Surface-activated microtiter-plate microarray for simultaneous CRP quantification and viral antibody detection

Microarrays are widely used in high-throughput DNA and RNA hybridization tests and recently adopted to protein and small molecule interaction studies in basic research and diagnostics. Parallel detection of serum antibodies and antigens has several potential applications in epidemiologic research, vaccine development, and in the diagnosis of allergies, autoimmunity, and infectious diseases. This study demonstrates an immobilization method for immunoassay-based microarray in conventional 96-well polystyrene plates for a serologic diagnostic method combined with quantitative C-reactive protein (CRP) assay. A synthetic peptide (HIV-1), a recombinant protein (Puumala hantavirus nucleocapsid), and purified virus preparations (Sindbis and adenoviruses) were used as antigens for virus-specific antibody detection and monoclonal anti-CRP antibody for antigen detection. The microarray was based on conventional enzyme immunoassays and densitometry from photographed results. Peptide and recombinant antigens functioned well, while whole virus antigens gave discrepant results in 1 out of 23 samples from the reference method, tested with human sera with various antibody responses. The CRP results were in concordance in the concentration range 0.5–150 mg/L with 2 commercially available CRP assays: ReaScan rapid test (R² = 0.9975) and Cobas 6000 analyzer (R² =0.9595). The results indicate that microtiter plates provide a promising platform for further development of microarrays for parallel antibody and antigen detection.     

Egy-Score Predicts Severe Hepatic Fibrosis and Cirrhosis in Egyptians With Chronic Liver Diseases: A Pilot Study

Background

Non-invasive methods for assessment of hepatic fibrosis are increasingly needed. Recent studies showed that combined elevation of tumor markers CA 19-9 and CA 125 is predictive of severe hepatic fibrosis or cirrhosis with high specificity.

Objectives

We aimed at developing a new panel of surrogate biomarkers for prediction of the stage of hepatic fibrosis by combining tumor markers with other known biomarkers of hepatic fibrosis.

Patients and Methods

A total of 92 patients with different types of chronic liver diseases (chronic hepatitis B, chronic hepatitis C and autoimmune hepatitis), were prospectively enrolled in our cohort. They were subjected to: ALT, AST, GGT, ALP, total bilirubin, INR, total cholesterol, albumin, platelet count, cancer antigen 19-9 (CA 19-9), cancer antigen 125 (CA 125), cancer antigen 15-3 (CA 15-3), haptoglobin, alpha-2-macroglobulin, apolipoprotein A1, abdominal ultrasound, liver biopsy and histological staging of hepatic fibrosis using the METAVIR system.

Results

Combined elevation of CA 19-9 and CA 125 with a summated value > 37 U/mL is predictive of severe hepatic fibrosis or cirrhosis (stage F3-F4 METAVIR) with a probability of 77.6%. Multivariate analysis showed that the most relevant collection of biomarkers for prediction of stage of hepatic fibrosis is: CA 19-9, age, alpha-2- macroglobulin, total bilirubin, platelet count & albumin. We developed a new score, named the “Egy-Score”, using a regression equation composed of this panel of biomarkers. Egy-Score could differentiate no or early fibrosis (stage F0-F2 METAVIR) from severe fibrosis or cirrhosis (stage F3-F4 METAVIR) with 83.7% accuracy.

Conclusions

Non-invasive assessment of hepatic fibrosis could be done using the Egy-Score. Egy-Score could differentiate no or early fibrosis (stage F0-F2 METAVIR) from severe fibrosis or cirrhosis (stage F3 - F4 METAVIR) with 83.7% accuracy.     

抗体筛查细胞抗原涵盖范围与电子配血技术的安全应用

目的探讨抗体筛查细胞抗原涵盖范围在保障电子配血技术安全应用中的意义。方法分别对供受血者进行AB0／RhD血型检测以及抗体筛查。患者二次血型鉴定结果一致,抗体筛查结果阴性,按电子配血规则进行电子配血,同时采用凝聚胺法对供受血者进行传统血清学交叉配血平行试验,分析抗体筛查细胞抗原涵盖范围与不规则抗体漏检情况以及对电子配血技术安全应用的影响。结果22790份患者血样,抗体筛查阳性164例,阳性率0．72％。15740份供血者血样,抗体筛查阳性11例,占0．07％。22540份血样均符合电子配血规则,由计算机实施电子配血未发现AB0／RhD血型不相容。用凝聚胺法进行传统血清学交叉配血平行检测,其中22534份血样配血相容,6份血样凝聚胺法交叉配血主侧不相容,未发现次侧不相容者。6份血样用谱细胞进行抗体特异性鉴定,结果全为MNS系统抗一Mur,漏检率为0．027％。结论目前临床所用抗体筛查细胞仅为血清学交叉配血而设计,抗体漏检率相对较高,不能充分保障电子配血技术的安全性。建议尽快制定电子配血技术标准,保障其在临床的推广应用。     

Polyphenolic antioxidants inhibit peptide presentation by antigen-presenting cells

Antigen-presenting cells (APC) provide two essential signals, e.g., antigenic peptides as well as costimulatory molecules for T-cell activation. Small molecules of smoke tobacco extracts (SM-STE) inhibited antigen presentation of A20 to OVAp-specific T-cell hybridomas. Pretreatment of A20 but not T hybridomas abrogates the APC function. Viability of APC and levels of MHCII, CD40 and B7 of APC were not affected by this treatment. The active principle, inhibiting APC was reproduced with pure tobacco polyphenols, quercetin and its glycoside, rutin. Antioxidant activity of rutin is relevant since rutin downregulated levels of reactive oxygen species (ROS) in phorbol ester-stimulated A20; moreover, another antioxidant, N-acetyl cysteine (NAC) also inhibited antigen presentation, albeit at a higher concentration. Other types of APC, such as bone marrow-derived mast cells (BMMC), MHCII-transfected fibroblast, and splenocytes are affected by tobacco polyphenols. We propose that polyphenols may affect redox-sensitive signal transduction pathway since APC function of PD 98059, MEK inhibitor-pretreated A20 were similarly abrogated. Taken together, we propose that maintaining appropriate intracellular redox of APC is crucial for its antigen-presenting function.     

SPECIFIC CYTOTOXICITY OF ANTIBODY TO YAL-198, A SPERM ANTIGEN PEPTIDE,TO MURINE ZYGOTE

Active immunization with the peptide segments rSMP-230 and YAL-198, corresponding to the hydrophilic extracellular domain of two human sperm antigens (rSMP-B and YWK-II, respectively), reduced fertility in female rats by different mechanisms. The anti-rSMP-230 antibody interferes with human and murine fertilization, and the anti-YAL-198 antibody blocks the development of mouse embryos. The authors examined in vitro at which stage the antibodies to rSMP-230 and YAL-198 were cytotoxic to murine embryos up to morula/blastocyst stage. Anti-rSMP-230 antibody was not cytotoxic to any stages. On the other hand, the anti-YAL-198 antibody arrested the growth of embryos at the 2-cell stage but not at more advanced developmental stages. When the anti-YAL-198 antibody was used, spotty staining was observed only on the surfaces of embryos that had arrested at the 2-cell stage. Unstained embryos, however, continued to develop normally. In contrast, the anti-rSMP-230 antibody stained murine sperm but failed to stain murine ova and embryos. The present results suggest that the human sperm components rSMP-B and YWK-II play important roles in sperm-egg interaction and early development of the embryo, respectively.     

大疱性系统性红斑狼疮的皮肤基底膜带相关抗原

免疫印迹和盐裂皮肤间接免疫荧光检测 ５例大疱性系统红斑狼疮（ＢＳＬＥ）血清，对照为 ５例获得性大疤性表皮松解症（ＥＢＡ）、２０例类天疱疮（ＢＰ）、２０例ＳＬＥ和１０例正常人血清。结果表明，３例（３／５）ＢＳＬＥ血清结合盐裂皮肤真皮侧和真皮提取物中２９０ ０００抗原，其中２例ＢＳＬＥ血清也结合表皮提取物中 １６５ ０００抗原，结果与 ＥＢＡ和部分ＢＰ血清相同。ＳＬＥ血清未结合 ２９０ ０００和 １６５ ０００抗原。提示ＢＳＬＥ血清中存在ＥＢＡ和ＢＰ抗体，推测ＥＢＡ和ＢＰ抗原可能是ＢＳＬＥ的皮肤基底膜带相关抗原。     

弗氏佐剂与抗原乳化方法的改良及其应用

目的改良弗氏佐剂与抗原的乳化方法并验证其效果。方法以微型电钻做为动力源,自制搅拌头,组装成佐剂搅拌器。利用此搅拌器乳化乙脑E蛋白和CFA/IFA的混合物,滴水实验、离心实验及琼脂糖双向扩散实验检测乳化效果。结果利用自制佐剂搅拌器制备出乙脑E蛋白和CFA/IFA的乳化剂,进行滴水实验,乳化剂滴于冰水上5～10min内完全保持完整不分散,成滴状浮于水面;进行离心试验,将乳化剂置于离心机中以3000r/min离心10min,未见分层现象;利用此乳化剂制备的免疫血清进行琼脂糖凝胶双向扩散实验,血清1︰16稀释后仍能观察到沉淀线出现。结论自制佐剂搅拌器制作简便,价格低廉,制备乳化剂时,混匀所需时间短,操作简单,安全高效,乳化效果好,为研究工作提供了便利,是一种值得推广的方法 。     

广州国境口岸出入境人员黄热病病毒血清学检测及感染风险预测

黄热病是由黄热病病毒引起的蚊媒传染病,主要发生在非洲亚撒哈拉地区和南美洲热带地区,是我国《国境卫生检疫法》规定的3种检疫传染病之一.为了解广州国境口岸入境人员黄热病病毒感染情况,本研究进行了黄热病毒抗原、抗体检测及其感染风险预测.     

New insights in mucosal vaccine development

Mucosal surfaces are the major entrance for infectious pathogens and therefore mucosal immune responses serve as a first line of defence. Most current immunization procedures are obtained by parenteral injection and only few vaccines are administered by mucosal route, because of its low efficiency. However, targeting of mucosal compartments to induce protective immunity at both mucosal sites and systemic level represents a great challenge. Major efforts are made to develop new mucosal candidate vaccines by selecting appropriate antigens with high immunogenicity, designing new mucosal routes of administration and selecting immune-stimulatory adjuvant molecules. The aim of mucosal vaccines is to induce broad potent protective immunity by specific neutralizing antibodies at mucosal surfaces and by induction of cellular immunity. Moreover, an efficient mucosal vaccine would make immunization procedures easier and be better suited for mass administration. This review focuses on contemporary developments of mucosal vaccination approaches using different routes of administration.     

The next generation recombinant human cytomegalovirus vaccine candidates—Beyond gB

Human cytomegalovirus (HCMV) infects the majority of the global population and persists within the infected host for life; infection of healthy adults rarely leads to severe acute clinical symptoms. In contrast, HCMV is a leading infectious cause of congenital disease and a common cause of complications in transplant recipients. A vaccine to prevent HCMV disease in these populations is a widely recognized medical need. We review recent advances in our understanding of the candidate vaccine antigens and published clinical trial data for the four most recent HCMV vaccine candidates: a gB subunit adjuvanted with MF59, a DNA vaccine expressing gB and pp65, alphavirus replicon particles (VRPs) expressing gB and a pp65–IE1 fusion protein, and a pp65 peptide vaccine. The candidates are safe, although some adverse events were reported for an adjuvanted variant of the pp65 peptide vaccine. The gB/MF59 vaccine elicited strong humoral responses with limited durability. The gB/pp65 DNA vaccine elicited cellular immunity, and the pp65 peptide vaccine elicited modest cellular immunity, but only when formulated with an adjuvant. Only the VRP vaccine expressing gB and pp65–IE1 elicited both humoral and cellular immunity. The gB/MF59 vaccine showed a short-term 50% efficacy at preventing infection of seronegative women and significantly reduced viremia and need for antivirals in solid organ transplant recipients, and the gB/pp65 DNA vaccine showed signs of clinical benefit in hematopoietic stem cell transplant recipients. Importantly, the partial efficacy of the subunit and DNA vaccines is new evidence that both humoral and cellular immunity contribute to controlling HCMV-related disease. These data show the clinical feasibility of a recombinant HCMV vaccine. We discuss areas for potential improvements in the next generation of vaccine candidates.