Increased specificity of diagnostic tests with recombinant major bee venom allergen phospholipase A2

Background In diagnosis of type I allergy recombinant allergens have potential advantages over conventional allergenic extracts, both regarding specificity and reproducibility. Objectives We therefore decided to study honey bee venom (BV) and its major allergen phospholipase A2 (PLA) in native and recombinant form for diagnosis of bee sting allergy. Method We investigated 85 patients with a history of a recent systemic allergic bee sting reaction and positive intracutaneous skin test to BV, and 21 controls with no history of allergic bee sting reaction and negative skin test to BV. Intracutaneous skin tests and determination of specific IgE by ImmunoCAP^R to BV, native PLA (nPLA) and recomhinant PLA (rPLA) were done in all patients and controls. Results In skin testing 84 (99%) of the 85 patients reacted to nPLA and 81 (95%) to rPLA, while none of the 21 controls was positive with nPLA or rPLA. Specific serum IgE to BV could be detected in 82 of the patients (96%), to nPLA in 73 (86%) and to rPLA in 66 (78%). Four (19%) of the controls had a positive CAP to BV, one (4.8%) to nPLA and none to rPLA. Analysis of discordant results in CAP showed, that most patients with specific IgE to BV, but not to nPLA and rPLA, had positive skin tests to both PLA preparations and low levels of BV specific IgE. Patients with specific IgE to nPLA but not to rPLA were usually sensitized to minor allergens of BV which contaminated the commercial nPLA. Conclusions PLA is the major allergen in BV. While diagnostic tests with BV are more sensitive, the specificity of tests with PLA, especially rPLA is clearly increased as compared with BV.     

Antigenic specificity of recombinant polypeptides, containing fragments of hepatitis E virus proteins]

Antigenic specificity of recombinant polypeptides HE40 and HE60 containing fragments of gene ORF2 and ORF3 protein products of hepatitis E, strain Burma, produced in E. coli cells, is analyzed. Blood sera from patients with acute hepatitis from an endemic region in Uzbekistan were tested for IgG to recombinant antigens by solid-phase enzyme immunoassay with a protein fragment coded by PRF3 gene, a synthetic peptide previously characterized in a commercial test system, as the positive control. 93% sera reacting with recombinant polypeptide HE60 and 32% reacting with HE40 reacted with the synthetic peptide. No antibodies to the studied polypeptides were detected in the sera of Moscow patients with hepatitis A, B, or C confirmed by laboratory findings. Antigenic specificity of recombinant polypeptide HE60 was confirmed by competitive enzyme immunoassay with the same peptide as the competitive antigen. Test system based on recombinant polypeptides HE40 and HE60 was used for deciphering the etiological structure of acute viral hepatitis which occurred in a hepatitis E endemic region of Uzbekistan in 1993.     

肿瘤坏死因子和白细胞介素2是对N-连接的寡糖特异的凝集素

采用固相结合试验证实:(1)rTNF-α和 rIL-2能以高度亲和力与多种含 N-连接寡糖的糖蛋白结合,其结合活性依赖于糖蛋白的糖基组分;(2)rTNF-α能识别各种类型的 N-连接糖链,而 rIL-2只能与高甘露糖型的 N-连接糖链结合;(3)甘露三糖特导地抑制 rTNF-α和 rIL-2与糖蛋白的结合;(4)rTNF-α和 rIL-2不能识别 O-连接的寡糖,由此表明,TNF 和 IL-2均为对 N-连接寡糖特异的凝集素,它们特异性识别的碳水化合物部位可能是 N-连接寡糖的核心结构.鉴于 N-连接的寡糖广泛存在于人类的分泌蛋白和膜糖蛋白,故单核,淋巴因子的碳水化合物结合活性在人类免疫反应调节中的作用将是一个值得探索的新领域.     

Circulating human antibodies against dengue NS1 protein: potential of recombinant D2V-NS1 proteins in diagnostic tests.

The dengue virus (DV) causes one of the most important arthropod-borne human viral diseases throughout the tropical and subtropical countries. However, the morbidity and mortality of DV infections could be reduced with an early hospitalization care and a rapid risk identification of developing the dengue haemorrhagic fever (DHF). The nonstructural glycoprotein 1 (NS1) has been pointed as a reagent for immune-assay diagnostic test optimization. To evaluate this potential, recombinant DV2-NS1 proteins (rNS1) were produced from Escherichia coli (NS1EC) and insect cells (NS1IC) expression. The tests were performed by analysis of a human serum panel reacted against different rNS1 forms. The results demonstrated high correspondence between the DV positive sera and the assay results using native or refolded forms of either NS1IC or NS1EC. Also, the IgG and IgM anti-rNS1 level profiles showed distinct distribution, depending on protein form and disease status. However, the IgM anti-rNS1 reactions did not show sensibility to detect the DV in primary infections. The data obtained from the paired serum samples reactivity comparison suggested a heterogeneous human immune response and absence of correspondence between the IgG and IgM profile levels. Moreover, a patient with negative reference test could be detected by specific IgG anti-rNS1 assays presented here. Therefore, these results sustain the usefulness of dengue nonstructural proteins, in particular the NS1, in diagnostic tests as a complementary reagent.     

巴斯德毕赤酵母表达系统表达重组蛋白的影响因素及优化

巴斯德毕赤酵母表达系统是一种优秀的重组蛋白表达系统,近年来被广泛应用于重组蛋白的表达。本文介绍了巴斯德毕赤酵母表达系统的特点及其用于外源蛋白表达的基本策略。随后重点讨论了发酵过程中影响外源蛋白表达的主要环境因素、影响机制及控制策略,这些影响因素包括诱导温度、甲醇浓度、溶解氧、氮源浓度和类型及诱导pH。它们影响重组蛋白的表达量和品质,并且这几种因素的作用是相互影响和制约的,因此需要系统优化。     

Utility: Sensitivity and specificity in developing diagnostic tests of combat-related post-traumatic stress disorder (PTSD)

This paper summarizes strengths and weaknesses of clinical utility of tests that diagnose Vietnam combat-related Post-Traumatic Stress Disorder (PTSD). Weaknesses reviewed are: excessive reliance upon one kind of measure of Index Diagnosis; failure to control for varying prevalence rates across samples; failure to compare accuracy across response modalities. Strengths that emerge from the review are that self-report measures have proven to be highly sensitive, and psychophysiological measures have been demonstrated as highly specific. Whereas one single "gold standard" measure of PTSD has yet to be devised, clinical researchers can continue to have confidence in the use of multiple measures.     

Sensitivity and specificity of rapid diagnostic tests for detection of group B streptococcal antigen in bacteremic neonates.

Latex particle agglutination (LPA) testing for antigen to group B streptococcus (GBS) has been useful in the diagnosis of GBS sepsis in newborns. However, recent reports have demonstrated that the sensitivity of LPA assays may be as low as 27 to 54%. The purposes of the present study were to directly compare the abilities of four urine antigen assays to detect GBS antigen with clinical urine samples from neonates with GBS bacteremia and to evaluate the effect of the urine concentration on the sensitivities and specificities of these assays. Urine samples were collected serially from neonates with blood cultures positive for GBS or on admission from healthy full-term infants. One milliliter of urine was removed, and the remainder was concentrated to a volume of 1 ml. Unconcentrated samples were serially diluted with normal saline and were assayed to determine the highest dilution which would produce a positive test result. The Wellcogen, Bactigen, and Directigen LPA tests and ICON immunoassay were directly compared by using concentrated and unconcentrated urine specimens and urine specimens with known titers. A total of 94 urine specimens, including 61 concentrated and 75 unconcentrated specimens, from bacteremic infants were available for sensitivity testing, and 220 urine specimens from uninfected infants were available for specificity testing. There were significant differences in sensitivity among the four assays when they were performed on concentrated urine specimens, as follows: Directigen, 98%; Bactigen, 92%; ICON, 89%; Wellcogen, 68%. When the assays were performed on unconcentrated urine specimens, the Directigen (84%) and Bactigen (76%) assays were each significantly more sensitive than the ICON (59%) or Wellcogen (43%) assay.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of porcine reproductive and respiratory syndrome virus GP5 glycoprotein N-linked glycans on immune responses in mice

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically significant viral diseases in the swine industry. Infection with PRRSV following vaccination is common, since protection is incomplete. Persistent infection may be one of the biggest obstacles to control of the disease. “Glycan shielding” was postulated to be a primary mechanism to explain evasion from neutralizing immune response, ensuring in vivo persistence of virus, such as HIV, SIV, and HBV. The objective of this study was to construct recombinant adenoviruses expressing single or multiple N-linked glycosylation site (NGS) mutant GP5 of PRRSV, and evaluate the expression in cell culture, and potential to induce immune responses in BALB/c mice. Six recombinant adenoviruses were constructed each expressing wild-type GP5 and 1-4 NGS mutants: N44S, N44/51S, N30/44/51S, N30/33/44/51S and N30/33S. Inoculation of BALB/c mice with all five recombinants expressing NGS mutant GP5 resulted in a significant neutralizing antibody responses which were significantly higher than that of recombinant adenovirus expressing wild-type GP5. But there were no significant difference in lymphocyte proliferation responses induced by wild type and NGS mutant GP5. It indicated that glycosylations of GP5 at residues N30, N33, N44 and N51 are critical for induction of neutralizing antibodies. These NGS mutant PRRSV GP5 will be useful to characterize the effects of glycosylation on immunogenicity in the natural host, and may lead to a new approach for the generation of PRRSV vaccines.     

Interpretive yields of screening Pap tests and diagnostic Pap tests

Recently, our laboratory has introduced the Medicare classification "diagnostic Papanicolaou (Pap) test" (DPT) for both conventional and liquid-based Pap tests. Because little is known regarding the diagnostic yield of DPT, we review our experience with these tests in a community with a low incidence of squamous intraepithelial lesions and contrast this with our experience with screening Pap tests (SPTs). A search of our laboratory's computerized data system identified all Pap tests classified as SPT and DPT from December 3, 2002 to December 2, 2003. The interpretations were tabulated and statistical comparisons were made. Between December 3, 2002 and December 2, 2003 63,626 Pap tests were interpreted (57,922 SPTs and 5704 DPTs). DPTs were far more likely to yield abnormal results (atypical squamous cells or worse, P<0.001) and were especially more likely to yield malignant results. These differences have numerous possible implications regarding screening, rescreening, and the design of studies that investigate the screening value of the Pap test.     

Several recombinant capsid proteins of equine rhinitis a virus show potential as diagnostic antigens

Equine rhinitis A virus (ERAV) is a significant pathogen of horses and is also closely related to Foot-and-mouth disease virus (FMDV). Despite these facts, knowledge of the prevalence and importance of ERAV infections remains limited, largely due to the absence of a simple, robust diagnostic assay. In this study, we compared the antigenicities of recombinant full-length and fragmented ERAV capsid proteins expressed in Escherichia coli by using sera from experimentally infected and naturally exposed horses. We found that, from the range of antigens tested, recombinant proteins encompassing the C-terminal region of VP1, full-length VP2, and the N-terminal region of VP2 reacted specifically with antibodies present in sera from each of the five experimentally infected horses examined. Antibodies to epitopes on VP2 (both native and recombinant forms) persisted longer postinfection (>105 days) than antibodies specific for epitopes on other fragments. Our data also suggest that B-cell epitopes within the C terminus of VP1 and N terminus of VP2 contribute to a large proportion of the total reactivity of recombinant VP1 and VP2, respectively. Importantly, the reactivity of these VP1 and VP2 recombinant proteins in enzyme-linked immunosorbent assays (ELISAs) correlated well with the results from a range of native antigen-based serological assays using sera from 12 field horses. This study provides promising candidates for development of a diagnostic ERAV ELISA.     

Expression of proteins of Mycobacterium tuberculosis in Escherichia coli and potential of recombinant genes and proteins for development of diagnostic reagents.

Recombinant plasmids containing DNA from Mycobacterium tuberculosis were transformed into Escherichia coli, and three colonies were selected by their reactivity with polyclonal antisera to M. tuberculosis. The three recombinant vectors contained DNA inserts of different sizes flanking a common 4.7-kilobase (kb) sequence. Each recombinant produced 35- and 53-kilodalton proteins (35K and 53K proteins, respectively) which were absent in the control E. coli. In Western blotting experiments, both proteins bound several antisera to M. tuberculosis but not antisera to other commonly isolated mycobacteria. Rabbits immunized with the recombinant 35K protein produced antisera which bound to both the 35K and 53K protein bands, a single 35K protein band present in a culture filtrate of M. tuberculosis, and single protein bands with differing molecular weights in whole-cell homogenates from other Mycobacterium spp. An additional recombinant vector containing a 2.2-kb subclone of the 4.7-kb sequence was constructed and, when used as a probe, demonstrated homology with various fragments of chromosomal digests of selected mycobacteria. Reactivity of this probe to Mycobacterium bovis and M. bovis BCG was indistinguishable from reactivity to M. tuberculosis. Immunoglobulin G reactivity to the 35K antigen was detected in antisera from 8 of 20 persons with active tuberculosis, 4 of 18 persons with leprosy, and none of 14 healthy controls. In contrast, reactivity to various proteins in M. tuberculosis culture filtrate was present in 18 of 20 patients with tuberculosis, 16 to 18 patients with leprosy, and 5 of 14 controls. The production of M. tuberculosis proteins by E. coli circumvents many difficulties encountered in the growth and manipulation of M. tuberculosis and may facilitate the development of better diagnostic and immunizing reagents.     

Age-stratified Bayesian analysis to estimate sensitivity and specificity of four diagnostic tests for detection of Cryptosporidium oocysts in neonatal calves

There is no gold standard diagnostic test for the detection of bovine cryptosporidiosis. Infection is usually highest in 2-week-old calves, and these calves also excrete high numbers of oocysts. These factors may give rise to variations in the sensitivity and specificity of the various diagnostic tests used to detect infection in calves of various ages. An age-stratified Bayesian analysis was carried out to determine the optimum diagnostic test to identify asymptomatic and clinical Cryptosporidium sp. infection in neonatal calves. Fecal samples collected from 82 calves at 1 week, 2 weeks, 3 weeks, and 4 weeks of age were subjected to the following tests: microscopic examination of smears stained with either phenol-auramine O or fluorescein isothiocyanate (FITC)-conjugated anti-Cryptosporidium monoclonal antibody, nested-PCR, and quantitative real-time PCR. The results confirmed a high prevalence of Cryptosporidium sp. infection, as well as a high level of oocyst excretion, in 2-week-old calves. The sensitivities of all the tests varied with the age of the calves. Quantitative real-time PCR proved to be the most sensitive and specific test for detecting infection irrespective of the age of the calf. The microscopic techniques were the least sensitive and exhibited only moderate efficiency with 2-week-old calves excreting large numbers of oocysts, the majority of which were diarrheic. It was concluded that, when interpreting the results of routine tests for bovine cryptosporidiosis, cognizance should be taken of the sensitivity of the tests in relation to the age of the calves and stage of infection.     

Immunodiagnosis of Ehrlichia canis infection with recombinant proteins

Ehrlichia canis causes a potentially fatal rickettsial disease of dogs that requires rapid and accurate diagnosis in order to initiate appropriate therapy leading to a favorable prognosis. We recently reported the cloning of two immunoreactive E. canis proteins, P28 and P140, that were applicable for serodiagnosis of the disease. In the present study we cloned a new immunoreactive E. canis surface protein gene of 1,170 bp, which encodes a protein with a predicted molecular mass of 42.6 kDa (P43). The P43 gene was not detected in E. chaffeensis DNA by Southern blot, and antisera against recombinant P43 (rP43) did not react with E. chaffeensis as detected by indirect fluorescent antibody (IFA) assay. Forty-two dogs exhibiting signs and/or hematologic abnormalities associated with canine ehrlichiosis were tested by IFA assay and by recombinant Western immunoblot. Among the 22 samples that were IFA positive for E. canis, 100% reacted with rP43, 96% reacted with rP28, and 96% reacted with rP140. The specificity of the recombinant proteins compared to the IFAs was 96% for rP28, 88% for P43 and 63% for P140. The results of this study demonstrate that the rP43 and rP28 are sensitive and reliable serodiagnostic antigens for E. canis infections.     

Multivariate random effects meta-analysis of diagnostic tests with multiple thresholds

Background  

Bivariate random effects meta-analysis of diagnostic tests is becoming a well established approach when studies present one two-by-two table or one pair of sensitivity and specificity. When studies present multiple thresholds for test positivity, usually meta-analysts reduce the data to a two-by-two table or take one threshold value at a time and apply the well developed meta-analytic approaches. However, this approach does not fully exploit the data.     

鸡IL-18在毕赤酵母中的表达与活性分析

目的:在毕赤酵母中表达鸡白介素18,并鉴定其活性.方法:应用PCR技术从重组质粒pMD18-T-ChIL-18中扩增出鸡IL-18成熟肽基因,亚克隆于毕赤酵母表达载体pPICZαA上,构建重组质粒pPICZαA-ChIL-18.经酶切、PCR和测序鉴定正确后,电转化入毕赤酵母菌X-33,筛选多拷贝单克隆进行诱导表达.表达产物纯化后用Western blot、ELISA和淋巴细胞转化试验分析其生物学活性.结果:重组菌正确表达了具有免疫原性的鸡IL-18,经分析该rchIL-18具有诱导淋巴细胞分泌γ干扰素和刺激淋巴细胞增殖的活性.结论:成功的在毕赤酵母中表达了具有生物活性的鸡IL-18.