Sensitization to poultry meat, fish, and coconut in a multiple food allergic young boy / Sensibilização a carne de aves, peixe e coco em um jovem com alergia alimentar múltipla

Poultry meat allergy is rare and may present as primary or secondary, in the context of bird-egg syndrome. Chicken meat is responsible for most of the reactions. Cross-reactive allergens (parvalbumins, enolases, aldolases) between fish and chicken meat have been described. Coconut allergy is also rare. Coc n2 (7S globulin) and Coc n4 (11S globulin) have been implicated. We present a complex multiple food allergy case report where investigation into fish and chicken meat allergies as well as coconut allergy is carried out.

A alergia à carne de aves é rara e pode apresentar-se como primária ou secundária, no contexto da síndrome ovo-ave. A carne de frango é responsável pela maioria das reações. Foram descritos alergênios com reação cruzada (parvalbuminas, enolases, aldolases) entre peixe e carne de frango. A alergia ao coco também é rara. Coc n2 (globulina 7S) e Coc n4 (globulina 11S) foram implicados. Apresentamos um relato de caso complexo de alergia alimentar múltipla, onde é realizada investigação sobre alergia a peixe e carne de frango, bem como alergia ao coco.

HIV nucleic acid results and follow-up of 73 WB negative and indeterminate samples in Dujiangyan area / 中华检验医学杂志

Objective:To test the HIV virus nucleic acid using immunoblot method (Western blotting, WB) and to follow-up with the negative and indeterminate samples in the Dujiangyan area, compare the WB and nucleic acid results before and after followed-up, and try to reduce the WB band′s false-negatives and false-positives.Methods:The 286 suspected HIV infection samples in the Dujiangyan region from January to October 2021 were confirmed by WB, the HIV virus load were tested for the samples that were WB negative and WB indeterminate, those patients were followed-up with epidemiological history and viral load results, and the results before and after tracking were compared.Results:In the 286 samples of suspected HIV infection included in this study, we reported 213 (74.48%) WB positive, 37 WB negative (12.94%), and 36 WB indeterminate (12.58%); 10 of 37 WB negative samples were followed-up; 18 of 36 WB indeterminate samples were followed-up. Among the followed-up WB negative and indeterminate samples, 17 of them had virus nucleic acid detection prior to the follow-up, and all of them turned positive after following-up. The others with no previous virus nucleic acid detection were confirmed to be negative.Conclusions:Among the followed-up samples, 2 samples were false-negative in WB negative results, and 3 were false-positive in WB indeterminate results. The viral nucleic acid must be tested and followed-up in WB negative and indeterminate samples.

Value and clinical application of multiplex bead-based flow fluorescent immunoassay in the detection of autoantibodies in primary biliary cholangitis / 临床肝胆病杂志

Objective To investigate the value of multiplex bead-based flow fluorescent immunoassay (MBFFI) in the diagnosis of primary biliary cholangitis (PBC) by analyzing its results in detecting antimitochondrial antibody M2 subtype (AMA-M2), gp210 antibody, and sp100 antibody. Methods A total of 340 patients who attended The Affiliated Hospital of Qingdao University from August 2018 to June 2020 and were diagnosed with PBC were enrolled as PBC group, and 143 patients with other diseases and 117 individuals undergoing physical examination were also enrolled. MBFFI and immunoblotting test (IBT) were used to detect AMA-M2, gp210, and sp100 autoantibodies in serum, and the Kappa-test was used to compare the consistency of the two methods in detecting the same autoantibody; the receiver operating characteristic (ROC) curve was used to evaluate the value of MBFFI detection of three antibodies in the diagnosis of PBC; the chi-square test was used to compare positive. Results In the PBC group, the two methods showed the best consistency in detecting AMA-M2(Kappa=0.874) and showed relatively good consistency in detecting gp210 and sp100 antibodies (Kappa=0.713 and 0.749). MBFFI had the highest sensitivity of 72.06% in detecting AMA-M2; it had a sensitivity of 44.71% in the combined detection of gp210 and sp100 antibodies, which was higher than its sensitivity in detecting each antibody alone; it had a sensitivity of 82.65% in the combined detection of AMA-M2, gp210, and sp100 antibodies, which was higher than its sensitivity in detecting each antibody alone. Combined detection of the three antibodies had the largest area under the ROC curve of 0.907 0 in the diagnosis of PBC. Conclusion MBFFI has good consistency with IBT in detecting autoantibodies associated with PBC, and the combined detection of AMA-M2, gp210, and sp100 antibodies by MBFFI has higher sensitivity and value in the diagnosis of PBC.

Value and clinical application of multiplex bead-based flow fluorescent immunoassay in the detection of autoantibodies in primary biliary cholangitis / 临床肝胆病杂志

Objective To investigate the value of multiplex bead-based flow fluorescent immunoassay (MBFFI) in the diagnosis of primary biliary cholangitis (PBC) by analyzing its results in detecting antimitochondrial antibody M2 subtype (AMA-M2), gp210 antibody, and sp100 antibody. Methods A total of 340 patients who attended The Affiliated Hospital of Qingdao University from August 2018 to June 2020 and were diagnosed with PBC were enrolled as PBC group, and 143 patients with other diseases and 117 individuals undergoing physical examination were also enrolled. MBFFI and immunoblotting test (IBT) were used to detect AMA-M2, gp210, and sp100 autoantibodies in serum, and the Kappa-test was used to compare the consistency of the two methods in detecting the same autoantibody; the receiver operating characteristic (ROC) curve was used to evaluate the value of MBFFI detection of three antibodies in the diagnosis of PBC; the chi-square test was used to compare positive. Results In the PBC group, the two methods showed the best consistency in detecting AMA-M2(Kappa=0.874) and showed relatively good consistency in detecting gp210 and sp100 antibodies (Kappa=0.713 and 0.749). MBFFI had the highest sensitivity of 72.06% in detecting AMA-M2; it had a sensitivity of 44.71% in the combined detection of gp210 and sp100 antibodies, which was higher than its sensitivity in detecting each antibody alone; it had a sensitivity of 82.65% in the combined detection of AMA-M2, gp210, and sp100 antibodies, which was higher than its sensitivity in detecting each antibody alone. Combined detection of the three antibodies had the largest area under the ROC curve of 0.907 0 in the diagnosis of PBC. Conclusion MBFFI has good consistency with IBT in detecting autoantibodies associated with PBC, and the combined detection of AMA-M2, gp210, and sp100 antibodies by MBFFI has higher sensitivity and value in the diagnosis of PBC.

Anaphylaxis to Agaricus bisporus ingestion / Anafilaxia por ingestão de Agaricus bisporus

ABSTRACT A 33-year-old male with house dust mite allergic rhinitis and asthma reported an episode of facial and lip angioedema, dyspnea, cough and dysphagia at the age of 25, minutes after eating a mushroom ( Agaricus bisporus ) pizza. He denied any drug intake, hymenoptera stings or other possible triggers, and no identifiable cofactors were present. Since then he avoided all types of mushrooms, however an accidental contact occurred with mushroom sauce that resulted in angioedema of the lip within minutes. The allergy workup included measurements of total IgE and specific IgE to mushroom, and skin prick test to aeroallergens sources, possible food allergen sources and mushroom extract, a prick to prick test with raw and cooked A. bisporus , in addition to a SDS-PAGE and immunoblotting assay. The study revealed a specific IgE to mushroom of 0.76kUA/L positive skin prick test to mushroom extract, and prick to prick test positive to white and brown A. bisporus (raw and cooked). The immunoblotting identified two IgE binding proteins with 10kDa and 27kDa. We report a case of A. bisporus anaphylaxis probably due to primary mushroom sensitization. We detected two IgE-reactive proteins with 10kDa and 27kDa as possible culprit allergens.

RESUMO Paciente do sexo masculino, 33 anos, com asma e rinite alérgica desencadeadas por ácaros, relatou episódio de angioedema facial e labial, dispneia, tosse e disfagia aos 25 anos, minutos após a ingestão de uma pizza de cogumelo ( Agaricus bisporus ). O paciente negou consumo de medicamentos, picadas de himenópteros, ou quaisquer outros possíveis desencadeadores ou cofatores que pudessem estar presentes. Desde então, evita todos os tipos de cogumelos, até a ocorrência de um contato acidental com molho de cogumelo, que resultou em angioedema labial minutos após. O estudo imunoalergológico incluiu doseamento de IgE total e específica para cogumelos, testes cutâneos para aeroalérgenos, possíveis alérgenos alimentares e extrato de cogumelos, teste prick to prick com A. bisporus cru e cozido e teste de SDS-PAGE immunoblotting . O estudo revelou IgE específica para cogumelos de 0,76kUA/L, teste cutâneo positivo para extrato de cogumelos e teste prick to prick positivo para A. bisporus branco e castanho (cru e cozido). O immunoblotting identificou duas proteínas de ligação de IgE, de 10kDa e 27kDa. Relatamos, assim, um caso de anafilaxia por ingestão de A. bisporus , provavelmente explicado por sensibilização primária a cogumelos. Detectamos duas proteínas IgE-reativas de 10kDa e 27kDa como os possíveis alérgenos responsáveis.

Two new mixed-ligand coordination polymers based on multi-N chelating ligand inhibit YAP expression and induce caspase-mediated spinal tumor cell apoptosis

Two new coordination polymers [Zn (bdc)(bpybzimH2)](DMF)0.5 (1, H2bdc=1,4-dicarboxybenzene, bpybzimH2=6,6′-bis-(1H-benzoimidazol-2-yl)-2,2′-bipyridine, DMF=N,N-dimethylformamide) and [Co (bpybzimH2)(sbc)]H2O (2, H2sbc=4-mercaptobenzoic acid) have been successfully prepared under solvothermal conditions using the multi-N chelating organic ligand bpybzimH2 as the foundational building block. In addition, the Cell Counting Kit-8 assay was conducted to evaluate the anti-proliferation activity of compounds 1 and 2 against human spinal tumor cells OPM-2. The cell viability curves showed that the two compounds have anti-proliferation activity on spinal tumor cells, and the activity of compound 1 is higher than compound 2. The annexin V-FITC/PI assay and western blot were used to detect the apoptotic percentage of OPM-2 cells incubated with compounds 1 and 2. The YAP protein expression and its role in cell apoptosis were further studied with qRT-PCR, immunoblotting, and flow cytometer.

Perfil imunogênico de vacinas inativadas contra agalaxia contagiosa em ovinos / Immunogenic profile of inactivated vaccines against contagious agalactia in sheep

Este trabalho teve como objetivo avaliar o perfil imunogênico em ovinos de três vacinas produzidas com linhagens brasileiras de Mycoplasma agalactiae. O perfil proteico do antígeno vacinal foi avaliado por SDS-PAGE e a imunogenicidade da vacina pela técnica de Western blot. A vacina foi inativada com formol, adsorvida em hidróxido de alumínio (Vacina 1), Montanide IMS-2215 (Vacina 2), Montanide Gel-01 (Vacina 3) e administradas em três doses. Entre a primeira e a segunda dose houve um intervalo de 21 dias, e entre a segunda e a terceira de 180 dias. O pool de soros de dez ovinos coletados nos períodos 0, 21, 35, 90, 150, 210, 270 e 360 dias pós-vacinação foram testados pela técnica de Western blot. A vacina 2 foi mais antigênica, com detecção de anticorpos 21 dias após a primeira dose. Para as vacinas 1 e 3, os anticorpos são verificados após 35 dias, com queda acentuada aos 90 dias; apenas anticorpos contra a proteína de 48 kDa apareceram após a terceira dose de forma discreta. Contra a vacina 2, ainda persistiram anticorpos contra as proteínas de 48, 55 e 80 kDa nos períodos 90, 150 e 210, que aumentaram após a terceira dose. Conclui-se que a vacina 2 induziu a resposta humoral de forma estável contra proteínas de M. agalactiae.

This study aimed to evaluate the immunogenic profile in sheep of three vaccines produced with a field Brazilian strains of Mycoplasma agalactiae. The vaccine protein profile was evaluated by SDS-PAGE and vaccine immunogenicity by Western blot. The vaccine was inactivated with formaldehyde and adsorbed onto three different adjuvants: with aluminum hydroxide (Vaccine 1), Montanide IMS 2215 (Vaccine 2), and Gel Montanide-01 (Vaccine 3). The vaccine was administered in three doses. Between the first and second dose there was an interval of 21 days, and between the second and the third one of 180 days. A pool of ten sera collected in 0, 21, 35, 90, 150, 210, 270 and 360 days after first vaccination were tested by Western blot techniques. The second vaccine was more antigenic with antibody detection 21 days after first dose. For both vaccines 1 and 3, antibodies were present 35 days after first dose, with a significant drop at 90 days; only antibodies against 48 kDa protein discreetly appeared after the third dose. Stimulation induced by vaccine 2 produced antibodies against 48, 55 and 80 kDa proteins that persisted until 90, 150 and 210 days after first dose, which rose again after third dose. It was concluded that the vaccine 2 induced stable humoral immunity against M. agalactiae proteins.

Confirmatory process and follow-up of 1 case with long-term HIV infection in Shandong / 中华预防医学杂志

Objective@#To describe the confirmation process and long-term follow-up results of 1 case of HIV with long term progression.@*Methods@#The subject was a HIV infected man aged 27 years old. The first HIV antibody positive was detected by ELISA in August 7th, 2013. Close contacts were identified as 3 homosexual partners who had been contacted before infection and the first sexual partner had been unable to get in touch. Adopting the first epidemiological survey questionnaire of AIDS comprehensive prevention and control information system in China, the investigators conducted face-to-face surveys on the general demographic characteristics and behavioral characteristics of the subject. After the first ELISA test result was positive, 4 rapid detections of colloid selenium, ELISA, western-blot, CD4+T and viral load test were followed up (August 14th, 21st, 30th and September 16th, 2013). Long term follow-up was performed to detect CD4+T and viral load to observe the progress of the case after the diagnosis of infection.@*Results@#The duration of sexual behavior was from 2011 to 2012 between the subject and his 1st sexual partner. During the study, repeated HIV antibody ELISA test results were negative. Sexual behavior maintained from January to April 2013 between the subject and his 2nd partner and the last one unprotected homosexual acts took place in April 2013. After the traceability survey, the 2nd sexual partner was an AIDS patient who had antiretroviral therapy in the anti HIV treatment module of AIDS comprehensive prevention information system. The subject and his 3rd partner maintained their sexual behavior from May to October 2013. The two ELISA tests of the 3rd partner were negative. Because of the need for hospital operation in August 7, 2013, the subject was tested for HIV antibody by ELISA and the result was positive while western blot test showed that the HIV-1 antibody was not confirmed (band type was gp160/gp120/p24). In the subsequent follow-up, 4 rapid detections of colloid selenium, ELISA and western-blot were conducted and all the results were positive (western-blot band type was gp160/gp120/gp41/p24/p17). Results of continuous follow-up for 5 years showed that the first four CD4+T cell counts were as follows: 520, 616, 834, 879. The following 22 CD4+T counts sustained at a high level and the median was 895 cells/μl. A total of 5 follow-up visits were conducted to detect viral load exceeding 1 000 copies/ml and the remaining 19 test results were lower than 1 000 copies/ml except that no viral load was detected in 2 follow-up visits. The result of homology analysis showed that the HIV types of the case and its 2nd sexual partner were all HIV-1 CRF_01AE. The similarity of gag region gene was 97.5%. So we inferred that the 2nd sexual partner was its source of infection, and the case was infected at the end of April 2013 with the last unprotected homosexual behavior.@*Conclusion@#The infected person was found to be an early HIV infection. Continuous follow-up test results indicated that the case belonged to a HIV long-term nonprogressor.

Significance of positive autoantibodies number in antinuclear antibody spectrum for diagbosing systemic lupus erythematosus / 重庆医学

Objective To evaluate the significance of positive autoantibodies number in antinuclear antibodies(ANAs) spectrum for the diagnosis of systemic lupus erythematosus(SLE).Methods A total of 1 297 patients were included in this study,among them 148 cases were SLE,317 cases were non-SLE rheumatic diseases,99 cases were nephropathy,210 cases were hematological diseases,and 523 cases were other diseases as the control group.The ANAs level in each group was detected by immunoblotting.The rate comparison adopted the chi square test.The value of positive autoantibodies number in diagnosing SLE was analyzed by receiver operating characteristic(ROC) curve.Results The positive autoantibodies number had good diagnostic value for SLE.The area under the curve was 0.934;the positive rate of autoantibodies(AAbs) in the SLE group was 93.92 ％,which was much higher than that in other groups(P＜0.01).When the number of positive-AAbs was ≥3,the positive likelihood ratio was greater than 10(72.78),showing a very good predictive value for the diagnosis of SLE.Conclusion Three or more kinds of positive-AAbs is highly suggestive of SLE.

Early Postnatal Valproic Acid Exposure Increase the Protein Level of Astrocyte Markers in Frontal Cortex of Rat

OBJECTIVE: In our previous study, it has been reported that valproic acid (VPA) effects gliogenesis and increases the number of glial precursor cells during the early postnatal period. However there is no specific report that whether this process is going on up to the age of mature brain development and the consequence effect of this ongoing gliogenesis process. METHODS: As an ongoing study, using Immunoblotting analysis, we checked the level of glial protein and glial-derived factor markers in the frontal cortex of a rat brain at postnatal day (PND) 21. RESULTS: The finding of the study suggests that, in the VPA group (p < 0.05), early exposure elicited significantly to increase the expression level of glial protein cells at PND 21 in the frontal cortex of rat brain. CONCLUSION: Therefore we suggest that, alter gliogenesis and abnormal number of glial cells modulate the neurobiological dysfunction and induces the risk of neurodevelopmental disorders.

Expression of phosphorylated extracellular signal-regulated kinase during development of hippocampus in mice / 吉林大学学报(医学版)

Objective: To investigate the expressions of phosphorylated extracellular signal-regulated kinase (pERK) in the hippocampus of the mice of embryonic (E) 18 d and postnatal (P) 1, 3, 7, 14, 21 and 28 d, and to discuss its possible role during the hippocampus development of the mice. Methods: The hippocampus tissues of the mice of E 18 d, P 1 d, P 3 d, P 7 d, P 14 d, P 21 d and P 28 d were obtained. Each time point was used as observation group. Immunohistochemistry, Western blotting and image analysis technique were used to analyze the expression levels of pERK protein in hippocampus tissue at each time point. Results: The immunohistochemistry results revealed that pERK protein mainly expressed in the nucleus of the nerve cells in the granular layer of dentate gyrus and pyramidal layer of Ammon' s horn in hippocampus. In the mice of E 18 d, the expression of pERK in DG and CA regions of hippocampus were slightly stained and arranged sparsely, and the expression level was low; and the pERK was deeply stained and the expression levels of pERK were increased gradually with the development of the hippocampus from E 18d to P 7 d; compared with other six time points, the expression level of pERK of the mice of P 7 d was the highest (F=34. 537, P0.05). The Western blotting results showed that pERK expressed at each time point, from E18 d to P7 d, the expression levels of pERK were increased gradually; compared with other six time points, the expression level of pERK on P 7 d was the highest (F = 33.856, P0.05). Conclusion: The hippocampus development is increased significantly at the early stage of the mice (E18 - P 7 d), and the expression levels of pERK are increased gradually in the meanwhile; the expression level peaks on P 7 d, and then it is gradually decreased until a stable level. pERK may take part in the cell proliferation in the development of hippocampus of the mice.

Comparative performances of serologic and molecular assays for detecting human T lymphotropic virus type 1 and type 2 (HTLV-1 and HTLV-2) in patients infected with human immunodeficiency virus type 1 (HIV-1)

ABSTRACT The present study evaluated several techniques currently available (commercial kits and in-house assays) for diagnosing human T lymphotropic viruses types 1 and 2 in two groups of patients enrolled at HIV/AIDS specialized care services in São Paulo: Group 1 (G1), n = 1608, 1237 male/371 female, median age 44.3 years old, majority using highly active antiretroviral therapy (HAART); G2, n = 1383, 930 male/453 female, median age of 35.6 years old, majority HAART naïve. Enzyme immunoassays [(EIA) Murex and Gold ELISA] were employed for human T lymphotropic viruses types 1 and 2 screening; Western blotting (WB), INNO-LIA (LIA), real-time PCR pol (qPCR), and nested-PCR-RFLP (tax) were used to confirm infection. Samples were considered human T lymphotropic viruses types 1 and 2 positive when there was reactivity using at least one of the four confirmatory assays. By serological screening, 127/2991 samples were positive or borderline, and human T lymphotropic virus infection was confirmed in 108 samples (three EIA-borderline): 56 human T lymphotropic virus type 1 [G1 (27) + G2 (29)]; 45 human T lymphotropic virus type 2 [G1 (21) + G2 (24)]; one human T lymphotropic virus type 1 + human T lymphotropic virus type 2 (G2); six human T lymphotropic virus [G1 (2) + G2 (4)]. Although there were differences in group characteristics, human T lymphotropic viruses types 1 and 2 prevalence was similar [3.1% (G1) and 4.2% (G2), p = 0.113]. The overall sensitivities of LIA, WB, qPCR, and PCR-RFLP were 97.2%, 82.4%, 68.9%, and 68.4%, respectively, with some differences among groups, likely due to the stage of human T lymphotropic virus infection and/or HAART duration. Indeterminate immunoblotting results were detected in G2, possibly due to the seroconversion period. Negative results in molecular assays could be explained by the use of HAART, the occurrence of defective provirus and/or the low circulating proviral load. In conclusion, when determining the human T lymphotropic virus infection, the findings highlight that there is a need to consider the blood samples with borderline results in screening assays. Of all the tested assays, LIA was the assay of choice for detecting human T lymphotropic virus type 1 and human T lymphotropic virus type 2 in human immunodeficiency virus type 1-infected patients.

Comparative performances of serologic and molecular assays for detecting human T lymphotropic virus type 1 and type 2 (HTLV-1 and HTLV-2) in patients infected with human immunodeficiency virus type 1 (HIV-1)

The present study evaluated several techniques currently available (commercial kits and in-house assays) for diagnosing human T lymphotropic viruses types 1 and 2 in two groups of patients enrolled at HIV/AIDS specialized care services in São Paulo: Group 1 (G1), n = 1608, 1237 male/371 female, median age 44.3 years old, majority using highly active antiretroviral therapy (HAART); G2, n = 1383, 930 male/453 female, median age of 35.6 years old, majority HAART naïve. Enzyme immunoassays [(EIA) Murex and Gold ELISA] were employed for human T lymphotropic viruses types 1 and 2 screening; Western blotting (WB), INNO-LIA (LIA), real-time PCR pol (qPCR), and nested-PCR-RFLP (tax) were used to confirm infection. Samples were considered human T lymphotropic viruses types 1 and 2 positive when there was reactivity using at least one of the four confirmatory assays. By serological screening, 127/2991 samples were positive or borderline, and human T lymphotropic virus infection was confirmed in 108 samples (three EIA-borderline): 56 human T lymphotropic virus type 1 [G1 (27) + G2 (29)]; 45 human T lymphotropic virus type 2 [G1 (21) + G2 (24)]; one human T lymphotropic virus type 1 + human T lymphotropic virus type 2 (G2); six human T lymphotropic virus [G1 (2) + G2 (4)]. Although there were differences in group characteristics, human T lymphotropic viruses types 1 and 2 prevalence was similar [3.1% (G1) and 4.2% (G2), p = 0.113]. The overall sensitivities of LIA, WB, qPCR, and PCR-RFLP were 97.2%, 82.4%, 68.9%, and 68.4%, respectively, with some differences among groups, likely due to the stage of human T lymphotropic virus infection and/or HAART duration. Indeterminate immunoblotting results were detected in G2, possibly due to the seroconversion period. Negative results in molecular assays could be explained by the use of HAART, the occurrence of defective provirus and/or the low circulating proviral load. In conclusion, when determining the human T lymphotropic virus infection, the findings highlight that there is a need to consider the blood samples with borderline results in screening assays. Of all the tested assays, LIA was the assay of choice for detecting human T lymphotropic virus type 1 and human T lymphotropic virus type 2 in human immunodeficiency virus type 1-infected patients. (AU)

Evaluation of the “shelf life” of nitrocellulose membranes immobilized with Paracoccidioides brasiliensis antigen for performing the immunodiagnosis of paracoccidioidomycosis / Avaliação do “tempo de prateleira” de membranas de nitrocelulose imobilizadas com antígeno de Paracoccidioides brasiliensis para o imunodiagnóstico da paracoccidioidomicose

Considerando-se que o immunoblotting para o imunodiagnóstico da paracoccidioidomicose (PCM) é uma metodologia in house e laboriosa envolvendo duas etapas iniciais, SDS-PAGE e Western blot, neste estudo foi avaliado o tempo de prateleira das membranas de nitrocelulose sensibilizadas com antígeno de P. brasiliensis, armazenadas a -20 oC durante 7, 15, 30, 45, 60 e 90 dias. Vinte e oito amostras de soro foram analisadas em dois grupos de membranas de nitrocelulose (membranas previamente bloqueadas com PBS-leite 5 % e as não-bloqueadas). Não houve diferença no padrão de reatividade quando os soros foram avaliados frente a ambos os grupos, especialmente para membranas armazenadas por 7, 15, 30, 45 e 60 dias. A boa estabilidade do antígeno utilizado para sensibilizar as membranas fez com que estas pudessem ser armazenadas a -20 °C até 60 dias. Estas características contribuem para efetuar o diagnóstico rápido da PCM, bem como as perspectivas dessas membranas sensibilizadas serem encaminhadas para os laboratórios, que não possuam infraestrutura necessária para executar as etapas que antecedem a realização de immunoblotting, como a produção de antígeno, as técnicas de SDS PAGE e Western blot. Este procedimento contribui substancialmente para melhorar o diagnóstico sorológico da PCM, pois poderá fornecer resultados reprodutíveis nas unidades componentes da Rede de Laboratórios.

The immunoblotting reaction for performing the paracoccidioidomycosis (PCM) immunodiagnosis is an in-house methodology; and being a laborious task involving two previous steps, SDS-PAGE and Western blot, we evaluated the shelf life of nitrocellulose membranes containing the immobilized P. brasiliensis antigens, stored at -20 oC for 7, 15, 30, 45, 60 and 90 days. Twenty-eight serum samples were analyzed on two nitrocellulose membranes groups: (a) membranes previously blocked with PBS-5 % non-fat dry milk and (b) the priory non-blocked membranes. No difference was detected in the reactivity pattern in serum samples evaluated in the both membrane groups, especially for those stored for 7, 15, 30, 45 and 60 days. It might be emphasized that a good stability of P. brasiliensis antigens, immobilized on the nitrocellulose membranes, enable them to be stored up to 60 daysat -20 oC. This finding contributes to the rapid diagnosis of PCM, and for sending them to other laboratories without adequate infrastructure for carrying out the steps that precede the immunodetection as the antigen production, SDS-PAGE and Western blot techniques. This scheme contributes substantially to improve the quality of PCM serodiagnosis, as it provides reproducible results in the units of the Laboratory Network.

Application value of CMIA in HCV infection validation in low risk population / 国际检验医学杂志

Objective To detect the reactive samples of enzyme-linked immunosorbent assay (ELISA1) by chemiluminescence microparticle immunoassay (CMIA),and to analyze the application value of CMIA in HCV infection validation of blood donors.Methods Nucleic acid 3-item combined testing (NAT),another ELISA2,HCV antibody supplementary test(Western Blot test,WB) and CMIA test supplemented in blood samples of 102 ELISA1 anti-HCV reactive blood donors were retrospectively analysed.Results Among 102 blood donors of anti-HCV positive,32 cases (31.37%,32/102) were HCV RNA reactive samples,50 cases (49.02%,50/102) were ELISA2/WB reactive simultaneously.With CMIA NAT results as the reference standard,CMIA was poorly correlated with HCV RNA (Spearman correlation coefficient rs=0.395,P<0.01),and the consistency between them was weak by Kappa test (Kappa=0.270,P<0.01).With ELISA2/WB detection results as the reference standard,CMIA was highly correlated with the results(Spearman correlation coefficient rs=0.713,P<0.01),and which showed high consistency by Kappa test (Kappa=0.674,P<0.01).Conclusion CMIA as a detection method of protein label after HCV infection has great value in the HCV infection confirmation in low-risk population.

Ubiquitin ligase Smurf1 promotes poly-ubiquitination of PDK1 / 军事医学

Objective To investigate the mechanism of 3-phosphoinositide-dependent protein kinase 1(PDK1)poly-ubiquitination.Methods Co-immunoprecipitation(Co-IP)and Western blot(WB)were used to analyze poly-ubiquitination of PDK1.It was confirmed that ubiquitin ligase smad ubiquitylation regulatory factor 1(Smurf1)inprove PDK1 poly-ubiquitination within MEF cells,site-directed mutagenesis and WB before PDK1 poly-ubiquitination sites were determined.Results We found that PDK1 could undergoes poly-ubiquitination,ubiquitin ligase Smurf1 was found to be a direct E3 ligase for PDK1 poly-ubiquitination and might rely on the ubiquitin ligase Smurf 1 K699 site activity.K304 was PDK1 poly-ubiquitination modification site point.Conclusion The ubiquitin ligase Smurf1 can promate poly-ubiquitination of PDK1.

Expressions of GLP-1R and CK17 in papillary thyroid cancer and clinical significance / 重庆医学

Objective To investigate the expressions of glucagon-like peptide-1 receptors (GLP-1R) and cytokeratin17 (CK17) in papillary thyroid cancer tissue and clinicopathological significance.Methods The immunohistochemistry and immunoblotting method were employed to detect the expressions of GLP-1R and CK17 in normal thyroid tissues (control group,50 cases) and papillary thyroid cancer tissue (observation group,80 cases).Then the correlation between the expressions of GLP-1R and CK17 with pathological factors of thyroid cancer was analyzed.Results Low expression of GLP-1R was found in 22 cases (44.00 ％) of the control group,while GLP-1R was expressed in 77 cases(96.25 ％) of the observation group,and the difference between both groups was statistically significant(P＜0.01).The expression of CK17 was positive in 8 cases(16.00％) of the control group,while 77 cases(96.25 ％) in the observation group were CK17 expression positive,and the difference between both groups was statistically significant(P＜0.01).The difference between GLP-1 positive expression and clinicopathologic factors had no statistical significance(P＞0.05).The difference between CK17 expression with sex and age had no statistical significance (P＞0.05),and was obviously related with the tumor diameter,clinical stages and cervical node metastasis of thyroid neoplasms(P＜0.05).Conclusion GLP-1R and CK17 all are highly expressed in papillary thyroid cancer tissue and CK17 expression is obviously related with the tumor diameter,clinical stages and cervical node metastasis of papillary thyroid cancer.

Comparative study on two kinds of allergen detection methods for allergic skin diseases / 国际检验医学杂志

Objective To investigate the clinical effect of different detection methods in the detection of allergens in allergic skin diseases .Methods Choose 74 cases of allergic skin disease patients in our hospital from March 2014 to March 2015 as the research object ,they were divided into two groups by random number method ,immunoblotting group and skin prick test group ,detected al-lergens of the two groups by immunoblotting and skin prick test before treatment .Results The positive rate was 70 .3% in immu-noblotting group ,and the positive rate was 56 .8% in skin prick test group .For allergens detection ,the positive rate was higher by using immunoblotting ,the differences between the two had significant difference(P<0 .05) .Conclusion Immunoblotting is better than the skin prick test for allergen detection in allergic skin diseases .

Study on comparation of three methods for anti-double-stranded DNA antibody and diagnostic value of joint detection in systemic lupus erythematosus / 国际检验医学杂志

Objective To analyse the differences of indirect immuno‐fluorescence (IIF) ,enzyme‐linked immunosorbent assay (ELISA) and immunoblotting technique(IBT) for the determination of anti‐dsDNA antibody ,and evaluate the value of joint detec‐tion of the three methods for diagnosing systemic lupus erythematosus(SLE) .Methods From January 2012 to March 2015 ,50 ca‐ses of patients with SLE ,100 cases of patients with other autoimmune disease(AID)and 100 healthy individuals were selected .Ser‐um levels of anti‐dsDNA antibody were detected by using IIF ,ELISA and IBT respectively .Then ,compared the sensitivity and spe‐cificity of the three methods ,and analysed the sensitivity and specificity of joint detection .Results The IIF method had the highest specificity(99 .5% ) ,while ELISA had the highest sensitivity(74 .0% ) .There were statistically significant differences in the positive detection rates of serum anti‐dsDNA antibody in patients with SLE between IIF and ELISA ,IIF and IRT ,ELISA and IBT(χ2 values were 11 .435 ,13 .994 and 4 .539 ;P<0 .05) ,and the Kappa values were 0 .411 ,0 .522 and 0 .278 respectively .The specificity of three methods joint in series was increased to 99 .5% ,and the sensitivity of parallel combined detection of the three methods was in‐creased to 82 .0% .Conclusion Among the three methods for detecting anti‐dsDNA antibody ,ELISA has the highest sensitivity , and IIF has the highest specificity .Moreover ,joint detection could increase the sensitivity and specificity .

Construction and identification of recombinant human neutrophil gelatinase-associated lipo1calinlinear multi-epitope peptide / 中华检验医学杂志

Objective The feasibility of predicting the B-cell epitopes of human Neutrophil Gelatinase-Associated Lipocalin (NGAL) was discussed by applicating bioinformatics technology.Linear epitope molecules that have diagnostic value were screened and these recombinant linear multi-epitope peptides were constructed,and expressed.The immunogenicity of the recombinant linear multi-epitope peptides were also identified.Methods NGAL amino acid sequence was got from GenBank in the Department of Clinical Laboratory of the Second Affiliated Hospital of Nanjing Medical University in July 2015,the Predicted,ABCpred,BepiPred,BcePred,and Lasergene softwares were used to predict the linear B cell epitope prediction.The predict epitopes were constructed and prokaryotic expressed,and then the single epitope antigens which could reacted with commercially available polyclonal NGAL antibody were screened out by Western blot.Finally,the multi-epitope peptide was constructed,expressed,and identified through immunoreactions.Results Eight possible epitopes were obtained after prediction.pET32a-N1-N8 prokaryotic expression vector were used to express the predict epitopes.After purification and Western blot analysis,three of the epitopes have strong antigenicity,and then a soluble fusion protein was expressed and obtained from the multi-epitope prokaryotic expression vector pET22b-Ngal_MEP1.The fusion protein was successfully purified by Ni2 + affinity column.Western blot analysis showed that the fusion protein had a strong antigenicity.Conclusions The constructed multi-epitope linear NGAL antigen peptides can obtain high soluble expression in prokaryotic expression system,and have a strong immunoreactivity,which can be used in subsequent antibody preparation.