scholarly journals Use of the 27-Kilodalton Recombinant Protein fromParacoccidioides brasiliensis in Serodiagnosis of Paracoccidioidomycosis

1998 ◽  
Vol 5 (6) ◽  
pp. 826-830 ◽  
Author(s):  
B. L. Ortiz ◽  
S. Díez ◽  
M. E. Urán ◽  
J. M. Rivas ◽  
M. Romero ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Healthy Subjects ◽  
Paracoccidioides Brasiliensis ◽  
Cell System ◽  
Etiologic Agent ◽  
Immunological Methods ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
High Coverage ◽  
Cross Reactions

ABSTRACT Paracoccidioidomycosis (PCM) is one of the most important endemic mycoses in Latin America; it is usually diagnosed by observation and/or isolation of the etiologic agent, Paracoccidioides brasiliensis, as well as by a variety of immunological methods. Although the latter are effective, two circumstances, cross-reactions with other mycotic agents and antigen preparation that is marked by extreme variability among lots, hinder proper standardization of the procedures. To circumvent this lack of reproducibility, molecular biology tools were used to produce a recombinant 27-kDa-molecular-mass antigen from this fungus; a sizable quantity of this antigen was obtained through fermentation of Escherichia coli DH5α, which is capable of expressing the fungal protein. The latter was purified by the Prep-Cell System (Bio-Rad); the recovery rate of the pure protein was approximately 6%. A battery of 160 human serum samples, consisting of 64 specimens taken at the time of diagnosis from patients with PCM representing the various clinical forms plus 15 serum specimens each from patients with histoplasmosis and aspergillosis, 10 each from patients with cryptococcosis and tuberculosis, 6 from patients with coccidioidomycosis, and 40 from healthy subjects, were all tested by an indirect enzyme-linked immunosorbent assay with the purified 27-kDa recombinant protein. The latter was used at a concentration of 1.0 μg/well; there were three serum dilutions (1:1,000, 1:2,000, and 1:4,000). The experiment was repeated at least twice. The average sensitivity for both experiments was 73.4%; in comparison with the healthy subjects, the specificity for PCM patients was 87.5% while for patients with other mycoses, it was 58.7%. Important cross-reactions with sera from patients with aspergillosis and histoplasmosis were detected. The positive predictive value of the test was 90.4%. These results indicate that it is possible to employ recombinant antigenic proteins for the immunologic diagnosis of PCM and, by so doing, achieve high coverage rates. Furthermore, antigen reproducibility can now be ensured, thus facilitating inter- and intralaboratory standardization.

scholarly journals Heterologous Expression, Purification, and Immunological Reactivity of a Recombinant HSP60 from Paracoccidioides brasiliensis

2002 ◽  
Vol 9 (2) ◽  
pp. 374-377 ◽  
Author(s):  
Daniela A. Cunha ◽  
Roseli M. Zancopé-Oliveira ◽  
M. Sueli ◽  
S. Felipe ◽  
Silvia M. Salem-Izacc ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Paracoccidioides Brasiliensis ◽  
High Sensitivity ◽  
Cross Reactivity ◽  
Antibody Responses ◽  
Healthy Individuals ◽  
Immunological Reactivity ◽  
Serum Samples ◽  
Recombinant Antigens

ABSTRACT The complete coding cDNA of HSP60 from Paracoccidioides brasiliensis was overexpressed in an Escherichia coli host to produce high levels of recombinant protein. The protein was purified by affinity chromatography. A total of 169 human serum samples were tested for reactivity by Western blot analysis with the purified HSP60 recombinant protein. Immunoblots indicated that the recombinant P. brasiliensis HSP60 was recognized by antibodies in 72 of 75 sera from paracoccidioidomycosis patients. No cross-reactivity was detected with individual sera from patients with aspergillosis, sporotrichosis, cryptococcosis, and tuberculosis. Reactivity to HSP60 was observed in sera from 9.52% of control healthy individuals and 11.5% of patients with histoplasmosis. The high sensitivity and specificity (97.3 and 92.5%, respectively) for HSP60 suggested that the recombinant protein can be used singly or in association with other recombinant antigens to detect antibody responses in P. brasiliensis-infected patients.

scholarly journals Enzootic bovine Leukosis: development of an indirect enzyme linked immunosorbent assay (I-Elisa) in seroepidemiological studies

1999 ◽  
Vol 30 (1) ◽  
pp. 37-42 ◽  
Author(s):  
Ester Teresa González ◽  
Estela Beatriz Bonzo ◽  
María Gabriela Echeverría ◽  
María Licursi ◽  
María Elisa Etcheverrigaray
Keyword(s):  
Core Protein ◽  
Leukemia Virus ◽  
Test Procedure ◽  
Immunological Response ◽  
Negative Control ◽  
Etiologic Agent ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Enzootic Bovine Leukosis ◽  
Bovine Leukosis

Bovine Leukemia Virus (BLV) is the etiologic agent of Enzootic Bovine Leukosis, a retrovirus exogenous to the bovine species. Once infected, there is no detectable viraemia but instead there is a strong and persistent immunological response to BLV structural proteins, essentially the gp51 envelope glycoprotein and the mayor core protein p24. We describe the test procedure of an indirect ELISA (I-ELISA) using polyclonal reagents for the detection of antibodies to BLV. For comparison, the sera were simultaneously tested by agar gel immunodiffussion (AGID) test, which is currently used as diagnostic standard for BLV infection. The antigen applied does not require a high degree of purification and the data from the analysis of the negative sera showed that the establishment of a cut-off level corresponding to 3 times the standard deviation (SD) above the mean for the negative control set of sera provided acceptable specificity, reducing the risk of false positives results. A comparison of the results obtained by AGID test and I-ELISA showed that considering a total of 465 serum samples, all of the 234 samples (50%) that were positive by AGID were positive to the I-ELISA. Of 225 serum samples which yielded negative results in the AGID test, 69 (15%) were found to be positive by the I-ELISA and 156 (33%) were negative by both techniques. Few sera (2%) that were non-specific by AGID were defined as negative or positive by I-ELISA.

scholarly journals Development of an Electrochemical Immunosensor for Specific Detection of Visceral Leishmaniasis Using Gold-Modified Screen-Printed Carbon Electrodes

Biosensors ◽  
2020 ◽  
Vol 10 (8) ◽  
pp. 81 ◽  
Author(s):  
Beatriz R. Martins ◽  
Yanne O. Barbosa ◽  
Cristhianne M. R. Andrade ◽  
Loren Q. Pereira ◽  
Guilherme F. Simão ◽  
...  
Keyword(s):  
Visceral Leishmaniasis ◽  
Electrochemical Immunosensor ◽  
False Positives ◽  
Cross Reaction ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Cross Reactions ◽  
Asymptomatic Patients ◽  
Screen Printed Carbon Electrode ◽  
Screen Printed

Visceral leishmaniasis is a reemerging neglected tropical disease with limitations for its diagnosis, including low concentration of antibodies in the serum of asymptomatic patients and cross-reactions. In this context, this work proposes an electrochemical immunosensor for the diagnosis of visceral leishmaniasis in a more sensitive way that is capable of avoiding cross-reaction with Chagas disease (CD). Crude Leishmania infantum antigens tested in the enzyme-linked immunosorbent assay (ELISA) were methodologically standardized to best engage to the sensor. The antibodies anti-Trypanosoma cruzi and anti-Leishmania sp. Present in serum from patients with diverse types of CD or leishmaniasis were chosen. A screen-printed carbon electrode modified with gold nanoparticles was the best platform to guarantee effective adsorption of all antigens so that the epitope of specific recognition for leishmaniasis occurred efficiently and without cross-reaction with the evaluated CD. The current peaks reduced linearly after the recognition, and still were able to notice the discrimination between different kinds of diseases (digestive, cardiac, undetermined Chagas/acute and visceral chronic leishmaniasis). Comparative analyses with ELISA were performed with the same groups, and a low specificity (44%) was verified due to cross-reactions (high number of false positives) on ELISA tests, while the proposed immunosensor presented high selectivity and specificity (100%) without any false positives or false negatives for the serum samples from isolated patients with different types of CD and visceral leishmaniasis. Furthermore, the biosensor was stable for 5 days and presented a detection limit of 200 ng mL−1.

scholarly journals Expression of Antibodies Directed to Paracoccidioides brasiliensis Glycosphingolipids during the Course of Paracoccidioidomycosis Treatment

2006 ◽  
Vol 14 (2) ◽  
pp. 150-156 ◽  
Author(s):  
Silvio Bertini ◽  
Arnaldo L. Colombo ◽  
Helio K. Takahashi ◽  
Anita H. Straus
Keyword(s):  
Immune Response ◽  
Paracoccidioides Brasiliensis ◽  
Immunoglobulin M ◽  
Antifungal Treatment ◽  
Primary Immune Response ◽  
Enzyme Linked Immunosorbent Assay ◽  
Carbohydrate Structure ◽  
Dimorphic Fungus ◽  
Serum Samples ◽  
Antibody Titers

ABSTRACT Paracoccidioidomycosis (PCM) is a granulomatous disease caused by the dimorphic fungus Paracoccidioides brasiliensis. The immunoglobulin classes and isotypes of antibodies directed to acidic glycosphingolipids (GSLs) and glucosylceramide of P. brasiliensis were determined by enzyme-linked immunosorbent assay of sera from 31 PCM patients. The reactivities of 38 serum samples were analyzed by considering the stage of treatment: before antifungal treatment (n = 10), during 1 to 4 months of treatment (T1-4; n = 9), during 5 to 12 months of treatment (T5-12; n = 9), and posttreatment (PT; n = 10). Sera from healthy subjects (n = 12) were used as controls. Only the GSL Pb-1 antigen, which presents the carbohydrate structure Galfβ1-6(Manα1-3)Manβ1, was reactive with the PCM patient sera. The PCM patient sera did not react with Pb-2, which lacks the Galf residue and which is considered the biosynthetic precursor of Pb-1, indicating that the Galf residue is essential for antibody reactivity. The Pb-1 glycolipid from nontreated patients elicited a primary immune response with immunoglobulin M (IgM) production and subsequent switching to IgG1 production. The IgG1 titer increased after the start of antifungal treatment (T1-4 group), and general decreases in the anti-Pb-1 antibody titers were observed after 5 months of treatment (T5-12 and PT groups). The Pb-1 antigen, an acidic GSL with terminal Galf residue, has potential application as an elicitor of the host immune response in patients with PCM.

scholarly journals Association of CCR5Δ32 Deletion and Human Cytomegalovirus Infection With Colorectal Cancer in Tunisia

2021 ◽  
Vol 12 ◽  
Author(s):  
Hanen Chelbi ◽  
Refka Jelassi ◽  
Sarra Belfkih ◽  
Amor Ben Amor ◽  
Nasreddine Saidi ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Human Cytomegalovirus ◽  
Healthy Subjects ◽  
Enzyme Linked Immunosorbent Assay ◽  
Potential Candidate ◽  
Serum Samples ◽  
Potential Candidate Gene ◽  
Tumor Tissues ◽  
Pcr Technology ◽  
Genetic Deletion

Background and objectives: Human cytomegalovirus (HCMV) and genetic polymorphisms of the chemokine receptor 5 have been suggested as factors associated with the progression of colorectal cancer (CRC). The aim of the study was to evaluate the associations of both CCR5Δ32 genetic deletion and/or HCMV virus infection with CRC in Tunisia. Materials and methods: The association between HCMV and CRC was validated by Nested PCR technology performed for HCMV and HCMV-specific serum IgG and IgM antibodies were investigated by enzyme-linked immunosorbent assay. Experiments were carried out on 40 tumor and 35 peri-tumor tissues, 100 blood from CRC patients and on 140 blood samples from healthy subjects and finaly serum samples of 80 patients with CRC and 100 healthy individuals. A conventional PCR has been optimized for the detection of CCR5Δ32 in100 CRC patients and 100 healthy subjects. Results: Our results show that HCMV is significantly active in 93% of patients compared to 60% in controls (p < 0.0001, OR = 8.85, 95% CI: 3.82 -20.50). Compared to the healthy controls, the titers of IgG and IgM antiCMV antibodies in CRC patients were significantly higher than in healthy subjects (p value < 0,0001 for IgG and IgM). Statistical analysis revealed a lack of association between CCR5Δ32 mutation and colorectal cancer (p = 0.788, OR = 1.265, 95% CI: 0.228-7.011). Conclusion: our data confirmed that the HCMV infection was related to the development of CRC and that CRC cells may be infected more favorably by HCMV. Given the importance of the CCR5 in inflammation and therefore CRC progression, further studies still needed to evaluate CCR5 role as a potential candidate gene for CRC susceptibility under other polymorphisms.

scholarly journals Detection of Immunoglobulin G Antibodies to Neospora caninum in Humans: High Seropositivity Rates in Patients Who Are Infected by Human Immunodeficiency Virus or Have Neurological Disorders

2006 ◽  
Vol 13 (1) ◽  
pp. 84-89 ◽  
Author(s):  
Janaína Lobato ◽  
Deise A. O. Silva ◽  
Tiago W. P. Mineo ◽  
Jodi D. H. F. Amaral ◽  
Gesmar R. Silva Segundo ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Immunoglobulin G ◽  
Neurological Disorders ◽  
Healthy Subjects ◽  
Neospora Caninum ◽  
Fluorescent Antibody ◽  
Antibody Test ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Immunodeficiency Virus

ABSTRACT Considering that little is known about the epidemiology of Neospora caninum infection in humans, particularly in populations with high Toxoplasma gondii infection rates, the present study aimed to investigate the presence of antibodies to N. caninum in T. gondii-seropositive and -seronegative individuals. A total of 256 serum samples divided into four groups (61 samples from human immunodeficiency virus [HIV]-positive patients, 50 samples from patients with neurological disorders, 91 samples from newborns, and 54 samples from healthy subjects) were assessed for N. caninum and T. gondii serologies by indirect fluorescent-antibody test, enzyme-linked immunosorbent assay, and immunoblotting (IB). Immunoglobulin G antibodies to N. caninum were predominantly detected in HIV-infected patients (38%) and patients with neurological disorders (18%), while newborns and healthy subjects showed lower seropositivity rates (5% and 6%, respectively). Seropositivity to N. caninum was significantly associated with seropositivity to T. gondii in both HIV-infected patients and patients with neurological disorders. Seroreactivity to N. caninum was confirmed by IB, with positive sera predominantly recognizing the 29-kDa antigen of N. caninum. The results of this study indicate the presence of N. caninum infection or exposure in humans, particularly in HIV-infected patients or patients with neurological disorders, who could have opportunistic and concurrent infections with T. gondii. These findings may bring a new concern for the unstable clinical health of HIV-infected patients and the actual role of N. caninum infection in immunocompromised patients.

scholarly journals Development of a Highly Specific RecombinantToxocara canis Second-Stage Larva Excretory-Secretory Antigen for Immunodiagnosis of Human Toxocariasis

2000 ◽  
Vol 38 (4) ◽  
pp. 1409-1413 ◽  
Author(s):  
Hiroshi Yamasaki ◽  
Kunioki Araki ◽  
Patricia Kim Chooi Lim ◽  
Ngah Zasmy ◽  
Joon Wah Mak ◽  
...  
Keyword(s):  
False Negative ◽  
Toxocara Canis ◽  
Recombinant Antigen ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Cross Reactions ◽  
Second Stage ◽  
Secretory Antigen ◽  
The Cross ◽  
Excretory Secretory Antigen

The specificity of the recombinant Toxocara canisantigen developed for the immunodiagnosis of human toxocariasis was compared with that of the excretory-secretory antigen from T. canis second-stage larvae (TES) by enzyme-linked immunosorbent assay. A total of 153 human serum samples from patients infected with 20 different helminths, including 11 cases of toxocariasis, were examined. No false-negative reactions were observed for the toxocariasis cases. When the TES was used at concentrations of 0.5 and 0.125 μg/ml, cross-reactions were observed in 79 (55.6%) and 61 (43.0%) of 142 cases, respectively. In contrast, when the recombinant antigen was tested at a concentration of 0.5 μg/ml, cross-reactions were observed in 19 (13.4%) of 142 cases. At a concentration of 0.125 μg/ml, however, the cross-reaction rate decreased sharply to only 2.1%, corresponding to 3 of 142 cases. The cross-reactions occurred with one case each of gnathostomiasis, paragonimiasis withParagonimus miyazakii, and spirometriasis, in which high antibody titers were detected. In addition, the recombinant antigen showed negative reactions with serum samples from patients infected with Ascaris and hookworms, which are the most common parasites in the world. These findings are also supported by experiments with animals infected with Ascaris and hookworm. From these results, the recombinant antigen is highly specific for toxocariasis and may provide more reliable diagnostic results than other methods.

Investigation of Parvovirus B19 IgM and IgG Positivity Rates in Pediatric Hematology Patients

2018 ◽  
Vol 13 (04) ◽  
pp. 274-276
Author(s):  
Ayşe Uğur ◽  
Mehmet Özdemir ◽  
Bahadır Feyzioğlu ◽  
Mahmut Baykan ◽  
Aysun Görkem
Keyword(s):  
Parvovirus B19 ◽  
Etiologic Agent ◽  
Immunoglobulin M ◽  
Outpatient Clinics ◽  
Assay Method ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Igg Antibody ◽  
Erythema Infectiosum ◽  
Pediatric Hematology

AbstractHuman parvovirus B19 is a frequent etiologic agent causing erythema infectiosum in children. It has recently been suggested that parvovirus B19 may be latent after infection and cause reactive infections especially in immunosuppressed patients with hematological problems. In this study, we aimed to investigate the parvovirus B19 immunoglobulin M (IgM) and immunoglobulin G (IgG) seropositivity rates of patients evaluated in a pediatric hematology clinic. We retrospectively screened the laboratory results of parvovirus B19 IgM and IgG antibody assays of children less than 18 years, who consulted pediatric in-and-outpatient clinics between 2013 and 2016. Parvovirus B19 IgM and IgG antibodies were investigated in serum samples by using enzyme-linked immunosorbent assay method in the Medical Microbiology Laboratory. Parvovirus B19 IgM antibodies were detected in 109 of 602 patients attending pediatric hematology clinics (18.1%). Parvovirus B19 IgG antibody was detected in 244 of 952 patients attending pediatric hematology clinics (25.6%). Parvovirus B19 IgM and IgG positivity in samples from pediatric in-and-outpatient clinics other than pediatric hematology were 2.8% and 35.7%, respectively. Parvovirus IgM and IgG positivity in serum samples sent from the pediatric hematology clinic and outpatients was statistically significant compared with those sent from pediatric clinics other than pediatric hematology (p = 0.0001 and p = 0.0008, respectively). The higher detection rate of serum parvovirus B19 IgM positivity in patients under the follow-up of pediatric hematology clinics suggests that immune suppression-related viral reinfection or persistence may occur in these patients.

scholarly journals First direct assay for intact human proinsulin

1998 ◽  
Vol 44 (7) ◽  
pp. 1514-1519 ◽  
Author(s):  
Paule Houssa ◽  
Bo Dinesen ◽  
Michelle Deberg ◽  
Bruce H Frank ◽  
Chris Van Schravendijk ◽  
...  
Keyword(s):  
Healthy Subjects ◽  
Limit Of Detection ◽  
Insulin Dependent Diabetes Mellitus ◽  
Dependent Diabetes Mellitus ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
C Peptide ◽  
Human Proinsulin ◽  
The Mean ◽  
A Chain

Abstract We describe a sensitive two-site sandwich enzyme-linked immunosorbent assay for the measurement of intact human proinsulin in 100 μL of serum or plasma. The assay is based on the use of two monoclonal antibodies specific for epitopes at the C-peptide/insulin A chain junction and at the insulin B chain/C-peptide junction, respectively. Cross-reactivities with insulin, C-peptide, and the four proinsulin conversion intermediates were negligible. The detection limit in buffer was 0.2 pmol/L (3 standard deviations from zero). The working range was 0.2–100 pmol/L. The mean intra- and interassay coefficients of variation were 2.4% and 8.9%, respectively. The mean recovery of added proinsulin was 103%. Dilution curves of 40 serum samples are parallel to the proinsulin calibration curve. Proinsulin concentrations in 20 fasting healthy subjects were all above the limit of detection: median (range), 2.7 pmol/L (1.1–6.9 pmol/L). Six fasting non-insulin-dependent diabetes mellitus and five insulinoma patients had proinsulin concentrations significantly higher than healthy subjects: median (range), 7.7 pmol/L (3.2–18 pmol/L) and 153 pmol/L (98–320 pmol/L), respectively.

Development and evaluation of a colloidal gold immunochromatographic assay based on recombinant protein CatL1D for serodiagnosis of sheep fasciolosis

2019 ◽  
Vol 94 ◽  
Author(s):  
W. Xifeng ◽  
Q. Mengfan ◽  
Z. Kai ◽  
Z. Guowu ◽  
L. Jing ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Sensitivity And Specificity ◽  
Colloidal Gold ◽  
Fasciola Hepatica ◽  
Fusion Gene ◽  
Immunochromatographic Assay ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Specificity And Sensitivity ◽  
Overlap Extension

Abstract Fasciolosis is a zoonotic parasitic disease that seriously endangers the development of animal husbandry and human health. In order to develop a rapid serological diagnostic method for fasciolosis in ruminants, the CatL1D and CatB4 genes of Fasciola hepatica were amplified by reverse transcription polymerase chain reaction (PCR) and cloned, respectively, and then the CatL-B fusion gene (MeCatL-B) was constructed by gene splicing by overlap extension PCR technique. The recombinant rCatL1D, rCatB4 and rMeCatL-B proteins were then prepared by prokaryotic expression, respectively, and the recombinant protein with high specificity and sensitivity was screened via indirect enzyme-linked immunosorbent assay. Using the selected recombinant protein rCatL1D as a diagnostic antigen, we developed a colloidal gold immunochromatographic assay (CGIA) for detecting F. hepatica-specific antibodies, and 426 serum samples of slaughtered sheep were used to evaluate the sensitivity and specificity of F. hepatica CGIA assay. The results showed that the sensitivity and specificity of rCatL1D protein (100%, 96.67%) were higher than those of rCatB4 (94.29%, 80%) and rMeCatL-B (91.43%, 90%). Compared with the gold standard post-mortem inspection, the specificity and sensitivity of the CGIA method was 100% and 97%, respectively, and the consistency rate between these two methods was 99.3%. These results confirmed that the CGIA method based on rCatL1D protein could be a promising approach for rapid diagnosis of sheep fasciolosis because of its high sensitivity and specificity.

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