Seroreactivity of purified Brugia malayi microfilarial soluble and excretory-secretory antigens in different clinical presentations of bancroftian filariasis

Brugia malayi microfilarial excretory-secretory (mf ES) and phosphate buffer saline soluble (mf S) antigens were fractionated by fast protein liquid chromatography (FPLC) on superdex 200 HR 10/30 gel filtration column. The active antigen fractions were identified and explored in comparison with whole mf ES and mf S antigens to detect filarial IgG antibodies in different groups viz microfilaraemics, acute, chronic and occult filarial cases of Wuchereria bancrofti infection and endemic and non-endemic normals. One of the fractions of mf ES antigen (ESF-6) and two fractions of mf S antigen (SF-2 & 3) were identified to be useful to detect filarial antibodies. A pooled preparation of these antigen fractions gave a sensitivity of 86.6% (for microfilaraemic cases) and a specificity of 95% to detect filarial IgG antibodies by indirect ELISA. The pooled FPLC purified mf antigens also showed 55-88% of cases of different grades of clinical filariasis and 65% of tropical pulmonary eosinophilia cases as positive for filarial antibodies. The pooled FPLC purified B. malayi mf antigens with higher specificity are preferable to whole mf ES and mf S antigens to detect active filarial infection in microfilaraemia and as well in different clinical entities of bancroftian filariasis.     

Humoral immune response and antigenemia in sheep experimentally infected with Schistosoma bovis. Cross-reactivity with Fasciola hepatica antigens.

Circulating antigen level, IgG antibody response to worm antigens and to excretory/secretory products (ES), and specificity to Fasciola hepatica antigens were determined in 6 Schistosoma bovis-infected sheep at weekly intervals for 15 wk. A noninfected control group was included. An enzyme-linked immunosorbent assay (ELISA) sandwich and a double-antibody ELISA test was used for antibody and antigen detection, respectively. The infection induced an early and relatively low IgG response to adult worm extract. This response was significantly elevated by 3 wk postinfection (PI), reached its maximum level at 9 wk PI, and was followed by a subsequent decrease. The response to ES antigens was slightly higher than that to adult worms, although the response started later, at 8 wk PI, and remained at its maximum level until 15 wk. A remarkable level of cross-reactivity was observed when adult F. hepatica extract was used. However, a low degree of cross-reactivity was found with ES antigen. The ELISA for circulating antigens was performed at weekly intervals for 8 wk. Antigens were detected as early as the first week of infection, although differences were statistically significant from week 5 onward. The highest values were observed at 7 week PI.     

Kinetics of antibodies and antigens in serum of mice experimentally infected with Echinostoma caproni (Trematoda: Echinostomatidae)

The present study reports on the kinetics of antibodies and antigens in serum of mice experimentally infected with 75 metacercariae of Echinostoma caproni during the first 12 wk postinfection (wpi). Antibody titers in the serum of mice were determined by an indirect enzyme-linked immunosorbent assay (ELISA) using excretory/secretory (ES) antigens of E. caproni. The early detection of antibodies against ES antigens of E. caproni is feasible using indirect ELISA. Mice developed significant antibody responses at 2 wpi, and the values progressively increased until the end of the experiment. This may be related to the intestinal absorption of adult worm antigens that induces humoral responses. The presence of E. caproni circulating antigens was determined by a capture ELISA based on polyclonal rabbit antibodies against ES antigens of E. caproni. High levels of seroantigens in mice were detected by 1-2 wpi, probably because of the local inflammatory responses in mice induced by the adult worms. A drop in circulating antigen levels was observed at 9 wpi, which could reflect changes in the intestinal tissues over the course of the infection.     

Characterization of a Toxocara canis species-specific excretory-secretory antigen (TcES-57) and development of a double sandwich ELISA for diagnosis of visceral larva migrans

This study describes the isolation of a Toxocara canis species-specific excretory-secretory (ES) antigen and the development of an enzyme-linked immunosorbent assay (ELISA) based on this antigen. Analysis of the ES antigens of T. canis, Toxocara vitulorum, Ascaris lumbricoides and Necator americanus larval antigen was performed by SDS-PAGE followed by western blotting. A 57 kDa T. canis-specific antibody fraction (TcES-57) was identified by western blotting and labelling with anti-Toxocara antibodies (from experimental rabbits and human patients) and tracing with anti-human or anti-rabbit peroxidase conjugate. No protein fraction of 57 kDa was detected in ES or larval antigens collected from T. canis, T. vitulorum, A. lumbricoides and N. americanus. Using TcES-57, a specific antiserum was produced in rabbits and a double sandwich ELISA was developed. This test was validated using known seropositive sera from toxocariasis patients, sera from A. lumbricoides or N. americanus patients, and 50 serum samples from cats. These tests revealed that TcES-57 antigen is specific to T. canis infection and does not cross react with sera of other related infections. Thus, ELISA based on TcES-57 antigen was proven to be an effective tool in the diagnosis of toxocariasis and studies on the role of T. canis in the epidemiology of human toxocariasis.     

Early stage-specific immune responses in primary experimental human hookworm infection

To determine the cellular immune response during different stages of hookworm infection, we infected two human volunteers with Necator americanus and followed their immune responses against stage-specific, crude antigen extracts through larval migration, pre-patency, and early patency. After chemotherapy, the volunteers were followed for an additional 6 months. Low-dose N. americanus infection resulted in mild clinical signs and peripheral blood eosinophilia occurred during the estimated time of arrival of juvenile worms in the intestine. After an initial rise in proliferative responses against larval and adult worm antigens, the cellular reactivity decreased until the end of pre-patency, rose again during patency, and dropped after chemotherapy. Before infection and during the course of infection, elevated concentrations of TNF-alpha were detected in supernatants of peripheral blood mononuclear cells (PBMC) stimulated in vitro with third-stage (L3) or adult worm excretory-secretory (ES) antigens, which dropped off after chemotherapy. After stimulation with L3 antigen, elevated concentrations of CCL17 were detected in supernatants during pre-patency and patency. Interestingly, a prominent rise in IL-10 secretion was detected in ES antigen-stimulated PBMC cultures during late pre-patency. During reinfection studies in endemic areas, such distinct cytokine and chemokine profiles might be additional markers to better classify egg-negative patients.     

Evaluation of a rapid ELISA technique for detection of circulating antigens of Toxoplasma gondii

To evaluate a modified rapid ELISA method for detecting CAg during Toxoplasma gondii infection, we analyzed the specificity and sensitivity of the ELISA method by using experimental Toxoplasma infection in rabbits and also tested this method in human samples including 5428 serum, 548 cerebrospinal fluid and two breast milk samples. We prepared PcAb, and used it for rapid one-step sandwich ELISA testing in which an incubation time in the regular ELISA procedure was omitted. This method detected CAg at the concentration of 31.2 ng/mL, and no cross-reaction was found with antigens of protozoa (Cryptosporidium parvum, Plasmodium falciparum), trematode (Schistosoma japonicum, Paragonimus sp.) and nematode (Brugia malayi, Ancylostoma duodenale, Ascaris lumbricoides and Trichinella spiralis). CAg was detected in rabbit serum 3 days after infection, and optical density values reached a peak 9-13 days after infection, then declined gradually. Among human serum samples, the positive rate of CAg was 2.11% in cerebral paralysis patients, whereas it was 0.22% or 0.71% in patients without neurological symptoms or in uncomplicated pregnant women. The difference among these three groups was statistically significant (P < 0.05). The positive rate of cerebrospinal fluid samples from cerebral paralysis patients was 10.58%. There is a statistically significant difference between the positive rates of meat-packing workers and blood donors (P < 0.01). In the retrospective analysis, CAg was detected in accordance with the onset of clinical symptoms, suggesting that CAg could reflect the clinical course in humans. Together with these results, CAg detected in the modified rapid sandwich ELISA could be a sensitive marker for acute and active infection of T. gondii.     

Effects of chromium on the immune system

The performance of integral membrane antigens (IMAs) of Mycobacterium habana TMC 5135 in detecting antimycobacterial antibodies in serum and body fluids of patients mainly of extrapulmonary tuberculosis was evaluated. The IMAs were recovered from the detergent phase during Triton X-114 treatment of the plasma membrane of M. habana. Antimycobacterial antibodies were detected by ELISA using IMAs in serum and body fluids of 42 patients and 62 control subjects. As authentic adjunct Mycobacterium tuberculosis antigens were also detected (by ELISA) in body fluids and circulating immune complexes using anti-M. tuberculosis H37Ra antibodies. Anti-M. habana IMA antibody detection increased the positivity rate from 26.% (11/42) and 10% (4/42) obtained by culture and smear microscopy, respectively, to 86% (36/42). M. tuberculosis antigens were also found in 29 out of 36 anti-M. habana IMA antibody-positive cases. Interestingly, all 11 culture-positive cases were also positive for anti-M. habana IMA antibodies. The mean antigen titres in 23 cases, positive for antigens in body fluids, were 2.34 times higher in those who were also positive for anti-IMA antibodies in serum than in those negative for these antibodies. M. habana IMAs may be promising non-tubercular candidate antigens in ELISA-based serodiagnosis of extrapulmonary tuberculosis with substantial sensitivity, specificity and safety.     

Detection of circulating antigens and antibodies in Toxocara canis infection among children in Chengdu, China

In order to determine the seroprevalence of Toxocara spp. infection in children from Chengdu, we performed an enzyme-linked immunosorbent assay (ELISA) and sandwich ELISA (S-ELISA) with excretory-secretory antigens isolated from second-stage larvae of Toxocara canis (TES-Ag ELISA). The seroprevalences of T. canis antibodies in the children from rural areas, urban districts, and urban districts with recent Ascaris lumbricoides infection were 17.7% (59/333), 2.1% (4/186), and 2.6% (1/38), respectively. Among 63 suspected patients with symptoms of T. canis infection, 31 had positive antibodies. The inhibition assay showed an apparent inhibiting capacity of TES-Ag for the antibody against T. canis larvae. The result of S-ELISA demonstrated that circulating antigens of T. canis larvae could be detected in part of the serum with positive antibodies and that the detection rate for circulating antigens in the sera could be improved by polyethylene glycol-acid treatment. This is the first epidemiological study to confirm the existence of T. canis infection and Toxocara-larvae migrans in Chengdu by the combination of TES-Ag ELISA and S-ELISA.     

SLE患者血清中SARS-CoV抗体阳性原因分析

为了探讨严重急性呼吸综合征(SARS)冠状病毒(SARS-CoV)抗体测定在系统性红斑狼疮(SLE)患者中的假阳性问题,应用酶联免疫吸附试验(ELISA)和荧光定量RT-PCR技术检测了66例正常对照和31例SLE患者血清中SARS-CoV抗体的阳性率。结果,66例正常对照中,IgM抗体均阴性,IgG抗体的阳性率为3.0%(2/66);31例SLE患者中,IgM抗体和IgG抗体阳性率分别为29%(9/31)和58.1%(18/31),IgG抗体和IgM抗体同时阳性为22.6%(7/31)。经RTPCR检测,上述阳性病例均为阴性。结论:用非纯化抗原制备的ELISA试剂盒测定SLE患者的SARS-CoV抗体,可能出现假阳性,两种抗体同时测定可降低诊断的假阳性率,提高诊断的特异性。在SLE患者中出现假阳性的原因可能与包被的抗原有关。     

Patterns and Risks of Trichinella Infection in Humans and Pigs in Northern Laos

Several outbreaks of trichinellosis associated with the consumption of raw pork have occurred in Laos since 2004. This cross-sectional study was conducted in four provinces of northern Laos to investigate the seroepidemiology of trichinellosis in the human population and determine the prevalence and species of Trichinella infection in the domestic pig population. Serum samples and questionnaire data were obtained from 1419 individuals. Serum samples were tested for Trichinella antibodies by ELISA using larval excretory–secretory (ES) antigens and a subset of 68 positive samples were tested by western blot. The seroprevalence of Trichinella antibodies was 19.1% (95% confidence interval (CI) = 17.1–21.1%). The risk of having antibodies detected by ELISA using ES antigens increased with age, being of Lao-Tai ethnicity, living in Oudomxay province and being male. Tongue and diaphragm muscle samples were collected from 728 pigs and tested for Trichinella larvae by the artificial digestion method. Trichinella larvae were isolated from 15 pigs (2.1%) of which 13 were identified as T. spiralis by molecular typing; the species of the two remaining isolates could not be determined due to DNA degradation. Trichinella spp. are endemic in the domestic environment of northern Laos and targeted preventative health measures should be initiated to reduce the risk of further outbreaks occurring.     

Immunoblot analysis of serum IgG, IgA and IgE responses against larval excretory-secretory antigens of Anisakis simplex in patients with gastric anisakiasis

To increase our understanding of the immune response to Anisakis infection, antigen specific IgG, IgA and IgE responses were identified using an immunoblot technique after polyacrylamide gel electrophoresis of excretory-secretory products from the larval stage of Anisakis simplex. Nine sera were drawn from proven cases of gastric anisakiasis within 3 days after symptoms had developed. The molecular weight of the major antigenic bands were distributed between 50 kDa and 120 kDa of the antigens. In nine cases of gastric anisakiasis, three of them were positive for IgG response, five for IgE, and six for IgA, respectively. None of control sera recognized the antigenic bands in IgA and IgE responses. In contrast, two controls had IgG antibodies against 1-2 proteins in the 65-95 kDa region. The antigenicity of the excretory-secretory products was lost following treatment by 0.2% trypsin, but not by 0.2 M periodic acid. Based on the results of reactivity to lectins, antigenic bands of the ES products possessed mucin type glycoconjugate residues in their protein portion. This indicates that the humoral responses of IgA and IgE antibodies to the larval ES antigens are a more reliable index of infection than that of the IgG response.     

A comparison of glycan expression and adhesion of mouse-adapted strains and clinical isolates of Helicobacter pylori

The role of Mycoplasma pneumoniae infection as a trigger for asthma exacerbations is well supported in previous studies. This study was designed to investigate the role of M. pneumoniae infection in acute exacerbation of asthma in children. A total of 150 patients (110 males, 40 females) were studied and immunoglobulin M (IgM) antibodies to M. pneumoniae were detected by enzyme-linked immunosorbent assay (ELISA), and PCR amplification was performed for the P1 gene to associate M. pneumoniae infection with asthma. As compared with 33 children with asthma, only two of the control subjects had positive IgM titers for M. pneumoniae , which was statistically significant ( P =0.002). A total of 15 children with asthma were positive by PCR for the P1 gene while none of the controls had a positive PCR. Of these positive cases, 24 cases were positive only by ELISA, six were positive only by PCR and nine patients were found to be positive by both ELISA and PCR. All the clinical characteristics of the patients at baseline were comparable between the moderate and the severe group of patients statistically, except for the peak expiratory flow rate. Mycoplasma pneumoniae infection was found to have a significant association with acute exacerbation in the moderate group of asthma patients by PCR ( P =0.01). These data suggest that M. pneumoniae infection may contribute to asthma exacerbation.     

SLE患者血清中SARS—CoV抗体阳性原因分析

为了探讨严重急性呼吸综合征(SARS)冠状病毒(SARS—CoV)抗体测定在系统性红斑狼疮(SLE)患者中的假阳性问题,应用酶联免疫吸附试验(ELISA)和荧光定量RT—PCR技术检测了66例正常对照和31例SLE患者血清中SARS—CoV抗体的阳性率。结果,66例正常对照中,IgM抗体均阴性,IgG抗体的阳性率为3．0％(2／66);31例SLE患者中,IgM抗体和IgG抗体阳性率分别为29％(9／31)和58．1％(18／31),IgG抗体和IgM抗体同时阳性为22．6％(7／31)。经RT—PCR检测,上述阳性病例均为阴性。结论：用非纯化抗原制备的ELISA试剂盒测定SLE患者的SARS—COV抗体,可能出现假阳性,两种抗体同时测定可降低诊断的假阳性率,提高诊断的特异性。在SLE患者中出现假阳性的原因可能与包被的抗原有关。     

Humoral response to selected antigens of Yersinia enterocolitica and Yersinia pseudotuberculosis in the course of yersiniosis in humans. I. Occurrence of antibodies to Yersinia lipopolisacharydes and Yop proteins by ELISA

The antibodies against the somatic antigens of Y. enterocolitica O3, O8, O9, O5,27,Y. pseudotuberculosis I, and released proteins Yop were detected using the ELISA in 1634 serum samples and 84 synovial fluids collected from 1290 persons suspected for yersiniosis, as well as 200 serum samples from healthy individuals (blood donors). The presence of antibody in diagnostically significant titres for somatic antigens of Yersinia were detected by ELISA in 20.5% and 50.6%, antibodies for released proteins Yop in 11.5% and 28.4% respectively of blood donors and patients suspected for yersiniosis. The antibody against the O3 antigen of Y. enterocolitica was the most frequently detected antibody while the most infrequent was the antibody for the antigen from the 08 serologic group. The results of the study showed that the humoral response picture to Yersinia antigens in the course of yersiniosis in humans is dependent on the age and sex of the patient, duration of the infection, and clinical manifestations. Most frequently the elevated antibody levels were detected among patients with erythema nodosum and patients with gastrointestinal symptoms. The frequency of occurrence of antibodies for most antigens of Yersinia, together with age increased reaching its peak, on the average, among individuals aged 21 - 40 years. Analysis of individual cases showed that by the end of the first week of infection, elevated levels of antibodies for somatic antigens of Yersinia are evident. On the other hand, antibodies for released proteins Yop as a matter of rule appear in the second week from the onset of clinical symptoms. Within this early phase of infection immunoglobulins of the A and M classes dominate reaching their highest level in the second to third week of the infection. In majority of the individuals studied antibodies of the IgG class reached their highest level much later in relation to those of the IgA and IgM classes. Significant differences were found in IgA antibody detection among individuals with clinical manifestations of stomachaches and arthritis. Nevertheless, among individuals with clinical symptoms of stomachaches, these immunoglobulins as a matter of principle disappear with a period of 2-3 months from the onset of clinical symptoms. In individuals with arthritis however the aforementioned immunoglobulins maintained at considerable levels even after a year. In joint-fluid samples obtained from patients with arthritis antibodies for Yersinia antigens were detected in similar levels just as obtained simultaneously serum from those individuals.     

Detection of Entamoeba histolytica antigen in stool samples in Mersin, Turkey

Entameoba histolytica, 1 of the 2 Entamoeba species with similar morphology that infect humans, causes invasive intestinal and extraintestinal diseases, whereas Entamoeba dispar is found commensally and is noninvasive. Because of their morphologic similarity, E. histolytica and E. dispar cannot be differentiated microscopically. The antigens of E. histolytica and E. dispar, however, may be detected by the ELISA method. Previous studies have found that the detection of antigens in the stool is as sensitive and specific as cultures and isoenzyme analyses. Stool samples from 272 patients with diarrhea in the province of Mersin, Turkey, were examined for the presence of Entamoeba species microscopically and for Entamoeba (E. histolytica/E. dispar) antigens using the ELISA method. An E. histolytica-specific ELISA test was used to examine 29 E. histolytica/E. disparpositive samples. Twenty-four (8.82%) of the samples tested positive for E. histolytica/E. dispar by trichrome staining, and 29 (10.6%) of the samples tested positive for E. histolytica/E. dispar by the Entamoeba screening test. Entamoeba histolytica was positive in 21 (7.72%) and E. dispar positive in 8 (2.94%) samples. The detection of true E. histolytica infection is possible with the use of E. histolytica-specific antigen ELISA tests. Thus, real cases of amoebiasis can be detected and treated, and overtreatment of the patients with E. dispar, which is the nonpathogenic species, will be prevented.     

Evaluation of Paracoccidioides brasiliensis Infection in Dairy Goats

Paracoccidioides brasiliensis is the etiological agent of paracoccidioidomycosis, a systemic mycosis that affects mainly rural workers in Brazil and other Latin American countries. The participation of domestic and wild animal species in the ecoepidemiology of paracoccidioidomycosis is not well understood. The objective of this study was to evaluate P. brasiliensis infection in dairy goats. The humoral immune response to the gp43 antigen, the main antigen used for paracoccidioidomycosis serodiagnosis and seroepidemiology, was evaluated in two goats immunized with inactivated P. brasiliensis yeast cells. Both animals produced antibodies against the P. brasiliensis gp43 antigen, detected by ELISA, 2 weeks after immunization. A total of 202 goat serum samples were analyzed by ELISA and the immunodiffusion test using P. brasiliensis gp43 and exoantigen as antigens. The seropositivity observed by ELISA was 26.2 % although no reactivity was detected by immunodiffusion. The animals over 18 months of age showed significantly higher positivity (40 %) than animals aged 6–18 months (14.8 %) and 0–6 months (2.6 %). Taking into account that cross-reactivity may occur with other pathogens, the serum samples were also analyzed by ELISA using Histoplasma capsulatum exoantigen as antigen and the positivity observed was 14.3 %. The low correlation (0.267) observed between reactivity to P. brasiliensis gp43 and H. capsulatum exoantigen suggests co-infection rather than cross-reactivity. This is the first report showing serological evidence of P. brasiliensis infection in goats and reinforces that domestic animals are useful epidemiological markers of paracoccidioidomycosis.     

肿瘤患者血清中SARS-CoV抗体阳性原因分析

探讨SARS冠状病毒(SARS—CoV)抗体在SARS病原学诊断中的特异性及其在肿瘤患血清中的假阳性问题。应用ELISA和荧光定量RT-PCR技术检测了111例正常对照和40例肿瘤患血清中SARS—CoV抗体的阳性率。在111例正常对照中,IgM抗体均阴性,IgG抗体的阳性率为3．6％(4／111);IgG抗体诊断SARS的特异性为96．4％,两种抗体同时阳性诊断SARS的特异性为100％。40例肿瘤患中,IgM抗体均阴性,IgG抗体阳性率17．5％(7／40)。经RT—PCR检测,上述肿瘤患阳性病例均为阴性。结果表明,同时测定SARS—CoV的两种抗体可降低诊断的假阳性率,提高诊断的特异性。用非纯化SARS—CoV抗原制备的ELISA试剂盒测定肿瘤患的SARS—CoV抗体,可能出现假阳性。在肿瘤患中出现假阳性的原因可能与包被的抗原有关。     

Identification of BCP-20 (FBXO39) as a cancer/testis antigen from colon cancer patients by SEREX

Cancer/Testis (CT) antigens are considered promising target molecules for immunotherapy. To identify potential CT antigens, we performed immunoscreening of a testis cDNA library with sera from colon cancer patients by SEREX. We isolated 114 positive cDNA clones comprising 90 different antigens, designated BCP-1 through BCP-90. Quantitative real-time and conventional RT-PCR analysis showed that BCP-20, -33, and -41 antigens were expressed strongly only in a normal testis and detected in 22 cases (39%), 12 cases (21%), and 17 cases (30%), respectively, from 57 colon tumors. BCP-20 was also detected in various cancer cell lines including breast, colon, hepatoma, renal, thyroid anaplastic, ovary, sarcoma, and lung. By ELISA analysis, anti-BCP-20 antibody was detected in 3 of 50 colon cancer and 1 of 24 gastric cancer patients while healthy donors were three positive (3/50). But the BCP-20 antibody levels of patients with colon cancer showed significantly higher titers than those of healthy donors. These data suggest that the BCP-20 gene is a new CT antigen and may be useful for diagnosis and immunotherapy.     

Serodiagnosis of human opisthorchiasis using cocktail and electroeluted Bithynia snail antigens

Cocktail and electroeluted antigens from Bithynia goniomphalos, the snail intermediate host of Opisthorchis viverrini, were extracted and purified. The performance of these two antigens in the antibody detection of human opisthorchiasis was evaluated by indirect ELISA. Serum samples from people whose stool was either: (i). positive for Opisthorchis eggs (n=61); or (ii). positive for at least one of 19 other species of parasite (n=125); or (iii). clear of parasites (n=30) were tested. The sensitivity, specificity, positive predictive value and negative predictive value of ELISA using cocktail antigen were 88.5, 88, 78.2 and 94%, respectively; those of ELISA using eluted antigen (53 kDa) were 91.8, 98.4, 96.5 and 96.1%, respectively. Cross-reaction with the eluted antigen was seen in only one of four cases of hymenolepiasis and only one of 10 cases of strongyloidiasis. The kappa coefficients for ELISA in relation to stool examination were 0.84 (cocktail antigen) and 0.87 (eluted antigen). This study showed that Bithynia snail antigen could be used to replace worm antigen in the antibody detection of human O. viverrini infection.