Improved canine and human visceral leishmaniasis immunodiagnosis using combinations of synthetic peptides in enzyme-linked immunosorbent assay

Background

Zoonotic visceral leishmaniasis (VL) is a severe infectious disease caused by protozoan parasites of the genus Leishmania and the domestic dogs are the main urban parasite reservoir hosts. In Brazil, indirect fluorescence antibody tests (IFAT) and indirect enzyme linked immunosorbent assay (ELISA) using promastigote extracts are widely used in epidemiological surveys. However, their sensitivity and specificity have often been compromised by the use of complex mixtures of antigens, which reduces their accuracy allowing the maintenance of infected animals that favors transmission to humans. In this context, the use of combinations of defined peptides appears favorable. Therefore, they were tested by combinations of five peptides derived from the previously described Leishmania diagnostic antigens A2, NH, LACK and K39.

Methodology/Principal Findings

Combinations of peptides derived A2, NH, LACK and K39 antigens were used in ELISA with sera from 44 human patients and 106 dogs. Improved sensitivities and specificities, close to 100%, were obtained for both sera of patients and dogs. Moreover, high sensitivity and specificity were observed even for canine sera presenting low IFAT anti-Leishmania antibody titers or from asymptomatic animals.

Conclusions/Significance

The use of combinations of B cell predicted synthetic peptides derived from antigens A2, NH, LACK and K39 may provide an alternative for improved sensitivities and specificities for immunodiagnostic assays of VL.     

Evaluation of a recombinant excretory secretory Haemonchus contortus protein for use in a diagnostic enzyme-linked immunosorbent assay

The nematode Haemonchus contortus (H. contortus) is one of the most pathogenic and economically important parasites of sheep. A 24 kDa protein is one of the important components in H. contortus excretory/secretory (ES), which was shown to have important biological function. In our research, the cDNA of its open reading frame (ES24) was obtained and analyzed. Then the ES24 was sub-cloned into pET-30a expression vector. The recombinant vector that codes hexahistidyl peptide fusion protein (His-ES24) was transformed into Escherichia coli BL21 (DE3) strain. After induction, a high expression level of His-ES24 was found at 6h taking about 26% of the total bacterial protein analyzed by gel thin-layer scanning. The expressed His-ES24 was purified and then used in an enzyme-linked immunosorbent assay (ELISA) to detect specific antibodies in serum samples. The ELISA was able to differentiate between H. contortus-infected sheep serum and Fasciola hepatica-infected sheep serum or non-infected sheep serum. No cross-reaction was observed in sheep sera that have been experimentally infected with F. hepatica. A total of 153 field sheep serum samples conserved in our laboratory were examined using the His-ES24 ELISA, and 82 (53.6%) of them were found seropositive to H. contortus. Our results demonstrate that the prokaryotic-expressed His-ES24 might be a useful diagnostic reagent for epidemiological studies of H. contortus in sheep.     

Development of enzyme-linked immunosorbent assay for detection of Mycoplasma pneumoniae antigens

The variant of enzyme-linked immunosorbent assay (ELISA) for detection of Mycoplasma pneumoniae (Mp) antigens in sera of patients with respiratory infections was developed. Sensitivity of detection of soluble antigens of Mp in modeling experiment varied from 1.5 to 1.0 ng/ml (on protein). Approbation of the assay was performed using 50 serum samples obtained from patients with confirmed diagnosis of respiratory mycoplasmosis. In the ELISA test Mp antigens were detected in 96% of samples. Obtained results were confirmed by testing of these serum samples and isolated from them circulating immune complexes (CICs) in immunoblotting using polyclonal antibodies labeled by horse-radish peroxidase. Mp antigenswere detected both in free state and as components of CICs. Specific reaction was observed with proteins, which molecular mass varied from 30 to 170 kDa (30, 37, 45, 56, 58, 72, 90, 130 and 170 (160) kDa). Obtained results point to appropriateness of use of developed assay for detection Mp antigens in sera of patients with respiratory infections.     

An enzyme-linked immunosorbent assay for the detection of canine antibodies to canine adenoviruses.

An enzyme-linked immunosorbent assay was used to detect canine immunoglobulin G antibodies specific for infectious canine hepatitis virus and the serologically related canine adenovirus Type 2. The sequential development of homologous and heterologous antibodies was measured by the enzyme-linked immunosorbent assay and serum neutralization tests in two groups of dogs which were experimentally infected with either infectious canine hepatitis virus or canine adenovirus Type 2. Both tests were comparable in their abilities to detect the development of homologous and heterologous antibodies. Homologous antibodies were detected earlier and to a higher titer in both tests. There was a 98% agreement between the serum neutralization test and the enzyme-linked immunosorbent assay when sera from 224 random-source dogs were examined for infectious canine hepatitis virus antibodies. The enzyme-linked immunosorbent assay was found to be a highly efficient and rapid test to determine the immune status of dogs to infectious canine hepatitis virus and canine adenovirus Type 2.     

Comparison of enzyme-linked immunosorbent assay systems using rift valley fever virus nucleocapsid protein and inactivated virus as antigens

Background

Rift Valley Fever (RVF) is a mosquito-borne viral zoonosis. To detect RVF virus (RVFV) infection, indirect immunoglobulin G (IgG) and immunoglobulin M (IgM) enzyme linked immunosorbent assays (ELISAs) which utilize recombinant RVFV nucleocapsid (RVFV-N) protein as assay antigen, have reportedly been used, however, there is still a need to develop more sensitive and specific methods of detection.

Methods

RVFV-N protein was expressed in Escherichia coli (E. coli) and purified by histidine-tag based affinity chromatography. This recombinant RVFV-N (rRVFV-N) protein was then used as antigen to develop an IgG sandwich ELISA and IgM capture ELISAs for human sera. Ninety six serum samples collected from healthy volunteers during the RVF surveillance programme in Kenya in 2013, and 93 serum samples collected from RVF-suspected patients during the 2006–2007 RVF outbreak in Kenya were used respectively, to evaluate the newly established rRVFV-N protein-based IgG sandwich ELISA and IgM capture ELISA systems in comparison with the inactivated virus-based ELISA systems.

Results

rRVFV-N protein-based-IgG sandwich ELISA and IgM capture ELISA for human sera were established. Both the new ELISA systems were in 100% concordance with the inactivated virus-based ELISA systems, with a sensitivity and specificity of 100%.

Conclusions

Recombinant RVFV-N is a safe and affordable antigen for RVF diagnosis. Our rRVFV-N-based ELISA systems are safe and reliable tools for diagnosis of RVFV infection in humans and especially useful in large-scale epidemiological investigation and for application in developing countries.

     

Immunodominant antigens of Leishmania chagasi associated with protection against human visceral leishmaniasis

Background

Protection and recovery from visceral leishmaniasis (VL) have been associated with cell-mediated immune (CMI) responses, whereas no protective role has been attributed to humoral responses against specific parasitic antigens. In this report, we compared carefully selected groups of individuals with distinct responses to Leishmania chagasi to explore antigen-recognizing IgG present in resistant individuals.

Methodology and Principal Findings

VL patients with negative delayed-type hypersensitivity (DTH) were classified into the susceptible group. Individuals who had recovered from VL and converted to a DTH+ response, as well as asymptomatic infected individuals (DTH+), were categorized into the resistant group. Sera from these groups were used to detect antigens from L. chagasi by conventional and 2D Western blot assays. Despite an overall reduction in the reactivity of several proteins after DTH conversion, a specific group of proteins (approximately 110–130 kDa) consistently reacted with sera from DTH converters. Other antigens that specifically reacted with sera from DTH+ individuals were isolated and tandem mass spectrometry followed by database query with the protein search engine MASCO were used to identify antigens. The serological properties of recombinant version of the selected antigens were tested by ELISA. Sera from asymptomatic infected people (DTH+) reacted more strongly with a mixture of selected recombinant antigens than with total soluble Leishmania antigen (SLA), with less cross-reactivity against Chagas disease patients'' sera.

Significance

Our results are the first evidence of leishmania proteins that are specifically recognized by sera from individuals who are putatively resistant to VL. In addition, these data highlight the possibility of using specific proteins in serological tests for the identification of asymptomatic infected individuals.     

An assay of collagenase activity using enzyme-linked immunosorbent assay for mammalian collagenase

A new method was developed for the measurement of collagenase activity using the enzyme-linked immunosorbent assay (ELISA). Rabbit colon wall collagenase, pepsin-soluble rat skin type I collagen, and its antisera were used in the present experiment. After the collagenase-degraded portion of the collagen coated on the microwell was released, the immunoreaction of the residual collagen on the microwell to anticollagen sera was determined by ELISA. This method was approximately 10 times more sensitive than the conventional assay procedure using [14C]-glycine-labeled reconstituted collagen fibrils as substrate. It was suitable for screening a large number of samples without radioisotopes.     

Immunodiagnosis of human dirofilariasis by enzyme-linked immunosorbent assay using recombinant DNA-derived fusion protein.

An enzyme-linked immunosorbent assay (ELISA) using antigenic beta-galactosidase-Dirofilaria immitis recombinant fusion protein (FP) obtained by the recombinant DNA technique provided a useful diagnostic tool for human dirofilariasis. D. immitis-infected human sera reacted strongly with FP that was immobilized with anti-beta-galactosidase monoclonal antibody on microplates. However, the FP did not react with sera from patients with other filariasis. In detection of anti-D. immitis IgG antibody. ELISA using FP was highly sensitive and specific compared to that using crude somatic antigen.     

Evaluation of the enzyme-linked immunosorbent assay for the serodiagnosis of tularemia

The usefulness of the ELISA using as antigen prepared in our laboratory supernatant obtained after centrifugation of sonicated F. tularensis cell suspension was compared with the tube agglutination test with commercial available antigen. Paired serum specimens obtained from 6 patients with ulceroglandular syndrome of tularemia were tested in both tests. The cut-off limit of serum antibodies was set at mean antibody titre determined in the sera of 115 blood donors exceeded by three standard deviations. Antibodies to F. tularensis in diagnostically significant titre were detected in all 12 serum samples by both tests. However the titres obtained in ELISA were several times higher than in tube agglutination test. In the second serum sample the level of IgA and IgM was lower but the level of IgG higher than in the first sample. We could not observe any difference in the level of antibodies between paired serum specimens in tube agglutination test.     

An enzyme-linked immunosorbent assay for bromodeoxyuridine incorporation using fixed microcultures

We report a quantitative method by which a single microculture can be examined for (i) cell morphology; (ii) cell number; (iii) DNA synthesis; and (iv) expression of cell antigens. This method first involves measuring by enzyme-linked immunosorbent assay (ELISA) the total bromodeoxyuridine (BrdU) incorporation into DNA by monolayer microcultures. The BrdU-ELISA measurement was followed by simultaneous immunostaining for BrdU-positive nuclei and for a cytoplasmic antigen. The method was applied to the measurement of mitogen-induced proliferation of rat sciatic nerve Schwann cell and cerebral astroglia microcultures. The ELISA measurement of BrdU incorporation compares favorably with measurements of tritiated thymidine incorporation and offers the additional advantages that the same microculture can subsequently be examined for cell number, for cell morphology, and for the percentage of cells having BrdU-labeled nuclei and other antigens.     

Serodiagnosis of canine Babesia gibsoni infection by enzyme-linked immunosorbent assay with recombinant P50 expressed in Escherichia coli

The entire P50 gene encoding a surface protein of Babesia gibsoni was cloned into the bacteria expression vector pGEX-4T-3 and subsequently expressed in Escherichia coli as a glutathione S-transferase fusion protein. The purified recombinant P50 was evaluated in an enzyme-linked immunosorbent assay (ELISA) for the serological diagnosis of B. gibsoni infection in dogs. ELISA was able to differentiate clearly among B. gibsoni-infected, Babesia canis-infected, and uninfected dog sera. The antibody response against the recombinant P50 was maintained at a high level until the chronic stage of infection in dogs experimentally infected with B. gibsoni. When serum samples collected from domestic dogs in Japan were examined for the diagnosis of B. gibsoni infection by the ELISA, 3 of 209 samples (1.4%) were positive for the antibody to B. gibsoni. This result was completely identical to those of Western blot analysis and the indirect fluorescent antibody test. These results indicate that the recombinant P50 expressed in E. coil is a useful diagnostic antigen for practical use in the diagnosis of B. gibsoni infection in dogs.     

Detection ofLegionella antibodies by enzyme-linked immunosorbent assay (ELISA) using whole cell and carbohydrate antigens

The systematic study ofLegionella as a human pathogen and a bacterium widely disseminated in the environment requires simplification of present methodology. We describe a highly sensitive enzyme-linked immunosorbent assay (ELISA) for the detection of serum antibodies that can also be used for the detection of antigen.Legionella pneumophila serogroups 1 and 3 (Philadelphia 2 and Bloomington 2),L. bozemanii (WIGA), andL. micdadei (TATLOCK) were grown in diphasic medium consisting of charcoal yeast extract agar (CYE) overlayed with yeast extract medium (YEM) for the production of whole cell antigen and CYE for the extraction of carbohydrate antigen. The whole cells were inactivated with 0.5% formalin. The carbohydrate was obtained from the supernatant of cells resuspended twice in phosphate buffered saline (PBS). The antigen was sterilized and concentrated by filtration and purified by chromatography through a Sepharose 4B column. The highest molecular weight fractions were used for chemical characterization, which confirmed the carbohydrate nature of the antigen, and for micro-ELISA. Titers ranging from 5×10³ to 3×10⁵ (inverse of serum dilutions) were obtained from rabbit sera collected after 1, 2, or 3 injections of whole cells. The titers were somewhat higher and more consistent with the higher of 2 antigen concentrations used (5 or 15g/ml protein or dry weight), and with the carbohydrate rather than the whole cell antigen. The reactions were serogroup and species specific and only low titers were obtained with some of the heterologous antigens. The sensitivity and specificity of the reactions were not diminished when as many as 4 antigens were mixed in the same well. Thus, the micro-ELISA can be used as a test of highly specific antigens as well as a screening test with mixtures of antigens. A preliminary test withLegionella containing water specimen concentrates and high-titer rabbit sera indicated that the micro-ELISA can also be used for the detection of antigen. This investigation appears to have paved the way for the simplification of the serological methodology for the study ofLegionella. On temporary leave from: Department of Microbiology, University of Maryland School of Medicine, 660 West Redwood Street, Baltimore, MD 21201.     

Efficacy of an enzyme-linked immunosorbent assay as a diagnostic tool for schistosomiasis mansoni in individuals with low worm burden

IgM-ELISA is an immunoenzymatic method useful for detection of IgM antibodies against a fraction of Schistosoma mansoni adult worm antigen (AWA) that is soluble in trichloroacetic acid (AWA-TCA). This method was applied to three groups of individuals with different clinical and epidemiological characteristics, and the results compared with those obtained by other diagnostic methods: immunofluorescence test for detection of IgM antibodies (IgM-IFT) or IgG antibodies (IgG-IFT), ELISA for detection of IgG antibodies (IgG-ELISA), and two parasitological methods, Kato-Katz and miracidium hatching. The IgM-ELISA presented a sensitivity of 98%, when the parasitologic fecal examination was defined as reference diagnostic method, and a specificity of 98 and 97.3%, respectively for the group of clinically healthy individuals and other helminth carriers. A comparative analysis between the results of IgM-ELISA and those obtained by other serologic tests showed a good degree of agreement, with Kappa indices ranging from 0.95 to 0.98. The diagnostic efficacy of 97.8%, as determined with schistosomiasis patients with low parasitic burden, suggests the excellent performance of the IgM-ELISA and its usefulness for the diagnosis of schistosomiasis when applied in low endemic areas.     

Immunization with a recombinant stage-regulated surface protein from Leishmania donovani induces protection against visceral leishmaniasis

Vaccination against visceral leishmaniasis has received limited attention compared with cutaneous leishmaniasis, although the need for an effective vaccine against visceral leishmaniasis is pressing. In this study, we demonstrate for the first time that a recombinant stage-specific hydrophilic surface protein of Leishmania donovani, recombinant hydrophilic acylated surface protein B1 (HASPB1), is able to confer protection against experimental challenge. Protection induced by rHASPB1 does not require adjuvant and, unlike soluble Leishmania Ag + IL-12, extends to the control of parasite burden in the spleen, an organ in which parasites usually persist and are refractory to a broad range of immunological and chemotherapeutic interventions. Both immunohistochemistry (for IL-12p40) and enzyme-linked immunospot assay (for IL-12p70) indicate that immunization with rHASPB1 results in IL-12 production by dendritic cells, although an analysis of Ab isotype responses to rHASPB1 suggests that this response is not sufficient in magnitude to induce a polarized Th1 response. Although both vaccinated and control-infected mice have equivalent frequencies of rHASPB1-specific CD4(+) T cells producing IFN-gamma, vaccine-induced protection correlates with the presence of rHASPB1-specific, IFN-gamma-producing CD8(+) T cells. Thus, we have identified a novel vaccine candidate Ag for visceral leishmaniasis, which appears to operate via a mechanism similar to that previously associated with DNA vaccination.     

The increased presence of repetitive motifs in the KDDR-plus recombinant protein,a kinesin-derived antigen from Leishmania infantum,improves the diagnostic performance of serological tests for human and canine visceral leishmaniasis

Visceral leishmaniasis (VL) is caused by protozoa belonging to the Leishmania donovani complex and is considered the most serious and fatal form among the different types of leishmaniasis, if not early diagnosed and treated. Among the measures of disease control stand out the management of infected dogs and the early diagnosis and appropriate treatment of human cases. Several antigens have been characterized for use in the VL diagnosis, among them are the recombinant kinesin-derived antigens from L. infantum, as rK39 and rKDDR. The main difference between these antigens is the size of the non-repetitive kinesin region and the number of repetitions of the 39 amino acid degenerate motif (6.5 and 8.5 repeats in rK39 and rKDDR, respectively). This repetitive region has a high antigenicity score. To evaluate the effect of increasing the number of repeats on diagnostic performance, we designed the rKDDR-plus antigen, containing 15.3 repeats of the 39 amino acid degenerate motif, besides the absence of the non-repetitive portion from L. infantum kinesin. Its performance was evaluated by enzyme-linked immunosorbent assay (ELISA) and rapid immunochromatographic test (ICT), and compared with the kinesin-derived antigens (rKDDR and rK39). In ELISA with human sera, all recombinant antigens had a sensitivity of 98%, whereas the specificity for rKDDR-plus, rKDDR and rK39 was 100%, 96% and 71%, respectively. When evaluated canine sera, the ELISA sensitivity was 97% for all antigens, and the specificity for rKDDR-plus, rKDDR and rK39 was 98%, 91% and 83%, respectively. Evaluation of the ICT/rKDDR-plus, using human sera, showed greater diagnostic sensitivity (90%) and specificity (100%), when compared to the IT LEISH (79% and 98%, respectively), which is based on the rK39 antigen. These results suggest that the increased presence of repetitive motifs in the rKDDR-plus protein improves the diagnostic performance of serological tests by increasing the specificity and accuracy of the diagnosis.     

Reactivity of purified and axenic amastigotes as a source of antigens to be used in serodiagnosis of canine visceral leishmaniasis

Although there is a great diversity of techniques and antigens used in the serodiagnosis of canine visceral leishmaniasis (CVL), total sensitivity and specificity have not yet been found. Since the use of amastigote forms in the indirect immunofluorescence assay has shown an improvement in the specificity of the test for the diagnosis of CVL, the performance of amastigotes forms of L. (L.) infantum chagasi as antigen source were evaluated in automatized ELISA test using crude antigen of axenic amastigote and purified amastigote from spleen of hamster chronically infected comparing with ELISA using total antigen produced with promastigote forms of L. (L.) infantum chagasi. One hundred and fifteen sera from dogs with positive parasitological diagnosis by PCR were used. The animals were classified into 2 groups: symptomatic (n = 67) and asymptomatic (n = 48) animals, in accordance with the clinical signs and laboratory tests were. As control, ninety-four sera from dogs with negative parasitological diagnosis were included. No significant difference was found in sensitivity, specificity, predictive values and accuracy between ELISA using whole antigens produced with both axenic and purified amastigotes in comparison with promastigotes forms. Correlation and concordance between the three total antigens tested in ELISA was observed. According to the similar performance among antigens, data pointed out to use antigen from promastigote forms for diagnosing canine leishmaniasis, especially due the easily in the production, lower cost and the abundance of correlative literature.     

Detection of Babesia caballi infection by enzyme-linked immunosorbent assay using recombinant 48-kDa merozoite rhoptry protein

The 48-kDa Babesia caballi merozoite rhoptry protein was expressed using a pGEX4T expression vector in Escherichia coli as glutathione S-transferase fusion protein (GST-BC48), and the expressed GST-BC48 was used in an ELISA to detect specific antibodies in serum samples. No cross-reaction was observed with sera from horses experimentally infected with Babesia equi. GST-BC48 ELISA was a highly sensitive and specific test when compared with the CFT. A total of 209 horse sera obtained from Central Mongolia were examined with the GST-BC48 ELISA and 46.4% (97/209) were found to be seropositive for B. caballi, suggesting that the GST-BC48 ELISA can be successfully used for both quarantine and epidemiological studies.