A Simple Outline of Methods for Protein Isolation and Purification

At the summer workshop of the Korean Endocrine Society held in 2016, some examples of protein experiments were discussed in the session entitled “All about working with proteins.” In contrast to what the title suggested, it was unrealistic to comprehensively discuss all protein analytical methods. Therefore, the goal was to outline protein experimental techniques that are useful in research or in bench work. In conversations with clinicians, however, I have always felt that researchers who do not engage in bench science have different demands than those who do. Protein research tools that are useful in bench science may not be very useful or effective in the diagnostic field. In this paper, I provide a general summary of the protein analytical methods that are used in basic scientific research, and describe how they can be applied in the diagnostic field.

Current status and prospects of quantitative immunoassay technologies / 中华检验医学杂志

Quantitative immunoassay technique is the common method of quantitative detection in clinical laboratory. Several important branches of quantitative immunoassay were formed by changing the tracer or the Antigen-antibody complex separation method, including radioimmunoassay, fluorescent immunoassay, enzyme immunoassay, chemiluminescence, colloidal gold, immuno-turbidimetric analysis and homogeneous immunoassay. Different immunoassay techniques have their own characteristics, also apply to different detecting conditions in clinic. This paper reviewed several common kinds of quantitative immunoassay technology, and discussed both their advantages and disadvantages, which provide reference for the application and development of clinical testing technology.

Evaluation of three commercially-available chikungunya virus immunoglobulin G immunoassays / Evaluación de tres inmunoensayos comercializados de inmunoglobulina G para el diagnóstico del virus del chikungunya

ABSTRACT The emergence of chikungunya virus in the Americas means the affected population is at risk of developing severe, chronic, rheumatologic disease, even months after acute infection. Accurate diagnostic methods for past infections are essential for differential diagnosis and consequence management. This study evaluated three commercially-available chikungunya Immunoglobulin G immunoassays by comparing them to an in-house Enzyme-Linked ImmunoSorbent Assay conducted by the Centers for Disease Control and Prevention (Atlanta, Georgia, United States). Results showed sensitivity and specificity values ranging from 92.8% - 100% and 81.8% - 90.9%, respectively, with a significant number of false-positives ranging from 12.5% - 22%. These findings demonstrate the importance of evaluating commercial kits, especially regarding emerging infectious diseases whose medium and long-term impact on the population is unclear.(AU)

RESUMEN Como consecuencia de la aparición del virus del chikungunya en las Américas, la población afectada corre el riesgo de padecer reumatismos crónicos graves, aun meses después de la infección aguda. Es fundamental contar con métodos precisos para diagnosticar los antecedentes de la infección a fin de elaborar un diagnóstico diferencial y abordar las manifestaciones de la fase crónica. Se han estudiado tres inmunoensayos comercializados de detección de inmunoglobulinas G para el diagnóstico del chikungunya, comparándolos con el enzimoinmunoanálisis de adsorción (ELISA) propio. Los resultados señalan valores de sensibilidad del 92,8% al 100% y de especificidad del 81,8% al 90,9%, así como un número significativo de falsos positivos, de entre el 12,5% y el 22%.(AU)

Evaluation of the VIDAS Anti-HCV Assay for Detection of Hepatitis C Virus Infection

BACKGROUND: Anti-hepatitis C virus antibody (anti-HCV) assays are recommended for screening HCV-infected persons. The VIDAS Anti-HCV Assay (bioMérieux, France), based on the enzyme-linked fluorescence test principle, was recently introduced in Korea. We evaluated the clinical performance of the VIDAS assay. METHODS: One hundred HCV-positive and 1,002 HCV-negative blood samples confirmed by Architect anti-HCV (Abbott Laboratories, USA) and COBAS TaqMan HCV real-time PCR (Roche Diagnostics, USA) or the Procleix Ultrio Plus Assay (Gen-Probe Incorporated, USA) were obtained from the Human Serum Bank (HSB) and tested by VIDAS. In case of discrepant results, we conducted a recombinant immunoblot assay (RIBA). RESULTS: The agreement rates for known HCV-positive and HCV-negative samples between the VIDAS assay and the HSB testing were 100% (95% confidence interval [CI]: 96.4-100%) and 99.5% (95% CI: 98.8-99.8%), respectively. One of the five discrepant samples was positive for Core 2+ and NS3-2 2+ reactivity, two samples were negative, and the other two were indeterminate regarding NS4 2+ reactivity in RIBA. We observed a significant but weak positive correlation between the titers of VIDAS and Architect assays (r=0.315, P<0.001). CONCLUSIONS: The VIDAS anti-HCV assay, developed on the VIDAS automated immunoassay platform based on the ready-to-use, single-sample test concept may be useful in small-to-medium-sized laboratories. It showed good agreement with Architect anti-HCV and COBAS PCR assays and is therefore useful for detection of HCV infection. Weakly test-positive (ambiguous) samples require additional testing by another anti-HCV, RIBA, or HCV RNA assay.

Diagnóstico da aspergilose invasiva:Aplicação das Técnicas de Reação em Cadeia da Polimerase (PCR) e Ensaio Imunoenzimático de Detecção da Galactomanana (EIA-GM®) / Diagnosis of invasive aspergillosis: Application of Polymerase Chain Reaction Techniques (PCR) and Enzyme-Linked Immunosorbent Assay for Detectionof Galactomannan (EIA-GM®)

Introdução: a aspergilose invasiva (IA) é uma infecção fúngica grave causada por espécies do gênero Aspergillus e acomete principalmente pacientes leucêmicos,diabéticos e aqueles receptores de transplante de células-tronco, que apresentem neutropenia. Os esporos dos fungos que colonizam o epitélio pulmonar podem invadir as células endoteliais de revestimento e o acesso vascular e, assim, disseminar-se paraoutros órgãos através do sangue. A elevada mortalidade da doença está relacionada à imunossupressão grave, à rápida progressão da infecção e, principalmente, à ausência de um diagnóstico precoce e eficiente. Portanto, o diagnóstico na fase inicial da infecção é adequado, proporcionando uma terapia mais eficaz, o que pode reduzir a taxa de mortalidade da doença. Objetivo: o presente estudo teve em vista avaliar a aplicabilidadeda técnica de reação em cadeia da polimerase (PCR) no auxílio do diagnóstico de AI, em comparação com os resultados gerados pelo ensaio imunoenzimático de galactomanana (EIA-GM®), este já validado comercialmente. Métodos: foram analisadas 245 amostras de pacientes tratados no hospital Santa Casa de Belo Horizonte. Entre essas amostras, 16% (N = 39) foram positivos nos testes EIA-GM®. Em seguida, essas 39amostras positivas foram analisadas pela técnica de PCR. Resultados: de acordo com os resultados, a técnica de PCR apresentou taxa de 97,44% de sensibilidade, 97,96% de acurácia e 100% de especificidade, quando comparada ao método EIA-GM®. Conclusão:a técnica de PCR pode auxiliar no diagnóstico da AI, sempre associando os seus resultados à clinica do paciente e aos testes de imunoensaios.

Introduction: invasive aspergillosis (AI) is a serious fungal infection caused by species of the genus Aspergillus that primarily affects leukemic and diabetic patients and those recipients of stem cell transplants, which have neutropenia. The fungi spores that colonize the lung epithelium may invade the endothelial cell lining and vascular access and thus, spread to other organs through the blood. The high mortality of the disease is related tosevere immunosuppression, rapid infection progression, and especially lack of an early and efficient diagnosis. Therefore, the diagnosis in the initial infection phase is beneficial,providing a more effective therapy that can reduce the disease?s mortality rate. Objective:this study aimed at evaluating the applicability of the polymerase chain reaction (PCR) cheganin assisting the diagnosis of AI compared to the resultsgenerated by galactomannan enzyme immunoassay (EIA-GM®) that is already commercially validated. Methods: 245 samples from patients treated in the Santa Casa de Belo Horizonte hospital were analyzed. Among these samples, 16% (N = 39) were positive in EIA-GM®tests. Subsequently, these 39 positive samples were analyzedby PCR. Results: According to the results, the PCR technique showed 97.44% sensitivity, 97.96% accuracy, and 100% specificity compared to EIA-GM®. Conclusion:the PCR technique may aid in the diagnosis of AI,always associating the results to the patient's clinicaland immunoassay tests.

Detection of Bacterial and Viral Pathogens in Stool Specimens Using Multiplex PCR

BACKGROUND: The rapid and accurate detection of diarrheal pathogens is essential to prevent the spread of diarrheal diseases. Recently, a multiplex PCR assay was developed to simultaneously detect various bacterial and viral diarrheal pathogens. In this study, we investigated the frequency of detection of various potential pathogens causing diarrhea by using multiplex PCR and compared the results to the results of stool culture tests for bacteria and enzyme immunoassays (EIAs) for rotaviruses and Clostridium difficile toxin B (CDTB). METHODS: We retrospectively analysed the results for multiplex PCR, culture tests, and EIA obtained from stool specimens submitted to the laboratory from May 2013 to September 2014. Multiplex PCR was performed using the Seeplex diarrhea ACE detection kit (Seegene, Korea), which detects five viruses and eight bacteria. RESULTS: Among 890 stool specimens, 408 (45.8%) were found to be positive by PCR. The PCR positivity rate for bacteria and viruses was 31.1% (277/890) and 18.9% (161/890), respectively. The relative frequencies of microorganisms or toxins detected by PCR were, in decreasing order, CDTB 24.0%, Clostridium perfringens 20.6%, norovirus-GII 15.8%, rotavirus 11.3%, Campylobacter spp. 7.5%, enteric adenovirus 5.7%, and Salmonella spp. 5.1%. The concordance rate of the results obtained using the PCR and culture tests was 99.2% for Salmonella spp., 95.7% for Campylobacter spp., and. 79.8% for C. difficile . The concordance rates for rotaviruses and CDTB were 99.7% and 83.6%, respectively. CONCLUSIONS: The multiplex PCR method showed a high detection rate and is useful for the simultaneous detection of various diarrheal pathogens.

Estudo de homogeneidade dos soros utilizados em controle de qualidade interno de testes imunodiagnósticos de HIV/Aids / AidsHomogeneity study of the internal quality control sera for immunodiagnosis of HIV/AIDS

Introduction: The present study reports the data from the first homogeneity assessment of samples composing the serum panels produced at the Immunology Center of Instituto Adolfo Lutz, São Paulo. These samples have been distributed to the public laboratories and those partaking in the Brazilian Unified Health System, and to the participants in the Internal Quality Control Program for human immunodeficiency virus (HIV) antibody (Ab) testing. Objective: To assess the homogeneity of serum samples in panels from different lots for HIV/acquired immunodeficiency syndrome (AIDS) immunodiagnosis by using the statistical method to ensure quality of the reference material. Method: Sera homogeneity was evaluated by means of enzyme-linked immunoassay/enzyme immunoassay (ELISA/EIA) for detection of HIV Ab, and the one-way analysis of variance was employed for analyzing the data. No statistically significant differences were found among the several serum vials. Conclusion: The sera dispensed in the vials were homogeneous in the respective lots...

Introdução: No presente estudo estão descritos os resultados das primeiras análises feitas sobre a avaliação da homogeneidade das amostras componentes de painéis de soros produzidos no Centro de Imunologia do Instituto Adolfo Lutz e distribuídos aos laboratórios públicos e conveniados ao Sistema Único de Saúde e participantes do Programa de Controle de Qualidade Interno para imunodiagnóstico de vírus da imunodeficiência humana/síndrome da imunodeficiência adquirida (HIV/AIDS). Objetivo: Avaliar a homogeneidade das amostras de soro componentes de painéis de diferentes lotes para imunodiagnóstico de HIV/Aids por meio de método estatístico para garantir a qualidade do material de referência. Material e método: A homogeneidade das amostras de soro foi avaliada por meio de enzyme-linked immunoassay/enzyme immunoassay (ELISA/EIA) para detecção de anticorpos anti-HIV, e os resultados foram submetidos à análise de variância fator único. Não foram encontradas diferenças significativas entre os resultados obtidos para os diversos frascos de soro. Conclusão: As amostras distribuídas nos frascos foram homogêneas entre si nos respectivos lotes...

Comparison of nuclear grade and immunohistochemical features in situ and invasive components of ductal carcinoma of breast / Comparação do grau nuclear e perfil imunoistoquímico nos componentes in situ e invasivo de carcinoma mamário

PURPOSE:To compare the prognostic and predictive features between in situ and invasive components of ductal breast carcinomas. METHODS:We selected 146 consecutive breast samples with ductal carcinoma in situ (DCIS) associated with adjacent invasive breast carcinoma (IBC). We evaluated nuclear grade and immunohistochemical expression of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), cytokeratin 5/6 (CK5/6), and epidermal growth factor receptor (EGFR) in both components, in situ and invasive, and the Ki-67 percentage of cells in the invasive part. The DCIS and IBC were classified in molecular surrogate types determined by the immunohistochemical profile as luminal (RE/PR-positive/ HER2-negative), triple-positive (RE/RP/HER2-positive), HER2-enriched (ER/PR-negative/HER2-positive), and triple-negative (RE/RP/HER2-negative). Discrimination between luminal A and luminal B was not performed due to statistical purposes. Correlations between the categories in the two groups were made using the Spearman correlation method. RESULTS:There was a significant correlation between nuclear grade (p<0.0001), expression of RE/RP (p<0.0001), overexpression of HER2 (p<0.0001), expression of EGFR (p<0.0001), and molecular profile (p<0.0001) between components in situ and IBC. CK 5/6 showed different distribution in DCIS and IBC, presenting a significant association with the triple-negative phenotype in IBC, but a negative association among DCIS. CONCLUSIONS: Our results suggest that classical prognostic and predictive features of IBC are already determined in the preinvasive stage of the disease. However the role of CK5/6 in invasive carcinoma may be different from the precursor lesions.

OBJETIVO: Comparar características prognósticas e preditivas entre os componentes in situ e invasivo de carcinomas ductais da mama. MÉTODOS: Selecionamos 146 amostras mamárias consecutivas com carcinoma ductal in situ (CDIS) associado com carcinoma invasivo (CI) adjacente. Avaliamos grau nuclear e a expressão imunoistoquímica de receptor de estrogênio (RE), receptor de progesterona (RP), receptor do fator de crescimento epidérmico humano 2 (HER2), citoqueratina 5/6 (CK5/6) e o receptor do fator de crescimento epidérmico (EGFR) em ambos componentes, in situ e invasor, e a porcentagem de células marcadas pelo Ki-67 no componente invasivo. CDIS e CI foram classificados nos tipos moleculares, determinados pelo perfil imunoistoquímico, como luminal (RE/RP-positivo/HER2-negativo), triplo-positivo (RE/RP/HER2-positivo), HER2-puro (RE/RP-negativo/HER2-positivo) e triplo-negativo (RE/RP/HER2-negativo). A discriminação entre luminal A e Luminal B não foi feita por motivos estatísticos. Correlações entre as categorias dos dois grupos foram feitas pelo método de correlação de Spearman. RESULTADOS: Houve significante associação entre grau nuclear (p<0,0001), expressão de RE/RP) (p<0,0001), superexpressão de HER2 (p<0,0001), expressão de EGFR (p<0,0001) e perfil molecular (p<0,0001) entre os componentes in situ e invasivo. CK5/6 mostrou distribuição distinta em CDIS e CI, apresentando significante associação com o fenótipo triplo-negativo em CI, mas uma associação negativa ente os CDIS. CONCLUSÕES:Nossos resultados sugerem que as características prognósticas e preditivas clássicas dos CI estão já determinadas no estágio pré-invasivo da doença. Entretanto, o papel da CK5/6 no carcinoma invasivo pode ser diferente daquele das lesões precursoras.

Comparison the effect of individual donation NAT and minipool of 16 donations NAT / 中华检验医学杂志

ObjectiveTo investigate the effect of individual donation-nucleic acid amplification test (ID-NAT) and minipool of 16 donations-NAT (P16-NAT) on the results of NAT of blood donors.Methods From February 2009 to June 2009,samples randomly collected from voluntary blood donors in Beijing were tested individually or in pooling of 16 donations by the PROCLEIX ULTRIO assay.For ID-NAT reactive samples with HBsAg,anti-HCV,or anti-HIV serologically unqualified,ID-NAT repeat reactive samples with serologically qualified,and P16-NAT reactive and followed resolution ID-NAT reactive samples,were performed for further discriminatory assays for HIV-1,samples and followed resolution ID-NAT reactive samples,were performed for further discriminatory assays for HBV,HCV and HIV-1discriminatory reagents.Samples which were HBV NAT + alone with serologically qualified were further quantified and confirmed of HBV DNA by Roche HBV quantitative PCR,analyzed by HBV serology and were diluted to simulate if they could be detected in P16-NAT.Results ( 1 ) Among 7613 samples tested by ID-NAT,26 were NAT positive,i.e.the ID-NAT positive rate was 0.34％ ( 26/7613 ). ( 2 ) Among 1004 P16 samples from 16 064 blood donations,27 were NAT positive,i.e.the P16-NAT positive rate was 0.17％ (27/16 064).(3)In serological qualified donations,ID-NAT yield rate (1 in 826,9/7438 ) was much higher than P16-NAT ( 1 in 7875,2/15 750) (x2 =11.880,P ＜ 0.05 ).All these 9 ID-NAT positive and 2 P16-NAT positive donations were discriminated as HBV NAT positive.There were no HCV NAT yield or HIV NAT yield samples. (4) Dilution assay showed only 2 of the 9 (22.22％ ) ID-NAT HBV yields were detected by P16-NAT.(5)Eight ID-NAT and 2 P16-NAT positive samples were quantified for HBV DNA and confirmed as HBV NAT yield,although the virus loads were very low:2 samples had HBV viral loads of 15 IU/ml and 472 IU/ml,6 samples ＜ 12 IU/ml,and 2 could not be detected in the original samples while had ＜ 12 IU/ml and 14.3 IU/ml in the 10 times concentrated samples.(6)Among 11 HBV NAT yield cases,3 (27.3％ ) were possible HBV window-period donors with all HBV seromarkers negative,the other 8 (72.7％ ) had occult HBV infections with anti-HBc or anti-HBe positive,however anti-HBc IgM negative.(7) The rate of initial P16-NAT reactive pools needed to be further tested by ID-NAT was 2.49％(25/1004).Initial P16-NAT reactive pools which caused by serologically qualified donations was 0.20％(2/1004).ConclusionsHBV NAT yield cases are detected at a higher frequency with ID-NAT than P16-NAT.In order to avoid samples with low viral loads would be undetected,NAT assay with high sensitivity should be selected and tested in minimized minipool donations or even with individual donation.

Rotavirus infection in a tertiary hospital: laboratory diagnosis and impact of immunization on pediatric hospitalization

BACKGROUND AND OBJECTIVES: Rotavirus (RV) is the main etiological agent of diarrhea in childhood; its laboratory diagnosis is crucial to guide the clinical management and prevention of its spread. RV immunization was introduced in Brazilian 6-month-old children in 2006. The present study was aimed to evaluate three methodologies used for human RV detection in stool samples obtained from patients hospitalized due to gastroenteritis in a teaching hospital and report the impact of RV immunization in hospitalization by diarrhea. METHODS: 293 stool samples collected in the 2001-2008 period were analyzed by enzyme immunoassay (EIA), latex agglutination (LA) and polyacrylamide gel electrophoresis (PAGE). RESULTS: Rotavirus was detected in 34.8 percent of samples by LA assay, 28.3 percent of samples by EIA assay and in 25.6 percent of samples by PAGE assay. Considering the PAGE method as gold standard, the sensitivity, specificity and accuracy of EIA were 94.6 percent, 94.4 percent and 94.5 percent, and to LA were 82.6 percent, 81.6 percent and 81.9 percent, respectively. CONCLUSION: These results indicate that antigen detection by EIA is a rapid, sensitive and specific method, and could be used in large-scale applications for screening stool samples suspected of RV infection. This study showed decreased incidence of RV infection in hospitalized children prior to the implementation of the national immunization program against RV.

The application value of T-cell spot of tuberculosis test in differential diagnosis of Crohn's disease and intestinal tuberculosis / 中华消化杂志

Objective To evaluate the value of T-cell spot of tuberculosis test (T-spot.TB) in differential diagnosis of Crohn's disease and intestinal tuberculosis.Methods From May 2010 to October 2010,in Xijing hospital,Fourth Military Medical University,the peripheral blood samples of 126 patients were collected and peripheral blood mononuclear cells were isolated with density gradient centrifugation.T-spot.TB was conducted according to the kit instructions.The clinical diagnosis of Crohn's disease and intestinal tuberculosis was according to clinical manifestations, imaging,endoscopy,pathology,laboratory tests and on empirical anti-TB treatment response.The sensitivity and specificity of T-spot.TB in diagnosis of Crohn's disease and intestinal tuberculosis was analyzed.Results Fifteen patients were diagnosed as Crohn's disease (11.9％,15/126),14 patients were intestinal tuberculosis (11.1％,14/126) and 40 patients were extraintestinal tuberculosis (31.7％,40/126).The positive rate of T- spot.TB in Crohn's disease,intestinal tuberculosis,extra-intestinal tuberculosis and other diseases was 1/15,12/14,70％ (28/40) and 0％ (0/57),respectively.Thedifference between the groups was statistically significant (P =0.00).There was statistically significant difference of T-spot.TB positive rate between Crohn's disease and intestinal tuberculosis (x2 =70.58,P=0.00).The sensitivity and specificity of T- spot.TB in Crohn's disease detection was 93.3％(14/15) and 87.5％(14/16),in intestinal tuberculosis was 85.7％(12/14) and 93.3％ (14/15).The negatively predictive value of Crohn's disease was higher [87.5％ (14/16)] than that of intestinal tuberculosis [12.5％ (2/16)].Conclusion T-spot.TB is helpful for differential diagnosis of Crohn's disease and intestinal tuberculosis.

The auxiliary diagnosis value of enzyme-linked immunosorbent spot assay and flow cytometry in detection of IFN-γ in tuberculous pleurisy / 中华检验医学杂志

Objective To explore the value of IFN-γ produced or secreted by CD+4 T Lymphocytes from pleural effusion mononuclear cells for the diagnosis of tuberculous pleurisy(plTB).Methods The PEMCs of 40 patients with tuberculous pleural effusion and 30 patients with malignancy pleural effusion were selected as the tuberculosis and disease control groups, then co-cultured with the early secretory antigenic target 6 (ESAT-6) and culture filtered protein 10 (CFP-10) fusion protein (E/C).The numbers of spot forming cells(SFC) secreting IFN-γ were enumerated by ELISpot and the ratios of cells producing IFN-γ were detected by flow cytometry and intracellular cytokine staining.Moreover, the two indicators were compared between tuberculosis and disease control groups to evaluate the 2 methods detecting IFN-γ in the diagnosis of plTB.Results After E/C stimulation, the numbers of SFC were 205(125-450)SFC/5×104 PEMC in tuberculosis group and 5(2-18)SFC/5×104 PEMC in disease control group by ELISpot.The difference between two groups was statistically significant (U= 20.00, P<0.01).The proportion of IFN-γ-secreting CD+4 T lymphocytes was 3.27% (1.81%-7.34%) in tuberculosis group and 0.12% (0.06%-0.46%) in control group detected by FCM. The difference between the two groups was statistically significant (U=45.00, P<0.01).The indicators of ELISpot in detection of IFN-γ which was secreted by PEMC after co-cultured with E/C were as follows: sensitivity 92.5% (37/40), specificity 80.0% (24/30), positive predictive value 0.86, negative predictive value 0.89, positive likelihood ratio 4.63, negative likelihood ratio 0.09 and accuracy 87.1%;and for FCM, they were 87.5% (35/40), 90.0% (27/30), 0.92, 0.84, 8.75 and 0.14, respectively and accuracy 88.6%.Conclusion After E/C stimulation, the assay for IFN-γ-secreting CD+4 T lymphocytes by FCM and ELISpot is highly sensitive and specific for diagnosis of plTB as an auxiliary method.

Comparison of an in-house tuberculosis-specific IFN-γ release assays with T-SPOT TB in latent tuberculosis infection diagnosis among HIV-infected individuals / 中华检验医学杂志

Objective To evaluate the diagnostic value of two tuberculosis-specific IFN-γ release assays in latent tuberculosis infection among HIV-infected individuals. Methods The levels of tuberculosis antigen-specific IFN-γin 102 HIV patients from AIDS Outpatient Clinic of Shenzhen Third People's Hospital were detected by in-house tuberculosis-specific IFN-γ ELISpot assay and commercial T-SPOT TB kit, and tuberculin skin test (TST) were done at the same time. There were 66 males and 36 females,and the average age was 35. Results Seventeen HIV infected patients were positive in both IFN-γ ELISpot and T-SPOT TB methods, the sensitivity, specificity positive predictive value(PPV), negative predictive value(NPV) and compliance rates of ELISpot were 94. 4% ,94. 0% ,77. 3% ,98. 8% and 94. 1% ,respectively. Three patients were positive in both IFN-γELISpot and T-SPOT TB methods, the sensitivity, specificity, PPV, NPV and compliance rates of TST were 16. 7%, 98. 8%, 75.0%, 84. 7% and 84. 3%, respectively. The average number of spots using three kinds of antigen ESAT-6, Pool A,Pool B obtained were 26. 89 ±5. 77,18. 96 ±4. 75 and 14. 51 ± 3.77, respectively. Only ESAT-6 and Pool B have a statistically significant difference (H=7.557,P = 0.022 9), no significant difference was shown between other groups. There was no significant difference between the positive rate and the CD4+ T cellls number(x2 =0. 860 8 ,P =0. 650 2) ,as the same as the T-SPOT TB (x2 = 1. 396 4, P = 0. 497 5 ). Conclusions The performance of this in-house tuberculosis-specific IFN-γ ELISPot assay was comparable to T-SPOT assay in diagnosis of latent tuberculosis infection, and the sensitivity and specificity of both these two assays were all much higher than TST. They canbe recommended in diagnosing latent tuberculosis infection in HIV infected patients.

Cross-genome HIV-specific cytotoxic T-lymphocyte responses among HIV-1 infected individuals with varied infection time and routes / 中华检验医学杂志

Objective To investigate and compare the features of the HIV-1-specific CTL responses among three HIV-infected groups with varied infection history. Methods Three HIV-infeeted groups were enrolled in this study, including two groups infected by blood transmission (one group has been infected for more than 10 years and the other for 1-2 years) and one group of the man who have sex with man. The HIV-1-specific CTL responses were quantified by an IFN-γ based ELISPot assay with a peptide matrix system containing overlapping peptides spanning the entire HIV-1 Clade B genomic consensus sequences. Results The responding rate of CTL responses against all 17 peptide pools among the group that infected 1-2 years,the group infected more than 10 years and the group of MSM were 40% ,65% ,23%. One way ANOVO analysis showed that the responding rate of CTL responses against all 17 peptide pools were statistical significant among the three groups (F=19.96, P＜0.01);the magnitude of CTL responses of the three groups were 0-5 835 SFCs/106 PBMC, 0-7 225 SFCs/106PBMC, 0-9 740SFCs/106pBMC, Kruskal-Wallis test showed that the magnitude of CTL responses were statistical significant among the three groups( H = 101.90 , P ＜0.01);the breadth of CTL were 7 ( 2-11 ), 11(9-14) and 4 (2-6) respectively and Kruskal- Wallis test showed that the breadth of CTL had no statistical significant among the three groups( H = 34. 75 ,P ＜0. 01 ). The sequence of responding rate, magnitude and breadth of CTL from high to low was the group that had been infected for more than 10 years, the group infected 1-2 years and the sex transmission group. The common characteristics of the CTL response among the three groups were that the responding rate and the magnitude of the peptide Nef and Gag was higher than other peptide's. The magnitude of CTL responses among three different CD4count groups (CD4 ＜ 200/μl, CD4 200-500/μl, CD4 ≥500/μl,) was 0-18 475 SFCs/106pBMC, 350-34 095 SFCs/106pBMC, 490-21 550 SFCs/106 PBMC and had no statistic difference among the three different CD4 groups(H=2.93, P=0.23) while the breadth of CTL was 3(0-8), 10(2-17), 10 (1-17)respoctively and the breadth of CTL was lower in the group of CD4 count less than 200/μl than the other two groups( H = 14. 72, P ＜ 0. 01 ). The magnitude of CTL responses among three different viral load (VL)groups (VL＜ LDL, LDL ＜ VL ＜ 1 × 104 copys/ml, VL≥1 ×104 copys/ml) was 490-18 475 SFCs/106pBMC, 0-24 115 SFCs/106pBMC, 770-34 095 SFCs/106 pBMC and had no statistic difference among the three different viral load groups ( H = 0.79, P=0.67) and the breadth of the three different viral load groups CTL was 8( 1-17), 11 (0-17), 8 (1-16) and Kruskal-Wallis test showed that there was no statistic difference among the three different viral load groups (H =5.27, P =0. 07). Conclusions All groups predominantly develope T cell immune responses against Nef and Gag proteins. With the elapse of HIV infection, the CTL responses are increased in both magnitude and responding rate. This information is important for vaccine development.

Evaluation and detection of CCP antigen-specific T cells by ELISpot assay in patients with rheumatoid arthritis / 中华检验医学杂志

Objective To optimize and establish ELISpot assay for CCP/AST which could secrete IFN-γ and IL-4, and explore the role and clinical significance of CCP/AST cells in occurrence and development of RA disease. Methods CCP was used as specific-stimulator with FLAG peptide as a control,the frequencies of positive SFC which could specifically secrete IFN-γ and IL-4 in 64 cases of RA, 64 cases of non-RA autoinunune diseases and 30 cases of healthy individuals were tested by ELISpot technique. The diagnostic value of CCP/AST cells was evaluated in patients with RA disease. Meanwhile, the relationships among the indexes above and patient joint symptoms as well as other laboratory parameters were further analyzed and discussed. Results The results showed that the mediam numbers of IFN-γ-SFC and IL-4-SFC were 39(12-77)/3 x 105 PBMC and 1 (1-3)/3 × 105 PBMC in RA patients, the positive rates were 81.3% and 18. 8% respectively. The median value of IFN-γ-SFC/IL-4-SFC ratio was of 15(5-39), the positive rate was of 78. 1%. Both IFN-γ-SFC and ratio of IFN-γ-SFC/IL-4-SFC were significantly higher than those of non-RA diseases (Z = - 7. 458, - 7. 019, P < 0. 01 ) and healthy control ( Z = - 6. 643, - 5. 760, P <0. 01 ), also both these parameters in RA patients with positive anti-CCP antibody and negative anti-CCP antibody were significantly higher than those patients with systemic lupus erythematosns ( Z = - 6. 573, - 6. 098, - 4. 552, - 4. 726, P < 0. 01 ), ankylosing spondylitis ( Z = - 3. 520, - 3. 326, - 2. 950,-2. 126, P<0. 01 or 0. 05), other autoimmune diseases (Z = -4. 838, -4. 418, - 3. 681, -3. 839,P < 0. 01 ) and healthy controls ( Z = - 6. 553, - 5. 578, - 4. 635, - 4. 163, P < 0. 01 ). Combining IFN-γ-SFC, IL-4-SFC with IFN-γ-SFC/IL-4-SFC for RA diagnosis, the area under curve of receiver operating characteristic( ROCAUC) and Youden index were 0. 910 and 0. 747. The diagnostic sensitivity and specificity were 87. 5% and 87.2%. Positive and negative predictive values were 82. 4% and 91.1%, respectively. Correlation analysis showed that ratio of IFN-γ-SFC/IL-4-SFC was not only significantly correlated with antiCCP antibody(r =0.393, P <0.01), but also correlated with joint symptoms in patients such as with number of joints swelling-pain ( r = 0. 429 , P < 0. 01 ), number of joints damage ( r = 0. 463, P < 0. 01 ),rheumatoid factor (r = 0. 166, P < 0.01) and erythrocyte sedimentation rate (r=0. 199,P<0.05).Conclusions There is widely existence of CCP/AST cells activation and abnormity in RA patients,indicating higher frequency of CCP-specific Th1 cells. These results provides a new experimental evidence for an objective understanding the function of specific cellular immune responses and cytokine network regulation mediated by citrullinated proteins in RA. So, the assay for CCP/AST cells has potential values in RA diagnosis and clinical application.

Retrospective analysis of hepatitis B virus serological markers in 70 582 inpatients / 中华检验医学杂志

Objective To analyze the detection rate of HBV serological makers in non-hepatic inpatients in the past six years. Methods Serum samples of 70 582 non-hepatic inpatients from three large hospitals were collected during 2003 to 2008. Serological markers of HBV ( HBsAg, anti-HBs, HBeAg, antiHBe and anti-HBc) were detected by the AxSYM MEIA system (Abbott Laboratories,Abbott Park,IL).Combining the test results of serological makers with other clinical data, several analysis models for this retrospective study were set up to evaluate the year-to-year changes in serological makers and the detection rates of each model. Results The order from high to low of detection rate of the 5 HBV serological markers was anti-HBc (55. 17% ), anti-HBs (49. 57% ), anti-HBe (28.42%), HBsAg ( 8. 92% ) and HBeAg (2. 12% ), and all of them had a downward trend in the past six years. The positive rate of HBsAg went down from 9. 30% (2003) to 8.70% (2008). The positive rate of HBsAg among people who were born after 1992 (2. 28% ) were significantly lower than that of the overall population (8. 92% ) and fell from 3.57%(2003) to 1.85% (2008). Each detection rate of all serological makers had male sexual side effect [HBsAg ( 12. 38%/7. 25% ), HBeAg ( 2. 72%/1.58% ), anti-HBc ( 56. 57%/53.43% ), anti-HBe (41.50%/28. 35% ) and anti-HBs (65.48%/50. 00% ), male/female]. The differences were statistically significant (Chi-square values of HBsAg, HBeAg, anti-HBc, anti-HBe and anti-HBs were 509.74,105.78, 69.66, 1 321.61 and 1 726.91, respectively; all P ＜ 0. 01).Twenty-six models of HBV serological makers from 70 582 inpatients were summed up, and 8 models had positive rates geater than or equal to 1%. The "All Negative" model ranked No. 1 and had no significant change from year to year. During the past six years, models representing "A11 Negative" and "anti-HBs Positive alone" were mainly in individuals younger than or equal to 20-year-old, while the models representing "anti-HBc and/or anti-HBe,anti-HBs Positive" were mostly in people older than 20-year-old. The distribution curve of models representing "HBsAg, HbeAg and anti-HBc Positive" and "HBsAg, anti-HBc, anti-HBe Positive"etc. showed a bell-shape, covering the population from 20-year-old to 70-year-old. Conclusions The slowlydescending tendency of the detection rates of HBV serological makers was observed during the past six years.The detection rates of HBV in the younger generation decreased significantly. However, the HBV infection rates of overall population is still high, so it is a high time that we made continuous improvement for the serum HBV screening technique in order to reduce the HBV infection ratess.

Comparison of five methods used for detection of Clostridium difficile infection / 中华检验医学杂志

Objective To evaluate five detection methods for the laboratory diagnosis of Clostridium difficile infection in the hospitals of USA, and explore a sensitive, specific, accuracy and rapid regimen for the early diagnosis of Clostridium difficile infection. Methods A total of 174 stool specimens submitted to the clinical microbiology laboratory for Clostridium difficile testing were separately tested by five methods including toxigenic culture (TGC), Premier Toxin A&B EIA( A/B-EIA), C. Diff Quick Chek Complete( DEIA), BD G eneOhm Cdiff assay(BD-PCR) and Laboratory-developed PCR(LD-PCR). The gold standard of TGC was used as a reference criterion, and the sensitivity, specificity, positive predictive value ( PPV )and negative predictive value (NPV) of A/B-EIA, D-EIA, BD-PCR and LD-PCR assays were determined. Results Among the 174 specimens studied, 24 were defined as true positives for Clostridium difficile infection by TGC assay, giving a positive rate of 13.8% (24/174). In comparison to the standard,the sensitivity, specificity, PPV and NPV were 62.5%, 99.3%, 93.8% and 94.3% for A/B-EIA;66.7%, 98.7%, 88.9% and 94.9% for D-EIA; 83.3%, 98.7%, 90.9% and 97.4% for BD-PCR;79.2%, 93.3%, 65.5% and 96.6% for LD-PCR. Among all tested specimens, 34 were positive by atleast one of five methods, and of which 15 were concordant by all five methods. The D-EIA results were divided into three groups depending on results of GDH and (or) toxins A/B: 18 were positive for both GDH and toxins A/B, 23 were positive for only GDH, and 133 were negative for both GDH and toxins A/B. Of 18 positive specimens by D-EIA assay, all were concordant with results of BD-PCR assay and 16 were agreement with results of TGC assay. Twenty-two of 24 positive specimens by TGC assay were included in 41 specimens that were positive for GDH. Among eight false negative specimens by D-EIA assay, four were differentiated as positive results by BD-PCR. According to the present study, the sensitivity, specificity,PPV and NPV of a two-step detection algorithm in combination with D-EIA and BD-PCR assays were 83.3%, 98.7%, 90.9% and 97.4%, respectively. Conclusions From the point of technological evaluation, BD-PCR is preferable. A two-step detection algorithm combining D-EIA with BD-PCR is proposed for the laboratory diagnosis of Clostridium difficile infection. This algorithm has demonstrated an excellent sensitivity and specificity, as well as decreased test turnaround time and test cost.

The value of the enzyme-linked immunospot assay detecting spot forming cells in the diagnosis of latent tuberculosis infection / 中华检验医学杂志

stinguish active tuberculosis and healthy cases with tuberculosis exposure history according SFC count.

Comparative assay of diagnostic value of Mycobacterium tuberculosis specific cellular immune responses assays and antibody test / 中华检验医学杂志

T-6 specific IFN-γ ELISPOT has higher specificity, sensitivity, the positive and negative predicative value. Therefore, the ELISPOT warrant for further improvement and clinical application.

Secreted expression of recombinant human autoantigen Sm B' in methylotrophic yeast Pichia Pastoris / 中华检验医学杂志

Objective To clone and express human autoantigen Sm B'in methylotrophie yeast Pichia Pagtoris.Methods The gene Sm B' was cloned bv PCR The PCR product wag inserted into the vector pPIC9k.The recombinant plasmid pPIC9k.Sm B' was transformed into yeast Sm D1168 by electroporation.The positive clones were screened in MD plates.The high copy number transformants were rapidly selected by using G418 and were induced by methan01.Supematants after induction were analyzed by SDS-PAGE and western blot.Sera collected from thirty patients with SLE.thirty patients with mixed connective tissue disease(MCTD)and thirty healthy volunteers were detected by immunodot and immunoblot.Results The PCR product wag about 700 bD in size which Wag in accordance with predicted 657 bp.The pPIC9k-Sm B'showed the same seqencing result with GenBank's report and restriction enzyme analysis confirmed our prediction.The pPIC9k-Sm B' positive clone produced a 32 000 protein which had natural immunogenicitv of human autoantigen Sm B'by SDS-PAGE and western blot.The positive rate of immunodot and IBT were 46.7%(42/90)and 51.1%(46/90),respectively.The agreement between immunodot and IBT was very close(Kappa value=0.911 2,P＜0.01).Conclusion Successfully cloning and expression of human autoantigen Sm B' in methylotmphic yeast Pichia Pagtoris hid a foundation for further research work.