Test For Detection Of Antibodies Research Articles

P1.05-016 Circulating BARD1 Antibodies for Early Detection of Lung Cancer

In a study of more than 100 NSCLC cases we previously showed that the expression of BARD1 isoforms is correlated with poor patient survival. BARD1 is a tumor suppressor acting with BRCA1 as ubiquitin ligase. BARD1 has also functions in mitosis and poly (ADP)-ribose signaling for DNA repair. In cancer cells BARD1 isoforms are generated by alternative splicing. SNP affecting splicing and cancer predisposition were identified in neuroblastoma. The alternatively spliced isoforms lack tumor suppressor functions and act as oncogenes. As the domain composition of cancer-associated isoforms predicts altered tertiary structures, we investigated whether BARD1 isoforms act as cancer antigens. ELISA assays were performed to detect antibodies generated against BARD1 isoforms in the serum of lung cancer patients. We used BARD1 protein fragments and short peptides for capturing autoimmune antibodies. Using fitted Lasso logistic regression methods, we developed an algorithm for the prediction of lung cancer based on a blood test for detection of BARD1 antibodies. Modeling values from 200 samples, shows a distinction of lung cancer and healthy controls with high sensitivity and specificity (AUC=0.961; Figure 1). Splitting the samples randomly and repeatedly into training sets and validation sets, confirmed an average AUC=0.964 for the training sets and AUC=0.861 for the validation sets. ROC curves for early and late stage lung cancers showed no difference in their AUCs. The BARD1 lung cancer test is highly specific and does not cross-react with other cancers. Lung cancer has a very long latent phase and is often discovered at an advanced and untreatable stage. Currently the detection by low dose CT scan is relatively expensive and not very specific. Therefore a blood test, such as the BARD1 test could i) help to detect cancers earlier, in particular by screening of risk groups, and ii) become a diagnostic aid in combination with CT scan.

Stability guidelines for dithiothreitol-treated red blood cell reagents used for antibody detection methods in patients treated with daratumumab

Daratumumab (DARA), a drug used to treat patients with multiple myeloma, causes interference in pre-transfusion testing. Samples from patients receiving DARA exhibit panreactivity in antibody detection and identification tests with red blood cells (RBCs). Many hospitals are sending these samples to reference laboratories. Dithiothreitol (DTT), a sulfhydryl chemical treatment of RBCs, negates this reactivity. This study investigated the stability of the antigens on DTT-treated RBCs to determine if large quantities of RBCs could be treated at one time, stored, and used for testing at a later time. Panel cells were treated with DTT and then stored as three sets. Set 1 DTT-treated RBCs were stored in Alsever's solution at 2°C to 8°C, washed daily, and suspended in pH 7.3 phosphate-buffered saline (PBS) prior to antigen typing. Set 2 DTT-treated RBCs were stored in pH 7.3 PBS. Set 3 DTT-treated RBCs were stored in Alsever's solution. Sets 2 and 3 were inspected daily for 14 days for observation of hemolysis. In Set 1, all antigen reactivity remained at ≥2+ with both single- and double-dose cells for 14 days. The Rh antigens gave stronger reactions longer, compared with those tested in the Duffy, Kidd, and MNS blood group systems. Sets 2 and 3 were monitored for hemolysis. On day 3, Set 2 began displaying hemolysis, with complete hemolysis by day 8. Set 3 did not display hemolysis in 14 days. In conclusion, a large volume of RBCs can be treated with DTT and stored in Alsever's solution for use without deterioration of the RBC antigens, saving institutions tech time, resources, and money.

Generation, characterization, and application in serodiagnosis of recombinant swine vesicular disease virus-like particles.

Swine vesicular disease (SVD) is a highly contagious viral disease that causes vesicular disease in pigs. The importance of the disease is due to its indistinguishable clinical signs from those of foot-and-mouth disease, which prevents international trade of swine and related products. SVD-specific antibody detection via an enzyme-linked immunosorbent assay (ELISA) is the most versatile and commonly used method for SVD surveillance and export certification. Inactivated SVD virus is the commonly used antigen in SVD-related ELISA. A recombinant SVD virus-like particle (VLP) was generated by using a Bac-to-Bac baculovirus expression system. Results of SVD-VLP analyses from electron microscopy, western blotting, immunofluorescent assay, and mass spectrometry showed that the recombinant SVD-VLP morphologically resemble authentic SVD viruses. The SVD-VLP was evaluated as a replacement for inactivated whole SVD virus in competitive and isotype-specific ELISAs for the detection of antibodies against SVD virus. The recombinant SVD-VLP assay produced results similar to those from inactivated whole virus antigen ELISA. The VLP-based ELISA results were comparable to those from the virus neutralization test for antibody detection in pigs experimentally inoculated with SVD virus. Use of the recombinant SVD-VLP is a safe and valuable alternative to using SVD virus antigen in diagnostic assays.

Separation of multiple antibodies by adsorption with allogeneic red blood cells.

Antibody detection and identification are processes that are commonly performed in the transfusion service before transfusion of allogeneic red blood cells (RBCs). Antibody identification usually follows the discovery of a positive antibody detection test, or other factors such as ABO serum/cell discrepancy or an incompatible crossmatch. Antibody identification is a necessary practice in blood banking to determine the suitability of blood products for transfusion on an individual basis. When the presence of multiple antibodies is suspected, several methods, including neutralization of patient's plasma, titration, elution, chemical or enzyme treatment of reagent RBCs, and adsorption with allogeneic RBCs, may be used to separate and properly identify other atypical antibodies that are present in a single serum or plasma sample. This review will focus on the use of allogeneic adsorption to identify antibody specificities in a patient's sample.

Two cases of the variant RHD*DAU5 allele associated with maternal alloanti-D

Rh is a complex blood group system with diverse genotypes that may encode weak and partial D variants. Standard serologic analysis may identify clinically significant D variants as D+; nevertheless, individuals with these D variants should be managed as D- patients to prevent antibody formation to absent D epitopes. Variant identification is necessary during pregnancy to allow for timely and appropriate Rh immune globulin (RhIG) prophylaxis for hemolytic disease of the fetus and newborn (HDFN) as D alloimmunization can occur with some D variants. Here, we describe two cases of the RHD*DAU5 allele associated with maternal alloanti-D in patients of African ancestry. Two obstetric patients were initially serologically classified as D+ with negative antibody detection tests on routine prenatal testing. Repeat testing at delivery identified anti-D in both patients with no history of RhIG administration or transfusion. DNA sequencing revealed that both patients possessed the RHD*DAU5 allele. Cord blood testing on both infants revealed positive direct antiglobulin test (DAT) results with anti-D eluted from the red blood cells (RBCs) of one of the infants. Despite the positive DAT, neither infant experienced anemia or hyperbilirubinemia. We document two cases of pregnant women whose RBCs expressed a partial D variant and were classified as D+ on the basis of standard serologic testing, resulting in subsequent failure to provide RhIG prophylaxis. Both cases were associated with alloanti-D formation but without significant HDFN. To our knowledge, these are the first reported cases of maternal alloanti-D associated with the RHD*DAU5 partial D variant.

Screening of the Cervidae family in Poland for Mycoplasma species

Abstract Introduction: Several Mycoplasma species can cause severe diseases in ruminant hosts, some of which are the diseases listed by the World Organisation for Animal Health (OIE). The role of the Cervidae family in carrying and transmitting ruminant mycoplasma infections in Poland is unknown. Material and Methods: Antibody and antigen detection tests for the main mycoplasma species that can affect wild ruminants were performed on 237 samples (serum, nasal swab, bronchoalveolar lavage, and lung) collected from 161 animals during 2011-2014. The samples were obtained from a cull of healthy population of deer which included: 96 red deer (Cervus elaphus elaphus), 19 fallow deer (Dama dama), and 46 roe deer (Capreolus capreolus). Results: Serological screening tests revealed positive reactions to Mycoplasma bovis in one sample and to Mycoplasma capricolum subsp. capripneumoniae in three samples; however, these three samples were negative by immunoblotting. Other antibody and antigen detection tests demonstrated negative results. Conclusion: Currently wild cervids in Poland do not play a significant role in transmitting mycoplasma infections to domestic animals, but they remain a potential risk.

Canine leishmaniosis: Serological comparison of a commercial rapid test with a quantitative enzyme-linked immunosorbent assay test for detection of anti-Leishmania infantum antibodies

Purpose: The aim of this study was to evaluate a serological immunochromatographic test for the detection of anti-Leishmania infantum canine antibodies. Methods & Materials: The rapid test was compared to a reference quantitative serological technique: ELISA in-house (Enzyme-Linked Immunosorbent Assay). This quantitative ELISA in-house was used to define the sera as positive or negative. One hundred canine serum samples were evaluated, 41 were considered negative and 59 were considered seropositive with different levels of anti-Leishmania antibodies: low levels (n=7), medium levels (n=28) and high levels (n= 24). Based on these results, the FASTest® LEISH (Diagsnostik Megacor, Austria) was evaluated: sensitivity, specificity, positive predictive value, negative predictive value, Kappa index and accuracy were calculated. Results: The sensitivity and specificity were 0.97 and 0.98, respectively. In an endemic area to Leishmania infantum infection with a low seroprevalence (10%), the positive predictive value was 0.82 and the negative predictive value was 1.00. By contrast, for a high seroprevalence area (25%), the positive predictive value was 0.93 and the negative predictive value was 0.99. For the Kappa index, FASTest® LEISH obtained 0.95 and the accuracy for this qualitative test was 0.97. Conclusion: The study showed that FASTest® LEISH is a reliable diagnostic test that complies with all requirements for a sensitive (relative sensitivity 97%) and specific (relative specificity 98%) rapid test. Compared to other commercial tests the FASTest® LEISH is a commercial rapid test with high sensitivity and specificity for early detection infected dog with a reasonable cost-benefit balance.

Generation and characterization of a monoclonal antibody against duck Tembusu virus envelope protein.

Duck Tembusu virus (DTMUV) is a newly emerging pathogenic flavivirus that has caused massive economic losses to the duck industry in China. Envelope (E) protein of DTMUV is an important structural protein, which is able to induce protective immune response in target animals and can be used as specific serological diagnosis tool. In this study, a novel monoclonal antibody, designated mAb 3E9, was generated against DTMUV E protein. It is positive in indirect ELISA against both His-E protein and the purified whole viral antigen. Also, this mAb showed positive reaction with DTMUV in Western blot and indirect immunofluorescence assay, and the isotype was IgG1. End-point neutralizing assay performed in BHK-21 cells revealed that the neutralization titer of 3E9 against DTMUV JS804 strain reached 1:50. Furthermore, functional studies revealed that 3E9 blocks infection of DTMUV at a step on viral attachment. The anti-E mAbs produced in the present work may be valuable in developing an antigen-capture ELISA test for antigen detection or a competitive ELISA test for antibody detection or therapeutic medicine for DTMUV in poultry.

Seroprevalencia de <i>Toxocara canis</i> en perros de las ciudades de Corrientes y Esperanza (Argentina)

<i>Toxocara canis</i> es un nematode de los caninos, que accidentalmente afecta al hombre provocando una enfermedad parasitaria zoonótica denominada toxocariasis. Los cachorros son responsables de la contaminación del ambiente a través de la eliminación de huevos infectantes al medio, mientras que las perras adultas representan un potencial reservorio, por ser capaces de transmitir verticalmente el parásito a sus crías. La infección en seres humanos constituye un problema sanitario de gran interés, principalmente por su impacto en niños. Los estudios coproparasitológicos en perros adultos han demostrado ser poco eficientes para detectar la infección. El objetivo de este estudio fue determinar valores de seroprevalencia de anticuerpos anti- <i>T. canis</i> en caninos de dos ciudades de Argentina. Se estudiaron perros de diferentes barrios de las ciudades de Corrientes y Esperanza. Se realizó el test de ELISA indirecto para detección de anticuerpos de tipo IgG específicos para <i>T. canis</i>. En Corrientes se estudiaron 119 perros. El análisis reveló que el 84,9% (n=101) de los canes presentaba serología positiva. En Esperanza se estudiaron 82 perros; el ELISA indirecto reveló que el 51,2% (n=42) del total de los canes presentaban serología positiva para este parásito. Los valores hallados son consistentes con los encontrados por otros autores en estudios similares, y no así con aquellos en los que se emplearon métodos directos para detección de huevos en heces, los cuales en general informan valores menores de prevalencia. La alta seroprevalencia hallada sugiere la necesidad de implementar medidas de control de la infestación en perros y de esa manera reducir el riesgo de transmisión al hombre.

Comparison between latex and microscopic agglutination test for detection of human leptospiral antibodies

Leptospirosis, notably Weil’s syndrome, is an often severe, acute febrile illness caused by microorganisms of the genus <em>Leptospira</em>. The microscopic agglutination test (MAT) is the basic method for the sero-diagnosis of leptospirosis, as the test has a high sensitivity and can be used for classification, but has some disadvantages. Therefore, we have explored latex agglutination test (LAT) for use as a practical and rapid for sero-diagnosis of human leptospirosis and compared the applicability of commercial tests MAT and LAT in the detection of specific antibodies against <em>Leptospira interrogans</em> in human.

A Study of Parasitic and Bacterial Pathogens Associated with Diarrhea in HIV-Positive Patients.

IntroductionDiarrhea is a common complication of acquired immune deficiency syndrome (AIDS), occurring in almost 90% of AIDS patients in developing countries like India. The present study was aimed to determine the prevalence and microbiological profile of pathogens associated with diarrhea in human immunodeficiency virus (HIV) positive patients and their relation to CD4 counts.Materials and methodsForty-five successive HIV-positive patients, 27 with diarrhea (study group) and 18 without diarrhea (control group), were included in the three-month study. The HIV infection was confirmed by three different antibody detection tests. The stool samples were collected on two consecutive days and were examined for parasites by microscopy using wet mount and modified Ziehl-Neelsen stain. They were examined for bacteria by Gram stain and conventional Ziehl-Neelsen stain and were inoculated on appropriate culture media. The isolates were identified by standard biochemical tests, followed by antibiotic susceptibility testing using the Kirby-Bauer disc diffusion method.Results Twenty-four pathogens were detected in diarrheal HIV-positive patients, including 14 parasites (58.33%), seven bacteria (29.17%), and three fungi (12.50%). Isospora sp. was the most common parasite (25.9%) followed by Cryptosporidium sp. (14.8%). Other parasites included Cyclospora sp., Strongyloides stercoralis, and Entamoeba histolytica (3.7% each).​ Escherichia coli (18.5%) was the most common bacterial isolate, of which, 80% were Enterotoxigenic E. coli (ETEC) while 20% were Enteropathogenic E. coli (EPEC). Other isolates included Shigella flexneri and Mycobacterium tuberculosis (3.7% each). The isolates were sensitive to furazolidone (94.11%), chloramphenicol (76.47%), and gentamicin (52.94%). The isolates from diarrheal patients showed resistance to norfloxacin (5.88% vs. 50%, p<0.05) as compared to those from non-diarrheal patients. The diarrheal HIV-positive patients had lower mean CD4 counts (202.6 cells/µL), as compared to those without diarrhea (239.28 cells/µL).Conclusion Isospora sp. is the most common parasite and Escherichia​ coli is the most common bacterium associated with diarrhea in HIV patients. The antibiotic sensitivity patterns should be monitored regularly to detect resistance to commonly used drugs. The prevalence of organisms in a region, various clinical manifestations, sensitivity patterns of isolates, and relation with CD4 count should be considered while instituting therapy in HIV patients with diarrhea.

P127 Detection of MICA antibodies: Comparison between MICA Screening and Single Antigen tests

Aim The presence of MICA antibodies is associated with worse graft outcome in solid organ transplant. Here, we aim to compare two MICA antibody detection tests: MICA Screening vs. Single Antigen Beads Based Test (SAB). Methods 20 sera with or without EDTA treatment were tested for MICA antibodies by screening (Onelambda LABScreen Mixed) or SAB tests (LABScreen MICA). Ratio of 2 in the screening test and MFI 1,000 in SAB test were used as the cutoff, respectively. Results We determined if EDTA treatment impact MICA SAB testing. Out of 15 samples, 7 samples displayed 23 MICA antibodies in EDTA treated or non-treated sera. In general, no significant difference of MFI (median fluorescence intensity) for MICA antibodies was observed between EDTA treated and non-treated sera. We did observe MFI of one MICA antibody increased from 9167 to 20566 upon EDTA treatment, suggesting this antibody showing prozone effect. We also found EDTA treatment increased MFI of positive control bead significantly (mean 6675 vs 9180). Next we compared results of MICA SAB testing on 20 EDTA treated sera with screening testing. We failed to identify the association between SAB test and screening test results (fig 1A). 4 sera, out of 8 MICA screening+/SAB- sera did not display HLA antibodies. 3 of these 4 patients had no sensitization events, suggesting positivity in these 3 patients is likely due to high background in MICA screening test. The mean ratio of screening testing on sera which were positive in SAB testing trended higher than sera having negative SAB test results, but the difference didn’t reach significance (fig 1B). Strong MICA antibodies (MFI>10,000) detected by SAB testing in two sera showed ratio >100 in screening testing, suggest both these assays can detect strong antibodies. Conclusions EDTA treatment may remove prozone effect for strong antibodies in MICA SAB testing. The screening testing tends to have high background, but strong MICA antibodies can be detected by both screening and SAB testing. The MICA SAB test is more specific than MICA screening test. Download : Download high-res image (71KB) Download : Download full-size image

Use of panel testing for detection of antinuclear antibody in a resource-limited setting: an appraisal

ABSTRACTObjectives: Despite an increase in the incidence of systemic connective tissue diseases (CTD), panel testing for detection of antinuclear antibodies (ANA) is not a routine practice in many health centers of the Indian subcontinent. Consequently, the data on its significance is scanty.Methods: To evaluate utility of panel testing, line immunoassay (LIA) and indirect immunofluorescence antinuclear antibody test (IIF-ANA) were performed in 321 cases of CTD.Results: Out of 321 serum samples screened by the above tests, 227 were positive and 18 were negative by both LIA and IIF-ANA. Additional 11/321 (3.4%) cases were picked up by LIA. SSA was most common specificity in these cases followed by SSA/SSB, SSB, Ro-52, Jo-1, dsDNA and nRNP/Sm.Conclusion: Use of LIA along with IF-ANA and ELISA improves sensitivity of CTD screening.

Rapid and Sensitive Multiplex Detection of Burkholderia pseudomallei-Specific Antibodies in Melioidosis Patients Based on a Protein Microarray Approach.

BackgroundThe environmental bacterium Burkholderia pseudomallei causes the infectious disease melioidosis with a high case-fatality rate in tropical and subtropical regions. Direct pathogen detection can be difficult, and therefore an indirect serological test which might aid early diagnosis is desirable. However, current tests for antibodies against B. pseudomallei, including the reference indirect haemagglutination assay (IHA), lack sensitivity, specificity and standardization. Consequently, serological tests currently do not play a role in the diagnosis of melioidosis in endemic areas. Recently, a number of promising diagnostic antigens have been identified, but a standardized, easy-to-perform clinical laboratory test for sensitive multiplex detection of antibodies against B. pseudomallei is still lacking.Methods and Principal FindingsIn this study, we developed and validated a protein microarray which can be used in a standard 96-well format. Our array contains 20 recombinant and purified B. pseudomallei proteins, previously identified as serodiagnostic candidates in melioidosis. In total, we analyzed 196 sera and plasmas from melioidosis patients from northeast Thailand and 210 negative controls from melioidosis-endemic and non-endemic regions. Our protein array clearly discriminated between sera from melioidosis patients and controls with a specificity of 97%. Importantly, the array showed a higher sensitivity than did the IHA in melioidosis patients upon admission (cut-off IHA titer ≥1:160: IHA 57.3%, protein array: 86.7%; p = 0.0001). Testing of sera from single patients at 0, 12 and 52 weeks post-admission revealed that protein antigens induce either a short- or long-term antibody response.ConclusionsOur protein array provides a standardized, rapid, easy-to-perform test for the detection of B. pseudomallei-specific antibody patterns. Thus, this system has the potential to improve the serodiagnosis of melioidosis in clinical settings. Moreover, our high-throughput assay might be useful for the detection of anti-B. pseudomallei antibodies in epidemiological studies. Further studies are needed to elucidate the clinical and diagnostic significance of the different antibody kinetics observed during melioidosis.

Low incidence of D alloimmunization among patients with a serologic weak D phenotype after D+ transfusion.

By consensus definition, the red blood cells (RBCs) of patients that react weak to 2+ in the serologic D test are classified as having the serologic weak D phenotype (weak D). The risk of D alloimmunization in patients with weak D is not well studied. This study retrospectively determined the incidence of D alloimmunization in patients with weak D who received D+ blood products. Patient records at four institutions were reviewed. Eligible patients had at least 1 D typing result with agglutination strength that was weak to 2+ result in gel or tube (Institutions 1-3) or weak to 2+ result in solid phase or a positive tube weak D test using antihuman globulin reagent (Institution 4). All patients received at least 1 D+ allogeneic RBC or platelet transfusion and had a subsequent antibody detection test performed at least 30 days after the first D+ transfusion. The incidence of alloanti-D formation was 0.15% (6/4011, at Institutions 1-3) and 5.1% (3/59, at Institution 4; these incidences were significantly different [p=0.0002]). There were 0.15% (6/4011) patients who had autoanti-D detected; all were at Institutions 1 to 3. Until RHD genotyping is widely available, these data support the fact that patients with serologic weak D may be transfused with D+ RBCs if an urgent transfusion is required. At Institution 4, inclusion of a higher proportion of black patients compared to the other institutions may have affected the incidence of alloimmunization by perhaps including partial D recipients who are at risk of forming D antibodies.

Development of enhancing agglutination reaction using gold nanoparticle for pre-transfusion testing.

To explore an alternative way for antibody detection testing, the examination of gold nanoparticle solution for enhancing unexpected antibodies for pre-transfusion testing was investigated. Exposure of foreign antigens on red blood cells from transfusion can trigger the immune system to produce unexpected antibodies. This immunological response may cause the complication to future transfusion. For detection of unexpected antibodies, the antibody screening test is performed approximately 30-60 min. To reduce turnaround time, enhancing reagent, low-ionic strength solution (LISS), is widely used. However, cost of enhancing reagent is an issue which has concerned in resource limited countries. Gold nanoparticle solution can increase red blood cells agglutination reaction. To solve this issue, study of gold nanoparticle solution was investigated. Samples were performed comparing between LISS and gold nanoparticle solution at antiglobulin phase. After reading the agglutination reaction, supernatants were collected and measured at the optical density at 760 nm by spectrophotometer. The optical density in the tube of gold nanoparticle solution was higher than in the tube of 2-5% cell suspension and monoclonal antibody. It has been observed that gold nanoparticle solution enhanced the reaction of agglutination 98% while LISS enhanced the agglutination only 60·8%. Employing a commercially available enhancing reagent, parallel samples confirmed results providing validation of the assay. It approximately costs $1 US dollars compared to $30 for a commercially available reagent. The low cost and yet effective time-consuming test for antibody screening is a practical and viable solution alternative way for performing in antibody screening test in resource limited countries.

Antibody detection tests for early diagnosis in tuberculous meningitis.

Tuberculous meningitis (TBM) is the most severe form of tuberculosis. Microbiological confirmation is rare and treatment is often delayed. Early diagnosis and immediate initiation of treatment are essential for effective TBM control. A systematic review was performed in this study to assess the diagnostic accuracy of detecting antibodies against Mycobacterium tuberculosis in the cerebrospinal fluid (CSF), according to standard methods. Test performance was summarized using a bivariate random-effects meta-analysis. Studies were identified by a search of the literature, up to July 25, 2015, in the EMBASE and MEDLINE databases via Ovid SP and PubMed. The Cochrane Library was also searched for original, peer-reviewed molecular epidemiology studies that reported the diagnosis of TBM based on antibody detection in the CSF. Thirty-six articles (58 studies) were identified. The sensitivity of antibody detection was 0.75 (95% confidence interval (CI) 0.66-0.82), specificity was 0.98 (95% CI 0.96-0.99), and the area under the receiver operating characteristic curve (AUROC) was 0.97 (95% CI 0.95-0.98). By subgroup analysis, the detection of anti-M37Ra was the highest (AUROC 0.99, 95% CI 0.98-1.00), followed by anti-antigen 5 (AUROC 0.99, 95% CI 0.97-0.99) and anti-M37Rv (AUROC 0.97, 95% CI 0.95-0.98). For the early diagnosis of TBM based on antibodies in the CSF, the detection of anti-M37Ra, anti-antigen 5, or anti-M37Rv provides the greatest sensitivity and specificity.

Comparison of analytical sensitivity of HIV diagnostic kits using dilutional panel

An array of tests have been developed over a period of time for screening and diagnosis of HIV infection which include tests for detection of antigen and antibody , viral nucleic acid and the virus itself. Amongst the available tests, serodiagnosis by ELISA is widely being used. It offers a relatively inexpensive procedure with ease of performance and ability to effectively screen a large number of specimens. Our current study is designed to compare the performance of two different HIV diagnostic kits having different coated antigens using HIV dilutional panel. Kit X had synthetic peptides (3rd generation), and the Kit Y had recombinant antigens (2nd generation) coated on the microtitre plates. 10 HIV-1 plasma panel members which were collected from various blood banks were used for comparison of their performance. The plasma samples were diluted in human plasma (base matrix) negative for anti HIV, anti HCV, HBsAg, VDRL and HTLV I/II. Serial dilutions in the range of 10 to10,000 fold for each sample were used for this study. These kits were compared in terms of antibody titre which is defined as the dilution at which the sample OD to cut off (S/Co) value is 1. It was seen that Kit X is more sensitive as compared to Kit Y for all the HIV panel members. This indicates that Kit X had better analytical sensitivity performance as compared to Kit Y since the S/Co ~ 1.0 of Kit X is at a higher dilution (1:1000) than of Kit Y (1:100).Thus this indicates that dilutional panel can help the manufacturers to design a diagnostic kit which is able to detect the infection at an early stage of disease. It was seen that the S/Co ~ 1.0 of kit X at a higher dilution.

Immunoproteomic and bioinformatic approaches to identify secreted Leishmania amazonensis, L. braziliensis, and L. infantum proteins with specific reactivity using canine serum

Leishmania spp have a wide range of hosts, and each host can harbor several Leishmania species. Dogs, for example, are frequently infected by Leishmania infantum, where they constitute its main reservoir, but they also serve as hosts for L. braziliensis and L. amazonensis. Serological tests for antibody detection are valuable tools for diagnosis of L. infantum infection due to the high levels of antibodies induced, unlike what is observed in L. amazonensis and L. braziliensis infections. Likewise, serology-based antigen-detection can be useful as an approach to diagnose any Leishmania species infection using different corporal fluid samples. Immunogenic and secreted proteins constitute powerful targets for diagnostic methods in antigen detection. As such, we performed immunoproteomic (2-DE, western blot and mass spectrometry) and bioinformatic screening to search for reactive and secreted proteins from L. amazonensis, L. braziliensis, and L. infantum. Twenty-eight non-redundant proteins were identified, among which, six were reactive only in L. amazonensis extracts, 10 in L. braziliensis extracts, and seven in L. infantum extracts. After bioinformatic analysis, seven proteins were predicted to be secreted, two of which were reactive only in L. amazonensis extracts (52kDa PDI and the glucose-regulated protein 78), one in L. braziliensis extracts (pyruvate dehydrogenase E1 beta subunit) and three in L. infantum extracts (two conserved hypothetical proteins and elongation factor 1-beta). We propose that proteins can be suitable targets for diagnostic methods based on antigen detection.

PARASITOLOGICAL AND MOLECULAR STUDIES ON TRYPANOSOMA EVANSI OF CAMELS IN EGYPT

This study was conducted on a total of 187 one humped camels (Camelus dromedarius). Blood samples were collected from 40 camels suspected forTrypanosoma infection from farms at Giza Pyramids area. In addition, 147 samples were collected from apparently healthy camels (97 and 50 from El-Bassatin and El-Warraq abattoirs respectively) for screening of Trypanosoma species infection. All samples were examined parasitologically by Giemsa stained blood smear and haematocrit centrifugation technique, serologically by card agglutination test (CATT) for detection of anti-trypanosomal antibodies and polymerase chain reaction (PCR) for DNA amplification with aspecific primers for detection of trypanozoan parasites. Out of 187 camels, 14 camels were positive by parasitological methods with a percentage of 7.49%, 21 positive samples were detected by anti- Trypanosoma antibodies using CATT with a percentage of 11.23%. Fourteen out of the positive blood samples by parasitological and serological techniques were used for PCR amplification. Thirteen out of 14 positive blood samples were PCR-positive and one was negative by using specific primers for T. evansi minicircle EVA1 and EVA2. The results of examined camels after using the polymerase chain reaction (PCR) for detection of DNA of trypanosomes in infected camels, showed 138 bp PCR product for the specific detection of Trypanosoma evansi. It was concluded that the use of PCR, beside parasitological and serological methods, is recommended for exact diagnosis in survey and control programmes ofTrypanosoma evansi.