Heterophilic antibodies in sera from individuals without loxoscelism cross-react with phospholipase D from the venom of Loxosceles and Sicarius spiders

Loxoscelism is a severe human envenomation caused by Loxosceles spider venom. To the best of our knowledge, no study has evaluated the presence of antibodies against Loxosceles venom in loxoscelism patients without treatment with antivenom immunotherapy. We perform a comparative analysis for the presence of antibodies capable of recognizing Loxosceles venom in a group of patients diagnosed with loxoscelism and in a group of people without loxoscelism. Methods The detection of L. laeta venom, Sicarius venom and recombinant phospholipases D from Loxosceles (PLDs) in sera from people with loxoscelism (Group 1) and from healthy people with no history of loxoscelism (Group 2) was evaluated using immuno-dot blot, indirect ELISA, and Western blot. Results We found naturally heterophilic antibodies (IgG-type) in people without contact with Loxosceles spiders or any clinical history of loxoscelism. Either serum pools or single sera from Group 1 and Group 2 analyzed by dot blot tested positive for L. laeta venom. Indirect ELISA for venom recognition showed titles of 1:320 for Group 1 sera and 1:160 for Group 2 sera. Total IgG quantification showed no difference in sera from both groups. Pooled sera and purified IgG from sera of both groups revealed venom proteins between 25 and 32 kDa and the recombinant phospholipase D isoform 1 (rLlPLD1), specifically. Moreover, heterophile antibodies cross-react with PLDs from other Loxosceles species and the venom of Sicarius spider. Conclusions People without contact with the spider venom produced heterophilic antibodies capable of generating a cross-reaction against the venom of L. laeta and Sicarius spiders. Their presence and possible interference should be considered in the development of immunoassays for Loxosceles venom detection.(AU)

Seroprevalence of bactericidal antibodies against serogroup B and C Meningococci in a University Hospital

Meningococcus serogroup B (MenB), clonal complex 32 (cc 32), was the Brazilian epidemic strain of meningococcal disease (MD) in the 1990’s. Currently, meningococcus serogroup C (MenC), cc 103, is responsible for most of the cases of the disease in Brazil. The aim of this study was to investigate the seroprevalence of bactericidal antibody (SBA) against representative epidemic strains of MenC, (N753/00 strain, C:23:P1.22,14-6, cc103) and MenB, (Cu385/83 strain, B:4,7:P1.15,19, cc32) in students and employees of a university hospital in the State of Rio Grande do Sul (RS, Brazil). A second MenC strain (N79/96, C:2b:P1.5-2,10, cc 8) was used as a prototype strain of Rio de Janeiro’s outbreak that occurred in the 1990’s. Our previous study showed a 9% rate of asymptomatic carriers in these same individuals. A second goal was to compare the SBA prevalence in meningococcal carriers and non-carriers. Fifty-nine percent of the studied population showed protective levels of SBA titers (log2≥2) against at least one of the three strains. About 40% of the individuals had protective levels of SBA against N753/00 and Cu385/83 strains. Nonetheless, only 22% of the individuals showed protective levels against N79/96 strain. Significantly higher antibody levels were seen in carriers compared to non-carriers (P≤0.009). This study showed that, similar to other States in Brazil, a MenC (23:P1.22,14-6, cc103) strain with epidemic potential is circulating in this hospital. Close control by the Epidemiological Surveillance Agency of RS of the number of cases of MD caused by MenC strains in the State is recommended to prevent a new disease outbreak.

Tratamento cirúrgico de 510 pacientes portadores de lipodistrofias secundárias ao uso de antirretrovirais / Surgical treatment of 510 patients with highly active antiretroviral therapy-associated lipodystrophy

INTRODUÇÃO O tratamento da Síndrome da Imunodeficiência Adquirida vem sofrendo importantes avanços desde a introdução da terapia antirretroviral altamente ativa, conhecida como HAART (high active antirretroviral therapy). Este tratamento levou à eliminação do vírus na corrente sanguínea e ao aumento na sobrevida, entretanto alterações metabólicas e estruturais tornaram-se evidentes. Uma dessas alterações é a redistribuição de gordura corpórea, também denominada lipodistrofia. Com uma das maiores casuísticas mundiais, o objetivo deste trabalho é demonstrar algumas das alternativas cirúrgicas, bem como os resultados obtidos na tentativa de minimizar o impacto da lipodistrofia. MÉTODO: No período de julho de 2005 a julho de 2013, 510 pacientes portadores de lipodistrofia secundária ao uso de HAART foram operados pela Clínica de Cirurgia Plástica do Hospital Heliópolis. Todos esses pacientes foram submetidos à prévia avaliação clínica e imunológica, sob auxílio da equipe de Infectologia. O presente trabalho foi aprovado pelo Comitê de Ética em Pesquisa da Fundação do ABC. RESULTADO: Dentre os 510 pacientes, 335 eram do sexo feminino e 175 do sexo masculino, com idades variando entre 16 e 74 anos. Quanto aos procedimentos, destacou-se lipoaspiração da giba e dorso, com 199 casos. Quanto à resposta estimulada através de questionário subjetivo, observou-se elevado grau de satisfação, aumento significativo da autoestima e maior adesão ao tratamento antirretroviral. CONCLUSÃO: A correção cirúrgica da lipodistrofia corporal comprovadamente melhora o aspecto estético do paciente que faz uso da HAART; porém, o efeito psicológico e social é ainda mais importante, elevando a autoestima, com diminuição dos estigmas, e proporcionando uma maior adesão ao tratamento antirretroviral.

INTRODUCTION The treatment of acquired immunodeficiency syndrome has undergone important advances since the introduction of highly active antiretroviral therapy (HAART). This treatment led to the elimination of the virus in the bloodstream and increased survival; however, metabolic and structural changes became evident. One of these changes is lipodystrophy, the redistribution of body fat. With one of the largest samples worldwide, the aim of this work was to present some of the various surgical alternatives as well as the results obtained for minimizing the impact of lipodystrophy. METHOD: From July 2005 to July 2013, 510 patients with HAART-associated lipodystrophy underwent surgery in the Clinic of Plastic Surgery, Heliópolis Hospital. All patients submitted to prior clinical and immunological assessments made with the aid of the infectious diseases team. The present study was approved by the Research Ethics Committee of the ABC Foundation. RESULTS: The 510 patients included 335 women and 175 men with an age range of 16-74 years. Liposuction of the cervicodorsal fat pad (buffalo hump) was predominant (199 cases). With regard to the response stimulated through a subjective questionnaire, a high degree of satisfaction was observed with a significant increase in self-esteem and greater adherence to antiretroviral treatment. CONCLUSION: The surgical correction of body lipodystrophy demonstrably improves the aesthetics of patients using HAART; however, its psychological and social effects are even more important since self-esteem increases and stigma decreases, which leads to better adherence to antiretroviral treatment.

A Recombinant Matrix Metalloproteinase Protein from Gnathostoma spinigerum for Serodiagnosis of Neurognathostomiasis

Neurognathostomiasis is a severe form of human gnathostomiasis which can lead to disease and death. Diagnosis of neurognathostomiasis is made presumptively by using clinical manifestations. Immunoblotting, which recognizes antigenic components of molecular mass 21 kDa and 24 kDa in larval extracts of Gnathostoma spinigerum (Gs 21/24), has high sensitivity and specificity for diagnosis of neurognathostomiasis. However, only very small amounts of the Gs 21/24 antigens can be prepared from parasites harvested from natural or experimental animals. To overcome this problem, we recently produced a recombinant matrix metalloproteinase (rMMP) protein from G. spinigerum. In this study, we evaluated this rMMP alongside the Gs 21/24 antigens for serodiagnosis of human neurognathostomiasis. We studied sera from 40 patients from Srinagarind Hospital, Khon Kaen University, Thailand, with clinical criteria consistent with those of neurognathostomiasis, and sera from 30 healthy control adults from Thailand. All sera were tested for specific IgG antibodies against both G. spinigerum crude larval extract and rMMP protein using immunoblot analysis. The sensitivity and specificity for both antigenic preparations were all 100%. These results show that G. spinigerum rMMP protein can be used as an alternative diagnostic antigen, in place of larval extract, for serodiagnosis of neurognathostomiasis.

Application of bispecific antibody against antigen and hapten for immunodetection and immunopurification

We present a bispecific antibody that recognizes an antigen and a hapten and can be applied to various biological assays, including immunoblotting and immunoprecipitation. In immunoblot analysis of serum, an anti-C5 x anti-cotinine bispecific tandem single-chain variable fragment (scFv)-Fc fusion protein and cotinine-conjugated horseradish peroxidase (HRP) generated a clean signal without the high background that was observed in a parallel experiment using HRP-conjugated goat anti-rabbit immunoglobulin G (Fc-specific) antibody. In immunoprecipitation analysis of serum, use of the bispecific tandem scFv-Fc fusion protein and cotinine-crosslinked magnetic beads significantly reduced the amount of protein contaminants compared with a parallel experiment done with protein A agarose beads. In subsequent immunoblot analysis, use of cotinine-HRP as the secondary probe instead of HRP-conjugated goat anti-rabbit IgG (Fc-specific) antibody successfully eliminated the band corresponding to the bispecific tandem scFv-Fc fusion protein.

Dot-Blot Immunoassay of Fasciola gigantica Infection using 27 kDa and Adult Worm Regurge Antigens in Egyptian Patients

The purpose of the present study was to evaluate the potential role of the 27-Kilodalton (KDa) antigen versus Fasciola gigantica adult worm regurge antigens in a DOT-Blot assay and to assess this assay as a practical tool for diagnosis fascioliasis in Egyptian patients. Fasciola gigantica antigen of an approximate molecular mass 27-(KDa) was obtained from adult worms by a simple elution SDS-PAGE. A Dot-Blot was developed comparatively to adult worm regurge antigens for the detection of specific antibodies from patients infected with F. gigantica in Egypt. Control sera were obtained from patients with other parasitic infections and healthy volunteers to assess the test and compare between the antigens. The sensitivity, specificity, positive and negative predictive values of Dot-Blot using the adult worm regurge were 80%, 90%, 94.1%, and 69.2% respectively, while those using 27-KDa were 100% which confirms the diagnostic potential of this antigen. All patients infected with Fasciola were positive, with cross reactivity reported with Schistosoma mansoni serum samples. This 27-KDa Dot-Blot assay showed to be a promising test which can be used for serodiagnosis of fascioliasis in Egyptian patients especially, those presenting with hepatic disease. It is specific, sensitive and easy to perform method for the rapid diagnosis particularly when more complex laboratory tests are unavailable.

Análise de uma heme oxigenase funcional em Trypanosoma cruzi / Analysis of a heme oxygenase functional in Trypanosoma cruzi

O Trypanosoma cruzi é o agente etiológico da doença de Chagas, transmitida através de insetos vetores triatomíneos durante a alimentação do hospedeiro vertebrado. Os triatomíneos ingerem numa única alimentação cerca de 10 mM de heme ligado à hemoglogina. O heme é uma importante molécula no metabolismo dos organismos. Um mecanismo intracelular importante no controle de sua homeostase é a degradação enzimática pela Heme Oxigenase (HO) formando biliverdina (Bv), monóxido de carbono e ferro. Como esta enzima não está presente no genoma de T. cruzi, esse trabalho tem por objetivo identificar uma atividade funcional de HO neste parasito, uma vez que dados do nosso laboratório mostram a presença de biliverdina nas incubações dessas células com heme. No presente trabalho testamos o efeito do SnPPIX (inibidor da HO-1), CoPPIX (indutor da HO-1) e Bv sobre a proliferação da forma epimastigota do parasito. A adição de SnPPIX diminuiu a proliferação do parasito tanto na ausência quanto na presença de heme. Quando a Bv foi adicionada à cultura esse efeito foi revertido; a Bv aumenta a proliferação celular na presença de heme. Por outro lado, a adição de CoPPIX não interferiu na proliferação. Posteriormente, mostramos através da técnica de immunoblotting, utilizando anticorpo monoclonal contra a HO-1, um aumento da expressão de uma proteína em resposta ao heme. Diferentemente das HO-1 já descritas que possuem massa molecular de 32 kDa, a única banda reconhecida pelo anticorpo apresenta 45 kDa. Analisamos também a expressão da HO-1 na presença de CoPPIX, SnPPIX e biliverdina, e somente o CoPPIX foi capaz de modular os níveis de expressão da HO-1. A análise estrutural através da técnica de imunocitoquímica mostrou uma maior expressão da enzima na presença de heme, e que a HO-1 de T. cruzi pode ter mais de uma localização, apresentando marcação citoplasmática e glicossomal. A fim de investigar a sequência da HO-1 de T. cruzi, o DNA genômico foi extraído para amplificação ...

Trypanosoma cruzi, the ethiologic agent of Chagas disease, is transmitted through triatomine vectors during their blood-meal on vertebrate host. These hematophagous insects ingest blood about 6 to 12 times its original weight, reaching in a single meal about 10mM heme bound to hemoglobin. Heme (iron protoporphyrin IX) is an important molecule in metabolism of all living organisms. One important intracellular mechanism to control heme homeostasis is its enzymatic degradation by heme oxygenase (HO). HO catalyzes the degradation of heme to biliverdin (Bv), carbon monoxide and iron. HO is absent in T. cruzi genome, thus we have been investigating the presence of a functional HO in this parasite, since our previous results showed a presence of biliverdin in heme-treated epimastigotes. In the present work, we evaluated the effect of SnPPIX, a HO-1 inhibitor, CoPPIX, a HO inducer, and Bv upon T. cruzi epimastigotes proliferation. The addition of SnPPIX decreased the parasite proliferation in the absence or in the presence of heme. When Bv was added to the culture this effect was reversed; Bv increases the parasite proliferation in the presence of heme. On the other hand, CoPPIX did not interfered on proliferation. Furthermore, we showed through immunoblotting, using an anti-HO-1 monoclonal antibody, an increase in the protein expression in heme-treated epimastigotes. Differently of described HO-1 that has a mass molecular of a 32 kDa, we showed a 45 kDa protein, the only band recognize by the HO-1 antibody. HO-1 expression analysis in the presence of CoPPIX, SnPPIX and biliverdin, showed that only CoPPIX was able to modulate its expression level. Ultrastructural immunocytochemistry analysis suggests a higher expression of the enzyme in heme-treated epimastigotes, and that T. cruzi HO-1 might have a dual distribution, since the anti-HO-1 antibody labeled both cytosol and glycosomes. In order to investigate the T. cruzi HO-1 gene sequence, we isolated genomic DNA ...

Immunological cross-reactivity of venoms from some Egyptian snakes and scorpions

The immunological cross-reactivity among the Elapidae, Viperidae and Buthdae venoms was detected in dot blot and western blot assays and quantified in ELISA as percentage of absorbance of heterologous versus homologous reaction. Mutual cross-reactivity between the Elapidae and Viperidae venoms was recognized, reflecting high inter-family relationship whereas only weak cross-reactions were observed between the scorpion and snake venoms. The anti-scorpion antivenom exceptionally binds a 30-35 kDa Cerastes cerastes venom component, revealing common antigenic components between scorpions and C. cerastes venom. Many of the moderate and weak cross reactivity in ELISA got unnoticed in the dot blot technique. This work primarily describes the commonality among venoms from remote source animals which is potentially important in the designation of venom detection kits and also in exploring common allergens responsible for cross-sensitization of patients to heterologous bites or stings leading to immediate hypersensitivity reactions

overview of various labeled assays used in medical laboratory diagnosis immune and non-immune assays

In this review, some light is thrown on various labeled immunoassays that depend on antigen-antibody [Ag-Ab] reactions, including immunofluorescence, radioimmunoassay, and enzyme immunoassay [EIA or ELISA]. Their definitions, principles, and applications are described, then they are discussed chronologically to show their stepwise development that led finally to full automation. Enzyme labeled immunoblot assays [Western blot, blot spot, and recombinant immunoblot assay], and luminescence [bioluminescence and chemiluminescence] are also discussed chronologically. Labeled assays, that do not involve Ag-Ab reaction but rather, utilizing biotin-steptavidin [BS] interaction and probe-target DNA interaction, and described, together with their applications for DNA/RNA detection and genotyping. Finally, included in the discussion were some luminescent labeled techniques that utilitze the immune Ag-Ab reaction together with non-immune BS reaction, such as the luminescent oxygen channeling immunoassay, and its commercialized AlphaLISA, both eliminate the washing steps without sacrificing high sensitivity, or wide dynamic range

Evaluation of Anti-dsDNA Antibody Tests: Crithidia luciliae Immunofluorescence Test, Immunoblot, Enzyme-linked Immunosorbent Assay, Chemiluminescence Immunoassay / 대한진단검사의학회지

BACKGROUND: Anti-double stranded DNA antibody (anti-dsDNA) test is useful for the diagnosis and monitoring of systemic lupus erythematosus (SLE). Although several methods are available, none of them is completely satisfactory and differences among them have been reported. We evaluated the diagnostic performance of 6 commercial kits for anti-dsDNA detection. METHODS: A total of 142 sera (SLE [N=74], other systemic rheumatic diseases [N=50], other diseases [N=18]) were tested by 6 different assay kits using different antigenic sources of DNA: Crithidia luciliae immunofluorescence test (CLIFT), salmon testes (immunoblot, IB), human (ELISA I), salmon testes with nucleosome linker (ELISA II), plasmid (ELISA III), and synthetic oligonucleotides (chemiluminescence immunoassay, CLIA). RESULTS: With manufacturers' cut-off values, 6 test kits showed sensitivities of 55.4-91.9%. ELISA I had a greater sensitivity than the other five assays (P0.05). With cut-off values set at 95% of specificity, ELISA II had a higher sensitivity than ELISA III (63.5% vs. 41.9%, P<0.05). IB had poor concordance rates with other assays (42.0-65.0%). Pearson correlation coefficients among 4 quantitative assays were 0.667-0.798. CONCLUSIONS: Six different assays showed various performances depending on the methods and cut-off values used. Except IB, the other five assays can be used for the detection of anti-dsDNA.

Evaluation of an enzyme-linked immunoelectrotransfer blot test for the confirmatory serodiagnosis of human toxocariasis

To improve the serodiagnosis of human toxocariasis, a sensitive and specific enzyme-linked immunoelectrotransfer blot (EITB-IgG) test was developed and evaluated using Toxocara canislarvae excretory-secretory antigens for detecting anti-Toxocara IgG antibodies. The EITB-IgG profile of toxocariasis was characterized by comparing 27 sera from patients with toxocariasis, 110 sera from healthy subjects and 186 sera from patients with other helminth diseases (ascariasis, ancylostomiasis, trichuriasis, enterobiasis, strongyloidiasis, hymenolepiasis, diphyllobothriasis, taeniasis, cysticercosis, hydatidosis and fascioliasis). Antigenic bands of 24, 28, 30, 35, 56, 117, 136 and 152 kDa were predominantly recognized in sera from all patients with toxocariasis. However, only bands of 24-35 kDa were highly specific for Toxocara infection (98.3 percent), whereas other antigenic bands observed displayed cross-reactivity. Additionally, when the results of the EITB-IgG test were compared to those of the ELISA-IgG test, a 100 percent concordance was observed for positive results in human toxocariasis cases. The concordance for negative results between the two tests for healthy subjects and patients with other helminth diseases were 96.3 percent and 53.7 percent, respectively, showing that the EITB-IgG test has a higher specificity than ELISA. In conclusion, the EITB-IgG test is a very useful tool to confirm the serological diagnosis of human toxocariasis.

Immunoblot analysis of sera in uncomplicated typhoid fever & with typhoid ileal perforation.

Background & objectives: Ileal perforation is a serious complication of typhoid fever. The exact reasons for the development of perforation in only a few of those infected with Salmonella Typhi is unknown, and it is likely that immunological factors are involved. Therefore we undertook this study to compare the antibody profile in patients with uncomplicated typhoid fever with those having ileal perforation by immunoblotting. Methods: Two groups of patients were included in the study. Group II comprised patients with uncomplicated typhoid fever (n=47), and group I with typhoid ileal perforation (n=33). The flagellar (H), lipopolysaccharide (LPS) and outer membrane protein (OMP) antigens of Salmonella Typhi were extracted and used to test patient sera for antibodies by immunoblotting Results: Immunoblotting using S. Typhi antigens enabled the detection of S. Typhi antibodies in the two groups of patients. A significant difference was seen in the response of these two groups of patients with respect to antibodies to flagella, lipopolysaccharide and outer membrane proteins. Antibodies to flagella were more pronounced among patients with uncomplicated typhoid fever, while anti-OMP antibodies were significantly associated with typhoid ileal perforation. Interpretation & conclusions: A comparison of antibodies in patients with uncomplicated typhoid fever and with ileal perforation revealed the differences in the antibody profiles of the two groups. Our study suggests that the difference in antibody response may in some way play a role in the pathogenesis of typhoid ileal perforation which can also potentially be exploited to develop suitable diagnostic tests.

Allergen Specific IgE Measurement with Polycheck Allergy: Comparison of Three Multiple Allergen Simultaneous Tests / 대한진단검사의학회지

BACKGROUND: The in vivo skin prick test (SPT) or in vitro detection of allergen specific IgE in serum is commonly used for the diagnosis of allergic disease. In this study, we evaluated the usefulness of a new multiple allergen simultaneous test (MAST) immunoblot assay, Polycheck Allergy (Biocheck GmbH, Germany). METHODS: A total of 100 patients with clinical findings of allergic diseases were tested by SPT and three different MAST assays: Polycheck Allergy (Biocheck GmbH, Germany), MAST CLA allergy system (Hitachi Chemical Diagnostics, USA) and Allergy Screen (R-biopharm, Germany). The results of MAST assays were compared with those of SPT. RESULTS: Concordance rates of MAST assays with SPT were 79-100% for Polycheck Allergy, 88.9-100% for MAST CLA and 72.7-98.3% for Allergy Screen. In ROC curve analysis, significant differences were observed in four of 25 allergens analysed: Alternaria, Birch, Hazelnut and D. farinae. For Alternaria and Birch, Polycheck Allergy (P0.05). CONCLUSIONS: Since Polycheck Allergy showed similar or superior result to the others, it can be used for the detection of allergen specific IgE antibodies.

Determination of antigenic patterns of three taenia extracts and their use for differential diagnosis of taeniasis by immunoblot

Immunoserological differential diagnosis of Taenia spp. is the major contribution of the present work. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE] was used to analyze the protein components of three Taenia extracts prepared from Cysticercus bovis, Taenia saginata and Taenia taeniaeformis. Antigenic components of the three taeniid antigens revealed by [SDS-PAGE] were: [I] Cysticercus bovis cystic fluid antigen: 180, 116, 90, 66, 45, 26, 17 and 10 kD. [II] Taenia saginata excretory secretory antigen [ES]: 198, 180, 146, 116, 55, 45, 35 and 26 kD. [III] Taenia taeniaeformis whole crude antigen 170, 140, 120, 116, 66, 55, 45, 35, 26, 17 and 10 kD. The characterized extracts were used as antigens in Western Blot [WB] technique. Sera from immunized rabbits with Cysticercus bovis fluid antigens were used in detecting a variety of immunogenic bands: [I] Cysticercus bovis: 160, 116, 86, 52, 45, 38 and 29 kD. [II] Taenia saginata: 86, 68 and 10 kD. [III] Taenia taeniaeformis: 170, 116, 86, 68, 52, 45 and 29 kD. The present results suggested that the diagnostic bands by Western Blotare 160 and 38 kD for Cysticercus bovis, 10 kD for Taenia saginata [ES] antigen and 170 kD for Taenia taeniaeformis

Sensitization of Children to Storage Mites in Kutahya, Turkey

Specific IgE against Acarus siro, Glycphagus domesticus, Tyrophagus putrescentiae, and Lepidoglyphus destructor have been investigated by ELISA in sera of 92 children. Of them, 41 were found to be specific IgE positive (> or = 0.35 IU/ml) against at least one of house dust mite species, Dermatophagoides pteronyssinus and Dermatophagoides farinae, by an immunoblot. In 65.9% of the dust mite-sensitized children, specific IgE against at least one of these mite species was found. Sensitization levels, including co-sensitization cases were found to be 35.7% against A. siro, 24.4% against T. putrescentiae, 31.7% against L. destructor, and 26.8% against G. domesticus. In non-sensitized children, dust mite sensitization level was found to be 25.5%. Breakdown of sensitization by individual species in this group was; against A. siro and T. putrescentiae at 7.8%, against L. destructor at 13.7%, and against G. domesticus at 9.8%. When all children were reckoned, 43.5% was found to be sensitized against at least one storage mite species, with sensitizations against A. siro at 18.5%, T. putrescentiae at 26.1%, L. destructor at 21.7%, and G. domesticus at 17.4%. In dust samples collected from the dwellings of children, distribution of species was found to be A. siro (17%), G. domesticus (23%), T. putrescentiae (29%), L. destructor (25%), and unidentified (6%). In Fisher's chi-square test on SPSS program, there was a relationship between dust mite sensitization and storage mite sensitization (P < 0.05), but no meaningful relationship was found on the basis of individual mite species.

Antigenemia and Specific IgM and IgG Antibody Responses in Rabbits Infected with Toxoplasma gondii

In this experiment, the correlation between antigenemia and specific antibody responses in Toxoplasma gondii-infected rabbits was assessed. We injected 1,000 T. gondii tachyzoites (RH) subcutaneously into 5 rabbits. Parasitemia, circulating antigens, and IgM and IgG antibody titers in blood were tested by ELISA and immunoblot. For detection of parasitemia, mice were injected with blood from rabbits infected with T. gondii and mice died between days 2 and 10 post-infection (PI). Circulating antigens were detected early on day 2 PI, and the titers increased from day 4 PI and peaked on day 12 PI. Anti-Toxoplasma IgM antibody titers increased on day 6 PI and peaked on days 14-16 PI. IgG was detected from day 10 PI, and the titers increased continuously during the experiment. The antigenic protein patterns differed during the infection period, and the number of bands increased with ongoing infection by the immunoblot analysis. These result indicated that Toxoplasma circulating antigens during acute toxoplasmosis are closely related to the presence of parasites in blood. Also, the circulating antigen levels were closely correlated with IgM titers, but not with IgG titers. Therefore, co-detection of circulating antigens with IgM antibodies may improve the reliability of the diagnosis of acute toxoplasmosis.

Detection of in vitro and in vivo released antigens of diagnostic interest in Mycobacterium tuberculosis by immunoblotting.

Identification of in vitro and in vivo released mycobacterial antigens are of considerable interest in diagnosis of Mycobacterium tuberculosis. Isolation of in vitro released antigen from M. tb excretory-secretory culture filtrate protein and in vivo released circulating tuberculous antigen from smear positive pulmonary tuberculosis sera by ammonium sulphate precipitation is reported. The antigens were resolved by SDS-PAGE and immunoblotting was performed using pooled serum of smear positive, smear negative pulmonary tuberculosis sera and normal sera to identify reactive tuberculous antigens. In vitro and in vivo released mycobacterial antigens showed reactivity at 100, 31, 43 and 20 kDa with smear positive and smear negative pulmonary tuberculosis patients. Further, the in vitro released antigen showed strong reactivity exclusively at 55 kDa antigen with smear positive and 24 kDa antigen with smear negative pulmonary tuberculosis sera. In vivo released antigen reacted exclusively at 170 and 16 kDa with smear positive and 19 kDa antigen with smear negative pulmonary tuberculosis patients. Antigens of 24 and 19 kDa which are reactive with sputum negative sera will be of diagnostic interest and need further study in patients with low bacillary load. The in vitro and in vivo released mycobacterial 100, 31,43 and 20 kDa antigens, reactive with patients sera are of diagnostic interest in tuberculosis.

Use of immunoblotting assay improves the sensitivity of paracoccidioidomycosis diagnosis

The purpose of this work was to evaluate two serological assays: double immunodiffusion (DI) and immunoblotting (IB) in immunodiagnosis of paracoccidioidomycosis (PCM). We evaluated by IB assay 23 sera samples from patients with clinical confirmation of PCM, all of them with negative DI results against culture filtrate from Paracoccidioides brasiliensis isolate 113. For IB, as well as for comparative DI assay, we employed soluble components of the cell wall outer surface (SCCWOS) from P. brasiliensis isolate 113 cultivated at 36°C in Fava-Neto's agar medium for 5 and 10 days. Among the 20 sera samples analyzed by DI, 13 (65 percent) were negative and 7 (35 percent) were positive against SCCWOS obtained on the 5th and 10th days. By IB assay, 95.4 percent and 100 percent of sera reacted against gp43 and gp70 present in SCCWOS from the 5th day and 95.6 percent recognized these fractions when evaluated against SCCWOS from the 10th day. Our results demonstrated that the use of an immunoenzymatic assay significantly improves the sensitivity of PCM immunodiagnosis and also suggests that at least two serological tests for antibody detection should be adopted in cases of questionable diagnosis.

Detection of molecular markers of toxoplasmosis among Egyptian patients with miscarriage using avidity IGG-ELISA and western blotting

A total of 54 miscarriage patients were divided into 3 groups. GI: 10 toxoplasmosis patients with +ve IgM-ELISA; GII 24 toxoplasmosis patients with +ve IgG-ELISA, and G III: 20 non-toxoplasmosis cross-matched females as a control. All groups were subjected to IgG-avidity ELISA and IgG-avidity immunoblotting. Avidity Indices [AI] by ELISA ranged from 22.6% to 73.3% in GI and from 9.6%-75.6% in GII AI were high [>40%] in 3 [30%] patients in G I and in 8 [33.3%] patients in G II. Sera of GI recognized the 20, 28,32,60,93 and l00 Kda bands with 55% reduction in the 38 and 60 Kda bands after treatment with 6 M urea solutions. Sera of G II recognized the 20, 28, 32, 38, 45, 95-97 and 106 Kda bands. There was 12.5%, 16.6% and 16.7% reduction in the 20, 32, and 106 Kda bands, respectively, after urea. The 38 and 60 Kda bands were identified as good diagnostic markers for the recent toxoplasmosis infection [GI]. The 20, 32 and 106 Kda bands were good markers of high avidity antibodies during the chronic toxoplasmosis [GIl]

Comparative genetic characterization between isolated pox virus from dove and some avian pox strains

Avipoxviruses from different geographic regions of the world have been characterized to study their genetic and biological properties, but so far, no such work has been performed on Egyptian isolates. Lesions suggestive of avian pox; found on Egyptian wild dove; were used for isolation of pox virus in a previous study. The resulting virus was propagated in chorio-allantoic membrane [CAM] of specific pathogen free [SPF] Embryonated chicken eggs [ECE]. PCR was carried out on the DNA of the dove poxvirus [DPV], pigeon poxvirus [PPV] and a vaccinal strain of fowl pox virus [FPV], then restriction fragment length polymorphism [RFLP] assay was carried out on the resulting amplicons of 578 bp length; using EcoRV and NlaIII restriction enzymes. The restriction profile revealed that the dove pox virus is identical to the PPV and both are different from FPV. The results of immunoblotting analysis of the 3 pox viruses against chicken anti FPV revealed that in spite of the minor antigenic differences observed between them the DPV is closely related to the PPV. In conclusion the Egyptian wild doves are found to play a serious role in the epidemiology of PPV among pigeon flocks