Recombinant VirB5 protein as a potential serological marker for the diagnosis of bovine brucellosis

The molecular tag vaccine against Brucella abortus and serological testing are the main methods of prevention of brucellosis used currently. They can discriminate vaccinated animals and humans from those naturally infected. In this study, we constructed a gene deletion mutant strain, B. abortus S19 virB5 with a molecular tag. Recombinant VirB5 was expressed and purified for evaluation as a diagnostic reagent for bovine brucellosis. In total, 400 sera samples were tested using a VirB5 antigen-based enzyme-linked immunosorbent assay (ELISA) and the results were compared with those of the standard tube agglutination test (SAT). This showed that the sensitivity was 88.2%, specificity was 97.8% and accuracy was 94.8%. Recombinant VirB5 could also be used to discriminate B. abortus-infected mice from mice infected with the B. abortus S19 virB5 mutant strain. It was concluded that recombinant VirB5 could be used as a potential antigen and serological marker for the diagnosis of bovine brucellosis.     

Indirect fluorescent antibody test versus enzyme-linked immunosorbent assay and agglutination tests in the serodiagnosis of patients with brucellosis

Anti-Brucella IgG, IgM and IgA in sera from patients with blood culture positive for B. melitensis and controls were measured by indirect fluorescent antibody (IFA) test and the findings compared with those of enzyme-linked immunosorbent assay (ELISA) and microagglutination test (MAT). Brucella melitensis and B. abortus antigens from three vendors (BioMerieux, Wellcome and Oxoid) and from reference strains (Ames, Iowa) were used in IFA and MAT while a whole cell heat-killed B. melitensis antigen was used in ELISA. Statistical analysis showed comparable results when using B. melitensis or B. abortus antigen, in IFA, from the same manufacturer but there were subtle differences among antigens from different manufacturers. Correlation between IFA and ELISA titers was poor, due to differences in the levels of these titers. However, the percentage of sensitivity, specificity, predictive positive, and predictive negative at different titers indicated the most reliable discriminative titers to be as follows: ELISA IgG 1 : 800 (100% for all), IgM 1 : 400 (100%, 93%, 100%, 100%, respectively) and IgA 1 : 200 (95%, 100%, 100%, 94%, respectively); IFA IgG 1 : 320 (95%, 93%, 95%, 93%, respectively) and IgM 1 : 80 (95%, 100%, 100%, 94%, respectively). IFA IgA showed either poor sensitivity or specificity at all titers. These findings and the subjective reading of IFA limit its value in Brucella diagnosis while the MAT showed high false negatives (5%–40%). Thus, ELISA proves to be the most reliable test for the diagnosis of patients with brucellosis.     

A repA-based ELISA for discriminating cattle vaccinated with Brucella suis 2 from those naturally infected with Brucella abortus and Brucella melitensis

The commonest ways of diagnosing brucellosis in animals include the Rose-Bengal plate agglutination test, the buffered plate agglutination test (BPA), the slide agglutination test, the complement fixation test, and the indirect enzyme linked immunosorbent assay (I-ELISA). However, these methods cannot discriminate the Brucella vaccine strain (Brucella suis strain 2; B. suis S2) from naturally acquired virulent strains. Of the six common Brucella species, Brucella melitensis, Brucella abortus, and B. suis are the commonest species occurring in China. To develop an ELISA assay that can differentiate between cows inoculated with B. suis S2 and naturally infected with B. abortus and B. melitensis, genomic sequences from six Brucella spp. (B. melitensis, B. abortus, B. suis, Brucella canis, Brucella neotomae and Brucella ovis) were compared using Basic Local Alignment Search Tool software. One particular gene, the repA-related gene, was found to be a marker that can differentiate B. suis from B. abortus and B. melitensis. The repA-related gene of B. suis was PCR amplified and subcloned into the pET-32a vector. Expressed repA-related protein was purified and used as an antigen. The repA-based ELISA was optimized and used as specific tests. In the present study, serum from animals inoculated with the B. suis S2 vaccine strain had positive repA-based ELISA results. In contrast, the test-positive reference sera against B. abortus and B. melitensis had negative repA-based ELISA results. The concordance rate between B. abortus antibody-negative (based on the repA-based ELISA) and the Brucella gene-positive (based on the ‘Bruce ladder’ multiplex PCR) was 100%. Therefore, the findings suggest that the repA-based ELISA is a useful tool for differentiating cows vaccinated with the B. suis S2 and naturally infected with B. abortus and B. melitensis.     

Evaluation of recombinant LigB antigen-based indirect ELISA and latex agglutination test for the serodiagnosis of bovine leptospirosis in India

Leptospirosis is a zoonotic disease caused by pathogenic spirochetes of the genus Leptospira, causing febrile infection characterized by multi-organ failure in humans and animals. Leptospiral Ig-like protein B (LigB) is a surface-expressed antigen that mediates host cell invasion or attachment. In this study, N-terminal conserved region of LigB protein (46 kDa) was evaluated for its diagnostic potential to detect anti-leptospiral antibodies in the sera of various animal species. Dot blot analysis revealed immunoreactivity of Leptospira-positive sera of cattle, buffalo, dog, sheep and goat to purified LigB protein. We have analyzed 1126 bovine serum samples, collected from Northern and Eastern part of India, by microscopic agglutination test (MAT) and recombinant LigB (rLigB) based ELISA and latex agglutination test (LAT). The sensitivity of rLigB based ELISA for 554 MAT positive sera was 96.9% and the specificity with 572 MAT negative sera was 91.08% whereas LAT showed sensitivity and specificity of 93.68% and 92.31%, respectively. Kappa values of 0.879 and 0.860 for recombinant antigen based ELISA and LAT indicate excellent agreement with the gold standard serological test, MAT, for the detection of anti-leptospiral antibodies in sera. Further, LAT based on rLigB antigen is a simple and rapid test, suitable for serodiagnosis of leptospirosis under field conditions, owing to its portability and longer shelf life.     

The role of glycosylated epitopes in the serodiagnosis of Strongyloides stercoralis infection

Carbohydrates of pathogen antigens have been disrupted by periodate oxidation, in order to reduce nonspecific bindings and improve serodiagnosis of parasite infections. In the present study, the enzyme-linked immunosorbent assay (ELISA) was carried out with filariform larvae antigen treated, or not treated, with sodium metaperiodate. Groups of sera from patients with Strongyloides stercoralis infection, with other intestinal parasites and a normal control, were used. The oxidation of Strongyloides stercoralis glycosylated epitopes reduced the seroreactivity of sera from patients with S. stercoralis infection as demonstrated by ELISA, with a decrease in sera optical densities. The number of cross-reactions of IgG and IgE-ELISAs increased by 12% and 16%, respectively, after antigen treatment with metaperiodate. This was more often observed in patients infected with Schistosoma mansoni and hookworm. Moreover, the IgG depletion from sera tested by IgE-ELISA led to the detection of previous false-negative samples from S. stercoralis–infected patients.     

重组汉坦病毒核蛋白抗原用于免疫滴金技术检测肾综合征出血热抗体的研究

目的　探索应用重组汉坦病毒核蛋白 (rNP)的免疫滴金法 (CGIDA)对肾综合征出血热 (hemorrhagicfeverrenalsyndrome ,HFRS)的诊断价值。 方法　制备纯化汉坦病毒核蛋白抗原 ,构建了HTN型汉坦病毒的核心区域 (334个碱基 ) ,克隆至原核表达载体 pGEX 4T 1中进行原核表达及纯化。在相同的CGIDA系统中 ,比较研究rNP与天然汉坦病毒核蛋白 (NP)的抗原功能。并与酶联免疫吸附试验 (ELISA)和间接免疫荧光法 (IFA)对比检测。结果　用rNP和天然NP平行检测HFRSIgM符合率达 89.7% ,HFRSIgG符合率达92 .3% ;CGIDA法检测HFRSIgM的敏感性为 75 .0 % ,特异性为 10 0 % ;CGIDA法检测HFRSIgG的敏感性为 83.1% ,特异性为10 0 %。结论　重组核蛋白的CGIDA对HFRS的早期诊断具有较好的应用价值。     

Development of an indirect ELISA with artificially synthesized N protein of PPR virus

The full-length gene encoding the nucleocapsid (N) protein of the virus (PPRV) responsible for an outbreak of peste des petits ruminants in Tibet in 2007 was synthesized in two stages using overlapping PCR without the need for viral genomic cDNA as template. The full-length N gene was successfully expressed in Escherichia coli, and the purified gene product bound to monoclonal antibody raised against PPRV N protein. Furthermore, it was able to replace recombinant B-N antigen as the coating antigen in a commercial ELISA kit prepared with another PPRV strain. Recombinant protein was employed as the coating antigen to develop an indirect ELISA for PPRV antibody detection in the sera of infected small ruminants. Antibody detection was optimal at a 1:200 serum dilution and an antigen concentration of 3.2 μg/ml, and the positive threshold (cutoff) value of the assay was 2.18. Analysis of 697 serum samples revealed the sensitivity and specificity of the indirect ELISA to be 96.7 and 96.1%, respectively, compared with a commercially available ELISA test.     

Serological response of patients infected with Salmonella enteritidis phage type 4

Sera from 14 patients infected with Salmonella enteritidis phage type (PT) 4 were used to examine the human serological response to S. enteritidis lipopolysaccharide (LPS). By immunoblotting 13 sera were found to contain IgM class antibodies directed at the O = 9 and O = 12 antigens on the LPS of S. enteritidis. With these sera antibodies of the IgM and IgA classes were detected with an enzyme-linked immunosorbent assay (ELISA) using S. enteritidis LPS. Levels of serum IgA antibodies correlated with the time interval between onset of diarrhoea and blood sampling. Sera from patients infected with S. thompson, S. javiana, S. typhimurium, S. stanley and S. senftenburg gave only low values in the S. enteritidis LPS ELISA. The protocols described may provide a valuable adjunct to established bacteriological methods for detecting evidence of infection with S. enteritidis.     

Serological response of patients with clinical typhoid

The techniques of immunoblotting and enzyme-linked immunosorbent assay (ELISA) were compared with the Widal test for examining the serum antibody response of patients with clinical typhoid. The Widal test detected antibodies in only four out of 16 patients. By ELISA and immunoblotting six patients were identified. With immunoblotting, patients' sera were found to contain antibodies binding to the 0=12 antigen of lipopolysaccharide (LPS), five of these sera also contained serum antibodies to H=d flagellar antigens. Serum antibodies to Salmonella typhi LPS, as detected by immunoblotting, correlated with high levels of antibodies to LPS as detected with a Salmonella enteritidis LPS ELISA. A rapid immunoblotting procedure was developed using S. enteritidis LPS and flagella prepared from S. meunchen, which could provide a serum test result within 1 day. This immunoblotting procedure might be a useful test to replace the Widal test as a diagnostic test for the serodiagnosis of typhoid.     

MgPa重组蛋白在生殖支原体血清学诊断中的应用

[目的]表达、纯化生殖支原体 （Mycoplasma genitalium,Mg） 黏附蛋白（MgPa）优势表位基因（MgPa＇,1075-1364aa）,探讨MgPa＇重组蛋白在Mg血清学诊断中的应用.[方法]IPTG诱导表达重组质粒pET-30a（＋）/MgPa＇,SDS-PAGE和免疫印迹鉴定表达结果;Ni-NTA亲和层析法纯化重组蛋白,间接酶联免疫吸附试验（Enzyme link immunosorbent assay,ELISA）检测人血清中特异性IgG抗体.[结果]SDS-PAGE检测诱导产物显示有一分子量约为37 000的特异蛋白带,免疫印迹检测其只与Mg抗血清发生特异反应;在311例有临床症状性传播疾病（STD）患者中用PCR法筛查出Mg阳性42例,阳性率13.5%（42/311）.重组蛋白用作ELISA包被抗原检测Mg阴阳性血清,重组蛋白能和Mg感染者阳性血清特异性结合,重组蛋白有良好的免疫反应性.[结论]表达的MgPa＇重组蛋白具有较好的免疫反应活性,可望用于生殖支原体的血清学诊断.     

Identification and characterization of Schistosoma japonicum Sjp40, a potential antigen candidate for the early diagnosis of schistosomiasis

The pathogenesis of schistosomiasis is mainly caused by egg-induced granuloma formation and subsequent fibrosis. If Schistosoma japonicum infections could be detected in the early stage, especially before the egg deposition in the host tissues, the development of severe pathologic lesions might be prevented efficiently. The present study identified and characterized S. japonicum Sjp40, a potential antigen candidate for the early diagnosis of schistosomiasis. From the S. japonicum cercariae cDNA library, a clone encoding Sjp40 was identified by screening with the pooled rabbit sera collected on day 21 postinfection. Then, the recombinant Sjp40 protein (rSjp40) and monoclonal antibodies (McAbs) anti-rSjp40 were developed. The expression profiles of Sjp40 at 3 stages of S. japonicum, including egg, cercariae, and adult, were also determined at both mRNA and protein level, which displayed that the expression pattern of Sjp40 varied at different stages. Quantification of circulatory anti-Sjp40 IgG in the infected mice sera by time-resolved fluoroimmunoassay showed a statistically significant increase on days 21, 28, 35, and 42 postinfection compared with the mice sera prior to infection and the control mice. It was further confirmed by Western blot that all 8 clones of anti-rSjp40 McAbs could react specifically with the native antigen in S. japonicum cercariae, and rSjp40 could be recognized by the pooled infected mouse sera on days 21, 28, 35, and 42 postinfection as well as the pooled patient sera with acute schistosomiasis japonica. These findings indicated that Sjp40 and its antibodies are detectable from the host at a relatively early phase (day 21 postinfection with S. japonicum) and suggested that Sjp40 is a potential antigen candidate for the early diagnosis of schistosomiasis.     

A gold nanoparticle strip for simultaneously evaluating FMDV immunized antibody level and discriminating FMDV vaccinated animals from infected animals

A gold nanoparticle strip was developed for rapidly evaluating FMDV type O antibody level and simultaneously discriminating FMDV vaccinated animals from infected animals. The strip was established depending on the colloidal gold nanoparticle labeling technique. Staphylococcal protein A colloidal gold nanoparticles were used as a probe. The epitope antigens of FMDV structural proteins and nonstructural proteins were dispensed on a nitrocellulose membrane as two test lines, respectively, and goat anti-pig antibody IgG was used as a control line. The assay was evaluated with FMDV immunized, infected sera and positive sera for another virus. The results showed the specificities of the T1 and T2 lines were 95.17% and 100% respectively. The sensitivity was in accordance with commercial ELISA kits. The coincidence rate of the new strip with 3ABC Mab-bELISA and LPB-ELISA was 95.5% and 93.13%, respectively. In summary, this experimental strip could provide a simple, inexpensive and rapid approach for onsite detection of FMDV type O antibody level and discrimination of FMDV vaccinated from infected animals without any expensive instrument.

A gold nanoparticle strip was developed for rapidly evaluating FMDV type O antibody level and simultaneously discriminating FMDV vaccinated animals from infected animals.     

Characterization of antibody response in patients with Borrelia meningitis

Western blot analysis was used to characterize the antibody response of 38 patients with Borrelia meningitis to different strains of Borrelia spirochetes. Eight strains of Borrelia spirochetes were analysed by SDS-PAGE which showed major proteins of 60, 41, 31·5–34 and 22–23 kD. Immunoblots of all sera, and all except one CSF from patients with clinically active disease showed IgG and/or IgM antibodies to at least one Borrelia protein. Antibodies to a 41 kD protein was the first to appear and patients with a longer duration of neurological disease had antibodies to as many as 19 different proteins. Some of the 40 controls showed weak bands, in some cases with the same location as in the Borrelia infected patients. In comparison with an ELISA assay based on a whole cell sonicate as antigen, western blot was more sensitive in detecting an early antibody response, especially in serum. We conclude that Western blot might be used as a complement for immunological diagnosis of Borrelia infection in selected cases with low or border-line ELISA titers. However, a more sensitive/specific ELISA assay might be developed with a single protein as antigen.     

Potential of recombinant flagellin fragment from Burkholderia thailandensis as an antigen for melioidosis antibody detection by indirect ELISA

Non-pathogenic Burkholderia thailandensis may be used as a model for Burkholderia pseudomallei due to the genetic similarity of these species. Moreover, the experimental manipulation of B. thailandensis is safer. In this study, we constructed recombinant flagellin protein fragments of B. thailandensis E264 (FLAG300, FLAG600, FLAG900, and FLAGFL fragments) and used fragments as the antigen to detect melioidosis antibodies by an indirect enzyme-linked immunosorbent assay (indirect ELISA). The serum samples consisted of serodiagnostic melioidosis sera (N = 52), septicemic sera caused by other bacteria (disease control) (N = 16) and healthy donor sera (N = 40). The area under the receiver operating characteristic (ROC) curve at optimal value (mean plus standard deviation) of all constructed fragments to melioidosis antibodies detection ranged from 0.654 to 0.953. The highest area under the ROC curve (AUROCC, 0.953) for FLAG300 fragment at 1600 serum titer revealed the best performance of melioidosis diagnosis. The indirect ELISA using this fragment as the antigen possessed 82.7% sensitivity and 94.6% specificity.     

AGGLUTINATION AFFINITIES OF THE ABORTUS-MELITENSIS GROUP OF BACTERIA WITH SPECIAL REFERENCE TO TWO HUMAN STRAINS

The conclusions to be drawn from this study are that while there appears to be evidence of serological distinctions between B. abortus and B. melitensis cultures as studied by earlier workers, the experiments reported in this paper show no serological distinctions among the six strains used in the study. B. melitensis II and B. melitensis III were found to be identical with the bovine and swine strains by means of agglutination and agglutinin absorption reactions in unheated sera, by agglutination in heated sera, and with the use of heated cultures in unheated and heated sera.     

梅毒血清学诊断用抗原Gpd蛋白的重组表达及鉴定

目的 利用基因工程技术重组表达梅毒螺旋体Gpd蛋白,探讨其在梅毒血清学诊断中的应用.方法 PCR扩增Gpd基因序列,构建表达载体pET-28b-Gpd,将表达载体转化感受态细胞E.coli BL21(DE3)并以IPTG诱导蛋白表达,经镍柱亲和层析纯化后,利用质谱和免疫印迹法鉴定重组蛋白.以重组蛋白建立间接ELISA法,并检测20份TPPA阳性和20份TPPA阴性血清去评价其在梅毒血清学诊断中的应用.结果 PCR扩增出约1.1 kb的Gpd片段,成功构建重组质粒pET-28b-Gpd.表达的重组蛋白的相对分子质量约41×103,主要以包涵体形式存在,占菌体总蛋白的40%.质谱和免疫印迹分析证实重组蛋白为Gpd蛋白,并能够与梅毒患者血清发生免疫学反应.间接ELISA法测定TPPA阳性血清和TPPA阴性血清的符合率分别为95%(19/20)和100%(20/20).结论 重组Gpd蛋白能够与梅毒患者血清发生特异性的免疫学反应,是一种可潜在应用于梅毒血清学诊断的新抗原.     

Diagnosis of chronic Pseudomonas aeruginosa infection in cystic fibrosis by ELISA for anti-pseudomonas LPS IgG antibodies

An enzyme-linked immunosorbant assay (ELISA) with urease enzyme was developed with either a polyvalent pseudomonas smooth lipopolysaccharide (LPS) extract vaccine (PEV-02) or rough LPS (R-LPS) from P. aeruginosa rough mutant PAC605. Each ELISA was able to differentiate between sera from cystic fibrosis (CF) patients chronically colonized with P. aeruginosa and sera from non-colonized patients. Sera from non-colonized and intermittently colonized CF patients seldom reacted with any of the Pseudomonas LPS, whereas sera from chronically colonized CF patients reacted strongly with most of the sixteen smooth O-serotype vaccine components and with the PAC605 R-LPS, indicating the presence either of a number of different serotype specific IgG antibodies and/or IgG antibodies directed to a common antigenic component of LPS rough core. Absorption studies and immunoblot analysis demonstrated that in sera from CF patients who were chronically colonized with P. aeruginosa a significant component of the anti-P. aeruginosa antibodies is specific for the core of P. aeruginosa LPS and cross reactive with a number of serotypes of P. aeruginosa LPS.     

Serological detection of patients infected with Salmonella enteritidis

Sera from 32 patients infected with Salmonella enteritidis and 20 sera from patients infected with Salmonellae other than S. enteritidis were used to establish serological tests for providing evidence of infection with this organism. With enzyme-linked immunosorbent assay (ELISA) and immunoblotting procedures with S. enteritidis lipopolysaccharide (LPS), and an immunoblotting procedure with g,m flagellar proteins, patients infected with S. enteritidis could be delineated from patients infected with other Salmonellae. The protocols described could be used to provide serological evidence of recent infection with S. enteritidis.     

Natural occurring IgG and IgM antibodies to gram-negative bacteria and lipopolysaccharides in human sera

In sera of 28 non-infected elderly individuals, relationships between natural occurring antibody populations against O-antigen deficient lipopolysaccharides (J5LPS, ReLPS and Lipid A), a mixture of twelve O-antigen containing lipopolysaccharides (“mixed LPS”) and five different gram-negative bacterial strains, were assessed in a quantitative ELISA. Concomitant measurement of total IgG and IgM concentrations (in radial immunodiffusion assays—RIDA) allowed for the expression of IgG and IgM antibody concentrations per mg IgG.ml⁻¹ and per mg IgM m⁻¹ respectively. Statistically significant positive correlations were found between IgM antibody concentrations to all mentioned antigens for each possible combination (r≥0·381; P<0·05). The strongest correlation was found between IgM concentrations to ReLPS and Lipid A (r=0·852; P<0·001). IgG antibody concentrations to ReLPS correlated statistically significant with IgG antibody concentrations to Lipid A (r=0·692; P<0·001), to J5LPS (r=0·409; P<0·05) and to “mixed LPS” (r=0·409; P<0·05). Beside the statistically significant correlations of IgG antibody concentrations to “mixed LPS” with ReLPS and with Lipid A (r=0·528; P<0·001), IgG antibody concentrations to “mixed LPS” also correlated statistically significant with three gram-negative enterobacterial strains. These data indicate that the application of “mixed LPS” as a capture antigen for the selection of donor sera with a high IgG antibody concentration to this polyvalent antigen, eventually will yield a pooled serum which also contains relatively high IgG antibody concentrations to O-antigen deficient lipopolysaccharide preparations as well as enterobacterial strains.     

Serological responses to eight Klebsiella pneumoniae capsular polysaccharide antigens in patients with bacteremia

Acute and convalescent sera from 13 patients with culture verified Klebsiella pneumoniae bacteremia were analysed for anticapsular polysaccharide (CPS) antibody levels using an enzyme-linked immunosorbent assay (ELISA). Eight of the patients were infected with strains expressing a CPS serotype identical with one of the eight CPS antigens used in the ELISA. In sera from seven out of eight patients, antibody responses homologous to the serotype of infecting strain was observed. Five sera exhibited a significant antibody rise (≥two-fold), while the remaining two pairs of sera showed high but not increasing titres. A concomitant antibody response to several of the heterologous CPS antigens was observed in six patients. Five additional patients were infected with K. pneumoniae expressing CPS serotypes different from the CPS antigen in the test system. All five patients made an antibody response to one or more of the heterologous antigens. The present findings indicate that structurally similar epitope(s) may be present on Klebsiella CPS antigens. Bacteremic infections may also induce a polyclonal B-cell activation, which may trigger an anamnestic response against other K. pneumoniae serotypes.