Littérature scientifique sur le sujet « IgE blotting »

Abstract In vivo studies suggested the possibility of an IgE-dependent regulation of high-affinity (FcRI) IgE receptor expression on basophils. The current studies extend these observations to in vitro cultures of human basophils. Incubation of basophils for 3 to 4 weeks resulted in a slow dissociation of IgE antibody, during which time FcRI expression decreased, as measured by flow cytometry using the anti-FcRIα monoclonal antibody, 22E7, or by measuring FcRIα mass by Western blotting of whole-cell lysates. Culture of basophils with IgE resulted in upregulation of FcRIα expression by both flow cytometry and Western blotting of whole-cell lysates. Upregulation followed a linear time course during 2 weeks of culture. The relative increase in FcRIα density depended on the starting density; with starting densities of FcRIα of 10,000 to 170,000 per basophil, the upregulation varied 20- to 1.1-fold, respectively. Upregulation occurred in high-purity basophils, was not influenced by IgG at concentrations up to 1 mg/mL, and was inhibited by dimeric IgE. Heat-inactivated IgE was less effective and the monoclonal antibody CGP51901 that prevents IgE binding to FcRIα blocked the ability of IgE to induce upregulation. The dose-response curve for IgE-induced upregulation had an effective concentration50 of 230 ng/mL. Although the receptor through which IgE induces this upregulation is not yet known, several characteristics suggest that the upregulation is mediated by IgE interacting through FcRIα itself.

In vivo studies suggested the possibility of an IgE-dependent regulation of high-affinity (FcRI) IgE receptor expression on basophils. The current studies extend these observations to in vitro cultures of human basophils. Incubation of basophils for 3 to 4 weeks resulted in a slow dissociation of IgE antibody, during which time FcRI expression decreased, as measured by flow cytometry using the anti-FcRIα monoclonal antibody, 22E7, or by measuring FcRIα mass by Western blotting of whole-cell lysates. Culture of basophils with IgE resulted in upregulation of FcRIα expression by both flow cytometry and Western blotting of whole-cell lysates. Upregulation followed a linear time course during 2 weeks of culture. The relative increase in FcRIα density depended on the starting density; with starting densities of FcRIα of 10,000 to 170,000 per basophil, the upregulation varied 20- to 1.1-fold, respectively. Upregulation occurred in high-purity basophils, was not influenced by IgG at concentrations up to 1 mg/mL, and was inhibited by dimeric IgE. Heat-inactivated IgE was less effective and the monoclonal antibody CGP51901 that prevents IgE binding to FcRIα blocked the ability of IgE to induce upregulation. The dose-response curve for IgE-induced upregulation had an effective concentration50 of 230 ng/mL. Although the receptor through which IgE induces this upregulation is not yet known, several characteristics suggest that the upregulation is mediated by IgE interacting through FcRIα itself.

Background. House dust mites are important allergen sources and some of these allergenic proteins may contain carbohydrate moieties, which are able to be isolated using lectins, as Concanavalin A (ConA). This study aimed to investigate allergenicity (IgE) and antigenicity (IgG1 and IgG4) of ConA-unbound and ConA-bound Dermatophagoides pteronyssinus (Dpt) crude extracts using sera of mite-allergic patients as well as inhibition capacity of antibody binding. Material and Methods. We obtained mannose-enriched and mannose-depleted fractions from Dpt by ConA affinity chromatography. Both ConA-bound and ConA-unbound fractions were evaluated by ELISA and Western Blotting for specific IgE, IgG1, and IgG4 reactivity with sera obtained from 95 mite-allergic patients (DP+) and 92 nonallergic (NA) subjects. Inhibition ELISA was used to assess cross-reactivity between Dpt extract and its fractions. Results. Among the DP+ patients, no difference was found between ConA-unbound and ConA-bound fractions regarding the levels of specific IgE, IgG1, and IgG4. Nonallergic subjects had the same levels of specific IgG1 to both ConA-unbound and ConA-bound fractions, although for specific IgG4, values were higher for ConA-bound. A positive correlation was found among specific IgE, IgG1, and IgG4 levels when Dpt was compared to ConA-unbound and ConA-bound fractions. Recognition of crude Dpt by IgE, IgG1, and IgG4 was highly inhibited by ConA-unbound and ConA-bound fractions. Western Blotting revealed a broad spectrum of bands ranging from 14 to 116 kDa recognized by specific IgE and IgG4. However, IgG1 reached higher frequency values on high molecular weight polypeptides. Conclusion. ConA-unbound and ConA-bound fractions derived from D. pteronyssinus crude extract revealed important components involved in the IgE recognition in allergic patients as well as IgG1 and/or IgG4 in allergic and healthy subjects.

Abstract Although FcεR have been detected on human eosinophils, levels varied from moderate to extremely low or undetectable depending on the donor and methods used. We have attempted to resolve the conflicting data by measuring levels of IgE, FcεRI, and FcεRII in or on human eosinophils from a variety of donors (n = 26) and late-phase bronchoalveolar lavage fluids (n = 5). Our results demonstrated little or no cell surface IgE or IgE receptors as analyzed by immunofluorescence and flow cytometry. Culture of eosinophils for up to 11 days in the presence or absence of IgE and/or IL-4 (conditions that enhance FcεR on other cells) failed to induce any detectable surface FcεR. However, immunoprecipitation and Western blot analysis of eosinophil lysates using mAb specific for FcεRIα showed a distinct band of approximately 50 kDa, similar to that found in basophils. Western blotting also showed the presence of FcR γ-chain, but no FcεRIβ. Surface biotinylation followed by immunoprecipitation again failed to detect surface FcεRIα, although surface FcRγ was easily detected. Since we were able to detect intracellular FcεRIα, we examined its release from eosinophils. Immunoprecipitation and Western blotting demonstrated the release of FcεRIα into the supernatant of cultured eosinophils, peaking at approximately 48 h. We conclude that eosinophils possess a sizable intracellular pool of FcεRIα that is available for release, with undetectable surface levels in a variety of subjects, including those with eosinophilia and elevated serum IgE. The biological relevance of this soluble form of FcεRIα remains to be determined.

The cause of the immunogenicity of Acanthopanax senticosus injection has been studied. The method used for the preparation of the injection includes boiling the plant material in water, precipitation with ethanol, and filtration to produce the extract. In the current study, this extract was treated with saturated ammonium sulfate to collect the protein from the extract. Indicators of systemic allergy, including total immunoglobulin (IgE), interleukin -4 (IL-4), and interferon-gamma (IFN-γ) levels in the antiserum of Guinea pigs sensitized with this protein extract were used to determine the immunogenicity of the protein extract. Testing was performed using Western blotting to identify IgE in antiserum binding in Guinea pigs sensitized by injection. The target protein band was further identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. The protein extract from A. senticosus caused positive reactions and significantly increased the levels of total IgE, IL-4, and IFN-γ in the serum of sensitized Guinea pigs. Western blotting showed that a group of proteins with a molecular weight of 60 000 in the A. senticosus protein extract acted as the major immunogenic protein by binding to serum IgE of Guinea pigs sensitized with A. senticosus injection. The protein was a pyruvate kinase homologous protein with high sequence similarity to the pyruvate kinase protein of Vitis vinifera. It was concluded that the allergic reaction caused by the injection of A. senticosus may be related to the interaction of these macromolecular proteins within the body.

Abstract One problem of conventional allergen-specific immunotherapy is the risk of anaphylactic reactions. A new approach to make immunotherapy safer and more efficient might be the application of engineered allergens with reduced IgE-binding capacity but retained T cell reactivity. Using overlapping dodeca-peptides, the dominant T cell epitopes of the timothy grass pollen allergen Phl p 5b were identified. By site-directed mutagenesis outside these regions, point and deletion mutants were generated. Allergen variants were analyzed for IgE-binding capacity with sera of different grass pollen allergic patients by Western blotting, Dot blotting, and EAST inhibition test, and for histamine releasing capacity with peripheral blood basophils from different patients. The deletion mutants revealed significantly reduced IgE reactivity and histamine releasing capacity, compared with the wild-type Phl p 5b. Furthermore, in vivo skin prick tests showed that the deletion mutants had a significantly lower potency to induce cutaneous reactions than the wild-type Phl p 5b. On the other hand, T cell clones and T cell lines from different allergic patients showed comparable proliferation after stimulation with allergen variants and wild-type Phl p 5b. Considering their reduced anaphylactogenic potential together with their conserved T cell reactivity, the engineered allergens could be important tools for efficient and safe allergen-specific immunotherapy.

Abstract The mAb BD6 is a monoclonal IgM that binds to the surface of RBL-2H3 cells and inhibits the binding of IgE to FcεRI, without reacting with this receptor. By cloning, mAb BD6 binding was resulted by the expression of the alpha1,3-galactosyltransferase gene. In binding assays on RBL-2H3 and alpha1,3-galactosyltransferase transfected PEAK cells there was partial competition between mAb BD6 and IB4, a galactose binding lectin. Both galactose and melibiose decreased the binding of mAb BD6 in a dose dependent manner on RBL-2H3 cells and abolished its binding on alpha1,3-galactosyltransferase transfected PEAK cells. MAb BD6 recognized a low molecular weight band by immunoblotting suggesting that it may be binding a lipid. This was confirmed by blotting lipids from RBL-2H3 cells separated by thin layer chromatography. By sucrose gradient analysis mAb BD6 bound to the lipid rafts fractions. RBL-2H3 variants were isolated completely deficient in mAb BD6 binding; these cells were also deficient in the ganglioside GD1b and had low expression of ganglioside GM1. However, these cells deficient in mAb BD6 binding and GD1b ganglioside still had normal FceRI induced degranulation. These results demonstrate that mAb BD6 inhibits IgE binding and reacts with a glycolipid present in lipid rafts; furthermore the ganglioside GD1b is not essential for degranulation. Supported by the Intramural Research Program, NIDCR, NIH.

Abstract The epsilon-chain of membrane-bound IgE on the surface of B lymphocytes is known to contain a membrane-anchoring peptide segment that is encoded by two membrane exons, me.1 and me.2. In analyzing pertinent segments in mRNA from human IgE-expressing B cells by using PCR methods and Northern blotting analyses, we have identified three species of mRNA of epsilon-chain with variations in the splicing of the membrane exons. The conventional species (m/s) contains the predicted me.1 and me.2; species m/1 harbors 156 extra nucleotides 5' of me.1 with unaltered reading frame; species s/t lacks me.1 and hence the segment encoding the hydrophobic transmembrane stretch and contains a shifted me.2 reading frame. Rabbit antibodies, which were prepared by immunization using a peptide of 36 amino acid residues representing an encoded segment unique to mRNA species m/l, could specifically bind to human IgE-expressing B cell lines and react with an epsilon-chain on Western immunoblots. These results indicate that there exists a previously unidentified isoform of human membrane-bound IgE.

Antigen detection during Western blotting commonly utilizes a horseradish peroxidase-coupled secondary antibody and enhanced chemiluminescent substrate. We utilized this technique to examine the impact of green tea-derived polyphenols on the binding of egg white protein-specific IgE antibodies from allergic human plasma to their cognate antigens. Our experiments unexpectedly showed that green tea-derived polyphenols, when stably complexed with egg white proteins, caused hyperactivation of horseradish peroxidase resulting in the appearance of white “ghost” bands. This study suggests that caution should be taken when evaluating polyphenol-bound proteins by enhanced chemiluminescence Western blotting using horseradish peroxidase and demonstrates that protein-bound polyphenols can be a source of “ghost” band artifacts on Western blots.

To improve diagnosis of Toxoplasma gondii in difficult patient groups, IgA- and IgE-immunosorbent agglutination assays (ISAGA), IgG avidity and Western blotting were developed and assessed. In the ISAGA, rabbit anti-human IgA or anti-human IgE (for the IgA-ISAGA and IgE-ISAGA respectively) was adsorbed onto microtitre plates; formaldehyde fixed tachyzoites were used to identify specific antibody. Both ISAGAs were specific; only 1/482 (0.2%) and 1/513 (0.19%) false positive results were recorded for the IgA and IgE-ISAGA respectively. Both were produced early in infection and were detected for up to 11 (IgA) and 10 (IgA) months. In the avidity ELISA, the performance of excretory/secretory, surface, cytoplasmic and mixed antigen was similar when tested with sera from patients with known duration of infection. Thirteen pregnant women were tested. Specific IgA was detected in all and so was not useful to time infection but specific IgE was absent in 5 women and may therefore have been useful to exclude recent infection. Specific IgE however, was not detected in one woman who seroconverted; detection of IgE may indicate severity of infection. High IgG avidity was measured in 4 patients and could have been useful in conjunction with IgE to exclude recent infection. In immunocompromised patients, IgA and IgE were detected with similar frequency to IgM, but their presence in some IgM negative patients makes them a useful addition to the repertoire of testing. High avidity in immunocompromised patients was not of use to improve diagnosis. Using Western blotting it was possible to differentiate between acute (< 4 months) and recent (4-10 months) infection. This test was less useful for timing infection of infection in pregnancy.

A alergia alimentar tem se tornado um problema crescente de saúde pública em muitos países e pode ser desencadeada por qualquer alimento. Em crianças os alimentos considerados mais alergênicos são leite de vaca e ovo e em adultos há uma alta frequência de alergia a frutos do mar, sendo camarão o marisco considerado mais alergênico. Clinicamente a alergia a camarão pode se manifestar em diferentes formas como dermatite atópica, angioedema ou até mesmo anafilaxia. Com base na história clínica e avaliação de resultados de exames complementares foram selecionados 18 indivíduos alérgicos a camarão e 9 não alérgicos como grupo controle. Foram realizados testes in vitro para dosagem de IgE especifica no soro e teste cutâneo. Para reações ocorridas há mais de um ano foi realizada provocação oral para confirmação diagnóstica. O soro de todos os indivíduos do estudo foram testados em Western Blotting 1D e 2D com extrato de carne de camarão e casca separadamente da espécie Xiphopenaeus kroyeri, que possui grande importância por ser altamente consumida em nosso país além de estar entre as dez estirpes mais capturadas no mundo. Ainda assim não está presente em nenhum teste comercial disponível e não há nenhum alérgeno descrito para o mesmo. A caracterização de suas moléculas alergênicas é importante para melhor auxilio no diagnóstico e tratamento de pacientes alérgicos. As proteínas que apresentaram reatividade IgE especifica foram submetidas a espectrometria de massas para identificação. Foi possível identificar alguns homólogos de alérgenos de outras espécies de camarão como, tropomiosina, arginina quinase, proteína sarcoplasmática de ligação ao cálcio, actina, cadeia leve e pesada de miosina e hemocianina. Encontramos um possível novo alérgeno denominado como citocromo c oxidase (subunidade I) que não foi descrito anteriormente como proteína alergênica. Analisando sensibilidade e valor preditivo positivo das nossas proteínas sugerimos que algumas delas podem ser boas candidatas como futuros marcadores diagnósticos para alergia a camarão. Quando comparamos o perfil de proteínas reativas em carne e casca de camarão não observamos diferenças significativas entre elas. Essa é a primeira descrição de alergia para a espécie de camarão X. kroyeri, popularmente conhecido como camarão sete barbas, mostrando reatividade a IgE especifica em pacientes alérgicos a camarão
Food allergy has become a growing problem of public health in many countries, and can be triggered by any food. In children the most allergenic foods are cow\'s milk and egg; in adults there are high frequencies of allergy to seafood, and shrimps are considered the most allergenic seafood. Clinically, shrimp allergy can manifest itself in different forms, such as atopic dermatitis, angioedema or even anaphylaxis. Based on the clinical history and evaluation of the tests, we selected eighteen subjects that were allergic to shrimp, and nine non-allergic to shrimp as a control group. In vitro tests were performed to IgE specific dosage and cutaneous test with commercial extract. When reactions occurred more than one year before oral provocation test was performed for diagnostic confirmation. The serum of all individuals were tested in Western Blotting 1D and 2D with the shrimp meat and shell extracts separately from the species Xiphopenaeus kroyeri, which has great importance since is highly consumed in the Southeast region of our country and is among the top ten captured species in the world. Nevertheless, there are no commercial clinical tests available for it, and there is no specific allergen described, both of them would help the diagnosis and treatment of allergic patients. Proteins that showed specific IgE reactivity were submitted to mass spectrometry for identification. We identified some homologues allergens from other shrimp species such as tropomyosin, arginine kinase, calcium-binding sarcoplasmic protein, actin, myosin heavy chain and hemocyanin. We founded a possible new allergen called cytochrome c oxidase (subunit I), that was never previously described as an allergenic protein. Analyzing sensitivity and positive predictive value of our proteins we suggest that some of them may be good candidates as future diagnostic markers for shrimp allergy. When comparing the reactive proteins profile in shrimp meat and shell, we did not observe significant differences between them. This is the first description showing reactivity to specific IgE for shrimp species X. kroyeri, popularly known as shrimp seven beards, in patients allergic to shrimp

Apesar das recomendações da Organização Mundial da Saúde, na reunião ministerial realizada em Amsterdam, Holanda em 2000, a tuberculose continua em ritmo crescente, atingindo principalmente os países em desenvolvimento e pessoas com o sistema imunológico comprometido, principalmente as infectadas pelo vírus da imunodeficiência adquirida. Os métodos utilizados no diagnóstico continuam os mesmos utilizados por muitos anos, cujas limitações impedem a ação rápida dos programas de saúde que buscam interromper a cadeia de transmissão da doença. Continuando uma linha de pesquisa do Laboratório de Soroepidemiologia e Imunobiologia do IMTSP, utilizando o método do Western Blotting, procuramos neste trabalho ampliar os conhecimentos da resposta imunológica de anticorpos em pacientes com tuberculose pulmonar, clinica e laboratorialmente definida, avaliando a participação das imunoglobulinas IgG, IgA e IgM, subclasses de IgG (IgG1 e IgG3) e a avidez da imunoglobulina IgG em amostras de soros colhidas no início e no final do tratamento. Nossos resultados mostraram que o melhor marcador imunológico foi a imunoglobulina IgG por apresentar melhor desempenho diagnóstico quando comparada com os resultados dos métodos microbiológicos e de dosagem de interferon gama, Quantiferon TB Gold. As frações protéicas que apresentaram melhor desempenho diagnóstico foram as de 38 e 30 KDa
Despite the recommendations of the World Health Organization, during the Ministerial meeting held in Amsterdam, Holland in 2000, tuberculosis continues at an important increasing rate, affecting mostly developing countries and people with severe compromised immune systems, especially those infected with human acquired immunodeficiency virus. The methods used for diagnosis are the same used for many years, whose limitations prevent the rapid action of countries health programs that seek to stop the chain of disease transmission. Continuing a line of research of the Laboratory of Seroepidemiology and Immunobiology of IMTSP using the method of Western blotting, in this study we tried to broaden the knowledge of the immune response of antibodies in patients with pulmonary tuberculosis, clinical and laboratory defined by evaluating the participation of IgG, IgA and IgM, IgG subclasses (IgG1 and IgG3) and immunoglobulin IgG avidity in serum samples collected in the beginning and end of treatment. Our results showed that the immunoglobulin IgG was best immunological marker when compared with the results of microbiological methods and determination of interferon gamma, QuantiFERON TB Gold. The proteins fractions that showed better diagnosis performance were 38 and 30 KDa

A sífilis adquirida e a congênita continuam aumentando e preocupando as autoridades sanitárias do mundo, ao verem que as metas estabelecidas para o seu controle estão longe de serem atingidas. Apesar dos esforços feitos na descoberta de novas ferramentas para o diagnóstico e monitoramento da sífilis, vemos um despreparo muito grande, principalmente entre os laboratoristas, em processar corretamente os reagentes disponíveis e melhor selecionar aqueles que apresentem qualidade para serem usados na rotina do laboratório. O que constatamos neste estudo é um espelho da realidade do diagnóstico da sífilis e mostra a dificuldade que os clínicos enfrentam ao receberem um laudo do laboratório. Trabalhando em colaboração com diferentes serviços e grupos de pesquisa, selecionamos aqueles que trabalhavam com: gestantes atendidas no pré-natal, doadores de sangue, pacientes infectados pelo HIV e pacientes de laboratório clínico e fizemos um estudo crítico da sorologia utilizada em cada serviço. Verificamos discrepância dos resultados obtidos nos testes não-treponêmicos VDRL e RPR, principalmente entre os soros de baixa reatividade, e nos treponêmicos FTA-abs, TPHA e ELISA. Decidimos aplicar o método de Western blotting e analisar o seu comportamento em todas as amostras de soros ensaiadas. Para obtenção de resultados reprodutíveis, fizemos a industrialização do método e formatamos um reagente na forma de um kit diagnóstico, WB Tp-IgG, que pudesse ser facilmente utilizado e interpretado. Os resultados obtidos mostraram que o WB Tp-IgG pode ser utilizado como método confirmatório da sífilis, substituindo o FTA-abs, tradicionalmente recomendado para essa finalidade e que pudesse fazer parte de uma proposta de algoritmo para o diagnóstico sorológico da sífilis.
Acquired and congenital syphilis have been increased and worried the worldwide health authorities, mainly because the WHO targets for syphilis control are far from to be held. Much effort had been made for development of new tools to be used in syphilis diagnosis and following up, however we noticed a lack of ability of laboratory workers in the correctly choosing and using the reagents in laboratory routine. In this study what we observed were the reality of syphilis diagnosis and the difficulties that physicians have in how to procedure when they received the results from the laboratory. Working in collaboration with different settings and research groups we choose some of them that attend pregnant women, blood donors, HIV patients and patients from clinical laboratory. With these group individuals we made a critical study of the serology methods used in each one. We verified high level of discordant results between nontreponemal tests VDRL and RPR, mainly in serum samples with low reactivity and between treponemal tests FTA-abs, TPHA and ELISA, in all services. To obtain reproducibility of the results we made the industrialization of the method and set up a reagent as a kit diagnosis, easily to performer. Appling the western blotting method we evaluated the performance of the test in all sera samples assayed. The results showed that the WB Tp-IgG can be useful as confirmatory test for syphilis, replacing FTA-abs, traditionally recommended for this and that could be included in algorithm propose for serological diagnosis of syphilis.

A tuberculose permanece como um dos maiores problemas de saúde pública mundial. O diagnóstico precoce e o tratamento rápido e eficiente dos indivíduos com tuberculose pulmonar ativa são medidas essenciais para a redução da morbidade, mortalidade e da incidência da tuberculose no mundo. As limitações encontradas nos métodos microbiológicos tradicionais, fizeram com que metodologias alternativas fossem desenvolvidas para melhorar o diagnóstico e o prognóstico da tuberculose humana. Neste trabalho verificamos o desempenho diagnóstico do Western blotting para pesquisa de anticorpos IgG anti - Mycobacterium tuberculosis, a utilização do teste QuantiFERON® - TB Gold e a detecção de moléculas de adesão celular (ICAM-1 e selectinas) como marcadores de prognóstico. Foram acompanhados até o final do tratamento 31 pacientes com tuberculose pulmonar diagnosticados por critérios clínicos e laboratoriais. Como controles, selecionamos população de indivíduos sadios, doadores de banco de sangue e indivíduos com outras pneumopatias. Os resultados mostraram que o Western blotting apresentou sensibilidade de 94% e especificidade de 96% no diagnóstico da tuberculose pulmonar, atendendo os requisitos da OMS para testes sorológicos. O QuantiFERON® - TB Gold apresentou sensibilidade de 83% e especificidade de 100%, após ajuste do limiar de reatividade. Os resultados das moléculas de adesão celular sugerem potencial para serem utilizadas como marcadores de prognóstico da doença. Ao associarmos os resultados do Western blotting ou do QuantiFERON® - TB Gold com a baciloscopia obtivemos sensibilidade superior a 95%; e quando associados à cultura a sensibilidade encontrada foi de 100%. O Western blotting mostrou ser uma ferramenta útil como auxiliar no diagnóstico da tuberculose pulmonar mesmo em pacientes com baciloscopia negativa.
Tuberculosis remains a major public-health problem. Rapid diagnosis and prompt treatment is the cornerstone to reduce morbidity, mortality and incidence of tuberculosis in the world. Alternative methods have been developed to overcome the limitations presented by conventional microbiological methods and to improve the diagnosis and prognosis of tuberculosis. In this study we verified the diagnostic performance of Western blotting for IgG anti-M.tuberculosis antibodies detection, QuantiFERON® - TB Gold and circulating adhesion molecules (ICAM-1 and Selectins) as prognosis markers. Thirty-one patients were followed-up during the treatment. Active pulmonary tuberculosis was diagnosed by clinical and laboratorial criteria. As group control healthy individuals, blood donors and patients with other lung diseases were included. Western blotting results showed a high performance with sensitivity of 94% and specificity of 96% for the diagnosis of pulmonary tuberculosis, attending WHO requirements for serological tests. After adjusting the threshold, QuantiFERON® - TB Gold showed sensitivity of 83% and specificity of 100%. The results of adhesion molecules suggested potential to use the test as prognosis markers. Combining Western blotting or QuantiFERON® - TB Gold with acid-fast bacilli (AFB) smear results, the overall sensitivity increase to more than 95%, and when combined with culture the overall sensitivity was 100%. Together, these findings, suggest that Western blotting could be a very useful supplementary tool for pulmonary tuberculosis, especially in patients with AFB smear negative.

Neurocysticercosis (NC) is a polymorphic disease and the immune response in human carrier is heterogenic. In this study, 35 primers were used for amplifications by Random Amplified Polymorphic DNA (RAPD) of Taenia solium metacestodes, from five different geographic areas in Brazil: 1) Distrito Federal (DF), Center West; 2) Barreiras (BA), Northeast and Southeast; 3) Hydro Basin of the Mosquito River (North of Minas Gerais, RM-MG), 4) São Paulo (SP) and 5) Uberaba (Minas Gerais, UB-MG). Metacestodes saline crude extracts of four populations (DF, BA, RM-MG e SP) were used for the detection of specific IgG antibodies by ELISA and Western Blotting (WB). A total of 157 serum samples of three groups, (G1): 49 NC patients; (G2): 68 patients with other helminthiasis: hydatidosis (10), taeniasis (20), strongyloidiasis (20), schistosomiasis (10) and hymenolepiasis (8); and (G3): 40 healthy individuals; were analyzed by ELISA. From these, the 98 serum samples were assayed by WB; G1 (49), G2 (39) and G3 (10). The genetic distances, in disagreement percentage, between the metacestode populations were calculated from of 15 RAPD markers and showed 49.5% (DF), 48% (BA), 38.5% (UBMG) and 28% (RM-MG and SP) of genetic distances. Six primers identified polymorphic fragments and the primers 26 (GGGTTTGGCA) and 29 (TCGCCAGCCA) allowed a better differentiation of populations. The fragments of 1000, 500 and 326 pb (pairs of bases) in the UB-MG and of 600 and 244 pb in RM-MG were amplified by primer 26. The fragments generated by primer 29 were 500, 800 and 1191 pb, 300 and 1377 pb, 1000 pb and 244 and 434 pb in SP, UB-MG, DF and BA populations, respectively. In G1, the positivity by ELISA was: 90% (DF), 69% (BA), 71% (MG) and 67% (SP). The DF extract was more antigenic than others (p=0.02). In WB, the 64-68 kDa antigens were recognized in all extracts, exclusively, in serum samples from active NC patients (p=0.001). Variation in banding pattern was detected between the extracts (p<0.05). In G2, the serum samples of hydatidosis patients presented from 70 to 90% positivity by ELISA in antigenic extracts (p<0.05); however, the bands recognition pattern in WB was different from that presented in G1 samples. The 77 kDa band was significantly identified in hydatidosis samples (p=0.0001). In conclusion, the T. solium populations analyzed showed genetic variability and antigenic differences.
A neurocisticercose (NC) constitui doença polimórfica, apresentando heterogeneidade da resposta imune no hospedeiro humano. Nesse estudo, o teste Random Amplified Polymorphic DNA (RAPD) foi utilizado com 35 primers na detecção de polimorfismo em metacestódeos de Taenia solium provenientes de cinco áreas geográficas distintas do Brasil: 1) Distrito Federal (DF), região Centro-Oeste; 2) Barreiras (BA), região nordeste e da região sudeste: 3) Bacia Hidrográfica do Rio Mosquito (norte de Minas Gerais, RM-MG), 4) São Paulo (SP) e 5) Uberaba (Minas Gerais, UB-MG). Os extratos salinos totais de metacestódeos de quatro populações (DF, BA, RM-MG e SP) foram utilizados na detecção de anticorpos IgG específicos pelo ELISA e Western Blotting (WB). As 157 amostras de soro de três grupos (G) de indivíduos: G1: 49 pacientes com NC; G2: 68 pacientes com outras helmintíases, sendo hidatidose (10), teníase (20), estrongiloidíase (20), esquistossomose (10) e himenolepíase (8) e G3: 40 indivíduos saudáveis (controles); foram analisadas pelo ELISA. Foram ensaiadas 98 amostras de soro: G1 (49), G2 (39) e G3 (10) pelo WB. As distâncias genéticas, por porcentagem de desacordo, foram de 49,5% (DF), 48% (BA), 38,5% (UB-MG) e 28% (RM-MG e SP) nas populações de metacestódeos, calculadas a partir de 15 marcadores de RAPD. Seis primers geraram fragmentos polimórficos nos isolados analisados, sendo que os primers 26 (GGGTTTGGCA) e 29 (TCGCCAGCCA) permitiram melhor diferenciação entre as populações, o primer 26 gerou os fragmentos de 1000, 500 e 326 pb (pares de bases) na amostra de UB-MG, e de 600 e 244 pb em RM-MG. O 29 gerou fragmentos em quatro das populações analisadas, sendo 500, 800 e 1191 pb em SP, 300 e 1377 pb em UBMG, 1000 pb no DF e 244 e 434 pb na BA. No G1, as freqüências de positividade no ELISA, foram: 90% (DF), 69% (BA), 71% (MG) e 67% (SP), sendo o extrato do DF mais antigênico que os demais (p = 0,02). No WB, o peptídeo de 64-68 kDa foi reconhecido em todos os extratos, exclusivamente, em amostras de pacientes com NC ativa (p=0,001). Detectou-se variação no padrão de reconhecimento de bandas entre os extratos (p<0,05). No G2, as amostras de soro de pacientes com hidatidose apresentaram de 70 a 90% de positividade no ELISA frente aos extratos analisados (p<0,05); porém, o padrão de reconhecimento de bandas no WB diferiu do apresentado pelas amostras do G1, sendo que a banda de 77 kDa foi significativamente identificada pelas amostras de pacientes com hidatidose (p=0,0001). Concluiu-se que as populações de T. solium analisadas nesse estudo, apresentaram variabilidade genética e diferenças de antigenicidade.
Doutor em Imunologia e Parasitologia Aplicadas

Le proteine della farina di frumento sono considerate gli allergeni responsabili del 60-70% dei sintomi respiratori che si manifestano nella malattia professionale denominata “asma dei panificatori”; inoltre il frumento è tra i 6 principali allergeni alimentari coinvolti in reazioni di ipersensibilità IgE-specifiche. Nel caso dell’allergia alimentare al frumento le manifestazioni variano da sintomi respiratori, gastrointestinali e cutanei fino a gravi reazioni sistemiche. Nonostante questa rilevanza allergologica, a livello molecolare la conoscenza degli allergeni del frumento è piuttosto scarsa. Una conseguenza di questa limitata conoscenza è che la diagnosi di malattie, quali l’asma del panificatore, può essere una difficile impresa. Si presuppone tuttavia che sia gli epitopi sequenziali che quelli conformazionali possano essere responsabili di queste reazioni allergiche. Lo Skin prick test (SPT) gioca un ruolo importante nella diagnosi allergologica ma i pazienti con asma del panificatore risultano positivi solo lievemente (dal 5 al 15%). Questa scarsa significatività dello SPT potrebbe essere una conseguenza della bassa solubilità di molte proteine del frumento così come un effetto di differenze nella standardizzazione degli estratti analizzati. OBIETTIVO Lo scopo di questo studio era quello di realizzare una caratterizzazione degli allergeni del frumento coinvolti nell’allergia alimentare e inalatoria. In particolare, l’obiettivo di questo studio era di esaminare la variabilità dei profili proteici riconosciuti dalle IgE di panificatori italiani sensibilizzati verso la farina di frumento e di identificare gli allergeni più frequentemente riconosciuti. Inoltre abbiamo voluto i) indagare quali epitopi fossero coinvolti nelle diverse forme di allergia al frumento ii) esaminare il reale contenuto allergenico negli estratti per lo SPT usati nella diagnosi dell’allergia al frumento mettendo in relazione la loro composizione con i risultati diagnostici prodotti. È stata sviluppata come strategia un’analisi proteomica del frumento seguita da IgE blotting cioè un approccio “allergonomico”. METODI per caratterizzare gli allergeni del frumento le proteine di farina di frumento, monocultivar Bolero, solubili in soluzione salina, sono state separate in elettroforesi monodimensionale (1-DE) e bidimensionale (2-DE) in condizioni ridotte e non ridotte. Inoltre, sono stati analizzati gli estratti di farina e di farina integrale usati per lo SPT. Le proteine IgE-specifiche sono state rivelate attraverso immunoblotting usando i sieri di 43 pazienti con allergia inalatoria e 9 con allergia alimentare al frumento. Dopo digestione triptica, sono state analizzate attraverso nano HPLC–ESI–MS/MS i peptidi di alcune proteine IgE-specifiche, frequentemente immunorivelate dalle IgE dopo elettroforesi bidimensionale. RISULTATI Gli immunoblot ottenuti con 43 sieri diversi utilizzati singolarmente presentavano una notevole eterogeneità. Sempre con questa metodica inoltre, sono stati realizzati profili di estratti in condizioni ridotte e non ridotte evidenziando il coinvolgimento di ponti disolfuro nella stabilizzazione dei determinanti. Infine è stato dimostrato che gli estratti usati per lo SPT sono solo parzialmente rappresentativi del reale contenuto allergenico del frumento e differenti lotti di questi estratti mostravano notevoli diversità. Si è inoltre osservato che la maggior parte degli allergeni non sono singoli spot proteici ma erano rappresentati da più isoforme proteiche con peso molecolare simile ma un punto isoelettrico diverso. Cinque dei principali spot proteici IgE-specifici sono stati identificati per mezzo della tecnica nano HPLC–ESI–MS/MS. In questo studio, abbiamo identificato un allergene inalatorio del frumento già riportato in letteratura [monomeric alpha-amylase inhibitor, 0.28]. Inoltre abbiamo identificato due allergeni inalatori del frumento, raramente descritti in letteratura [triosephosphate-isomerase e thioredoxin peroxidase], e abbiamo scoperto due nuovi allergeni mai riportati come tali [glucose and ribitol dehydrogenase homolog – barley e una heat shock protein]. Nel caso dell’allergia alimentare al frumento, sorprendentemente, non ci sono IgE-specifiche per le proteine del frumento in 8 casi su 9 (88%). CONCLUSIONI. La rilevanza clinica dei due nuovi allergeni identificati dovrà essere ulteriormente investigata ma certamente i nostri risultati potrebbero contribuire ad aumentare la specificità dei saggi diagnostici. L’IgE-blotting non ha rilevato allergeni alimentari perciò, nel prossimo futuro, sarà necessario sviluppare un approccio alternativo per lo studio di queste patologie.
Wheat flour proteins are allergens involved in 60% to 70% of workplace-related respiratory-symptoms of bakers, furthermore wheat belongs to the six major food allergens inducing IgE-mediated hypersensitivity-reactions. In the case of wheat food-allergy the manifestations range from cutaneous, gastrointestinal, and respiratory symptoms to severe systematic reactions. Despite this allergological relevance the knowledge of wheat allergens at molecular level is scanty. It is nevertheless assumed that conformational and sequential epitopes might be both responsible for wheat allergic reactions. As a consequence of this limited knowledge the diagnosis of wheat allergy is sometime a difficult task. Skin prick tests (SPTs) play an important role in the allergological diagnosis but baker’s asthmatic patients result positives in a limited (from 5 to 15%) range. This scarce SPT predictability may be a consequence of the low solubility of numerous wheat proteins as well as an effect of differences in test-extracts standardization. OBJECTIVE The aim of the present study was to achieve a more detailed and comprehensive characterization of the wheat allergens involved in food allergy and baker’s asthma. In particular, the aim of the study was to investigate the variability of IgE antibody patterns of wheat flour-sensitized Italian bakers and to identify the most frequently recognized allergens. Furthermore i) we tried to verify whether different epitopes are involved in the different forms of wheat allergy ii) we tested the real allergen content in SPT used in wheat allergy diagnosis in relationship to their performance in clinical practice. The employed strategy was the proteomic analysis of wheat followed by IgE blotting i.e. an “allergenomic” approach. METHODS To characterize wheat allergens, water/salt-soluble wheat flour proteins from the monocultivar Bolero were separated by using mono dimensional (1-DE) and 2-dimensional (2-DE) gel electrophoresis under reducing and non-reducing conditions. Furthermore, using 1-DE SPT-solutions, wholemeal wheat flour and flour extracts were separated. IgE-binding proteins were detected by immunoblotting using the sera of 43 patients with inhalant allergy and 9 with food allergy to wheat. After tryptic digestion, the peptides of some IgE-binding proteins, frequently recognized by IgE on 2-DE, were analyzed by nano HPLC–ESI–MS/MS. RESULTS The IgE immunoblots obtained with 43 different sera exhibited a remarkable inter-individual heterogeneity. Furthermore, the immunodetected profiles were very different under reducing and non-reducing condition. The analyzed SPT-solutions demonstrate to be only partially representative of the real allergenic content of wheat and different batch of these extracts show remarkable differences. It was also demonstrated that the majority of the allergens were not single polypeptide spots, but consisted of up to ten isoforms of similar molecular mass but with different isoelectric points. Five of the predominant IgE-binding protein spots were identified by using nano HPLC–ESI–MS/MS. In this study, we identified one already reported wheat inhalant allergen [monomeric alpha-amylase inhibitor, 0.28] by a database search. Moreover we identified two wheat inhalant allergens, rarely described in literature [triosephosphate-isomerase and thioredoxin peroxidase], and we found out two never reported wheat inhalant allergens [glucose and ribitol dehydrogenase homolog – barley and a heat shock protein]. In the case of food allergy, surprisingly, no specific IgEs for wheat proteins were detected in 8 out of 9 cases (88%). CONCLUSIONS. The clinical relevance of the identified 2 new allergens will be further investigated in the near future but certainly our findings could contribute to increase the specificity of diagnostic assays. IgE-blotting did not reveal food allergens for this reason, in the future, an alternative approach to allergomic must be developed.

The goal of this study was to map the glycosylation pattern and antigenic properties of the salivary proteins of two closely related sand fly species, Phlebotomus perniciosus and P. orientalis. Affinity blotting with commercially available lectins revealed that many salivary proteins of these species are N-glycosylated, while the presence of O-glycosylation could not be confirmed. The level of N-glycosylation of most of these proteins is quite low, a larger number of potential N-glycosylation sites were found only in the amino acid sequences of P. orientalis hyaluronidase and endonucleases of both species tested. Four antigens from P. perniciosus salivary glands were selected for expression in a bacterial expression system; two of these proteins (PpeSP01 and PpeSP01B) were not glycosylated and the glycosylation level of the remaining two (PpeSP03B and PpeSP07) was low. The antigenic properties of the four chosen recombinant proteins were subsequently tested using immunoblot and ELISA. During the initial experiments with the sera of dogs experimentally bitten by P. perniciosus, two proteins (rSP07 and rSP01B) were proven unsuitable and they were excluded from further experiments. Recombinant proteins rSP03B and rSP01 were recognized by the same IgG antibodies as the native forms of these proteins...

It has been reported that the tissue factor(TF) of monocytes and leukemic leukocytes is one of the trigger substances of disseminated intravascular coagulation(DIC) in leukemia patients. To assess the properties of TF of leukemia cells, their TF was analyzed by the method of Western blotting. Placenta TF was purified using Concanavalin-A affinity chromatography. Briefly, human placenta TF was extracted from placenta acetone powder using Triton X-100 extraction and purified by Concanavalin-A affinity chromatography and SDS-preparative PAGE. Hie final product of the purified placenta TF-apoprotein exhibited a molecular weight (MW) approximately 46 kD in non-reduced condition and 47 kD in reduced condition and showed single band on analytical SDS-PAGE. Activity of purified placenta apo-TF after relipidation with placenta phospholipid was 4500 units/mg and was 6300 times purified from the starting material. Serum IgG fraction was separated from rabbits after 8 times weekly injections of purified placenta TF. This anti-TF-IgG fraction showed single precipitin line against purified placenta apo-TF and inhibited procoagulant activity of saline extract of the placenta as well as those of homogenates of the endotoxin(LPS)-stimulated Molt-4 cells and U-937 cells. Using this antibody, analysis of TF of cultured leukemia cells(Molt-4 and U-937) and acute promyelocytic leukemia(APL) cells was done by the Western blotting technique as follows: LPS-stimulated Molt-4, U-937 or APL cells were homogenized, delipidated and solubilized with 1% SDS-PAGE. Thereafter, Western blotting was done by the method of Towbin and TF of the blotted protein was immunologically identified. The nitrocellulose paper was soaked with anti-TF-IgG for 18 hours’, then peroxidase-conjugated anti-rabbit-goat IgG was used as an indicator of the antibody and o-dianisidine as substrate. In either case of APL cells, LPS-stimulated Molt-4 or U-937, single band which showed procoagulant activity was detected and the MW of the protein was approximately 48 kD in non-reduced condition. These results indicate that TF of all these leukemic leukocytes has common antigenic determinant and equal MW to placenta TF.

Chronic idiopathic thrombocytopenic purpura (ITP) is a clinical syndrome characterized by destruction of platelets by antiplatelet antiboodies. The precise pathogenic mechanism of platelet destruction in ITP is not known, although many investigators have reported that platelet-associated IgG (PAIgG) is increased in this desease. We have evaluated PAIgG in 66 patients with ITP by a competitive solidphase microenzyme immunoassay and investigated its specificity aganist antiplatelet antibody in 24 patients with ITP by Western blotting. PAIgG values were elevated in most ITP patients with platelet counts of under 50,000/μl, but within normal range in most patients with platelet count of over 50,000/μl. PAIgG values were also elevated in ITP patients with megakaryocyte counts of over 200/μl, and within normal range in most patients with normal megakaryocyte counts. Western blotting was carried out by SDS-PAGE of whole platelet lysate or platelet membrane lysate and trasfer of the platelet fraction onto nitrocellulose strips. Bound immunoglobulins were detected with an avidin-biotin-peroxidase system. Several bands of bound immunoglobulins were detected in the whole platelet lysates of ITP patients, but most of these could not be detected in platelet membrane lysates. This finding suggests that some immunoglobulins from ITP patients may bind to cytoplasmic proteins in whole platelet lysate. These observations are consistent with the hypothesis that the pathogenesis of ITP involves an immunological mechanism.

Previous immunochemical studies have demonstrated that the introduction of a chain crosslinks by Factor XIII begins immediately after fibrin aggregation. While there area data to indicate that regions Aα #241-476 and Aα #518-584 are specifically involved in this early crosslinking process, identification of the exact glutamine and lysine residues that participate in the reaction remains to be determined. In this study monoclonal antibodies, whose specificity for these two regions has been defined (anti-Aα #259-276, F-105; anti-Aα #540-554, F-102), were used to isolate preparations of partially degraded fibrinogen molecules which could then be characterized for C00H-terminal Aα chain heterogeneity and crosslinking capacity to obtain a more precise localization of residues involved in early crosslinking. In order to assess the relative contribution of sites within the COOH-terminal region that includes 1 potential GLN at Aα #565 and 4 potential LYS at Aα #556, 562, 580 and 585, fibrinogen molecules that were missing these residues were isolated from preparations of purified fibrinogen using F-102-Sepharose immuno-affinity adsorption. Characterization of the partially degraded material (i.e., non-binding) by Western Blotting using F-102 and F-105 confirmed the presence of Aα remnants (29K-66K) that shared the structure Aα #1-276 but differed in the extent to which regions between ∼#276 and ∼#559 were preserved. When the crosslinking capacity of these partially degraded molecules was examined, using Western Blotting to monitor the appearance of crosslinked a chains during in vitro clotting, effective formation of early crosslinked species ( ∼100K, ∼ 200K) as well as eventual accumulation of a polymers, could be demonstrated. These findings indicate that at least one early a chain crosslink involves activity on the part of LYS and GLN residues located proximal to Aα #540. While these data do not rule out the existence of additional crosslinking sites situated more distally (i.e., within Aα #540-584), they do indicate that degraded fibrinogen molecules, circulating under pathophysiologic conditions, may undergo adequate fibrin stabilization despite a loss of at least 70 COOH-terminal Aα chain residues.

Monoclonal antibodies were raised against synthetic peptides corresponding to the N-termini of the 90kd and 80kd subunits of human FVIII. Preliminary screening was performed directly against the peptides (linked to albumin) coated onto polystyrene wells. IgG was purified by Protein A-Sepharose and affinity purified using the immunogen peptides linked to Sepharose. Immunoreactivity with both plasma and recombinant FVIII was compared by Western blotting, two-site ELISA employing a neutralizing rabbit anti-FVIII IgG as capture antibody, and inhibition in a fluid phase FVIII activity assay. None of the antibodies neutralized clotting or amidolytic activities associated with either FVIII protein. All of the antibodies immunoblotted intacl or thrombin digested FVIII polypeptides according to their anticipated epitope recognition sites; this was useful in determining identity between the two types of FVIII protein. Furthermore, when either FVIII protein was bound to polyclonal IgG coated onto polystyrene microtiter wells, the 80kd antipeptide antibody was capable of detecting both antigens with equal affinity. These results, coupled with results using the 90k antipeptide suggest that epitope accessibility for these antipeptide antibodies is the same for plasma and recombinant FVIII under the conditions tested.

Limulus clotting factor, factor C, is a lipopolysaccharide (LPS)-sensitive serine-protease zymogen present in the hemocytes. It is a two-chain glycoprotein (M.W. = 123,000) composed of a heavy chain (M.W. = 80,000) and a light chain (M.W. = 43,000) T. Nakamura et al. (1986) Eur. J. Biochem. 154, 511-521 .On further studies of this zymogen, a single-chain factor C (M.W. = 123,000) was identified by Western blotting technique. The heavy chain had an NH2-terminal sequence of Ser-Gly-Val-Asp-, which was consistent with the NH2-terminal sequence of the single-chain factor C, indicating that the heavy chain is located in the NH2-terminal part of the zymogen. The light chain had an NH22-terminal sequence of Ser-Ser-Gln-Pro-. Incubation of the two-chain zymogen with LPS resulted in the cleavage of a Phe-Ile bond between residues 72 and 73 of the light chain. Concomitant with this cleavage, the A (72.amino acids) and B chains derived from the light chain was formed. The complete amino acid sequence of the A chain was determined by automated Edman degradation. The A chain contained a typical segment which is similar structuraly to those a family of repeats in human β2 -glycoprotein I, complement factors B, Clr, Cls, H, C4b-binding protein, 02, coagulation factor XIII b subunit, haptoglobin a chain, and interleukin 2 receptor. The NH2-terminal sequence of the B chain was Ile-Trp-Asn-Gly-. This chain contained the serine-active site sequence of -ASP-Ala-Cys-Ser-Gly-Asp-SER-Gly-Gly-Pro-.These results indicate that limulus factor C exists in the hemocytes in a single-chain zymogen form and is converted to an active serine-protease by hydrolysis of a specific Phe-Ile peptide bond. The correlation of limulus factor C and mammalian complement proteins was also suggested.

Calmodulin (CaM)-binding proteins have been identified in human platelets using Western blotting techniques and 125I-CaM. Ten proteins of 245, 225. 175, 150, 90. 82(2), 60 and 41(2) kilodaltons (kDa) bind 125I-CaM in a Ca2+-dependent manner; the binding is blocked by both trifluoperazine and nonradiolabeled CaM. The 225 and 90 kDa proteins are labeled by antisera against myosin light chain kinase (MLCK); the 60 kDa and one of the 82 kDa proteins have been identified as the CaM-dependent phosphatase (calcineurin) and caldesmon. The other proteins are presumed to be other Ca2+/CaM regulated enzymes and proteins which may be important in platelet function. Most of the CaM-binding proteins are degraded upon addition of Ca2+ to a platelet homogenate; the degradation may be blocked by either EGTA, leupeptin or N-ethylmaleimide which suggests that the degradation is due to a Ca2+-dependent protease. Activation of intact platelets under conditions which promote platelet aggregation (i.e. stirring with extracellular Ca2+) also results in limited proteolysis of CaM-binding proteins including those labeled with anti sera against MLCK and the phosphatase. In vitro studies utilizing purified phosphatase and calpain I indicate that the phosphatase is irreversibly activated upon Ca2+-dependent proteolysis. The proteolytically-activated enzyme is insensitive to either Ca2+ or Ca2+/CaM; in addition, its activity in the absence of Ca2+ is even greater than the activity of the unproteolyzed enzyme in the presence of Ca2+ and CaM. Proteolytic stimulation of the phosphatase is accompanied by degradation of the 60 kDa subunit of the enzyme (subunit A) to 56, 52 and 45 kDa fragments, sequentially; proteolysis results in the loss of CaM binding to the enzyme. These results suggest that the Ca2+-dependent protease may have a physiological role in platelet activation as an irreversible activator of Ca2+/ CaM-dependent reactions. Supported by NIH grant HL29766.

Monoclonal antibodies (MCA’s) were prepared against human protein C (PC) according to Köhler & Milstein, and those that recognize the Ca2+-dependent PC conformers were screened by direct ELISA in the presence of 2 mM either CaCl2 or EDTA. Out of nine MCAߣs thus screened, five MCA's designated as HPC-1˜5, respectively, were found to react with PC in the presence of Ca2+ but not EDTA. By SDS-PAGE coupled with Western Blotting performed in the presence of 2 mM CaCl2, we found that two MCA’s HPC-1 and 2, recognized the light chain, and two others, HPC-3 and 4, recognized the heavy chain of PC. But another MCA, HPC-5 was found to react with only non-reduced antigens. Further study showed that HPC-1 and 5 failed to react with the Gla-domainless PC, i.e. PC from which the N-terminal Gla-domain of the light chain had been cleaved off by α-chymotrypsin. However, all the other three MCA's retained the reactivity with the antigen in the presence of Ca2+ even after the Gla-domain had been removed. The binding of these MCA’s to PC in the presence of Ca2+ was found to be saturable with respect to the Ca2+ concentration and the half maximal binding for each MCA was calculated to be about 0.5+mM. Moreover, many other divalent cations such as Mg2+, Mn2+ , Ba2+, Zn2+, Co2+, Sr2+, were found to substitute for Ca2+ in inducing the metal ion-dependent but Gla-domain-independent conformer of PC.Cross-reactivity to other vitamin K-aependent plasma proteins was examined by direct ELISA; HPC-2 and 3 reacted solely to PC, but HPC-1 and 4 also reacted with prothrombin and HPC-5 with both prothrombin and factor X.These findings indicated that there are two or more metal binding sites besides the Gla-domain, possibly one in the light chain and the other(s) in the heavy chain. The presence of these metal binding sites may contribute to the unique conformer of vitamin K-dependent plasma proteins including protein C.

The Ethanol Gelation Test(=EGT) is a well-documented simple,specific and frequently used test to detect plasma soluble fibrin(Godal & Abildgaard:Gelation of soluble fibrin in plasma by ethanol.Scand.J.Haanat.3,342,1966) .If soluble fibrin present in plasma amounts to 1% or more of the plasma-f ibrinogen conc.,the admixing of 0.15 ml 50% ethanol to 0.5 ml plasma in a test tube will-(subsequent to incubation for 10 min at 20°C)-upon tilting the test tube semi-horizontally produce a characteristic,(upwardly) convex gel. Although earlier studies have confirmed the validity and specificity of EGT as a means to detect soluble fibrin,we found it of interest to see to which degree such soluble fibrin is FXIII-stabilized EGT-positive(from patients with Disseminated Intravascular Coagula-tion(DIC) and EGT-negative plasma was studied as follows: The EGT test was performed as above,and the entire content of the test tube emptied upon a nylon micro-meshed membrane.Applying slight suction underneath the nylon manbrane the fluid was removed,leaving the ethanol precipitated material which was immediately dissolved in SDS (1%)-urea(5M)-Tris-HCl (0.15M,pH8.6). After incubation at 100°C for 1 min the material was SDS-electrophoresed on flat-bed agarose(2%) Subsequent to Western-blotting onto nitrocellulose and gelatine-blocking, the fibrin(ogen)-related pattern was reacted with either: a)polyclonal antibodies to fibrinogen,b)plyclonal antibodies to FPA or c)monoclonal antibody to FPA(gift from Dr.Nieuwenhuizen, Leyden, Holland) .Then,the fibrin(ogen)related pattern was developed using peroxidase-conjugated secondary antibodies.From the specificity of the primary antibodies used,it could be deduced that:1)A substantial amount of the soluble fibrin content of DIC-plasma was present in an oligomeric form(up to 6-mers) .2)These oligomers contained fibrinogen, i.e. thus representing FXIII-1 inked fibrinogen/fibrin hybride molecules .3) Even normal plasma contained some detectable oli-gomers(up to 3-mers) .4) Col lecting blood with all appropriate thrombin- and FXIII-inhibitors did not change the patterns obtained and described above.It may be concluded that soluble fibrin occurs mainly in a FXIII-stabilized,oligomeric form which contains fibrinogen Due to the nature of the polymerization process,the fibrinogen moiety of these hybride molecules must be end-located representing a physiological means to inhibit further polymer growth.