Antigenic Bands Research Articles

Serodiagnosis of nasal myasis in camels (Camelus dromedaries) in Egypt using third larval instar affinity-purified glycoprotein

The larvae of Cephalopina titillator cause nasopharyngeal myiasis in camels, which parasitize the living tissues of the nasal and paranasal sinuses, pharynx, and larynx. C. titillator infestation adversely affects camel health, meat, and milk production, and can even cause death. In our study, to improve the immunodiagnosis of camel nasal myiasis, a sensitive and specific enzyme-linked immunosorbent assay (ELISA) was developed and evaluated using the Concanavalin-A (Con-A) affinity purification for the C. titillator-N-acetylglucosamine (Ct-GlucNAc) glycoprotein fraction from third larval instars as an antigen for detecting C. titillator antibodies. Crude antigens were prepared from larval instars of C. titillator and evaluated by indirect ELISA. The third C. titillator larval antigen (L3Ct) had the highest protein content (P < 0.001) and the best diagnostic value; chi-square = 235 (P < 0.001). Four glycoprotein fractions were purified separately from the L3Ct antigen by Con-A purification and evaluated. The Ct-GlucNAc glycoprotein fraction was the fraction of choice with the highest diagnostic accuracy (P < 0.05). Using Ct-GlucNAc as a coating antigen, indirect ELISA showed a 99.3% sensitivity for positive results in camel myiasis samples and 100% specificity for negative results in healthy camel samples. The diagnostic accuracy was 99.7%, and no cross reactivity was detected for other parasitic diseases. The indirect ELISA results were confirmed by the western immunoblotting which was characterized by comparing sera from naturally infested dromedary camels with C. titillator, sera from healthy camels and sera from camels with other parasitic infections (Echinococcus granulosus, Fasciola gigantica, Hard ticks; Hyalomma dromedarii, Trichostronglid sp., Eimeria spp., and Cryptosporidium sp.). Immunoreactive antigenic bands of 63, 50, 30 and 18 kDa were predominantly detected in sera from camels with nasopharyngeal myiasis and didn’t react with healthy and camel’s sera from other parasitic infections. However, seven immunoreactive bands appeared at 120, 70, 63, 48, 35, 29, and 19 kDa in the crude L3Ct antigen. In addition, a positive rate of C. titillator immunodiagnosis was detected by indirect ELISA (48.6%, chi-square = 483, P < 0.001), which was significantly greater than that of postmortem diagnosis (31%). In conclusion, the current study introduces a new diagnostic immunoaffinity glycoprotein fraction of C. titillator 3rd larval instar-based ELISA as a highly accurate, simple and fast method to detect specific antibodies of nasal myiasis in camels.

Characterization of antigenic proteins of the Taenia solium postoncospheral form

Neurocysticercosis is the leading cause for acquired epilepsy worldwide, and it is caused by the larval stage of the parasite Taenia solium. Several proteins of this stage have been characterized and studied to understand the parasite-host interaction, however, the proteins from the early cysticercus stages (the postoncospheral form) have not yet been characterized. The study of the postoncospheral form proteins is important to understand the host-parasite relationship in the early stages of infection. The aim of this work was to identify postoncospheral form antigenic proteins using sera from neurocysticercosis patients. T. solium activated oncospheres were cultured in HCT-8 cells to obtain the postoncospheral form. Soluble total and excretory/secretory proteins were obtained from the postoncospheral form and were incubated with both pool sera and individual serum of neurocysticercosis positive human patients. Immunoblotting showed target antigenic proteins with apparent molecular weights of 23 kDa and 46–48 kDa. The 46–48 kDa antigen bands present in soluble total and excretory/secretory postoncospheral form proteins were analyzed by LC-MS/MS; proteins identified were: nuclear elongation factor 1 alpha, enolase, unnamed protein product/antigen diagnostic GP50, calcium binding protein calreticulin precursor and annexin. The postoncospheral form expresses proteins related to interaction with the host, some of these proteins are predicted to be exosomal proteins. In conclusion, postoncospheral proteins are consistent targets of the humoral immune response in human and may serve as targets for diagnosis and vaccines.

Paraneoplastic Neurologic Syndromes.

Progress is ongoing in understanding paraneoplastic neurologic disorders, with new syndromes and antibodies being described and more detailed evidence available to guide workup for diagnosis and treatment to improve outcomes. Many excellent reviews have summarized the molecular features of different antibodies, but this article emphasizes the clinical features of each syndrome that may help guide initial diagnosis and treatment, which often should occur before an antibody or cancer is found to confirm the diagnosis. Recent findings include updated diagnostic criteria with validated sensitivity and specificity, discovery of novel antibodies, and clinical findings that increase the likelihood of an underlying paraneoplastic disorder. Suggestive syndromes that have been recently identified include faciobrachial dystonic seizures and pilomotor auras in anti-leucine-rich glioma inactivated protein 1 encephalitis, extreme delta brush on EEG in N-methyl-d-aspartate (NMDA)-receptor encephalitis, déjà vu aura in anti-glutamic acid decarboxylase 65 (GAD65) encephalitis, and sleep disturbances in several disorders. In addition, there is confirmed utility of brain positron emission tomography (PET) and CSF markers, including carcinoembryonic antigen and oligoclonal bands, as well as improved tests for the presence of leptomeningeal cancer cells in CSF. Associations of cancer immunotherapies with paraneoplastic syndromes and herpes simplex virus encephalitis (and COVID-19) with NMDA-receptor encephalitis have been described. All neurologists should be aware of advances regarding paraneoplastic neurologic syndromes, as patients can present with a wide variety of neurologic symptoms and earlier diagnosis and treatment can improve outcomes.

Profiling of Humoral Immune Response in Typhoid Patients against Differentially Extracted Whole Cell Bacterial Protein Derived from S. typhi and S. spp.

Typhoid fever is a multiorgan infectious disease caused by Salmonella typhi. It is transmitted through fecal oral route and can be fatal without proper treatment. Therefore, early diagnosis of typhoid fever is crucial. In the previous study, we have developed TYPHOIDYNE EIA, which showed excellent synergy between the genus conserved and species-specific antigens in the serodiagnosis of typhoid fever. TYPHOIDYNE EIA can effectively detect and differentiate typhoid patients, typhoid vaccinated subjects, healthy subjects, and subjects with other febrile illnesses. Following the successful development of TYPHOIDYNE EIA, in this report, we further characterize the antigenic components of differentially extracted S. typhi and S. spp recognized by IgM, IgG, and IgA antibody isotypes in typhoid patients and possible typhoid carrier by the western blot (WB) assay. The WB characterization revealed a dynamic pattern of recognition, with significant variations in the number of antigenic bands observed between the differentially extracted arrays of antigens. The reactivity of patient's sera was divided into 3 regions, with region 1 (≥55 kDa) showing the strongest reactivity followed by region 2 (54 kDa-34 kDa) and region 3 (<34 kDa). Overall, the good synergy expressed in these bands suggests the potential role of these proteins in differentiating typhoid patients with possible typhoid carrier. The antigenic bands highlighted in this study are also identified as prospective biomarkers for diagnostic use and vaccine development.

Secretory Excretory and Somatic Immunogenic Antigens Profiles of Adult Fasciola spp.

Fascioliasis is a common human-animal disease that is reported in most parts of the world. Fascioliasis is also prevalent in different provinces of Iran. Since it has done no study on the excretory/secretory and somatic immunogenic antigens profiles of adult Fasciola in Iran, the present study was performed on the Fasciola spp. collected from Mazandaran province. For this purpose, the Fasciola worm was isolated from the liver of infected sheep, then its excretory/secretory and somatic antigens were prepared from adult worms. The protein of the samples was measured by the Lowry method. Then, somatic and secretory excretions were examined by SDS-PAGE and the protein profile of the two substances was determined. To evaluate the immunogenicity, the somatic and secretory excretions antigens of Fasciola spp. were injected into white rabbits and after boosting, the blood serum of the rabbits was collected and then Western blotting was performed on them and the results were evaluated. According to the results of Western blotting, 11 somatic antigen bands with a molecular weight of 149, 122, 99, 85, 75, 65, 50, 46, 40, 37, 30 kDa and 12 protein bands of excretory/secretory antigens with molecular weights of 100, 82, 75, 70, 58, 55, 47, 40, 38, 37, 30,25 kDa were observed in adult Fasciola spp. that immunogenic, which appear to have a protective effect or can be used to prepare a diagnostic kit.

Proteomic analysis of the excretory-secretory products from Strongyloides venezuelensis infective larvae: new insights for the immunodiagnosis of human strongyloidiasis.

Serodiagnosis of human strongyloidiasis is a practical alternative to parasitological methods due to its high sensitivity. However, cross-reactivity with other helminth infections limits its utility, and this problem is due to the use of homologous or heterologous somatic extracts of the parasite as an antigen source. Excretory-secretory (E/S) products from Strongyloides infective larvae can be used to improve the serodiagnosis. The combined use of western blot and proteomics became an interesting strategy to identify immunological markers for the serodiagnosis of strongyloidiasis. The present study describes the proteomic analysis of the antigenic components from E/S products of S. venezuelensis infective larvae that were recognized by IgG antibodies from patients with strongyloidiasis. Our results showed that IgG antibodies from patients with strongyloidiasis recognized between 15 and 16 antigenic bands in the E/S products from S. venezuelensis that were incubated in PBS or in RPMI culture medium, respectively. Overall, antigenic bands of low and high molecular weight were more specific than those of intermediate molecular weight, which were cross-reactive. A 36-kDa antigenic band was 93% sensitive and 100% specific (a probably arginine kinase of 37kDa), while other antigenic bands were highly sensitive but low specific. Proteomic analysis revealed differences between the protein profiles from E/S-RPMI and E/S-PBS since only one-third of all proteins identified were common in both types of E/S products. Bioinformatic analysis showed that more than 50% of the proteins from E/S products are secreted within extracellular vesicles and only a small percentage of them are actually released by the classical secretory pathway. Several components from the E/S products were identified as plasminogen-binding proteins, probably used as an immune evasion mechanism. The data provided here provide valuable information to increase understanding of E/S products from S. venezuelensis infective larvae. This may help us to find new targets for the immunodiagnosis of human strongyloidiasis.

Characterization of antigens of Enterobius vermicularis (pinworm) eggs

Enterobiasis (pinworm infection) caused by Enterobius vermicularis is a common parasitic infection prevalent worldwide especially in children. Infection is diagnosed by microscopic detection of E. vermicularis eggs on perianal swabs. This study aimed to characterize the antigens of E. vermicularis eggs as a preliminary step towards identifying diagnostic targets for detection in infected individuals. The study was conducted between October 2019 and February 2020, following approval from Ethics Review Committee of the Faculty of Medicine, University of Colombo (EC-19-034). E. vermicularis eggs were harvested from perianal swabs using acetone and purified with 1× PBS (pH 7.2). A portion of eggs was used for preparing antigen slides, while the rest were sonicated and vortexed with glass beads and inoculated subcutaneously (with weekly booster doses) into a Wistar rat for developing antibodies. Blood drawing from rat was done weekly for 5 weeks. Confirmation of the presence of antibodies was done by surface immunofluorescence against eggs on the antigen slides. Protein bands were determined using SDS-PAGE assay and immunogenic antigen bands were determined by reacting with antiserum after immunoblotting. The band sizes of the proteins were determined against corresponding bands of a protein ladder. Surface immunofluorescence was positive with serum obtained from day 14 post-inoculation from the Wistar rat as well as that obtained from a person with chronic enterobiasis. The most prominent and immunogenic protein bands identified from egg antigens were 21 kDa, 66 kDa, 83 kDa, 96 kDa, 112 kDa, 121 kDa, 140 kDa and 151 kDa. Methods used in this study were effective in obtaining E. vermicularis egg antigens which were immunogenic. Furthermore, surface antigens of intact eggs reacted with antibodies developed against crushed egg antigens. These findings may pave the way for the development of effective immunodiagnostics.

Using IBMR3 Monoclonal Antibodies for the Detection of the Expression Antigens Profile on 3T3 and HT29 Cancer Cell Line after Analysis

The detection of several types of cancer is possible by using monoclonal antibody (MAb). Studies have shown that different types of cancer have related specific abnormal proteins, which are identified by MAb. IBMR3 is a MAb which was raised against synthetic peptides that are associated with the selected amino acid sequence of human interleukin-4 receptors. Aims: The aim of this study was to evaluate the effectiveness of specific monoclonal IBMR3 antibodies expression in the cancer cell lines of 3T3 and HT29, in order to recognize specific antigen and make evaluation, using immunoblotting technique which is widely use in this filed. Methods: Protein extracts were extracted from these cancer cell lines and used for western bloat. The Immunoblotting were consequently subjected to densitometry analysis using bio imaging machine. This bio imaging process will facilitate to measure the molecular weights, peak height and raw volume of the protein bands for the 3T3 and HT29 cancer cell lines, which helps in diagnosis of any pathogenic antigen. Results: The bands obtained from bio imaging were exposed on the PVDF membrane. In the 3T3 bio imaging process revealed four bands and molecular weight were 299.58, 87.90, 41.67 and 23.54 KDa. However, bio imaging results for HT29 revealed also four bands with molecular weights: 90.11, 41.31, 23.87 and 20.86 KDa. The results of Peak height densitometry for IBMR3 antigen bands for 3T3 were: 1856.985, 551.769, 394.164, and 216.185. However HT29 peak height results were: 281.544, 101.711.202.668, 757.213. The raw volume (amount of protein bands of IBMR3 Ag for 3T3) were: 1460168.75, 206078.47, 161406.89, and 219583.16. However HT29 raw volume results were: 110197.11, 76106.84, 98632.59, and 221395.34. Negative protein staining for 3T3 and HT29 cancer cell line bands were done by using mouse IgM serotype. IgM serotype was not indicated that means no specific antigens for negative control IgM. Conclusion: The bio imaging revealed ........

A Seroepidemiological Investigation of Toxocariasis in Forestry Workers in Bulgaria

Determination of professional activities, which are connected with higher risk of toxocariasis, is very important for well-timed diagnosis and prevention. That is why our research aimed to investigate the spread of toxocariasis in people, whose professional activities are related with forests. Thirty-three people are included in the study – eight employees of Forest Research Institute at Bulgarian Academy of Sciences, Sofia, and 25 forestry workers from three different locations in South Bulgaria. The specific parasitological diagnosis of toxocariasis is performed with ELISA and Western blot. Presence of anti-Toxocara IgG antibodies in ELISA was established in eleven forestry workers. This reveals very high seroprevalence of toxocariasis among this profession – 33.3%. Moreover, the examination of 15 sera (11 with positive and 4 with borderline ELISA results for toxocariasis) with Western blot showed the presence of two and more antigen bands in 11 samples (33.3%). These results show that professional activities in theforest could be defined as risk factor for development of toxocariasis.

Comparison of Rapid and Automated Antigen Detection Tests for the Diagnosis of SARS-CoV-2 Infection.

SARS-CoV-2 viral antigen detection may be an interesting alternative to RT-PCR for the diagnosis of SARS-CoV-2 infection as a less laborious or expensive method but requires validation. This study aimed to compare the performance of the DiaSorin™ LiaisonXL automated quantitative antigen test (QAT) and the AAZ™ rapid antigen test (RAT) to the DiaSorin™ MDX RT-PCR assay. A total of 242 nasopharyngeal samples were tested at La Pitié-Salpêtrière University Hospital (Paris, France). Performances for the detection of variants of SARS-CoV-2 were further investigated. RATs were visually read for qualitative results and band intensity was determined. Overall sensitivity was 63.2% for QAT and 58.6% for RAT. For RT-PCR Ct value 25, sensitivity was 89.8% for both tests. Both tests showed comparable sensitivity for detection of variants. There was a strong relationship between antigen concentration and band positivity. On the same set of samples these tests share similar performances.

The Evaluation of Humoral Immune Response to Contagious Bovine Pleuropneumonia in Cattle with Respiratory Disorders by Western Blot Technique, Competitive Enzyme Linked Immunosor-bent Assay and Complement Fixation Test

In this study 253 serum samples from cattle showing respiratory disorders were tested by confirmatory Western Immunoblotting test (IBT), complement fixation test (CFT) and competitive enzyme linked immunosorbent assay (c-ELISA). Two (0.8%) out of 253 serum samples were found to be positive and two were (0.8%) doubtful by CFT while 7 (2.8%) serum samples were found as positive and 25 (9.9%) of them were found to be doubtful by c-ELISA. On the other hand, a core profile of antigenic bands needed to be identified at 110, 98, 95, 62/60 and 48 kDa was not detected in any of the test serum sample. Immunoblot analysis some of serum samples displayed the number of bands between one or three and some of them were highly faint. In order to detect similarities between MmmSc strains related to their electrophoretic profiles, whole cell proteins of Mycoplasma species isolated from cattle and MmmSC reference strains (Mycoplasma mycoides subsp. mycoides SC Botswana (African strain), Mycoplasma mycoides subsp. mycoides SC PG1 (reference strain), Mycoplasma mycoides subsp. mycoides SC V5 (Australian strain), M. bovigenitalium, M. bovirhinis, M. capricolum subsp. capricolum, M.canadense, M.alkalescens and M. bovis) were analysed by sodium dodecyl sulphate polyacrilamide gel elektrophoresis (SDS-PAGE). The result of this analysis indicated that antigens of 220, 78, 82, 68, 24 kDa were common among all tested strains. According to these results, it was thought that positive results by both CFT and c-ELISA, also non specific band patterns observed by IBT might be caused by a cross reaction between tested mycoplasma strains. As conclusion, it is recommended that all the positive results by CFT and c-ELISA be confirmed by IBT.

Promising proteins detected by Western blot from Echinococcus granulosus protoscoleces for predicting early post-surgical outcomes in CE-affected Tunisian children

BackgroundCystic echinococcosis (CE) affects predominantly young patients in highly endemic areas. Improved serological methods are needed for the follow-up of CE cases, especially given the high rates of post-surgical relapse that require detection as soon as possible.MethodsWe designed a study to investigate the value of antigenic proteins extracted from Echinococcus granulosus (E. granulosus) protoscoleces, and of recombinant B2t and 2B2t proteins, for assessing the efficacy of surgical treatment carried out on CE-affected children. This study was performed on 278 plasma samples collected from 59 Tunisian children surgically treated for CE and monitored for 3 years post-surgery. The patients were classified according to post-surgical outcomes into a “non-relapsed” (NRCE) and a “relapsed” (RCE) group. We performed in-house ELISAs to measure anti-B2t and anti-2B2t IgG and immunoblotting for the detection of IgG against SDS-PAGE-resolved E. granulosus protoscoleces-specific antigens. The Wilcoxon test was applied to assess anti-B2t and anti-2B2t IgG levels. We applied the Cochran Q test to compare the distribution of immunoblotting antigenic bands between 1-month and 1-year post-surgery.ResultsThe probability of being “relapse-free” when a decrease in antibody titers occurred between 1 month and 1 year post-surgery was 81% and 75%, respectively, for anti-B2t and anti-2B2t IgG. We identified five protoscolex protein bands of 20, 26/27, 30, 40 and 46 kDa as highly immunoreactive by immunoblot for both RCE and NRCE patients at 1 month post-surgery, and significantly lower immunoreactivity after 1 year (p < 10–4) for NRCE compared to RCE patients. The proteins at 26/27 and 40 kDa displayed the best performance in predicting the outcome, with an 84% probability of being relapse-free when the reactivity against the 40 kDa antigen, the doublet at 26/27 kDa, or both was absent or disappeared between 1 month and 1 year post-surgery, and a 93% probability of being relapsed when both bands remained reactive or increased in intensity between the two time points.ConclusionsThe B2t protein could be useful for the prediction of CE early post-surgical outcomes. The proteins of E. granulosus protoscoleces, especially the doublet P26/27 and P40, could be promising predictive biomarkers for the post-surgical follow-up of CE cases as well.Graphical

Characterization of Antigenic Property of Adult Spirocerca lupi Collected from Esophageal Nodules in Dogs

Spirocerca lupi, the esophageal nematode of dog , causes a potentially fatal disease in domestic dogs. Immunological techniques can identify the parasite proteins which elicit an immune response. However, the antigenic properties of S. lupi adult worms have not been fully understood. The immuno-reactivity of the naturally infected dog sera with the S. lupi somatic antigens showed 7 prominent immunoreactive bands of distinct sizes at 199, 148, 100, 87, 49, 16, and 12 kDa, whereas 8 prominent bands at 230, 200, 100, 60, 49, 29, 16, and 12 kDa against hyper immune serum raised in the rabbits. Common antigen bands for both types of serum were observed at 100, 49 , 16 and 12 kDa. Intriguingly, the current study was able to indicate the detailed antigenic profile with specific molecular moieties which might be a manifesto for future investigation. Identified specific antigens merit further analysis as potential tools for the evaluation of treatment success and prognosis of spirocercosis, and development of a sensitive and specific diagnostic test for early detection of S. lupi in infected dogs.

Performance of a Point-of-Care Test for the Rapid Detection of SARS-CoV-2 Antigen.

The rapid detection of infections caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is necessary in the ongoing pandemic. Antigen-specific point-of-care tests (POCT) may be useful for this purpose. Here, such a POCT (SARS-CoV-2 NADAL® COVID-19 Ag) was compared to a laboratory-developed triplex real-time polymerase chain reaction (RT-PCR) designed for the detection of viral nucleoprotein gene and two control targets. This RT-PCR served as a reference to investigate POCT sensitivity by re-testing upper respiratory tract (URT) samples (n = 124) exhibiting different SARS-CoV-2 loads in terms of RT-PCR threshold cycle (Ct) values. The optical intensities of the antigen bands were compared to the Ct values of the RT-PCR. The infectivity of various virus loads was estimated by inoculating Vero cells with URT samples (n = 64, Ct 17-34). POCT sensitivity varied from 100% (Ct < 25) to 73.1% (Ct ≤ 30); higher SARS-CoV-2 loads correlated with higher band intensities. All samples with a Ct > 30 were negative; among SARS-CoV-2 free samples (n = 10) no false-positives were detected. A head-to-head comparison with another POCT (Abbott, Panbio™ COVID-19 Ag Rapid Test) yielded similar results. Isolation of SARS-CoV-2 in cell-culture was successful up to a Ct value of 29. The POCT reliably detects high SARS-CoV-2 loads and rapidly identifies infectious individuals.

Plasma skeletal muscle myosin phenotypes identified by immunoblotting are associated with pulmonary embolism occurrence in young adults

BackgroundPurified skeletal muscle myosin (SkM) binds factor Xa and is procoagulant. The molecular forms of SkM in human plasma have not been characterized. MethodHuman plasma SkM heavy chain (HC) isoforms of different molecular weights were detected by a newly developed immunoblotting protocol. In this pilot study, the distribution of SkM HC antigen isoforms in plasmas of healthy subjects and young adult patients with venous thrombosis was analyzed. ResultsMultiple SkM HC antigen bands were detected in human plasmas, corresponding to full-length SkM HC, heavy meromyosin, or the S1 fragment. Plasma immunoblots of healthy subjects displayed three major phenotypes: Type I with two primary bands for full-length SkM and heavy meromyosin, and two lesser bands including S1 fragment (54%); Type II with bands primarily for full-length SkM HC (34%); and Type III with only a band for the S1 fragment (12%). Plasma SkM HC antigen Type II phenotype was associated with an increased occurrence of isolated pulmonary embolism in younger patients, respectively (≤50 years old). ConclusionsThree SkM HC antigen phenotypes were identified in human plasma by immunoblotting, and Type II phenotype was correlated with the occurrence of isolated pulmonary embolisms in younger patients.

A simple and efficient purification method of native immunoreactive antigen for diagnosis of camel hydatidosis.

Background:Cystic echinococcosis (CE), a zoonotic disease that affects animal and human health, is of increasing economic importance due to high morbidity rates and high economic losses in the livestock industry.Aim:The present study was conducted to purify the antigen from hydatid cyst fluid (HCF) with high diagnostic efficacy of camel hydatidosis using indirect enzyme-linked immunosorbent assay (ELISA).Materials and Methods:The HCF antigen was purified using Sephacryl S-300 column chromatography. Characterization of fractions was performed using reducing and non-reducing sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. Further, antibodies against Echinococcus granulosus cysts in camel serum were detected using indirect ELISA.Results:The purification process resulted in three fractions of antigens: FI, FII, and FIII. Indirect ELISA showed that higher diagnostic efficacy was observed in FI than in FII and FIII. Indirect ELISA, in which FI was utilized, showed 88% sensitivity and 91.7% specificity. Non-reducing SDS-PAGE showed that FI had two bands of molecular weights 120 and 60 kDa. Western blot analysis of FI demonstrated that 60, 38, and 22 kDa were antigenic bands when reacted with naturally infected camel sera with E. granulosus cysts. Using indirect ELISA, F1 recorded an infection percentage of 81.7% in randomly collected camel serum samples.Conclusion:FI is a promising antigen for accurate diagnosis of camel CE using indirect ELISA.

Molecular characterization and immunodiagnostics of Dicrocoelium dendriticum species isolated from sheep of north-west Himalayan region.

Despite its extensive presence among grazing ruminants, dicrocoeliosis, also known as 'small liver fluke' disease, is poorly known and often underestimated by researchers and practitioners in many countries. The accurate identification and prepatent diagnosis of Dicrocoelium dendriticum infection is an essential prerequisite for its prevention and control. In the present study, the morphologically identified specimens isolated from the bile ducts of sheep (Ovis aries) were validated through molecular data. The sequence analysis of the second internal transcribed spacer (ITS-2) of our isolates showed a high degree of similarity with D. dendriticum using the BLAST function of the National Center for Biotechnology Information (NCBI). The phylogenetic analysis of our isolates showed a close relationship with previously described D. dendriticum isolates from different countries. The antigenic profiles of somatic and excretory/secretory (E/S) antigens of D. dendriticum were revealed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting using sera from sheep naturally infected with D. dendriticum. By SDS-PAGE, 16 distinct bands were revealed from crude somatic fraction. Immunoblotting analysis of these proteins with positive sera exhibited six seroreactive bands ranging from 27 to 130 kDa. Among these, the 84 and 130 kDa bands were quite specific, with high diagnostic specificity and sensitivity. The E/S fraction comprised nine distinct bands, as revealed by SDS-PAGE analysis. Immunoblotting analysis of these proteins with positive sera exhibited five antigenic bands ranging from 27 to 130 kDa. Among these, the 130 kDa band was found to be quite specific, with high diagnostic specificity and sensitivity. The present study concludes that the protein bands of 84 and 130 kDa in somatic fraction and 130 kDa in E/S fraction can be used for the immunodiagnostic purpose for this economically important parasite, which may also encourage further studies regarding their vaccine potential.

Characterization of Glycoproteins of Native 19kDa C-Terminal Merozoite Surface Protein-1 from Native Antigen of Plasmodium falciparum

Plasmodium falciparum is the protozoan parasite which causes malignant malaria of medical concern. Prime candidates for recombinant vaccine development are asexual stage antigens of P. falciparum, for example, merozoite surface proteins (MSP1 and MSP2) not given satisfactory results to date. In this study, the 19kDa C-terminal of MSP1, a vaccine candidate was purified in its native form in the ring stage, and its glycoproteins studied. The study was carried out at the Biochemistry Department of Pasteur Institute of Iran in the years 2015-2016. Large scale culture of P. falciparum was performed in vitro with 80% ring stage parasitemia. Isopycnic ultracentrifugation with 36% sucrose and analytical SDS-PAGE on the supernatant and precipitate performed, and the 19kDa antigen was obtained by cutting it from strips of preparative SDS gels. Purified protein was concentrated and analyzed by SDS-PAGE and immunoblotting, using antibodies raised to recombinant C-terminal MSP1. The purified protein gave a single band of 19kDa antigen as shown by silver staining of SDS-PAGE and a single bond in immunoblotting. Bioinformatics also confirmed the likelihood of the presence of glycans on the antigen. The presence of N and O-glycoproteins were detected by Q proteome kit. This work was done on the ring stage, and earlier workers confirmed the presence of glycoproteins on MSP1 in the other stages. This glycosylation is present in all stages, and maybe incomplete protection elicited by recombinant MSP1 antigens is due to lack of N and O-glycoproteins.

Western Immunoblotting for the Diagnosis of Enterococcus faecalis and Streptococcus gallolyticus Infective Endocarditis

Blood culture-negative endocarditis (BCNE) remains a diagnostic challenge. In our center, despite a systematic and exhaustive microbiological diagnostics strategy, 22% of patients with BCNE remain without an identified etiology. In an effort to determine the relevance of using Western blot (WB) for the etiological diagnosis of BCNE in patients with early antibiotic use, we developed specific assays for the major infective endocarditis (IE) causative agents, namely, Staphylococcus aureus, Enterococcus faecalis, Streptococcus anginosus, and Streptococcus gallolyticus. Our technique was effective to identify the antigenic profiles of the four tested agents, but cross-reactions with S. aureus and S. anginosus antigens were frequent. A scoring method was developed for the diagnosis of E. faecalis and S. gallolyticus IE using the presence of reactivity to at least two antigenic bands for each bacterium and the positivity to at least one of the Ef300, Ef72, or Ef36 proteic bands for E. faecalis, and positivity for the two Sg75 and Sg97 proteic bands for S. gallolyticus. We tested these diagnostic criteria in a prospective cohort of 363 patients with suspected IE. Immunoblotting for the diagnosis of E. faecalis IE showed a sensitivity of 100% and a specificity of 99%. The positive and negative predictive values were 73 and 100%, respectively. Regarding S. gallolyticus infection, immunoblot had a sensitivity of 100% and a specificity of 95%. However, the positive predictive value was 22%, whereas the predictive negative value was 100%. Using WB, we identified a potential etiological agent in 4 of 14 BCNE cases with no identified pathogen. In conclusion, WB constitutes a promising and helpful method to diagnose E. faecalis or S. gallolyticus IE in patients with early antibiotic uptake and negative blood cultures.

Molecular Characterization and Immunodiagnostic Potential of Various Antigenic Proteins of Fasciola Gigantica Species Isolated from Sheep of North West Himalayan Region.

SummaryThe control of the digenetic trematode Fasciola gigantica has been the major challenge in both cattle and small ruminants as there is a paucity of an effective and commercial vaccine. Thus, the accurate identification and prepatent diagnosis of F. gigantica is an essential prerequisite for its successful prevention and control. In the present study, the morphologically identified specimens isolated from the liver and bile ducts of sheep (Ovis aries) were validated through molecular data. The sequence analysis of ITS2 of our isolates showed high degree of similarity with F. gigantica and F. hepatica using BLAST function of NCBI. The phylogenetic analysis of our isolates showed a close relationship with previously described F. gigantica and F. hepatica isolates from different countries. The antigenic profile of somatic and E/S antigens of F. gigantica were revealed by SDS–PAGE and immunoblotting using sera from sheep naturally infected with F. gigantica. By SDS-PAGE, 20 distinct bands were revealed from crude somatic fraction. Immunoblotting analysis of these proteins with positive sera exhibited 8 sero-reactive bands ranging from 14 to 97 kDa. Among these 38 and 44 kDa bands were quite specific with high diagnostic specificity and sensitivity. The E/S fraction comprised 7 distinct bands, as revealed by SDS-PAGE analysis. Immunoblotting analysis of these proteins with positive sera exhibited 6 antigenic bands ranging from 23 – 54 kDa. Among these 27 and 33 kDa were found to be quite specific with high diagnostic specificity and sensitivity. The present study concludes that the protein bands of 38 and 44 kDa in somatic fraction and 27 and 33 kDa in E/S fraction can be used for the immunodiagnostic purpose for this economically important parasite, which may also entice further studies regarding their vaccine potential.