Comparison of two IgG4 assay formats (ELISA and rapid dipstick test) for detection of brugian filariasis.

Brugia malayi infection is endemic in several Asian countries. Filaria-specific IgG4 antibody detection based on BmR1 recombinant antigen has been shown to be sensitive and specific for the diagnosis of brugian filariasis. Two formats of the test has been reported ie indirect ELISA (BE) and rapid dipstick test (BR). Since different test formats use different amounts of sample and reagents which may affect its sensitivity and specificity, this study was performed to compare these two test formats in the detection of B. malayi. A total of 264 blinded serum samples from India and Malaysia were employed. Group 1 comprised 164 samples from actively infected individuals and group 2 comprised 100 samples from filaria non-endemic areas. Sensitivity was 96.3% (158/164) and 90.8% (149/164) for rapid test and ELISA respectively; chi-square p=0.00. Both test formats demonstrated 100% specificity. Therefore the rapid test format was equally specific but more sensitive than the ELISA format. The ELISA format would be able to demonstrate decline in IgG4 titer post-treatment while the rapid test would be very useful for screening and diagnosis in the field.

Immunoblot analysis of Schistosoma japonicum egg antigens with sera from patients with acute and chronic schistosomiasis japonica.

Humoral immune responses of IgG, IgM, IgA, IgE and IgG subclass antibodies to Schistosoma japonicum egg antigens were determined by immunoblotting with serum samples from individuals in China with acute (n=24) or chronic (n=35) schistosomiasis. In general, IgM, IgA, and IgE in sera from acute patients exhibited strong binding to antigens but binding was much weaker in chronic cases. Reaction of IgG4 of chronic cases was stronger than that of IgG4 of acute cases. The recognition profile of each antibody isotype in sera was analyzed for 11 major antigen molecules (antigens with apparent molecular weights of 82, 76, 61, 57, 53, 46, 40, 32, 27, 10 and less than 6.5 kDa). Except for the 10 kDa molecule, they were well-recognized by IgA and IgE in sera of acute cases. In other combinations of antibody class and clinical phase, recognition patterns against these molecules differed among individuals. Notably, the 10 kDa molecule was specifically recognized by total IgG and IgG4 in sera from most of the chronic patients, but in sera from only one acute case. This result suggests that the 10 kDa molecule is one of the major target antigens of IgG4 and may be useful as a marker antigen to characterize the clinical phases of S. japonicum infection.

Recombinant expression of Toxocara canis excretory-secretory antigen TES-120 in Escherichia coli.

The gene encoding the excretory-secretory antigen TES-120 of dog ascarid worm Toxocara canis was cloned into the bacterium Escherichia coli. The specificity of the recombinant TES-120 antigen produced by the bacterium was investigated. A total of 45 human serum samples from patients infected with differenthelminthes and protozoa, including 8 cases of toxocariasis, were tested against the recombinant antigens in immunoblot assays. The results from the assays revealed that the recombinant TES-120 antigen reacted with sera from toxocariasis patients only. This highly specific recombinant TES-120 antigen can potentially be used for the development of an inexpensive serodiagnostic assay for human toxocariasis.

Immunodiagnosis of human fascioliasis using an antigen of Fasciola gigantica adult worm with the molecular mass of 27 kDa by a dot-ELISA.

Immunodominant antigens of an approximate molecular mass of 27 kDa (FG 27) were obtained from an excretory-secretory product of adult Fasciola gigantica by a simple continuous-elution method. A dot-ELISA using the FG 27 antigen was developed for the detection of specific antibodies from patients infected with F. gigantica. Control sera were obtained from patients with other parasitic infections and healthy volunteers. The accuracy, sensitivity, specificity, and positive and negative predictive values were 98.2%, 100%, 97.4%, 76.9% and 100%, respectively. This dot-ELISA is a specific, sensitive and easy to perform method for the rapid diagnosis of fascioliasis, particularly when more complex laboratory tests are unavailable.

Evaluation of crude antigen of Dirofilaria immitis third-stage larva for detection of antibody against Wuchereria bancrofti infection by indirect ELISA.

Dirofilaria immitis is an important heart worm in dogs. An immunodiagnostic test is frequently applied to use an alternative antigen from other parasites. A crude antigen from infective third stage larva (L3) of D. immitis was employed in detecting the antibody to Bancroftian filariasis in humans by indirect ELISA. It was shown that 25 cases of Bancroftian filariasis (76%) at a cut-off value of 0.230, were positive. Cross-reactivity was tested using available sera of other helminthic infections. These sera were 47% (23/49) positive. They comprised a major intestinal helminthic infection, 7 from 15 (46%) strongyloidiasis sera, none from 5 (0%) hookworm infection sera, 6 from 10 (60%) trichinosis sera, 2 from 10 (20%) cysticercosis sera and 8 from 9 (88%) gnathostomiasis sera. The mean OD of sera from Bancroftian filariasis patients was not significantly different from that of the other helminthic infections (p>0.05). In this study, crude antigen may be valuable for the serodiagnosis of Wuchereria bancrofti when subjects do not have tissue helminth infections. However, the crude antigen should be purified to obtain a better sensitivity and specificity of the test.

Improved antigens for IgG-ELISA diagnosis of strongyloidiasis.

Two preparations of antigens for the diagnosis of strongyloidiasis were prepared from an extract of the infective larvae of Strongyloides stercoralis: a crude antigen (CA) and a molecular weight cut-off antigen (MWCOA). Both antigens were analysed by indirect ELISA against the sera of strongyloidiasis (26 cases), other helminthiases (167) and normal controls (30). The larvae were obtained from fecal culture by a modified polyethylene tube technique after screening tests by triple simple smears per case. The larvae were extracted with distilled water and further sonicated to obtain a supernatant, the CA. A part of the CA was separated for an antigen containing molecules of lower than 30 kDa by an ultrafree-MC centrifugal filter tube (PLTK): this was designed as the MWCOA. The CA gave 96.15% sensitivity and 40.12% (67/167) specificity at a cut-off value of 0.980 (5SD); false positives were produced by 19 of 20 different helminthiases. The MWCOA produced 96.15% sensitivity at cut-off value of 0.71 (4SD); the specificity of the test was 78.44% (131/167), higher than that of CA. False positives also appeared with 15 other helminthic infections. This study suggests that MWCOA is more specific than CA. A purified MWCOA will be necessary in order to reduce cross-reactivity and provide the suitable diagnosis of strongyloidiasis.

Immunoblot evaluation of the specificity of the 29-kDa antigen from young adult female worms Angiostrongylus cantonensis for immunodiagnosis of human angiostrongyliasis.

The antigenic components of Angiostrongylus cantonensis young adult female worm somatic extract (FSE) were revealed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. The sera tested were from patients with proven angiostrongyliasis, other parasitic diseases, and healthy adults. Both the sera and cerebrospinal fluid (CSF) were tested from patients with clinical angiostrongyliasis. The CSF from patients with other neurological diseases were also included. Using SDS-PAGE, we found that the FSE comprised more than 30 polypeptides. Immunoblot analysis revealed at least 12 or 13 antigenic bands in patients with proven or clinical angiostrongyliasis, respectively. The patterns of reactivity recognized by the serum and CSF antibodies against FSE were similar. These antigenic components had molecular masses ranging from less than 14.4 to more than 94 kDa. The prominent antigenic band of 29-kDa might serve as a reliable marker for the diagnosis of angiostrongyliasis. The sensitivity, specificity, positive and negative predictive values of immunoblot analysis in this antigenic band were 55.6%, 99.4%, 83.3% and 97.4%, respectively.

Affinity purified oval antigen for diagnosis of Opisthorchiasis viverrini.

Monoclonal antibodies (MAb) were raised against an oval antigen of the liver fluke Opisthorchis viverrini which is the causative agent of a parasitosis, i.e. opisthorchiasis in Thailand. The antibodies were used in an affinity column to purify the O. viverrini oval antigen from a crude extract of adult parasites by chromatography. The oval antigen was then used in a membrane (dot) ELISA for detecting antibodies in serum samples of parasitologically confirmed Opisthorchis viverrini infected individuals (adult parasites were found in stools after praziquantel treatment and salt purgation), as well as of individuals infected with other parasites and parasite-free controls. The MAb-based dot-ELISA using the affinity purified O. viverrini oval antigen revealed 100% sensitivity, specificity and accuracy for detecting O. viverrini infection. The test is simple, rapid and highly reproducible. Several samples can be tested at the same time without the requirement for special equipment or much increase in testing time; thus it is suitable for mass screening for O. viverrini exposure, especially in new endemic areas. Furthermore using serum specimens could increase patient and community compliance compared to the conventional parasitological survey which uses stool samples for the detection of O. viverrini ova, without treatment and subsequent salt purgation, this conventional method shows a low sensitivity and is also unpleasant to both the sample donors and the laboratory technicians which has historically shown a further negative impact on the final outcome.

Wuchereria bancrofti: detection of microfilariae in asymptomatic microfilaremic individuals with Setaria digitata antigens.

A dot-ELISA for detection of microfilariae of Wuchereria bancrofti in an endemic area was developed. This test can differentiate the endemic normals from the microfilaraemic asymptomatic individuals. Antigens of molecular weight 130 and 52 kDa of the cattle filaria worm Setaria digitata were used for this test. It was observed that these two antigens were also present in the serum of asymptomatic microfilaraemic individuals.

Differential serodiagnosis of cystic and alveolar echinococcosis using native and recombinant antigens in Japan.

Our group at Asahikawa Medical College has established differential serodiagnosis for zoonotic larval cestodiases such as alveolar echinococcosis (AE), cystic echinococcosis (CE) and neurocysticercosis (NCC) using purified specific antigens. In this brief review, we introduce (a) four imported CE cases in Japan, easily identified serologically, (b) most recent advances in serology for differentiation of AE and monitoring of prognosis of AE in Japan. It includes application of affinity purified Em18 and prototype of a recombinant Em18 antigen. Serology using affinity purified Em18 antigens is showing much higher sensitivity for detection of AE cases which are usually undetectable by the ongoing serology for AE authorized in Hokkaido, Japan. As serology for AE, CE or NCC is still not popular in the majority of Asian countries, we expect that this review paper stimulates researchers who are interested in serology or serodiagnosis for these larval cestodiases including AE, CE and NCC.

A preliminary analysis of Wuchereria bancrofti microfilarial antigens for potential use in diagnosis.

Several antigens from the microfilarial stage of Wuchereria bancrofti have been identified using immunoblots of microfilarial antigens and screening with immune sera and tropical pulmonary eosinophilia (TPE) sera. This analysis revealed an array of antigens with apparent molecular weights of 14kDa, 35kDa, 42kDa, 63kDa, 88kDa, 97kDa and 200kDa. Among these only the 14kDa and 42kDa antigens were consistently recognized by most of the immune sera. A 132kDa antigen was recognized only by TPE sera. Analysis of rabbit immune sera revealed that the 42kDa antigen was shared by two developmental stages of W. bancrofti, namely L3 and mF. This antigen could become a potential vaccine candidate. The 14kDa antigen seems specific for the microfilarial stage and therefore could be a diagnostic marker for active infection. The 132kDa antigen could aid in the diagnosis of TPE.

Human antibody isotype responses to Schistosoma japonicum egg antigen.

Schistosoma japonicum-infected subjects from Hubei province of China were investigated to determine the class and subclass of the antibody response to soluble egg antigen (SEA), using an enzyme-linked immunosorbent assay. The subjects were 50 acute and 55 chronic cases. In acute cases, the mean OD values for IgA, IgE and IgG3 were very high, while the positive ratios of IgA and IgE were only 78% and 74%, respectively. The positive ratios of IgG, IgM, IgG1, IgG3 and IgG4 were all above 90%. In chronic cases, the mean OD values for IgG, IgG3 and IgG4 were very high, and the positivity rates of IgG, IgG1, IgG3 and IgG4 were all above 90%. Comparing the two study groups, the mean OD values of IgM, IgA, IgE were higher in acute cases than those of chronic cases (p < 0.0001), while the mean OD values of IgG, IgG4 were higher in chronic cases than in acute cases (p < 0.05). The mean OD values of IgG3 in both groups were high and those of IgG2 in both groups were low.

A dot-ELISA test using monoclonal antibody-purified antigens for the diagnosis of paragonimiasis caused by Paragonimus heterotremus.

A dot enzyme-linked immunosorbent assay (dot-ELISA) using antigens purified by monoclonal antibody-affinity chromatography was developed for detecting antibodies to Paragonimus heterotremus in four groups of subjects. They consisted of 30 patients with P. heterotremus infection, 93 patients with other parasitic infections, 18 patients with pulmonary tuberculosis and 30 normal, healthy controls. Sensitivity, specificity, as well as positive and negative predictive values of the test were 100, 97, 88, and 100%, respectively.

A study on the culture medium antigens of Cystcercus cellulosae for detecting antibodies of cysticercosis by means of ABC-ELISA.

Two antigens of Cysticercus cellulosae, cystic fluid antigen (CFA) and the culture medium antigen (CMA), were used in Avidin-Biotin Peroxdase Complex-ELISA (ABC-ELISA) to detect IgG antibodies in 45 cases of cysticercosis treated with praziquantel. The results revealed the total positive rates as 51.11% with CMA and 82.22% with CFA. The positive rates in the cases treated within 2 courses of treatment were 79.17% for CMA and 87.50% for CFA, and only 19.05% for CMA and 71.43% for CFA in the cases treated for more then 3 courses. The fact that the positive rates decreased as the courses of treatment increased showed that the sensitivity of CMA might be related to the vital conditions of the worms in the body, whether alive or dead. It is, therefore, recommended that CMA has the potential to be employed in ABC-ELISA both as an indicator for diagnosing cysticercosis and as a reference for the evaluation of the treatment.

Partially purified antigens of Paragonimus heterotremus for serodiagnosis of human paragonimiasis.

The preparative crude extract of Paragonimus heterotremus was fractionated by isoelectric focusing. Fractions at pH 5 which contained a specific antigen with a relative molecular weight of 31.5 kDa were pooled and used in an indirect enzyme-linked immunosorbent assay (ELISA) and immunoblot analysis for diagnosis of human paragonimiasis. The sensitivity and specificity of ELISA were found to be 100% and 99% respectively. The band of 31.5 kDa antigenic component was found to give consistent reaction with paragonimiasis sera. The sensitivity, specificity and predictive value (positive and negative) of immunoblot analysis for the 31.5 kDa band were all 100%.

Immunodiagnosis of swine trichinellosis.

A rapid, sensitive and specific serologic test has been developed for the diagnosis of swine trichinellosis. The ELISA based test utilizes L1 stichosome antigens recovered as excretory-secretory (ES) products from in vitro cultivated muscle larvae. Field studies conducted with 20,000 commercial swine using crude ES antigen demonstrated that the test could detect 98% of the medically significant infections. The test had a false-positive rate of less than 3%. Because of difficulties in regulating the quality and quantity of ES antigen and the need to continually maintain infected laboratory animals for producing the diagnostic reagent, efforts have been made to clone and express the gene(s) encoding the immunodominant ES antigens. To date a cDNA sequence, designated TsA-12, which codes in part for a 53-kDa ES antigen, has been identified and expressed in bacteria. Results demonstrate that TsA-12 is recognized by immune sera and further suggest that the immunodominant 45-, 48- and 53-kDa ES proteins which share antigenic epitopes are distinct glycoproteins.

A recombinant immunodiagnostic antigen for bovine cysticercosis.

The 70% ammonium sulfate-soluble fraction of the cyst fluid of Taenia hydatigena (designated ThFAS) was previously shown to have potential as an immunodiagnostic reagent for bovine cysticercosis. Western blot analysis indicated that the specific reactivity with antibodies in sera of T. saginata-infected cattle was associated with a 10 kDa component. Rabbit antiserum to ThFAS identified a homologous antigenic protein from the cestode Taenia crassiceps. Consequently, a cDNA expression library was constructed in lambda gt11 using poly A mRNA purified from T. crassiceps metacestodes and screened with rabbit antiserum to ThFAS. One strongly reactive clone (designated lambda TCA-2) produced a 123 kDa beta-galactosidase fusion protein which reacted in Western blot with sera from calves experimentally-infected with T. saginata and did not react with sera from uninfected calves or from cattle infected with Fasciola hepatica or with common gastrointestinal cattle parasites.

Serological differentiation of human small fluke infections using Opisthorchis viverrini and Haplorchis taichui antigens.

Sera from 642 inhabitants of Vientiane Province (Laos) were examined by enzyme-linked immunosorbent assay (ELISA) using cytoplasmic and membranous antigens prepared from adult worms. Worms of Opisthorchis viverrini originated from liver of dissected cats, Haplorchis taichui were obtained from a stool specimen of a Laotian patient after praziquantel treatment. The sera were divided into five groups according to the intensity of infection expressed as egg count per gram of patients stool (EPG). Correlation between intensity of infection and the level of antibodies in serum was recorded. Reactions obtained using the cytoplasmic antigens were more sensitive and more specific compared to those with membranous antigens. Cross-reactions between antigens of both helminth species were found. Highly positive sera were examined using electroimmunotransfer blots (EITB) with cytoplasmic antigens of both species, which enabled the species differentiation. Antigens of both species yielded several shared fractions; however, differences between them were found: homologous sera reacted specifically with O. viverrini antigen in the area of 70 kDa and with H. taichui antigen in the area of 10 kDa. Thirty-one of 122 tested sera had specific antibodies against O. viverrini, 77 sera against H. taichui and 14 sera against both species. The results confirmed our assumption about predominant occurrence of heterophyid flukes in the human population living in studied area, compared with the occurrence of opisthorchid flukes. Hence, serology seems to be helpful tool for correct diagnosis of small fluke infections.

Practicable IFAT slide antigen preparations for detection of Gnathostoma spinigerum antibodies in rabbits.

This study was designed to determine which stage of Gnathostoma spinigerum and which method of the preparation of test antigens are the most suitable for the detection of antibodies in serum of rabbits infected with advanced third stage larvae (AL3) of G. spinigerum by the indirect fluorescent antibody test (IFAT). Antigens from parasite ova and first stage larvae (L1) were obtained from freshly preserved specimens and affixed to glass slides with egg albumin. AL3 antigens consisted of paraffin sections, cryostat sections and pellets of crude worm soluble extract. Slides of adult male and female worms were prepared in cryostat sections. Pellets of crude worm soluble extract (AL3) smeared onto slides gave the best positive reaction followed by AL3 cryostat sections and L1.

Purification of antigen for serodiagnosis of echinococcosis.

Antigen for serodiagnosis of Echinococcosis is purified by chromatography on DEAE-Cellulose and Sephadex G-200 from hydatid cyst fluid. The antigen is electrophoretically pure and found sensitive and specific for Echinococcus granulosus. The antigen is thermostable and is apparently a lipo protein.