Performance of a Western immunoblot assay to detect specific anti-Toxoplasma gondii IgG antibodies in human saliva

Toxoplasma gondii represents the most prominent infectious parasitic organism found in humans. While normally asymptomatic in healthy individuals, toxoplasmosis can cause abortion in patients during pregnancy, or can be fatal in immunosupressed individuals such as persons suffering from acquired immunodeficiency syndrom (AIDS). Toxoplasma gondii infection in humans is routinely assesssed by serological means. Here, we show that detection of anti-T. gondii IgG is also possible using a non-invasive methodology employing saliva. Sera and saliva of 201 healthy volunteers were investigated for the presence of anti-T. gondii-IgG antibodies by immunoblotting. The sera of 59 (29.4%) individuals showed IgG antibodies against T. gondii by ELISA, Vidas, and immunoblotting; 58 (98.3%) of these were also positive for anti-T. gondii IgG in the saliva immunoblot, with diagnostic relevant bands of Mr of 32-35 kDa and 40-45 kDa. The saliva immunoblot test exhibits a specificity of 100% and a sensitivity of 98.5%. Thus, saliva could be used as an alternative, non-invasive means for the detection of specific anti-T. gondii IgG in humans.     

Genetic polymorphism in Taenia solium metacestodes from different Brazilian geographic areas

The aim of the present study is to investigate genetic polymorphisms in Taenia solium metacestodes from different Brazilian geographical areas and to relate them to antibody recognition in serum samples of neurocysticercosis (NC) patients. Metacestodes were obtained from the Distrito Federal (DF), Bahia, Minas Gerais (MG) and S?o Paulo (SP) regions of Brazil. Samples of human sera from 49 individuals with NC, 68 individuals with other helminthiasis and 40 healthy volunteers were analysed (157 individuals in total). Antigens were prepared and used in enzyme-linked immunosorbent assay and western blotting assays to detect specific immunoglobulin G antibodies. Genetic distances between metacestode populations were analysed using random amplified polymorphic DNA (RAPD) analysis. Our results show that there was a higher frequency of reactivity in the DF region in the sera from NC patients (p < 0.05), while discrimination between active and inactive NC was seen only in extracts from the MG and SP regions (p < 0.05). Using RAPD, the sample from the DF region presented a greater increase compared to the other regions. A relationship between genetic polymorphisms among T. solium metacestodes from different areas in Brazil and the differences in antibody detection in patients with NC were established.     

In vitro hydroxylation of proline in the collagen of the cysticercus of Taenia solium

1. In vitro hydroxylation of proline in cysticercus collagen was carried out using two different vertebrate enzymes. 2. Chick embryo enzyme is more active on cysticercus collagen than new-born rabbit enzyme. 3. Hydroxylation of cysticercus collagen is more efficient at 40 than at 37 degrees C. 4. No prolyl hydroxylase activity was detected in cysticercus extracts. 5. Collagen from the adult tapeworms Taenia solium and T. saginata lack hydroxyproline. 6. SLS crystallites from T. solium and T. saginata collagen show the same band pattern as cysticercus collagen.     

An ocular cysticercosis in Bali, Indonesia caused by Taenia solium Asian genotype

An ocular cysticercosis case of a nine-year-old Balinese girl in Indonesia is reported. She presented with redness and pain in the left eye and showed a cysticercus in the anterior chamber in December 2010. Morphological feature of the cysticercus removed from the anterior chamber indicated that it was an immature cysticercus of Taenia species with no hooklets. However, mitochondrial DNA analysis using a piece of histopathological specimen revealed it a cysticercus of Taenia solium Asian genotype. Serology by immunoblot and ELISA highly specific to cysticercosis was negative.     

Immunoglobulins anti-Anisakis simplex in patients with gastrointestinal diseases

The nematode Anisakis simplex causes anisakidiasis, a disease that often mimics other gastrointestinal diseases. Patients with digestive haemorrhaging, Crohn's disease, digestive cancer and appendicitis were analysed for antibodies to A. simplex. Antibody detection was carried out by enzyme-linked immunosorbent assay (ELISA) and immunoblotting using crude extract (CE) antigen and excretory-secretory (ES) products. Total immunoglobulin (Igs), IgG, IgM, IgA and IgE were studied. The highest percentage was obtained when Igs were tested against CE antigen. A higher percentage of positivity was observed with the appendicitis group. The Crohn's disease group showed the highest levels of IgG against the ES antigen. Using immunoblotting, 24% and 48% of sera from patients with symptoms of Crohn's disease and digestive haemorrhaging, respectively, showed a positive immunorecognition pattern of CE antigen. The prevalence of detectable antibodies against A. simplex is higher in patients with digestive disorders than in the healthy population. A linear correlation was observed between prothrombin activity and Igs-CE, IgA-CE and IgA-ES but not between IgE-CE and the other immunoglobulin levels. Specific IgA is associated with a higher activity index of Crohn's disease. Specific antibodies were observed against A. simplex in patients with appendicitis and gastrointestinal cancer, indicating a higher rate of positivity for IgA.     

Seroepidemiological studies of HIV-1 infection in large Brazilian cities

The prevalence of HIV-1 antibodies in selected groups of individuals from Rio de Janeiro, S?o Paulo and Santos was determined retrospectively. These groups and respective prevalences were: hemophiliac patients from Rio de Janeiro (1983-1984) 98.0%; polytransfused hemodialysis patients from S?o Paulo (1985-1986) 3.0% and (1987) 7.7%; intravenous drug addicts from S?o Paulo and Rio de Janeiro (1986-1987) 15.9%; male prisoners from S?o Paulo (1988) 12.5%, and pregnant women from Santos (1988-1989) 3.6%. These data stress the magnitude of AIDS in Brazil.     

A monoclonal antibody-based ELISA for the detection of circulating excretory-secretory antigens in Taenia saginata cysticercosis.

A series of monoclonal antibodies (MoAb) produced against excretory and secretory products from 10- and 20-week-old Taenia saginata cysticerci were tested for their ability to detect circulating antigen in a double antibody sandwich enzyme-linked immunosorbent assay (ELISA). Two MoAb, 12G5 and 2H8, proved to be highly reactive with the tegument of viable T. saginata cysticerci and recognized antigenic components of 65, 87 and 100 kDa in immunoblotting. The detection limit of the assay using 12G5 as trapping antibody and 2H8 as a biotinylated indicator antibody was 0.1 ng protein per ml. Although the sensitivity of the test varied from one animal to another, the minimum number of living cysticerci, which could be detected by the ELISA, was 88. Animals harbouring only dead cysticerci gave similar reactions as non-infected control animals. Cross-reactions were only observed with taeniid parasites. The test was able to detect circulating antigen also in sheep and pigs, respectively infected with T. ovis and T. solium and in the serum samples of confirmed cases of human T. solium cysticercosis.     

Age of appearance of IgG, IgM, and IgE antibodies specific for Loa loa in Gabonese children

IgG, IgM, and IgE antibodies against the filaria Loa loa were measured in umbilical cord blood and in blood from young Gabonese children by an ELISA technique using a homologous metabolic antigen. For children in eight consecutive age groups and adults the percentage of the population positive for each of the antibody classes was determined. The number of children with maternal IgG decreased until one year of age when new synthesis began to become apparent. IgM antibodies were detected only after six months, probably indicating an early infancy as opposed to a fetal infection. The percentage of individuals positive for IgM or IgE reached a peak between two and three years old, followed by a slight decline. Over half of the individuals over one year of age had IgM antibody against L. loa, indicating long-term synthesis of this class of immunoglobulin in many people. In the first two years of life, IgE antibodies were usually accompanied by L. loa-specific IgM. This specific IgE did not appear to trigger the synthesis of nonspecific IgE. By the age of two, 95% of the population had some antibodies against L. loa and by five the percentage of individuals positive for each antibody class had reached adult levels.     

Antibodies against Toxoplasma gondii in the Pacific bottlenose dolphin (Tursiops aduncus) from the Solomon Islands

Serum samples from 58 Pacific bottlenose dolphins (Tursiops aduncus) from the Solomon Islands were tested for the IgG antibody to Toxoplasma gondii by the latex agglutination test (LAT), enzyme-linked immunosorbent assay (ELISA), and immunoblotting. The ELISA cut-off value was taken as OD > or = 0.276, and the final dilution ratio, recognized as positive, was represented by the end titer. In 25 of 58 samples, no antibody activity was detected by LAT and ELISA. In 8 of 58 samples, anti-T. gondii IgG antibodies were detected by both LAT and ELISA, with titers of greater than 1 : 64 and 1 : 160, respectively. By immunoblotting, the 8 serum samples producing higher titers showed specific antibody IgG binding to several antigens on the T. gondii lane, but not on the Neospora caninum lane. No specific bands were noted on the lanes for either parasite in the 25 serum samples for which no antibody activity was detected. The specific binding of IgG antibodies to T. gondii antigens observed for serum samples producing higher titers suggests that Pacific bottlenose dolphins from the Solomon islands are exposed to T. gondii.     

Population genetic structure of Taenia solium from Madagascar and Mexico: implications for clinical profile diversity and immunological technology

Taenia solium is a cestode parasitic of humans and pigs that strongly impacts on public health in developing countries. Its larvae (cysticercus) lodge in the brain, causing neurocysticercosis, and in other tissues, like skeletal muscle and subcutaneous space, causing extraneuronal cysticercosis. Prevalences of these two clinical manifestations vary greatly among continents. Also, neurocysticercosis may be clinically heterogeneous, ranging from asymptomatic forms to severely incapacitating and even fatal presentation. Further, vaccine design and diagnosis technology have met with difficulties in sensitivity, specificity and reproducibility. Parasite diversity underlying clinical heterogeneity and technological difficulties is little explored. Here, T. solium genetic population structure and diversity was studied by way of random amplified polymorphic DNA in individual cysticerci collected from pigs in Madagascar and two regions in Mexico. The amplification profiles of T. solium were also compared with those of the murine cysticercus Taenia crassiceps (ORF strain). We show significant genetic differentiation between Madagascar and Mexico and between regions in Mexico, but less so between cysticerci from different localities in Mexico and none between cysticerci from different tissues from the same pig. We also found restricted genetic variability within populations and gene flow was estimated to be low between populations. Thus, genetic differentiation of T. solium suggests that different evolutionary paths have been taken and provides support for its involvement in the differential tissue distribution of cysticerci and varying degrees of severity of the disease. It may also explain difficulties in the development of vaccines and tools for immunodiagnosis.     

Measurement of 150 kDa protein of Taenia solium metacestodes by antibody-sandwich ELISA in cerebrospinal fluid of neurocysticercosis patients.

An antigenic protein in cystic fluid of Taenia solium metacestodes (CF) of 150 kDa was measured by antibody-sandwich ELISA in serum and cerebrospinal fluid (CSF) of neurocysticercosis patients. Capture antibodies were rabbit antisera against CF (RACF) and a monoclonal antibody (MAb) against 150 kDa protein in CF. Lower limit of antibody-sandwich ELISA was 8 ng/ml of the protein. Except CF, no tested helminths extracts reacted. Levels of the protein in 351 sera from 255 patients (55 surgery confirmed and 202 antibody and CT/MRI confirmed) were below sensitivity of the assay. Of 276 CSF from 212 patients, 31 samples (11.2%) showed positive findings. This assay, therefore, was not sensitive enough to be a diagnostic. Instead, the 150 kDa protein appeared in CSF in such situations as in 2 days after praziquantel treatment, or as in a patient infected with a racemose cysticercus with degenerated cyst wall. Of cases whose follow-up CSF were assayed, 2 cases showed that the protein appeared intermittently. These results suggest strongly that appearance of free 150 kDa protein is associated with cyst wall rupture. In CSF which contained the 150 kDa protein over 61 ng/ml, the protein was recognized in SDS-PAGE before and after immunoprecipitation.     

Antileishmanial IgG and IgE antibodies recognize predominantly carbohydrate epitopes of glycosylated antigens in visceral leishmaniasis

The specificity of human antileishmanial IgG and IgE antibodies to glycosylated antigens of Leishmania chagasi was evaluated. An ELISA was performed with soluble leishmanial antigen (SLA) and a panel of 95 sera including samples from patients with subclinical infection (SC) and visceral leishmaniasis (VL), subjects cured of visceral leishmaniasis (CVL), and from healthy individuals from endemic areas (HIEA). Antileishmanial IgG were verified for 18 (40%) of 45 SC subjects (mean absorbance of 0.49 +/- 0.17). All nine sera from VL patients had such antibody (0.99 +/- 0.21), while 11 (65%) of 17 CVL individuals were seropositive (0.46 +/- 0.05). Only three (12%) of 24 HIEA controls reacted in IgG-ELISA. Antileishmanial IgE was detected in 26 (58%) of 45 SC patients (0.35 +/- 0.14), and in all VL patients (0.65 +/- 0.29). These antibodies were also detected in 13(76%) of 17 CVL subjects (0.42 +/- 0.14) while all HIEA controls were seronegative. There was no correlation between antileishmanial IgG and IgE antibody absorbances. Mild periodate oxidation at acid pH of SLA carbohydrates drastically diminished its antigenicity in both IgG and IgE-ELISA, affecting mainly the antigens of 125, 102, 94, and 63 kDa as demonstrated by western immunoblotting.     

High SARS-CoV-2 seroprevalence in persons experiencing homelessness and shelter workers from a day-shelter in São Paulo,Brazil

Brazil presents one of the highest COVID-19 death tolls in the world. The initial SARS-CoV-2 epicenter was São Paulo city. As of 2019, the homeless population of São Paulo city was estimated at 24,344 individuals, the largest national homeless population. The present study aimed to concomitantly assess the molecular and serological prevalence and associated risk factors of SARS-CoV-2 infection in a homeless population and related shelter workers from a day-shelter. Serum samples, nasopharyngeal and oropharyngeal swabs of persons who are homeless and shelter workers collected from August 25^th to 27^th, 2020 were tested for the presence of anti-SARS-CoV-2 IgM and IgG antibodies by ELISA and SARS-CoV-2 RNA by RT-qPCR, respectively. All swab samples tested negative by RT-qPCR. Seropositivity of IgM and IgG was 5/203 (2.5%) and 111/203 (54.7%) in persons who are homeless, and 5/87 (5.7%) and 41/87 (47.1%) in shelter workers, respectively, with no statistical differences between groups. The high seroprevalence found herein indicates early environmental and urban spreading of SARS-CoV-2, associated with sociodemographic and economic vulnerability.     

Neurocysticercosis case with a single cyst in the brain showing dramatic drop in specific antibody titers within 1 year after curative surgical resection

We report on one Japanese case subject with neurocysticercosis (NCC) who had a single cyst in the brain and had undergone curative surgical resection. Pathological examination revealed that it was a cysticercus of Taenia solium. Serological examination of the pair serum samples just before and 1 year after surgery revealed that antibody responses against the glycoproteins, highly specific to NCC and detected in the serum sample just before surgery by both immunoblot and ELISA became negative within 1 year after the surgery. It is, therefore, strongly suggested that this case had a single cysticercus in his whole body and the presence of a single cysticercus was sufficient to evoke antibody responses against it.     

Reproductive behavior and seasonal occurrence of Psecas viridipurpureus (Salticidae, Araneae)

The reproductive behavior and the seasonal occurrence of Psecas viridipurpureus were studied at the Esta??o Ecológica do Noroeste Paulista, a small conservation area in the northwest region of S?o Paulo State, Brazil (49 degrees 22'50"W and 20 degrees 48'36"S). P. viridipurpureus occurred on "gravatá", a bromeliaceous plant (Bromelia balansae, Bromeliaceae) which does not accumulate rain water. During the courtship display the couple occupied the median region of the "gravatá" leaves, with the male always located in a higher position than the female. The males of P. viridipurpureus showed a complex courtship behavior, which included five motor patterns. The courtship and mating behavior occurred preponderantly during the rainy season and the juvenile recruitment between December and July. The retreat of P. viridipurpureus differs from the Salticidae pattern, since the egg sacs are covered with a plain silk cover and are not wrapped in cocoons.     

A comparative study of biochemical and immunological properties of triosephosphate isomerase from Taenia solium and Sus scrofa

We produced the Taenia solium triosephosphate isomerase (TPI) in Escherichia coli and compared its biochemical and immunological properties with those of the commercial TPI from Sus scrofa. Taenia solium TPI is a homodimer composed of two 27-kDa monomers, with a specific activity of 5,683 U/mg and a Km value of 0.758, and S. scrofa TPI is also dimeric with similar monomeric molecular weight, specific activity of 4,227 U/mg, and a Km value of 0.51. The catalytic parameters for the isomerization of glyceraldehyde 3-phosphate, affinity between TPI monomers, and kinetic thermal denaturation and inactivation were similar for both enzymes. Anti-T. solium TPI antibodies cross-react weakly with Schistosoma mansoni TPI but do not cross-react with S. scrofa, human, or protozoan TPIs. These antibodies inhibited T. solium TPI activity but did not affect S. scrofa enzymatic activity. Immunizations with 1 microg of the T. solium TPI reduced 52% of cysticerci in a mouse-Taenia crassiceps model 1 mo after challenge. Our findings show that T. solium and S. scrofa TPIs possess similar biochemical and enzymatic properties but do not share immunological properties because anti-T. solium TPI antibodies did not recognize S. scrofa TPI. Inhibition of enzyme activity by anti-TPI antibodies suggests that they can be used as inhibitors of the enzyme.     

Human serum antibody response to Helicobacter pylori whole cell antigen in an institutionalized Bangladeshi population

AIMS: To use a commercial ELISA kit and an immunoblot assay to investigate the antibody levels of selected members of the Bangladeshi population to Helicobacter pylori protein antigens. METHODS AND RESULTS: Using immunoblotting, high seroprevalence rates were observed in all age groups, although the subjects within the 1-9 years age group had the highest seroprevalence of antibodies to H. pylori antigens. By ELISA, the highest level of seroprevalence was observed in those over the age of 20 years. CONCLUSION: On the basis of these results the overall prevalence rate of H. pylori infection for the whole population was 77.4%; 77.9% for orphan boys and 76% for carers. CagA antibodies were detected in 86% of those with high levels of antibodies to H. pylori antigens. SIGNIFICANCE AND IMPACT OF THE STUDY: A combination of immunoblotting and ELISA was the most efficient means of detecting serum antibodies to H. pylori antigens and could be applied to the screening of human sera for H. pylori-specific antibodies.     

NADP-dependent isocitrate dehydrogenase of Astyanax scabripinnis (Pisces, Characidae) from three altitudes at Grande Stream, Campos do Jord?o, SP.

Electrokinetic, thermic, and kinetic properties of products of NADP-dependent isocitrate dehydrogenase (IDHP; EC 1.1.1.42) loci of Astyanax scabripinnis (Pisces, Characidae) collected at three different altitudes (700 m, 1,800 m, and 1,920 m) of Grande Stream at Campos do Jord?o, State of S?o Paulo, Brazil, were analyzed. Two IDHP bidirectionally divergent loci, a single skeletal muscle, the IDHP-A*, and a single liver IDHP-B*, both polymorphic, were detected in the three different altitude populations. The variant allele *128 at the IDHP-A* locus, had its highest frequency detected in the 1,920 m population (0.494). Among the nine variant alleles detected at the IDHP-B* locus (*37, *57, *69, *79, *85, *114, *119, *124, and *140), the *37 and 79 were detected only in 1,800 m population. Chi-square values showed that only the 700 m population is not in Hardy-Weinberg equilibrium for the IDHP-A* locus, while for the IDHP-B* locus, no population is. Homogeneity Chi2 test indicated that the populations are significantly different in their A and B phenotype frequencies. Wright's FST mean value (0.036 and 0.32, IDHP-A* and IDHP-B*, respectively) was 0.178 for the three altitude populations which means that 82% of total genetic diversity was found among individuals of each one of the populations. Stability at environmental temperatures (16 degrees to 21 degrees C), and apparent Km and Vmax values of each A-phenotype skeletal muscle crude extract suggest different roles of A-isoforms during the increased lipogenesis that occurs in fish at low temperatures.     

Taenia solium: molecular cloning and serologic evaluation of 14- and 18-kDa related, diagnostic antigens

We are attempting to design a simpler assay based on synthetic or recombinant antigens to replace the labor-intensive enzyme-linked immunoelectrotransfer blot (EITB-C), which is currently used to diagnose Taenia solium cysticercosis. From the lentil lectin-bound fraction of cyst glycoproteins (the LLGP fraction used in the EITB-C), we previously identified and purified 2 related polypeptides of 14- and 18-kDa that demonstrated diagnostic usefulness. Using degenerate oligonucleotide primers corresponding to amino acid sequences of these polypeptides and a cDNA library prepared from T. solium cysticerci, we amplified cDNA clones that represent the 14- and 18-kDa polypeptides. These clones share sequence homology at the nucleotide and amino acid levels. Synthetic polypeptides that represented the full-length, mature proteins (sTS14 and sTS18) were assessed for serologic potential using an ELISA. sTS14, but not sTS18, demonstrated utility as a diagnostic antigen. sTS14 was recognized by antibodies in a majority of the sera from patients with cysticercosis and none of the sera from persons with other helminth infections or uninfected human sera. Furthermore, polyclonal antibodies to sTS14 reacted with 6 discrete proteins present in the LLGP cyst fraction, suggesting that TS14 is a subunit of other previously described antigens used for diagnosing cysticercosis.     

Monoclonal antibodies against distinct determinants of histone H5 bind to chromatin

A series of monoclonal antibodies specific for distinguishable epitopes in chromosomal protein histone H5 were obtained from mice immunized with either free H5 or H5 . RNA complexes. The antibodies elicited by H5 could be distinguished from those elicited by H5 . RNA by their binding to native or acid-denatured H5, by their interaction with the globular region of H5, and by their cross-reactivity with H1o. The specificity of the antibodies was assessed by enzyme-linked immunosorbent assay (ELISA) and immunoblotting experiments. The antibodies could distinguish between H5 and the closely related histones H1 and H1o. The binding of some of the antibodies to the antigens was dependent on the type of assay used, suggesting nonrandom binding of the antigen to the solid supports used in ELISA and immunoblotting. Competitive ELISA experiments indicate that 8 of the 11 antibodies characterized bind to distinct epitopes. Three monoclonal antibodies bind to epitopes which are in close spatial proximity, causing mutual steric hindrance. The monoclonal antibodies bind to nuclei of fixed cells and to isolated chromatin, indicating that the epitopes are present both in the purified protein and in chromatin-complexed H5. These monoclonal antibodies can be used to study the organization of distinct regions of histones H5 and H1o in chromatin and chromosomes.