.7%) the percentage of lymphocytes bound by platelets was significantly different from pregnant women (56.1 ± 15.9%) (P < 0.0001). Serial sampling of blood in five women undergoing IVF/ET who had normal pregnancies showed the detection of PIF by 4 days after transfer. The lymphocyte/platelet binding assay was not influenced by hCG, progesterone and estradiol. The interassay and intraassay variabilities were <3%. CONCLUSIONS: The lymphocyte/platelet binding assay is a simple, reproducible, specific and cost efficient assay for measurement of PIF. Application of this assay will provide investigative and diagnostic tools for identifying and monitoring early pregnancy events.     

Performance of a commercial Chicken-Ovo-transferrin-ELISA on the serum of brown layer chickens infected with Gallibacterium anatis and Streptococcus zooepidemicus

To evaluate Ovo-transferrin (OTF), a positive acute-phase protein in chickens, as a diagnostic biomarker of selected bacterial infections we checked the performance of a commercial Chicken-OTF-ELISA (ICL, Inc., Portland, OR, USA) by analytical and overlap performances using two groups of serum samples obtained from 26 Gallibacterium anatis-infected and 20 Streptococcus zooepidemicus-infected brown layer chickens. In addition, sera from 14 apparently healthy and 19 negative control chickens were analysed in the Gallibacterium group whereas sera from 20 healthy and 11 negative control chickens from the Streptococcus group were analysed. All calibration curves revealed high coefficients of determination (≥0.97) between optical density (OD_450nm) and concentrations of OTF (mg/ml). OTF concentrations in high, medium and low pools (made of sera from a combination of infected and/or non-infected birds) were >6.4, >3.8 to <4.5 and <1.6 mg/ml in the Gallibacterium group, and >6.7, >3.5 to <3.7 and <1.1 mg/ml in the Streptococcus group, respectively. For each pool, low coefficients of intra-assay (7.8, 5.7 and 5.3) and inter-assay (15.8, 18.0 and 18.0) variations were obtained in the Gallibacterium study. In the Streptococcus study only the intra-assay variation was low (3.7, 3.8 and 6.2, respectively). The linearity check was acceptable demonstrating a straight line with slope and intercept, not deviating from one and zero, respectively, using the Gallibacterium sera, whereas the Streptococcus sera deviated from the linear line. Detection limits were low (Gallibacterium, 0.01 mg/ml; Streptococcus, 0.32 mg/ml). OTF concentrations (mean ± standard error of the mean) in overlap performances were elevated in the sera of infected chickens (Gallibacterium, 4.4 ± 0.3 mg/ml; Streptococcus, 3.2 ± 0.4 mg/ml) compared with negative controls (1.7 ± 0.1 mg/ml) (P < 0.05). In conclusion, the Chicken-OTF-ELISA can be used to measure reproducible serum OTF concentrations in brown layer chickens as a response to G. anatis infections, whereas an adjustment of dilution process is proposed to optimize to use in S. zooepidemicus-infected chickens.     

Two evolutionary models for the interactions of dietary organic cyanogens,hemoglobins, and falciparum malaria

Significant regional and ethnic variations in hemoglobin S frequencies among 485 adult nonpregnant Liberian women uniformly exposed to holoendemic falciparum malaria suggest that an additional major factor may influence the distribution of this hemoglobinopathy and the severity of the infectious disease with which it is causally associated. The differential consumption of organic cyanogen-rich cassava (Manihot esculenta) foodstuffs and subsequent dosage-dependent in vivo exposure to sublethal CN^?, SCN^?, and CNO^? may directly interact with hemoglobin S by inhibiting sickling diathesis, and, at higher intakes, this dietary factor may adversely affect Plasmodium survival and antigenicity. In this study, low dietary organic cyanogen intakes (0.3 mg CN^?/kg body wt/day) in NW and W geographical areas are associated with higher regional hemoglobin S gene frequencies (11%; phenotypic incidence 20%), lower mean (± SD) positive Plasmodium antibody titers (861.6 ± 102.4), and a higher mean (± SEM) prevalence of clinical falciparum malaria [3.04 times (± 0.09) within the previous 12 months]. In contrast, high dietary organic cyanogen intakes (1.5 mg CN^?/kg body wt/day in SE and C geographical areas) are associated with lower hemoglobin S gene frequencies (2%; phenotypic incidence 4%), higher mean (± SD) positive Plasmodium antibody titers (968.7 ± 160.5), and a lower mean (± SEM) prevalence of clinical falciparum malaria [1.73 times (± 0.11) within the previous 12 months]. No other significant malaria-linked genetic variation exists between regions or ethnic groups. Two evolutionary models are hypothesized to suggest how 18 generations of differential dietary organic cyanogen intakes could produce two distinct patterns of change in hemoglobin S gene frequencies, modifying both the cadence and direction of evolution.     

Human epidermal growth factor receptor 2 (HER2) immunoreactivity: specificity of three pharmacodiagnostic antibodies

Schrohl A‐S, Pedersen H C, Jensen S S, Nielsen S L & Brünner N
(2011) Histopathology 59 , 975–983 Human epidermal growth factor receptor 2 (HER2) immunoreactivity: specificity of three pharmacodiagnostic antibodies Aims: The availability of specific antibody‐based test systems is essential to testing of HER2 protein expression. Here, we mapped epitopes recognized by three pharmacodiagnostic HER2 antibodies and investigated their specificity towards peptides and fusion proteins homologous to the intracellular domains of HER1, HER2, HER3 and HER4. The investigated antibodies were PATHWAY^® HER2 (clone 4B5; Ventana Medical Systems Inc., Tucson, AZ, USA), HercepTest? (Dako Denmark A/S, Glostrup, Denmark), and Oracle^® HER2 (clone CB11; Leica Microsystems GmbH, Wetzlar, Germany). Methods and results: Epitopes were mapped using the alanine scanning method. Specificity was investigated in immunohistochemical stainings, competitive enzyme‐linked immunosorbent assay (ELISA) and immunoblotting. All three antibodies reacted with HER2 proteins and peptides in immunohistochemical stainings, ELISA and immunoblotting. PATHWAY^® HER2 also stained HER4‐expressing cells, reacted with HER4 peptide in ELISA and detected HER4 fusion protein in an immunoblot. Oracle^® HER2 weakly detected HER4 in immunohistochemical stainings, whereas the HercepTest? antibody showed no cross‐reactivity with other HER proteins. Conclusion: Our study shows that the PATHWAY^® HER2 antibody can bind HER4 peptides and fusion proteins in three different experimental settings. This should be investigated further to determine whether binding of HER4 also occurs in tissue samples and if such binding would have implications for therapy decisions for breast cancer patients.     

Calreticulin is a B cell molecular target in some gastrointestinal malignancies

Calreticulin, upon translocation to the cell surface, plays a critical role in the recognition of tumour cells and in experimentally induced cellular anti‐tumour immunity. However, less is known about anti‐calreticulin antibodies and their role in malignancies. Using enzyme‐linked immunosorbent assay (ELISA), we found immunoglobulin (Ig)A and/or IgG anti‐calreticulin antibodies in sera of approximately 63% of patients with hepatocellular carcinoma (HCC), 57% of patients with colorectal adenocarcinoma (CRA) and 47% of patients with pancreatic adenocarcinoma (PACA), while healthy controls, patients with viral hepatitis C and with chronic pancreatitis reached only 2%, 20% and 31% seropositivity, respectively. We found significantly elevated mean levels of IgA anti‐calreticulin antibodies (P < 0·001) in patients with HCC (78·7 ± 52·3 AU, mean ± standard deviation), PACA (66·5 ± 30·9 AU) and CRA (61·8 ± 25·8 AU) when compared to healthy controls (41·4 ± 19·2 AU). Significantly elevated mean levels of IgG anti‐calreticulin antibodies (P < 0·001) were detected in patients with HCC (121·9 ± 94·2 AU), gall bladder adenocarcinoma (118·4 ± 80·0 AU) and PACA (88·7 ± 55·6 AU) when compared to healthy controls (56·7 ± 22·9 AU). Pepscan analysis revealed a large number of antigenic epitopes of calreticulin recognized by both IgA and IgG antibodies of patients with HCC and PACA, indicating robust systemic immune response. Moreover, significantly elevated levels of antibodies against peptide KGEWKPRQIDNP (P < 0·001) in these patients, tested by ELISA, confirmed the distinct character of antibody reactivity against calreticulin. The high occurrence and specificity of serum anti‐calreticulin autoantibodies in the majority of patients with some gastrointestinal malignancies provide the evidence for their possible clinical relevance.     

Development of an ELISA specific for Listeria monocytogenes using a polyclonal antibody raised against a cell extract containing internalin B

We have developed a new enzyme-linked immunosorbent assay (ELISA) that is specific to the foodborne pathogenic micro-organism Listeria monocytogenes. It is based on an antibody raised against an L. monocytogenes cell preparation optimized for extraction of internalin B. Only in a sandwich ELISA format was the protein A-purified antibody specific to L. monocytogenes. In a competitive ELISA format, the antibody recognizes other Listeria species. The sandwich ELISA shows no recognition of L. innocua, L. ivanovii, L. welshimeri, L. seeligeri, or L. grayii. It has a minimum detectable level for L. monocytogenes of log₁₀?6.37 cfu ml^?1 in pure culture, is reproducible, and is unaffected by the presence of high numbers (approximately log₁₀?8.0 cfu ml^?1) of the other Listeria species. Possible reasons for the format-dependent specificity are discussed. When the ELISA was applied to milk samples inoculated with L. monocytogenes reference material (5 cfu ml^?1), there was a strong response to the enrichment cultures. The new assay may prove useful in detection of L. monocytogenes in enrichment cultures of food samples.     

Carbohydrate supplementation delays DNA damage in elite runners during intensive microcycle training

The aim of this study was to evaluate the effect of carbohydrate supplementation on free plasma DNA and conventional markers of training and tissue damage in long-distance runners undergoing an overload training program. Twenty-four male runners were randomly assigned to two groups (CHO group and control group). The participants were submitted to an overload training program (days 1–8), followed by a high-intensity intermittent running protocol (10 × 800 m) on day 9. The runners received maltodextrin solution (CHO group) or zero energy placebo solution as the control equivalent before, during, and after this protocol. After 8 days of intensive training, baseline LDH levels remained constant in the CHO group (before: 449.1 ± 18.2, after: 474.3 ± 22.8 U/L) and increased in the control group (from 413.5 ± 23.0 to 501.8 ± 24.1 U/L, p < 0.05). On day 9, LDH concentrations were lower in the CHO group (509.2 ± 23.1 U/L) than in the control group (643.3 ± 32.9 U/L, p < 0.01) post-intermittent running. Carbohydrate ingestion attenuated the increase of free plasma DNA post-intermittent running (48,240.3 ± 5,431.8 alleles/mL) when compared to the control group (73,751.8 ± 11,546.6 alleles/mL, p < 0.01). Leukocyte counts were lower in the CHO group than in the control group post-intermittent running (9.1 ± 0.1 vs. 12.2 ± 0.7 cells/μL; p < 0.01) and at 80 min of recovery (10.6 ± 0.1 vs. 13.9 ± 1.1 cells/μL; p < 0.01). Cortisol levels were positively correlated with free plasma DNA, leukocytes, and LDH (all r > 0.4 and p < 0.001). The results showed that ingestion of a carbohydrate beverage resulted in less DNA damage and attenuated the acute post-exercise inflammation response, providing better recovery during intense training.     

Antibodies to β2 microglobulin in the sera of patients with systemic lupus erythematosus

The present study reports the detection of antibodies to β₂ microglobulin in the sera of patients with systemic lupus erythematosus (SLE). Using a Farr-type ammonium sulphate precipitation assay, test sera were reacted with ¹²⁵Iβ₂ microglobulin, and immunoglobulins precipitated by 50% saturated ammonium sulphate. Increased β₂ microglobulin binding activity (normal values: mean±2 sd = 35.5 ±7.8) was detected in 18 of 42 SLE sera. Anti-HLA sera did not reveal increased binding activity, suggesting that the antibody in SLE serum was directed toward free β₂ microglobulin. Direct validation was done by reacting ¹²⁵Iβ₂ microglobulin with 4 SLE sera having increased ¹²⁵Iβ₂ microglobulin binding activity, and subjecting the reactants to sucrose density gradient ultracentrifugation. Two peaks were obtained, one corresponding to free β₂ microglobulin, and the other to 7S material complexed to β₂ microglobulin. Normal sera demonstrated only one peak corresponding to unbound β₂ microglobulin. Assays of β₂ microglobulin binding activity on protein fractions obtained by Sephadex G200 column chromatography also showed the presence of increased binding activity with 7S fractions. Using a double antibody assay, the 7S material reactive to β₂ microglobulin was demonstrated to be IgG. It was also shown that sera with abnormal β₂ microglobulin binding activity had higher titres of antinuclear antibody compared to those lacking such activity (t = 3.18; P<0.01), indicating the pathogenetic relationship of this antibody to increased disease activity. This antibody may be responsible for some of the abnormalities of cell-mediated function previously described in SLE patients.     

Elevated Epstein–Barr virus loads and lower antibody titers in competitive athletes

Epstein–Barr virus (EBV) is a persisting herpesvirus which is controlled by the adaptive immune response after primary infection and maintained in a latent state. However, reactivation or persistent replication is observed in situations where the immune response is compromised. Since intensive physical training has been reported to diminish immune function, increased EBV load may be a cause of reduced performance and decreased ability to sustain high training loads in competitive athletes. Samples drawn from 209 athletes during their regular follow‐up appointments were tested. One hundred sixty‐five individuals of similar age not active in competitive sports served as case–controls. EBV load was quantified in peripheral blood leucocytes (PBLs) by real‐time PCR, and EBV antibodies were detected in plasma by ELISA and immunoblot analysis. EBV DNA was detectable in 25 of 209 athletes and in 26 of 165 controls. Of note, the EBV load per 10⁵ PBLs was 6.44 ± 1.75 in the case and 1.67 ± 0.44 copies in the controls, yielding a high significant difference (P < 0.0001). However, EBV‐specific IgG titers were significantly lower in athletes (150.4 ± 10.73 U ml^?1 vs. 241.6 ± 18.59 U ml^?1). As monitored by immunoblotting, primary infections were detected with low prevalence, three in the case group and one in the control group. These findings demonstrate that EBV is present at higher levels in athletes, but the antibody response is lower in athletes than in the controls. J. Med. Virol. 82:446–451, 2010. © 2010 Wiley‐Liss, Inc.     

Measurement of Typhi Vi antibodies can be used to assess adaptive immunity in patients with immunodeficiency

Vaccine‐specific antibody responses are essential in the diagnosis of antibody deficiencies. Responses to Pneumovax II are used to assess the response to polysaccharide antigens, but interpretation may be complicated. Typhim Vi^®, a polysaccharide vaccine for Salmonella typhoid fever, may be an additional option for assessing humoral responses in patients suspected of having an immunodeficiency. Here we report a UK multi‐centre study describing the analytical and clinical performance of a Typhi Vi immunoglobulin (Ig)G enzyme‐linked immunosorbent assay (ELISA) calibrated to an affinity‐purified Typhi Vi IgG preparation. Intra‐ and interassay imprecision was low and the assay was linear, between 7·4 and 574 U/ml (slope = 0·99–1·00; R² > 0·99); 71% of blood donors had undetectable Typhi Vi IgG antibody concentrations. Of those with antibody concentrations  > 7·4 U/ml, the concentration range was 7·7–167 U/ml. In antibody‐deficient patients receiving antibody replacement therapy the median Typhi Vi IgG antibody concentrations were  < 25 U/ml. In vaccinated normal healthy volunteers, the median concentration post‐vaccination was 107 U/ml (range 31–542 U/ml). Eight of eight patients (100%) had post‐vaccination concentration increases of at least threefold and six of eight (75%) of at least 10‐fold. In an antibody‐deficient population (n = 23), only 30% had post‐vaccination concentration increases of at least threefold and 10% of at least 10‐fold. The antibody responses to Pneumovax II and Typhim Vi^® correlated. We conclude that IgG responses to Typhim Vi^® vaccination can be measured using the VaccZyme Salmonella typhi Vi IgG ELISA, and that measurement of these antibodies maybe a useful additional test to accompany Pneumovax II responses for the assessment of antibody deficiencies.