Highly sensitive serological approaches for Pepino mosaic virus detection / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Pepino mosaic virus (PepMV) causes severe disease in tomato and other Solanaceous crops around globe. To effectively study and manage this viral disease, researchers need new, sensitive, and high-throughput approaches for viral detection. In this study, we purified PepMV particles from the infected Nicotiana benthamiana plants and used virions to immunize BALB/c mice to prepare hybridomas secreting anti-PepMV monoclonal antibodies (mAbs). A panel of highly specific and sensitive murine mAbs (15B2, 8H6, 23D11, 20D9, 3A6, and 8E3) could be produced through cell fusion, antibody selection, and cell cloning. Using the mAbs as the detection antibodies, we established double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), Dot-ELISA, and Tissue print-ELISA for detecting PepMV infection in tomato plants. Resulting data on sensitivity analysis assays showed that both DAS-ELISA and Dot-ELISA can efficiently monitor the virus in PepMV-infected tissue crude extracts when diluted at 1:1310720 and 1:20480 (weight/volume ratio (w/v), g/mL), respectively. Among the three methods developed, the Tissue print-ELISA was found to be the most practical detection technique. Survey results from field samples by the established serological approaches were verified by reverse transcription polymerase chain reaction (RT-PCR) and DNA sequencing, demonstrating all three serological methods are reliable and effective for monitoring PepMV. Anti-PepMV mAbs and the newly developed DAS-ELISA, Dot-ELISA, and Tissue print-ELISA can benefit PepMV detection and field epidemiological study, and management of this viral disease, which is already widespread in tomato plants in Yunnan Province of China.

Highly sensitive serological approaches for Pepino mosaic virus detection / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Pepino mosaic virus (PepMV) causes severe disease in tomato and other Solanaceous crops around globe. To effectively study and manage this viral disease, researchers need new, sensitive, and high-throughput approaches for viral detection. In this study, we purified PepMV particles from the infected Nicotiana benthamiana plants and used virions to immunize BALB/c mice to prepare hybridomas secreting anti-PepMV monoclonal antibodies (mAbs). A panel of highly specific and sensitive murine mAbs (15B2, 8H6, 23D11, 20D9, 3A6, and 8E3) could be produced through cell fusion, antibody selection, and cell cloning. Using the mAbs as the detection antibodies, we established double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), Dot-ELISA, and Tissue print-ELISA for detecting PepMV infection in tomato plants. Resulting data on sensitivity analysis assays showed that both DAS-ELISA and Dot-ELISA can efficiently monitor the virus in PepMV-infected tissue crude extracts when diluted at 1:1 310 720 and 1:20 480 (weight/volume ratio (w/v), g/mL), respectively. Among the three methods developed, the Tissue print-ELISA was found to be the most practical detection technique. Survey results from field samples by the established serological approaches were verified by reverse transcription polymerase chain reaction (RT-PCR) and DNA sequencing, demonstrating all three serological methods are reliable and effective for monitoring PepMV. Anti-PepMV mAbs and the newly developed DAS-ELISA, Dot-ELISA, and Tissue print-ELISA can benefit PepMV detection and field epidemiological study, and management of this viral disease, which is already widespread in tomato plants in Yunnan Province of China.

Standardization and validation of Dot-ELISA assay for Paracoccidioides brasiliensis antibody detection

Background Paracoccidioidomycosis (PCM) is a neglected systemic mycosis caused by a dimorphic fungus of the Paracoccidioides genus. The standard diagnosis is based on isolation of the fungi in culture, and by microscopic visualization of characteristic multiple budding yeast cells in biological samples. However, in some situations, access to the site of injury prevents the collection of biological material. A variety of immuno-serological techniques has proven useful for allowing inferring diagnosis with a certain degree of certainty, thus optimizing time. The aim of this study was to standardize and validate the Dot-ELISA (DE) assay, comparing it with the serological standard, double immunodiffusion (DI). Methods In order to standardize the DE assay, 143 serum samples were used. Out of those, 23 were from apparently healthy patients, 77 were from patients with confirmed PCM and 43 were from patients with other lung infections (tuberculosis, aspergillosis and histoplasmosis). To validate the DE technique, 300 serum samples from patients with PCM clinical suspicion (probable and possible cases) were employed, and these results were compared with those of DI. Results The DE assay showed sensitivity of 91%, specificity of 95.4%, positive predictive value of 96%, negative predictive value of 98.2%, accuracy of 93%, and great precision (k = 0.93). In addition, the nitrocellulose membranes have proved to be viable for using at least 90 days after P. brasiliensis B-339 antigen sensitization. Conclusion Dot-ELISA method was found to be an extremely promising tool as serologic screening technique, because of its high sensitivity. Furthermore, Dot-ELISA shows the prospect of being transferred to laboratories of mycoserology including those with fewer resources or even to be used directly in the field. It has an excellent shelf life - membranes coated with antigen can be used for testing without changes in the pattern of reactivity among laboratories - and presents reliable values of sensitivity, specificity, predictive values, accuracy and a high correlation with the serological standard methodology. Based on the present findings, it possible to state that this technique constitutes a remarkable option to be used in routine diagnosis for public health centers.(AU)

Standardization and validation of Dot-ELISA assay for Paracoccidioides brasiliensis antibody detection

Abstract Background Paracoccidioidomycosis (PCM) is a neglected systemic mycosis caused by a dimorphic fungus of the Paracoccidioides genus. The standard diagnosis is based on isolation of the fungi in culture, and by microscopic visualization of characteristic multiple budding yeast cells in biological samples. However, in some situations, access to the site of injury prevents the collection of biological material. A variety of immuno-serological techniques has proven useful for allowing inferring diagnosis with a certain degree of certainty, thus optimizing time. The aim of this study was to standardize and validate the Dot-ELISA (DE) assay, comparing it with the serological standard, double immunodiffusion (DI). Methods In order to standardize the DE assay, 143 serum samples were used. Out of those, 23 were from apparently healthy patients, 77 were from patients with confirmed PCM and 43 were from patients with other lung infections (tuberculosis, aspergillosis and histoplasmosis). To validate the DE technique, 300 serum samples from patients with PCM clinical suspicion (probable and possible cases) were employed, and these results were compared with those of DI. Results The DE assay showed sensitivity of 91%, specificity of 95.4%, positive predictive value of 96%, negative predictive value of 98.2%, accuracy of 93%, and great precision (k = 0.93). In addition, the nitrocellulose membranes have proved to be viable for using at least 90 days after P. brasiliensis B-339 antigen sensitization. Conclusion Dot-ELISA method was found to be an extremely promising tool as serologic screening technique, because of its high sensitivity. Furthermore, Dot-ELISA shows the prospect of being transferred to laboratories of mycoserology including those with fewer resources or even to be used directly in the field. It has an excellent shelf life membranes coated with antigen can be used for testing without changes in the pattern of reactivity among laboratories and presents reliable values of sensitivity, specificity, predictive values, accuracy and a high correlation with the serological standard methodology. Based on the present findings, it possible to state that this technique constitutes a remarkable option to be used in routine diagnosis for public health centers.

Padronização da metodologia dot-ELISA para detecção de anticorpos IgG anti-Toxoplasma gondii em saliva / Standardization of dot-ELISA methodology for detection of IgG antibodies anti-Toxoplasma gondii in saliva

O diagnóstico da infecção pelo T. gondii é usualmente feito pelas técnicas sorológicas, mas a amostra (soro ou plasma) pode ser restrita em determinados grupos protegidos, em que a coleta de sangue é considerada agressiva e invasiva. Os anticorpos são encontrados em outros materiais biológicos, de coleta não invasiva, como a saliva. Os métodos de detecção de anticorpos no mercado estão padronizados para utilizar amostras de soro, e há metodologias alternativas de maior sensibilidade utilizando-se saliva, mas estas requerem equipamentos de difícil uso no campo. Dot-ELISA tem alta sensibilidade e leitura visual sem equipamentos, que facilita a execução de ensaio em campo utilizando-se técnica de triagem rápida e eficiente. Neste contexto, foi padronizado o dot-ELISA de alta sensibilidade para detecção de anticorpos anti-T. gondii em saliva e soro, utilizando-se amostras de 20 voluntários adultos. A sensibilidade e a especificidade do dot-ELISA padronizado foram semelhantes em soro e saliva, com exata distinção de amostras positivas e negativas, mesmo na ocorrência de baixas concentrações de anticorpos como na saliva. A saliva mostra ser material biológico adequado para detecção de anticorpos anti-T. gondii em estudos epidemiológicos da toxoplasmose em crianças ou outros grupos protegidos, em que a coleta de sangue é restrita...

Desenvolvimento e padronização do Dot-ELISA usando peptídeos recombinantes para o diagnóstico sorológico de Neospora caninum / Development and standardization of Dot-ELISA using recombinant peptides for serological diagnosis of Neospora caninum

A neosporose é reconhecida como uma das maiores causas de aborto e perdas neonatais em bovinos de leite e corte em todo o mundo. Nos últimos anos esta doença tem atraído o interesse de pesquisadores com foco na epidemiologia e métodos eficazes de diagnóstico desta doença. No presente estudo objetivou-se desenvolver e padronizar um teste Dot-ELISA para o diagnóstico sorológico de Neospora caninum com um peptídeo recombinate como antígeno, visando o desenvolvimento de um kit para diagnóstico a campo. O peptídeo recombinante (rNcGRA1) foi desenhado com base na metodologia de genética reversa de epítopos antigênicos originados de uma proteína de grânulos densos de N. caninum, e sintetizado pela GenScript (USA). Produzido mediante o processo fermentativo em leveduras Pichia pastoris KM71. Para a padronização do Dot-ELISA, membranas de nitrocelulose de 0.22µm foram sensibilizadas com 1µL do antígeno e posteriormente os soros foram diluídos em solução de lavagem e incubados durante 1 hora. A revelação foi feita mediante a adição de Proteína G marcada com peroxidase por 30 minutos, seguido da solução reveladora a base de 3,3’-Diaminobenzidine (DAB). Logo após a padronização foram testados 44 soros bovinos diagnosticados por imunofluorescência indireta (RIFI), obtendo-se uma concordância nos resultados do teste de 95,5% e uma sensibilidade e especificidade de 100% e 92% respectivamente. Quanto ao Kit para diagnóstico a campo na Plataforma Tecnológica RapidFlow-Through Miriad®, o peptídeo rNcGRA1 apresentou marcações visíveis ao reagir com os soros positivos, e não apresentou marcações usando os soros negativos. Este estudo é o primeiro a utilizar peptídeos recombinantes e mostrar-se eficiente para o diagnóstico sorológico de bovinos naturalmente infetados por N. caninum...

Neosporosis is recognized as a major cause of abortion and neonatal loss in cattle worldwide, both for dairy cattle and beef cattle. In recent years this disease has attracted the interest of researchers and studying the epidemiology and effective methods of diagnosis of this disease. The present study aimed to develop and standardize on a Dot-ELISA for the serological diagnosis of Neospora caninum by using recombinant peptide as antigen for the development of a diagnostic kit used on the field. The recombinant antigen (rNcGRA1) was designed based on the method of reverse genetics derived antigenic epitopes of dense granules protein of N. caninum and synthesized by GenScript (USA). It was produced by the fermentation in yeasts Pichiapastoris KM71. The serological technique was used for the Dot-ELISA detection of IgG specific for N. caninum in which 0.22μm nitrocellulose membranes were sensitized with 1μL of antigen and subsequently the plasmas were diluted in a washing solution and incubated for 1 hour. The results will revealed by the addition of Protein G labeled with peroxidasse for 30 minutes, followed by the developing solution based on 3,3’-Diaminobenzidine (DAB). Soon after standardization tested 44 bovine plasmas were diagnosed by indirect immunofluorescence assay (IFA), agreeing with the results on a 95.5% and a sensitivity and specificity of 100% and 92% respectively. In regard to the diagnostic kit for the Technology Platform Rapid Flow-Through Miriad®, the peptide presented rNcGRA1 visible markings to react with positive plasma, and showed no markings using the negative plasma. This study is the first to use recombinant peptides and prove to be efficient for the serological diagnosis of cattle naturally infected...

Humoral Response against Native Plasmodium berghei Hemozoin and Synthetic Hemozoin in BALB/c Mice.

Aims: Malaria is a disease caused by protozoan parasites of the genus Plasmodium. One of the malaria mechanisms of adaptation to the host is the digestion of hemoglobin by the trophozoite stage. This mechanism provides the amino acids needed by the parasite and is carried out during the erythrocytics schozogony phase, which results in the formation of in soluble pigment crystals named hemozoin (Hz). Hz is responsible for many of the immune pathological complications of malaria, given that this pigment accumulates in various organs in severe cases of the disease. Here, we evaluated the humoral response in BALB/c mice against native Plasmodium berghei Hz (PbHz) and synthetic Hz (SHz). Place and Duration of Study: Laboratory of Immunology of Infection Diseases. Department of Cell Biology, Simón Bolívar University, Caracas, Venezuela. This study was performed between January 2012 and June 2012. Methodology: We determined the humoral response of SHz and PbHz by an enzyme linked immunosorbent assay (ELISA), using hyper-immune sera from mice experimentally infected with P. berghei or Plasmodium yoelii. In addition, SHz was evaluated as antigen by Western Blot and dot-ELISA. Results: When SHz was employed as antigen, we showed by indirect ELISA that the sera from mice immunized with SHz generated higher titers than sera obtained from mice infected with either Plasmodium species. Moreover, the sera from human infections also recognized SHz as antigen, but showed a better recognition by dot- ELISA or Western Blot than by indirect ELISA. Conclusion: In summary, our results indicated that SHz can be used as a rapid and successful diagnostic antigen for natural malaria infections by indirect ELISA, dot-ELISA and Western Blot techniques.

An evaluation of the dot-ELISA procedure as a diagnostic test in an area with a high prevalence of human Toxocara canis infection

The aim of this work was to evaluate a dot-enzyme-linked immunosorbent assay (dot-ELISA) using excretory-secretory antigens from the larval stages of Toxocara canis for the diagnosis of toxocariasis. A secondary aim was to establish the optimal conditions for its use in an area with a high prevalence of human T. canis infection. The dot-ELISA test was standardised using different concentrations of the antigen fixed on nitrocellulose paper strips and increasing dilutions of the serum and conjugate. Both the dot-ELISA and standard ELISA methods were tested in parallel with the same batch of sera from controls and from individuals living in the problem area. The best results were obtained with 1.33 µg/mL of antigen, dilutions of 1/80 for the samples and controls and a dilution of 1/5,000 for the anti-human IgG-peroxidase conjugate. All steps of the procedure were performed at room temperature. The coincidence between ELISA and dot-ELISA was 85 percent and the kappa index was 0.72. The dot-ELISA test described here is rapid, easy to perform and does not require expensive equipment. Thus, this test is suitable for the serological diagnosis of human T. canis infection in field surveys and in the primary health care centres of endemic regions.

Diagnóstico de leptospirosis canina por medio de las técnicas Dot-ELISA y MAT en perros con enfermedad renal en Bogotá / Diagnostic of canine leptospirosis by Dot – ELISA and MAT tests in dogs with renal disease in Bogota

La leptospirosis es una enfermedad zoonótica, causada por especies del género Leptospira(orden Spirochaetales, familia Leptospiraceae), de gran importancia mundial, debido a su amplia distribución y diversidad de serogrupos y serovares que afectan a varias especies. Una de las especies más afectada por esta bacteria es la canina, en la cual esta bacteria desencadena una infección renal o hepática aguda. La falla renal crónica es una consecuencia común de la infección y los abortos pueden ocurrir en hembras preñadas. En los últimos años, la leptospirosis se ha catalogado como uno de los posibles diagnósticos diferenciales más comunes para perros que presentan signos de enfermedad renal aguda o hepática. En el presente trabajo se estudiaron treinta caninos con enfermedad renal, a los cuales se les realizó una prueba de diagnóstico serológico para leptospirosis: la técnica de aglutinación microscópica (MAT) para seis serovares de Leptospira interrogans, y una prueba de diagnóstico en orina por medio de la prueba Dot-Elisa para los serovares canicola, icterohaemorrhagiae, pomona y grippotyphosa. Se diagnosticaron como positivos a Leptospira, como causa de la enfermedad renal, utilizando los resultados de las dos pruebas, diez caninos (33,3%), los cuales presentaron títulos a MAT para los serovares icterohaemorrhagiae, canicola y grippotyphosa principalmente. Nueve de los diez perros fueron positivos a Dot-Elisa con una distribución homogénea en los cuatro serovares que se manejaron. Los veinte perros restantes (66,7%) fueron negativos. La asociación entre la prueba Dot-Elisa y la prueba MAT fue altamente significativa (P < 0,01).

Leptospirosis is a zoonotic disease that is caused by species of genus Leptospira (Order Spirochaetales, Family Leptospiraceae). This is very important on a global level, due to its widespread distribution and diversity of serogroups and serovars that affect an extensive group of animal species. Canines are one of the most affected species, where this bacterium generates an acute renal or hepatic infection. Chronic kidney disease is a common consequence of the infection and miscarriage can also happen in pregnant females. During the past few years, Leptospirosis has been catalogued as one of the most common differential diagnostics for dogs with acute renal and hepatic disease symptoms. Thirty (30) dogs with renal disease were evaluated during this project, undergoing serological testing for Leptospirosis: the Microscopy Agglutination Test (MAT) for six Leptospira interrogans serovars and a diagnostic urine test through Dot–ELISA for serovars canicola, icterohaemorrhagiae, pomona and grippotyphosa. The results of both tests came out positive for Leptospira as the cause of the renal disease in ten (10) dogs (33.3%), which showed titles on MAT mainly in serovars icterohaemorrhagiae, canicola and grippotyphosa. Dot–ELISA was positive in 9 of the 10 dogs, with a homogeneous distribution in the 4 serovars. The remaining 20 dogs (66.7%) came out negative. The association between the Dot–ELISA test and the MAT test was highly significant (P < 0.01).

Expression and activity identification of a human nasopharyngeal carcinoma I50 anti-idiotype antibody / 中南大学学报(医学版)

Objective To obtain I50 anti-idiotype antibody and identify its activity in vitro.Methods I50 anti-idiotype (Id) antibody gene was amplified from the template of fuse 5-I50 by PCR to construct a prokaryotic expression vector pET25b-I50. The expression of pET25b-I50 in E. coli BL21(DE3) was induced by isopropylthio-β-D-galactopyranoside (IPTG) and was confirmed by SDS-PAGE and Western blot with Ab1(FC2) monoclonal antibody and an anti-hexahistidine tag antibody. The method of dialysis refolding was used to restore the activity of I50 anti-Id antibody, which was measured by Dot-ELISA and lymphocyte proliferation assay. Results The recombinant vector was successfully constructed and the recombinant protein was successfully expressed and purified with 90% purity. The relative molecular weight of the expressed protein was 15 kD, which was in accordance with expectation. The activity of I50 anti-Id antibody could be restored and could promote the proliferation of lymphocyte in a dose-dependent manner. Conclusion These results suggested that I50 anti-Id protein vaccine is likely an option in the therapy against nasopharyngeal carcinoma in vivo.

Comparison of dot-ELISA and standard ELISA for detection of Neisseria meningitidis outer membrane complex-specific antibodies

Dot-ELISA using the outer membrane complex antigens of Neisseria meningitidis as a target was standardized for rapid detection of meningococcal-specific antibodies in human serum. We investigated the level of meningococcal-specific IgG, IgA, and IgM in serum using dot-ELISA with outer membrane antigens prepared from Neisseria meningitidis serotype B:4.19:P1.15,3,7,9 (a strain isolated from a Brazilian epidemic). The dot-ELISA is based on the same principles as the standard ELISA and is useful for detection of anti-N. meningitidis B antibodies in serum of patients with meningococcal infections. For the assay, outer membrane complexes (OMCs) were absorbed by nitrocellulose membrane and blocked with a 5 percent skim milk solution. Serum samples were drawn upon hospital admission and during convalescence from patients with meningococcal septicemia, and single samples were drawn from uninfected controls. We retrospectively examined a total of 57 serum samples: 35 from patients infected with N. meningitidis B, 12 from patients infected with Haemophilus influenzae b, and 10 from health individuals. When performed at room temperature, dot-ELISA took approximately four hours to perform, and the optimum antigen concentration was 0.42 µg per dot. The specificity of IgG, IgM, and IgA demonstrates that dot-ELISA using OMCs from N. meningitidis B as a target is suitable for serologic verification of clinically suspected meningococcal disease in patients and for titer determination of antibodies produced during different phases of natural infection. Furthermore, the sensitivity of dot-ELISA was comparable to that of standard ELISA. Overall, dot-ELISA is simple to perform, rapid, and low cost. Further validation of the test as a screening tool is required.

Frequency of human toxocariasis in a rural population from Cajamarca, Peru determined by DOT-ELISA test / Freqüência de toxocaríase humana numa população rural de Cajamarca, Peru, mediante o uso do teste DOT-ELISA

The aim of this study was to estimate the frequency of human toxocariasis in Cauday district, Cajamarca, Peru, using a dot-ELISA test. From June to October 2005, a total of 256 adult subjects were studied. Blood samples were collected for serology by a dot-ELISA test and for hematological examination. Parasitological examination was also carried out in stool samples to check cross-reactions in the dot-ELISA. The frequency observed was 44.92%, with a significant higher proportion of positivity in male subjects. From subjects with positive serology, 45.6% had respiratory symptoms, 40.44% abdominal pain, 32.35% hepatic symptoms, 14.7% cutaneous signs, 13.23% ocular manifestations, 43.38% eosinophilia, and all of these were statistically associated to serology. Among the population evaluated, 90.23% (231/256) were parasitized. From subjects with positive serology, 92.17% had at least one intestinal parasite and the most frequent were: Blastocystis hominis (68.38%), Giardia lamblia (28.68%), Hymenolepis nana (20.0%), Ascaris lumbricoides (15.65%), Entamoeba histolytica/E. dispar (13.24%), Cyclospora cayetanensis (4.41%), Cryptosporidium sp. (1.47%), Enterobius vermicularis (0.87%), Strongyloides stercoralis (0.87%), Taenia sp. (0.87%), and Trichuris trichiura (0.87%). The rate of false positives in the dot-ELISA test was improved by serum absorption each with A. suum antigens, with a decrease of cross-reactions. In conclusion, human toxocariasis is highly frequent in this population and some risk factors like dog/cat ownership, presence of pets within house, and previous history of geophagia were observed in the present study.

O propósito do presente estudo foi estimar a freqüência da toxocaríase no distrito de Cauday, Cajamarca, Peru, usando o dot-ELISA teste. Entre junho e outubro de 2005, um total de 256 pessoas foram avaliadas. Coletaram-se amostras de sangue para o teste de dot-ELISA e para o exame hematológico e amostras de fezes para exame parasitológico. A freqüência geral de anticorpos anti-Toxocara observada foi de 44,92%, com maior proporção significativa de positividade em pessoas do sexo masculino. Das pessoas com sorologia positiva, 45,6% apresentavam sintomas respiratórios, 40,44% dores abdominais, 32,35% moléstias hepáticas, 14,7% sinais cutâneos, 13,23% manifestações oculares, 43,38% eosinofilia e todos estes fatores foram estatisticamente associados à sorologia. Entre as pessoas avaliadas 90,23% estavam parasitadas e 92,17% das pessoas com sorologia positiva tinham algum parasito intestinal, sendo os mais freqüentes: Blastocystis hominis (68,38%), Giardia lamblia (28,68%), Hymenolepis nana (20,0%), Ascaris lumbricoides (15,65%), Entamoeba histolytica/E. dispar (13,24%), Cyclospora cayetanensis (4,41%), Cryptosporidium sp. (1,47%), Enterobius vermicularis (0,87%), Strongyloides stercoralis (0,87%), Taenia sp. (0,87%) e Trichuris trichiura (0,87%). A taxa de falsos positivos no teste dot-ELISA foi melhorada pela absorção dos soros com antígenos de A. suum, com diminuição das reações cruzadas. Em conclusão, a toxocaríase humana é altamente freqüente nesta população e fatores de risco como ter um cão/gato, presença dos animais dentro de casa e estória prévia de geofagia foram observados durante o presente estudo.

Dot-Elisa for evaluation of hydatid cyst wall, protoscoleces and hydatid cyst fluid antigens in the serodiagnosis of cystic echinococcosis / Dot-Elisa para avaliação da parede do cisto hidático, protoscoleces e antígenos do fluido do cisto hidático no sorodiagnóstico da equinococose cística

The aim of the present study is to evaluate cyst wall and protoscolex as an alternate source of antigen in serodiagnosis of cystic echinococcosis (CE). A total of 90 blood samples, 30 each of confirmed CE cases, disease controls and healthy controls were collected. Dot-ELISA using cyst wall, protoscolex and cyst fluid were used to demonstrate anti-hydatid antibodies. The sensitivity of Dot-ELISA using cyst wall, protoscolex and cyst fluid was 96.66 percent, 86.66 percent and 93.33 percent respectively and the specificity of the assay was 70 percent for Dot-ELISA using cyst fluid, protoscolex and cyst wall antigens. Results of the present study show that cyst wall and protoscolex can also be an useful source of antigen in detection of hydatid antibodies in the serodiagnosis of CE.

O propósito do presente trabalho é avaliar a parede cística e protoscolex como fontes alternativas de antígeno no sorodiagnóstico de equinococose cística (CE). De um total de 90 amostras de sangue, foram coletadas 30 de casos CE confirmados, 30 de controles de doença e 30 controles saudáveis. Dot-Elisa usando parede cística, protoscolex e fluido cístico foi utilizada para demonstrar anticorpos anti-hidáticos. A sensitividade de Dot-Elisa usando parede cística, protoscolex e fluido cístico foi de: 96,66 por cento, 86,66 por cento e 93,33 por cento respectivamente e a especificidade do ensaio de 70 por cento para Dot-Elisa usando fluido cístico, protoscolex e antígeno da parede cística. Resultados do presente estudo mostram que parede cística e protoscolex podem ser fontes úteis de antígeno na detecção de anticorpos hidáticos no sorodiagnóstico do CE.

Canine ehrlichiosis: clinical, hematological, serological and molecular aspects

The aim of the present study was to compare the direct detection methods of Ehrlichia canis (blood smears and nested PCR), serological tests (Dot-ELISA and Immunofluorescent Antibody Test - IFAT), and demonstrate the most suitable test for the diagnosis of different stages of infection. Blood samples and clinical data were collected from 30 dogs examined at the Veterinary Teaching Hospital, UNESP, Jaboticabal, SP, Brazil. The clinical signs most frequently observed were apathy, anorexia, pale mucous membrane, fever, lymphadenopathy, splenomegaly, hemorrhages and uveitis. Evaluating the humoral immune response, 63.3 percent of the sera were IFAT positive, while 70 percent were Dot-ELISA positive. By nestedPCR 53.3 percent of the samples were positive. Comparing these techniques it was concluded that serology and nPCR are the most suitable tests to confirm the diagnosis of canine ehrlichiosis, however it should be always treated as a complementary data to clinical and hematological evaluation. Serology has an important role in the subclinical and in the chronic phase, nPCR is recommended in the acute stage, and, especially, to identify the ehrlichia specie.

O objetivo deste estudo foi comparar técnicas para detecção direta de Ehrlichia canis (detecção de mórulas em esfregaço sangüíneo e nested PCR), testes sorológicos (Dot-ELISA e Reação de Imunofluorescência Indireta - RIFI) e identificar o teste mais adequado para o diagnóstico de diferentes fases da infecção. Amostras sangüíneas e dados dos prontuários clínicos foram colhidos de 30 cães examinados no Hospital Veterinário, UNESP - Jaboticabal, SP. Os sinais clíncos mais freqüentemente observados foram apatia, inapetência, palidez de mucosas, febre, linfadenopatia, esplenomegalia, hemorragias e uveíte. Na avaliação da resposta imune humoral, observou-se que 63,3 por cento das amostras foram positivas na RIFI, e 70 por cento no Dot-ELISA. Na nPCR, foram detectadas 53,3 por cento de amostras positivas. Ao comparar estas técnicas, concluiu-se que a sorologia e a nPCR são testes adequados para a confirmação do diagnóstico da erliquiose canina. Entretanto, os resultados destas técnicas devem sempre ser complementares ao exame clínico e hematológico. A sorologia tem um importante papel nas fases subclínica e crônica da doença, por isso recomenda-se a nPCR para o diagnóstico na fase aguda e, especialmente, para a identificação da espécie de erliquia envolvida.

APPLICATION OF PVC-FAST-DOT-ELISAFOR THE DIAGNOSIS OF CLONORCHIASIS / 中国人兽共患病学报

Aim In order to understand infective conditions of Clonorchis sinensis in different populations in Nantong.Methods The antibodies of Clonorchis sinensis in fishermen area and cooks and shopkeepers et al. were investigated with PVC-Fast-Dot-ELISA. Results The average detective rate of Clonorchis sinensis antibodies was 3.92％ (17/434),the detective rate of Clonorchis sinensis in residents of fishermen area, cooks and shopkeepers (6.45％ ,2/31,5.59％ ,9/161、4.29％ ,3/70)was obviously higher than that of in the normal control group(0. 86％ ,1/115). There was a significant difference in the statistics (X2 = 3.52,X2 = 4.01,X2 = 2.28,P＜0. 01 ). Conclusions ①There is human infection of Clonorchis sinensis in Nantong area. ②The data emphasizes that the infection of Clonorchis sinensis has a strong association to do with occupation and contacting with raw fishes and opportunity of eating raw fishes and shrimps. ③It is recommended that the people of common eating raw fishes and shrimps must be examined and treated regularly.

Improving the affinity of an anti-TNF? scFv by error prone PCR and staggered extention process(StEP) / 中国免疫学杂志

Objective:To improve the affinity of an anti TNF? scFv.Methods:Starting from an anti TNF? scFv gene a mutant phage antibody library was generated by error prone PCR.Affinity improved clones were selected and subjected to staggered extension process to shuffle the mutated sites.Mutants with further improved affinity were selected by bio panning.Affinity was judged by dot blot ELISA and thiocyanate elusion ELISA.Results:Seven affinity improved mutants were obtained from library constructed by error prone PCR.By StEP mediated shuffling of these 7 clones and via bio panning,mutants with further improved affinity were obtained.Conclusion:Combination of error prone PCR and StEP could be used to improve the affinity of antibodies. [

Construction, expression and characterization of a mono-specific bivalent diabody derived from an anti-anti-idiotypic monoclonal antibody NP48 of Schistosoma japonicum / 中国血吸虫病防治杂志

Objective To construct a mono-specific bivalent diabody (scFv dimer) gene derived from an anti-anti-idiotypic monoclonal antibody NP48 of Schistosoma japonicum and to express and characterize the protein.MethodsThe mono-specific diabody gene (D) was constructed by SOE (splicing by overlap extension) and using Gly_4Ser as a linker to join the C-terminus of the V_H to the N-terminus of the V_L.D was linked with prokaryotic expression vector pBAD/g. The target protein expression in E.coli TOP10 was induced by arabinose. Then a purification procedure for the target protein was carried out. The antigen binding activity of expressed product was detected with Dot-ELISA. ResultsThe V_H-G_4S-V_L (D) gene was confirmed by sequencing. The pBAD/g-D recombinant were determined by digesting with endonucleases and expected bands were identified. There were less soluble target proteins in the supernantes and higher target proteins in the pellets as inclusion body when separating the D expression proteins. And the insoluble fraction was recovered as a soluble, correctly processed protein by solubilising with 8 mol/L Urea. The molecular weight of the target protein was about 27 kD. The binding activity of the target protein to anti-idiotypic monoclonal antibody NP30 of Schistosoma japonicum was verified by Dot-ELISA. ConclusionThe purified protein from the constructed recombinant pBAD/g-D could interact specifically with antigen NP30. So the constructed mono-specific diabody has the part characteristics of anti-anti-idiotypic monoclonal antibody NP48 of Schistosoma japonicum.

Testing the Human Serum for HBsAg by Ultrasensitive Chemiluminescent Photographic Detection-Dot-ELISA Technique / 中国输血杂志

The most new ultrasensitive chemiluminescent photographic detectiondot-ELISA (CPD-Dot-ELISA) technique was developed by combining the chemiluminescence technique with Dot-ELISA. The sensitivity of testing for pure HBsAg by CPD-Dot-ELISA was 30 and 60 times higher than by general Dot-ELISA and plate ELISA, respectively. 243 clinical serum specimens had been tested for HBsAg by both plate ELISA and CPD-Dot-ELISA, indicating that of 243 specimens tested for HBsAg by the former, 149 were positive, while of 243 specimens tested for HBsAg by the latter, 194 were positive. Of 149 positive specimmens tested by the former, only Ⅰ wasn't detected by the latter. 14 specimens randomly sampled from the additional 45 positive serum specimens detected by the latter, and 2 serum specimens which proved to be positive by both methods, had then been subject to neutralized test for HBIG, indicating that all 16 mentioned above specimens were positive. The results showed that the CPD-Dot-ELISA technique not only had its high sensitivity, good specificity and repro-ducibility, but also it was simple in manipulation, economically practical and worthy to be widely spread.

Application of Dot Immunogold Filtration Assay for Detecting Serum Antibodies in Clonorchiasis Patients / 中国寄生虫学与寄生虫病杂志

0^05). The negative rate of DIGFA in healthy people was 100%(40/40). The cross reaction rate in 20 cysticercosis cases and 25 schistosomiasis cases were 5%(1/20) and 4%(1/25), respectively. Both coincidence rates comparing DIGFA with dot\|ELISA were 90^9%(50/55). Conclusion DIGFA is as sensitive and specific as the dot\|ELISA,and has the advantages of simplicity and without specific equipment.

THE RAPID DETERMINATION OF ABO BLOOD GROUP IN BODY FLUIDS (OR STAINS)BY DOT-ELISA USING ENZYME-LABELLED MONOCLONAL ANTIBODY / 中国法医学杂志

One step dot-ELISA method for the rapid determination of ABO blood groups in humam body fluids(or stains)was established using enzyme-labelled anti-A,--B and anti--H monoclonal antibody (McAb).The sample was applied on the nitrocellulose membrance as a dot. After washing, the appropriate McAb wasadded on top of the dot, and then followed by adding 3, 3'-diaminobenzidine(DAB). The brown color indi-cated the positive reaction. The ABO blood typing of 521 saliva samples including both secretor and non-se-cretor were carried out. All the results were correct. The advantages of this method are accurate, sensitive,rapid, easy to perform, as well as not time consuming. It is more sensitive than the conventional hemaggluti-naton inhibition test.