Circulating Antigen Research Articles

Excretion patterns of Schistosoma mansoni antigens CCA and CAA by adult male and female worms, using a mouse model and ex vivo parasite cultures.

Assays which enable the detection of schistosome gut-associated circulating anodic (CAA) and cathodic (CCA) antigen in serum or urine are increasingly used as a diagnostic tool for schistosome infection. However, little is known about the production and clearance of these circulating antigens in relation to the sex and reproductive maturity of the parasite. Here we describe CAA and CCA excretion patterns by exploring a mouse model after exposure to 36 male-only, female-only and mixed (male/female) Schistosoma mansoni cercariae. We found that serum and urine CAA levels, analysed at 3 weeks intervals, peaked at 6 weeks post-infection. Worms recovered after perfusion at 14 weeks were cultured ex vivo. Male parasites excreted more circulating antigens than females, in the mouse model as well as ex vivo. In mixed infections (supporting egg production), serum CAA levels correlated to the number of recovered worms, whereas faecal egg counts or Schistosoma DNA in stool did not. No viable eggs and no inflammation were seen in the livers from mice infected with female worms only. Ex vivo, CAA levels were higher than CCA levels. Our study confirms that CAA levels reflect worm burden and allows detection of low-level single-sex infections.

Upconversion nanoparticle-assisted single-molecule assay for detecting circulating antigens of aggressive prostate cancer.

Sensitive and quantitative detection of molecular biomarkers is crucial for the early diagnosis of diseases like metabolic syndrome and cancer. Here we present a single-molecule sandwich immunoassay by imaging the number of single nanoparticles to diagnose aggressive prostate cancer. Our assay employed the photo-stable upconversion nanoparticles (UCNPs) as labels to detect the four types of circulating antigens in blood circulation, including glypican-1 (GPC-1), leptin, osteopontin (OPN), and vascular endothelial growth factor (VEGF), as their serum concentrations indicate aggressive prostate cancer. Under a wide-field microscope, a single UCNP doped with thousands of lanthanide ions can emit sufficiently bright anti-Stokes' luminescence to become quantitatively detectable. By counting every single streptavidin-functionalized UCNP which specifically labeled on each sandwich immune complex across multiple fields of views, we achieved the Limit of Detection (LOD) of 0.0123 ng/ml, 0.2711 ng/ml, 0.1238 ng/ml, and 0.0158 ng/ml for GPC-1, leptin, OPN and VEGF, respectively. The serum circulating level of GPC-1, leptin, OPN, and VEGF in a mixture of 10 healthy normal human serum was 25.17 ng/ml, 18.04 ng/ml, 11.34 ng/ml, and 1.55 ng/ml, which was within the assay dynamic detection range for each analyte. Moreover, a 20% increase of GPC-1 and OPN was observed by spiking the normal human serum with recombinant antigens to confirm the accuracy of the assay. We observed no cross-reactivity among the four biomarker analytes, which eliminates the false positives and enhances the detection accuracy. The developed single upconversion nanoparticle-assisted single-molecule assay suggests its potential in clinical usage for prostate cancer detection by monitoring tiny concentration differences in a panel of serum biomarkers.

Early diagnosis of experimental Trichinella spiralis infection by nano-based enzyme-linked immunosorbent assay (nano-based ELISA)

Trichinellosis is a serious foodborne zoonotic disease. It is an important threat to public health all over the world. Although anti-Trichinella IgG detection is the most widely used method for diagnosis of trichinellosis, but there is an obvious window between clinical symptoms and positive serology. Gold nanoparticles (AuNPs) can be conjugated with antibodies affording them promising applications for bio-chemical detection. Herein, AuNPs-based ELISA was evaluated for the first time in the detection of Trichinella spiralis circulating antigen (CAg) for its potential as a diagnostic tool of experimental infection. Swiss Albino mice were orally inoculated with 100 muscle larvae/mouse. Animals were sacrificed 6, 8, 10, 12, 14, 16, 22 and 28 day-post infection (dpi). Blood samples were tested for CAg by both standard ELISA and nano-based ELISA using anti-rabbit polyclonal IgG conjugated with AuNPs. CAg was only detected by nano-based ELISA 6, 8, 10 dpi and by both formats 12–28 dpi. Nano-based assay recorded a statistically significant high sensitivity (58.33%, 91.67%) and accuracy (72.22%, 94.44%) 8 and 10 dpi, respectively in comparison to standard ELISA. Both assays showed high sensitivity and accuracy 12–28 dpi. Thus, nano-based ELISA could be considered as an early sensitive diagnostic method for experimental trichinellosis.

DEPRESSION AUGMENTS THE PRODUCTION OF HIGH-AFFINITY ANTIBODIES AGAINST ESTROGEN METABOLITE-RECEPTOR COMPLEX IN PROSTATE CANCER PATIENTS

BackgroundDepression is a common symptom associated with prostate cancer (PC), elevated levels of 16α-hydroxyestrone (16α-OHE1) have been linked to increased risk of PC and estrogen receptor (ER) had been expressed in prostate tissue. This study was carried out to know whether depression augments the production of anti-bodies against 16α-OHE1-ER in PC patients. MethodsForty-six depressed PC (DPC) (out of total 60 PC) patients and 40 control subjects (who have normal circu-lating prostate-specific antigen [PSA] levels) were checked for the presence of antibodies by ELISA (direct binding and competition) and quantitative precipitin titration. ResultsAntibodies from DPC patients demonstrate high binding to 16α-OHE1-ER in comparison to overall PC patients (p<0.05) and controls (p<0.001). Although, PC sera showed high binding to 16α-OHE1-ER in com-parison to ER (p<0.05) or 16α-OHE1 (p<0.001). The relative affinity of IgGsfrom DPC and PC patients was 1.01×10−7 and 1.19×10−7 M, respectively. ConclusionsDepression triggers high-affinity antibodies against this antigen in PC patients through inflammatory conditions. It also increased the release of pro-inflammatory cytokine (IL-6) that further enhanced antibodies in PC patients.

A Large Cohort of Neurocysticercosis in Shandong Province, Eastern China, 1997-2015.

Neurocysticercosis (NCC) has a wide spectrum of neurologic and psychiatric manifestations, including epileptic seizures, high intracranial pressure, cognitive dysfunction, and meningoencephalitis. This study presents the clinical diagnosis of 2539 NCC patients in Shandong Province, Eastern China, from 1997 to 2015. The diagnosis was based primarily on clinical features, neuroimaging, immunology, and electroencephalogramy studies. In all age groups, seizures were found to be the most common manifestation, followed by headaches, memory loss, and limb numbness, and disability. After antiparasitic treatment, computed tomography and magnetic resonance imaging showed that most of the lesions had been completely absorbed in 2106 (82.95%) patients, most of the lesions had been absorbed and a small proportion was converted into calcifications in 433 (17.05%) cases. Indirect hemagglutination (IHA), enzyme-linked immunosorbent assay (ELISA), and circulating antigen (CAg) serum tests initially gave 76.45%, 86.37%, and 80.66% positive results, respectively. After antiparasitic therapy, the positive rates of the IHA, ELISA, and CAg tests were 74.77%, 84.70%, and 3.75%, respectively, showing no significant difference in antibody levels (p > 0.05), but a significant difference in CAg levels (p < 0.01). The clinical antiparasitic treatment of NCC with three to four courses of a combination of albendazole and praziquantel achieved satisfactory therapeutic efficacy.

Factors Associated with Wuchereria bancrofti Microfilaremia in an Endemic Area of Mali.

Although Wuchereria bancrofti (Wb), the causative agent of lymphatic filariasis, is endemic throughout Mali, the prevalence of Wb microfilaremia (Mf) can vary widely between villages despite similar prevalence of infection as assessed by circulating antigen. To examine this variation, cross-sectional data obtained during screening prior to an interventional study in two neighboring villages in Mali were analyzed. The overall prevalence of Wb, as assessed by Wb CAg (circulating antigen), was 50.3% among 373 participants, aged 14-65. Wuchereria bancrofti Mf-positive and negative individuals appeared randomly distributed across the two villages (Moran's I spatial statistic = -0.01, Z score =0.1, P>0.05). Among the 187 subjects positive for Wb CAg, 117 (62.5%) had detectable Mansonella perstans microfilaremia (Mp Mf) and 64 (34.2%) had detectable Wb microfilaremia. The prevalence of Mp microfilaremia was 73.4% in the Wb Mf-positive group (as compared to 56.9% in the Wb Mf-negative group; p=0.01), and median Wb Mf load was increased in co-infected subjects (267Mf/ml vs 100 Mf/ml; p<0.001). In multivariate analysis, village of residence, Mp Mf positivity and gender were significantly associated with Wb Mf positivity. After controlling for age, gender, and village of residence, the odds of being Wb Mf positive was 2.67 times higher in Mp positive individuals (95% confidence interval [1.42-5.01]). Given the geographical overlap between Mp and Wb in Africa, a better understanding of the distribution and prevalence of Mp could assist national lymphatic filariasis control programs in predicting areas of high Wb Mf prevalence that may require closer surveillance.

Seroprevalence and genotype of Toxoplasma gondii in pigs, dogs and cats from Guizhou province, Southwest China

BackgroundToxoplasma gondii is an obligate, intracellular protozoan that infects almost all warm-blooded animals, including humans, domesticated and wild animals. Recent studies of Toxoplasma gondii isolates from animals in different regions of China have shown a limited genetic diversity with the dominance of the ToxoDB PCR-RFLP genotype #9 named as “Chinese 1”. However, there is not much published information regarding its prevalence in domestic animals from Guizhou province, a subtropical region in Southwest China. The objectives of this study were to determine seroprevalence and genetic diversity of T .gondii in pigs, dogs and cats in Guizhou province, Southwest China.FindingsThe anti-T. gondii IgG were detected in 70.0%(49/70) pigs, 20.56%(22/107) dogs and 63.16(12/19) cats. The anti-T. gondii IgM were found in 0.93%(1/107) dogs, 21.53%(4/19) cats, but not in pigs. In addition, the toxoplasma circulating antigen (CAG) were detected in 16.9%18/70)pigs, 13.1% (14/107) dogs and 10.5%(2/19) cats. The T. gondii DNA were detected in 31.5%(22/70) pigs, 3.7%(4/107) dogs and 52.63%(10/19) cats. Five T. gondii isolates were obtained(3 from pigs and 2 from cats). The genotype of these five isolates belonged to the predominant genotype “Chinese 1”.ConclusionsThe high prevalence of T. gondii infection in pigs,cats and dogs indicated that the T. gondii infection is common in Guizhou province. Additionally, the T. gondii genotype “Chinese 1” was dominant in Southwest China.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-015-0809-2) contains supplementary material, which is available to authorized users.

Detection of circulating antigens in serum samples of mice experimentally infected with Trichinella spiralis by a sandwich ELISA based on IgY

Abstract In the present study, a sandwich ELISA based on IgY (egg yolk immunoglobulin) was developed for detection of circulating antigens (CAg) in sere of mice experimentally infected with Trichinella spiralis. The IgY-sandwich ELISA assay involved the use of chicken antibody IgY against excretory-secretory (ES) antigens of Trichinella spiralis muscle larvae as a capture antibody and mouse polyclonal antibody IgG to ES antigens as a detecting antibody. This method was able to detect as little as 3 ng/ml of ES antigens added to normal mouse serum. A group of sixteen mice was orally inoculated with 500 T. spiralis muscle larvae per animal. The serum samples from the infected mice were taken during 1–35 days post-infection (dpi). The CAg was detectable as early as 8 dpi in the sera of infected mice. The level of CAg increased dramatically during 13–15 dpi and reached a peak at 22 dpi and remained a plateau for 3 days, then declined gradually. Another peak of CAg occurred at 31 dpi. The anti-Trichinella antibodies was first detected in 14.3 % of the infected mice at 2 weeks post-infection (wpi), and reached a peak positive rate of 100 % at 5 wpi. Moreover, the infected mice were treated with abendazole at 5 wpi and the serum CAg levels increased significantly during 2–6 days posttreatment (dpt) and then declined rapidly during 8–14 dpt. By 42 dpt, the CAg levels decreased to the undetected level, but the detection rate of antibodies was still 100 %. The IgY-sandwich ELISA appears to be a sensitive for detection of antigenemia of T. spiralis and valuable to judge the efficacy of chemotherapy in trichinellosis.

Using specific synthetic peptide (p176) derived AgB 8/1-kDa accompanied by modified patient’s sera: A novel hypothesis to follow-up of Cystic echinococcosis after surgery

Cystic echinococcosis (CE) is caused by the larval stage of the tapeworm Echinococcus granulosus. Until now, CE does not have an effective follow-up after surgery. To date, CE follow-up is conducted based on either antibody or antigen detection assays by double antibody sandwich ELISA. Unlike antigen detection, antibodies to imply exposure to an Echinococcus infection while their titration could remain for a longer period (1–10years) after surgery. Likewise, antibody respond may be related to the location of a mature hydatid cyst. Antigen detection shows presence of CE infection, it is extremely important and necessary in follow-up of CE after surgery. The circulating antigen (CAg) titration decrease faster than circulating antibody (during 1–3weeks) after operation. Location of hydatid cyst in detecting antigen is affected less than antibody also. Regarding this subject, antigen detection has several limitations that lead to be used less in CE follow-up. Although, AgB 8/1-kDa subunit is considered as a principle and immunogenic CAg but sensitivity of CAg detection compared to with antibody has variable range, between 33% and 85% which owing to formation of circulating immune complexes (CICs) in result of antigen – antibody complex. The another problem is non using specific CAg (AgB 8/1-kDa subunit) for production of specific paratopes (rabbit hyper immune antiserum) against AgB 8/1-kDa which is used as capture (primary) antibody in double antibody sandwich ELISA assay. The designation of synthetic peptides from conserved regions of AgB 8/1-kDa can help to this problem. These regions (motifs) should be selected for allelic, dominant, immunogenic and conserved without any genetic variation. The first part of this hypothesis suggests which patient’s sera should be treated with acidic buffers such as boric acid, acetic acid, glycine/HCl, polyethylene glycol (PEG) or combination of each of them accompanied by boiling patient’s sera which causes breaking Ag-Ab complexes and in result of releasing AgB 8/1. These modifications are effective to releasing CAg from CIC. To date, the synthetic peptides have been widely used in CE serodiagnosis based on circulating antibody detection only. In second part of this hypothesis suggests the using synthetic peptide of p176 derived AgB 8/1-kDa subunit containing conserved specific epitopes for preparation of specific paratopes (rabbit hyper immune antiserum) based on CAg detection. So, there is no need any native antigens for preparation of non-specific rabbit hyper immune antiserum. These novel improvements can help to decrease the cross-reactivity with other parasitic diseases (specificity). Increasing antigen detection could make a chance in sensitivity of patient’s sera and in result of the best and suitable tool for CE follow-up.

Analysis of a novel cathepsin B circulating antigen and its response to drug treatment in Trichinella-infected mice

In this paper, we cloned a novel full-length cDNA that encodes a Trichinella spiralis cathepsin B-like protease gene (TsCPB) using 3'-RACE PCR. The recombinant mature TsCPB protein (rTsCPB) was then expressed in an Escherichia coli expression system and purified with Ni-affinity chromatography. Real-time quantitative PCR revealed that TsCPB was expressed across all development stages of the parasite but had the highest expression level during the adult stage. Furthermore, rTsCPB was detected in Trichinella excretory-secretory products with anti-rTsCPB rabbit polyclonal antibodies. Interestingly, rTsCPB was strongly recognized by the T. spiralis-infected sera in Western blotting, implying that TsCPB protein appeared in the peripheral blood of Trichinella-infected mice as circulating antigens (CAg). We then analyzed the dynamic levels of TsCPB CAg and its antibodies in T. spiralis-infected sera by using an improved double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) and indirect ELISA, respectively. The results showed that TsCPB CAg can be detected much earlier compared to antibody detection in Trichinella-infected mice. In addition, we monitored the effects of albendazole drug therapy (a dosage of 370mg/kg body weight, twice a day) on T. spiralis-infected mice by detecting the levels of TsCPB CAg and its antibody in the sera of drug-treated mice. The results showed that the levels of CAg dramatically decreased after successful drug treatment, while the antibody level remained unchanged. Overall, the novel Trichinella antigen TsCPB could be a promising novel circulating antigen molecule for the detection of Trichinella infection and for monitoring the efficacy of drug treatment of trichinellosis.

Detection of circulating antigen in serum of mice infected with Trichinella spiralis by an IgY–IgM mAb sandwich ELISA

In this study, a sandwich ELISA based on IgY (egg yolk immunoglobulin) and IgM monoclonal antibody (mAb) against excretory-secretory (ES) antigens of Trichinella spiralis muscle larvae was developed for detection of circulating antigens (CAg) in serum from mice infected with T. spiralis. The IgY-IgM sandwich ELISA involved the use of chicken antibody IgY as a capture antibody and mouse IgM mAb 5A7G4 as a detecting antibody. This assay was able to detect as little as 1 ng/ml of ES antigens added to normal mouse serum. Two groups of BALB/c mice infected with T. spiralis larvae were used: heavily infected mice (20 mice infected with 300 larvae) and lightly infected mice (20 mice infected with 100 larvae) and 10 normal mice as control. The CAg was detectable as early as 4 days post infection (dpi) in the sera from both groups of infected mice, then increased rapidly, reached a peak with detection rate of 100% in heavily infected mice at 10 dpi and 80% in lightly infected mice at 22 dpi, respectively. The anti-Trichinella IgM antibodies was first detected in 40% of heavily infected mice and 20% of lightly infected mice at 8 dpi, and reached a peak positive rate of 100% in heavily infected mice at 16 dpi and in lightly infected mice at 26 dpi, respectively. The novel assay appears to be sensitive for detection of antigens of T. spiralis and valuable to the early diagnosis of trichinellosis.

Prevalence of Toxoplasma gondii antibodies, circulating antigens and DNA in stray cats in Shanghai, China

BackgroundToxoplasma gondii is prevalent in most areas of the world and may cause abortions or neonatal complications in humans. As the only definitive host, cats play an important role in the epidemiology of the disease. Infection rates in cats, especially stray or free-living cats, are considered to be the best sentinels of the level of T. gondii in the environment. The T. gondii infection can be diagnosed in different ways with different methods depending on the target. However, little information on T. gondii infection in cats was available in Shanghai, China. Moreover reports on prevalence of circulating antigens, antibodies and DNA of T. gondii in the same study are rare.MethodsIn the present study, the presence of antibodies (Ab), circulating antigens (CA), and/or DNA of Toxoplasma gondii in samples from 145 stray or unwanted cats from 6 animal shelters in Shanghai (China) was determined in order to estimate the prevalence of T. gondii infection, by Ab-ELISA, CA-ELISA, and nested-PCR, respectively.ResultsThe positive rates for the antibodies, circulating antigen and DNA of T. gondii were 11.7% (17 of 145), 5.5% (8 of 145) and 5.71% (2 of 35), respectively. No cat tested was positive by both the Ab-ELISA and the CA-ELISA, but the results of the PCR were consistent with the CA-ELISA assay. Therefore, the overall estimated prevalence of toxoplasmosis was 17.2% (25 of 145). According to our results, the positive rates of specific antibodies and circulating antigen of T. gondii were significantly different between adult cats (>1 year old) and juvenile cats (≤1 year old); the former was 13.5% versus 3.9% by Ab-ELISA, while the latter was 1.7% versus 23.1% by CA-ELISA. From the results obtained with all three detection methods used in this study, the rate of infection was not significantly different between male and female cats (P ≥0.05); and the overall rate was 17.9% for males versus 16.4% for females.ConclusionsThe results suggest that detection of circulating antigens (CA) is necessary in surveys of T. gondii infection, especially for juvenile cats. Our investigation revealed that the prevalence of T. gondii infection in stray cats in Shanghai is high. Control programs are needed for stray cat populations in order to reduce the risk of zoonotic transmission of toxoplasmosis to other domestic animals and humans, especially females.

Detection of Trichinella spiralis Circulating Antigens in Serum of Experimentally Infected Mice by an IgY-mAb Sandwich ELISA

In this study, a sandwich enzyme-linked immunosorbent assay (ELISA) based on IgY (egg yolk immunoglobulin) and monoclonal antibody (mAb) against excretory-secretory (ES) antigens of Trichinella spiralis muscle larvae was developed for detection of circulating antigens (CAg) in serum from mice infected with T. spiralis. The IgY-mAb sandwich ELISA involved the use of chicken antibody IgY as a capture antibody and mouse mAb 35B9 as a detecting antibody. This method was able to detect as little as 1 ng/mL of ES antigens added to normal mouse serum. A group of 15 mice was orally inoculated with 500 T. spiralis muscle larvae per animal and the serum samples were daily taken during 1-49 days post-infection (dpi). The level of CAg was detectable as early as 3 dpi in the sera from infected mice, increased gradually, and reached two peaks with detection rate of 40% at 13 dpi and 100% at 24 dpi, respectively. The anti-Trichinella antibodies was first detected in 33.3% of the infected mice at 3 week post-infection (wpi), and reached a peak positive rate of 100% at 5 wpi. Moreover, the infected mice were treated with abendazole at 5 weeks post-infection, and the serum levels of CAg in treated group began to increase rapidly at 2 days post-treatment (dpt) and reached a peak with detection rate of 100% (10/10) at 8 dpt, and then decreased gradually. By 42 dpt, the CAg levels decreased to the undetected level, but the anti- Trichinella antibodies were still detected in 100% of the infected mice. The novel assay appears to be sensitive for detection of antigens of T. spiralis and should be valuable to the early diagnosis and evaluation of the efficacy of chemotherapy in trichinellosis.

Identification and profiling of circulating antigens by screening with the sera from schistosomiasis japonica patients

BackgroundSchistosomiasis is a chronic disease caused by trematode flatworms of the genus Schistosoma. The disease remains a serious public health problem in endemic countries and affects at least 207 million people worldwide. A definite diagnosis of the disease plays a key role in the control of schistosomiasis. The detection of schistosome circulating antigens (CAs) is an effective approach to discriminate between previous exposure and current infection. Different methods have been investigated for detecting the CAs. However, the components of the schistosome CAs remain unclear. In this study, we analyzed the CAs in sera of patients infected with Schistosoma japonicum.MethodsThe parasites were collected from the infected rabbits for preparing the adult worm antigen (AWA). The hyline hens were immunized subcutaneously with AWA to produce anti-AWA IgY. The IgY was purified by water-dilution and ammonium sulfate precipitation method and identified by ELISA and Western blotting. After purification and characterization, IgY was immobilized onto the resin as a capture antibody. The circulating antigens were immune-precipitated from patients′ serum samples by direct immunoprecipitation. The precipitated proteins were separated by one-dimensional electrophoresis and analyzed by LC-MS/MS.ResultsFirstly, the IgY against AWA was produced from the eggs of immunized hens by AWA, which gave a titer of 1:12800. The purified IgY was used as the capture antibody to enrich the CAs in sera of S. japonicum infected patients through immunoprecipitation. The CAs were determined by LC-MS/MS. There were four proteins, including protein BUD31 homolog, ribonuclease, SJCHGC06971 protein and SJCHGC04754 protein, which were identified among the CAs.ConclusionsWe developed a novel method based on IgY for identification and profiling CAs in sera of S. japonicum infected patients. Four new CAs were identified and have potential value for further development of an antigen assay.

Study on distribution of cohere antigen of 12D5 and 21B7 monoclonal antibodies of Angiostrongylus cantonensis and its application of immnodiagnosis

Objective To detect infection of Angiostrongylus cantonensis and examine effection of treatment to prepare monoclonal antibodies(McAbs). Methods Six-week-old BALB/c mice were imrnunized by the intraperitoneal injection of e/s antigens of Angiostrongylus cantonensis. Fusion of splecn cells from immunized mice with prepared SP2/0-Ag14 myeloma cells was performed in RPMI 1640. Fused cells were suspended in RPMI 1640 containing 1% HAT and 20% fetal calf serum and dispensed into 96-well cell culture plates. The supernatants of clones were screened by ELISA with sera of patients of angiostrongyliasis.Distribution of cohere antigen of 12D5 and 21B7 monoclonal antibodies was analyzed with immunohistochemistry. Two McAbs ( 12D5 and 21B7) were applied to detect the circulating antigen (CAg) in the sera of rats infected with A. cantonensis and angiostrongyliasis patients respectively by double antibody sandwich ELISA.Results 12D5 McAb was identified as IgG1 and 21 B7 McAb was IgM. Western blot result showed two McAbs could used to identified 55 × 103 protein of adult worms of A. cantonensis. Cohere antigen of 12D5 and 21B7 monoclonal antibodies were distributed on intestine surface of A. cantonensis. The detection rates of CAg in the sera of infected rats 100% (48/48), the detection rates of CAg in the sera of angiostrongyliasis patients was 100% (32/32). No cross-reaction to sera of patients with other infection of parasites, such as clonochiasis, fasiolopsiasis, ancylostomiasis, trichinosis, anisakiasis as well as schsitosomiasis, and health srea did not reacted with 12D5 and 21B7 McAbs,and detaction rate of antibody of angiostrongyliasis patients only reached 75% (24/32) with antigen of A. cantonensis. Conclusion Cohere antigen of 12D5 and 21B7monoclonal antibodies were antigens of enteric epithelium. Sandwich ELISA with 12D5 and 21B7 McAbs showed high specificity act as detecting CAg of A. cantonensis in sera of infection animal and patients. It is apparent that Sandwich ELISA with 12D5 and 21 B7 is not only rapid and simple without requirement of special instrument, but also rather sensitive and specific for the detection of current infection with A. cantonensis. Key words: Angiostrongylus cantonensis; Monoclonal antibodies; Sandwich ELISA; Antigen distribution analysis

Development of a Double Antibody Sandwich Elisa Assay for the Diagnosis of Angiostrongyliasis

With the use of 2 monoclonal antibodies (MAbs) against excretory/secretory (ES) antigens of adult Angiostrongylus cantonensis, a new method was developed for double antibody sandwich ELISA for the detection of circulating antigens (CAg). To evaluate the sensitivity of the new procedure, the CAg in sera of rats (80) and mice (15) infected with A. cantonensis, as well as CAg in sera of clinically confirmed angiostrongyliasis patients (70), were evaluated. Cross-reaction testing was used to determine the specificity of serum from patients infected with Ascaris lumbricoides, Trichinella spiralis , Toxoplasma gondii , Schistosoma japonicum, Paragonimus westermani, Clonorchis sinensis, Echinococcus granulosus, Spirometra, and Taenia solium, as well as normal healthy people. The results proved that the sensitivity and the specificity of the new method were totally effective for the detection of A. cantonensis CAg. The assay is highly sensitive, specific, and reproducible, with easy handling and excellent cost effectiveness, and thereby provides a new method for the accurate diagnosis of angiostrongyliasis.

Myelitis Caused by Infection of Angiostrongylus cantonensis

A 32-year-old man presented to our hospital on July 5, 2006, after the onset of headache, paresthesias of the left upper limb for 10 days, and weakness for 7 days before admission. He had eaten an inadequately cooked Pomacea canaliculata 20 days previously. Laboratory testing indicated a normal white blood cell count of 6,700/mm3 with mild eosinophilia of 7.8% (523/mm3). A lumbar puncture test showed an opening pressure of 220 mm H2O and 160 cells with 23% eosinophils, and cerebrospinal fluid (CSF) cultures were negative. We detected the circulating antigens (CAg) of Angiostrongylus cantonensis by double antibody sandwich enzyme-linked immunosorbent assay (ELISA), and they tested positive. This method had a high sensitivity (86.4%), and no cross-reactions with sera from patients with many other parasites were observed.1 Therefore, the result was helpful for diagnosis. Spinal magnetic resonance imaging (MRI) showed a lesion with high signal intensity in the cervical spinal cord on both sagittal and transverse T2-weighted imaging (T2WI) (Figures 1 and ​and2)2) at 9 days after admission. Figure 1. A lesion in the cervical spinal cord presented as hyperintense on a sagittal T2WI. Figure 2. A lesion in the cervical spinal cord presented as hyperintense on a transverse T2WI. On the basis of history, clinical presentation, and examinations, a diagnosis of angiostrongyliasis was made,2 and the patient was treated with a combination of albendazole and dexamethasone. Symptoms of headache and paresthesia resolved within 14 days, and spinal-cord lesions completely resolved by a 1-month follow-up (Figures 3 and ​and44). Figure 3. The abnormally high signal on a sagittal T2WI completely disappeared. Figure 4. The abnormally high signal on a sagittal T2WI completely disappeared.

Seroprevalence of Angiostrongylus cantonensis Infection in Humans in China

A seroepidemiological survey was carried out in China during 2009-2010 to determine the extent of circulating antigens (CAg) for Angiostrongylus cantonensis in the Chinese population using the gold immunochromatographic assay, with the objective of elucidating the nationwide prevalence of angiostrongyliasis in China. A total of 1,730 blood samples was collected and assayed from the general adult population (the "general group"), and those involved in aquaculture or processing of snails Achatina fulica and Pomacea canaliculat (the "occupational group") from 5 provinces (Fujian, Hunan, Guangdong, Guangxi, and Zhejiang) and 1 municipal city (Beijing). The overall seroprevalence for the "occupational group" was 7.4% (40/540), which was significantly higher (P < 0.001) than that of the "general group" (0.8%, 9/1,190). The seroprevalence in males (9.5%) was significantly higher than in females (4.2%) (P < 0.05). These results demonstrate that angiostrongyliasis represents a significant zoonotic disease in China, requiring the strengthening of food safety for control of this food-borne disease.

Detection of circulating antigen of Angiostrongylus cantonensis by 12D5 and 21B7 monoclonal antibodies

To detect the rate of Angiostrongylus cantonensis infection and to study the effects of treatment so as to prepare monoclonal antibodies (McAbs), and gold immunochromatography assay (GICA) with 12D5 and 21B7 McAbs could be prepared in advance. Two McAbs (12D5 and 21B7) were applied to detect the circulating antigen (CAg) in the sera of rats infected with A. cantonensis and angiostrongyliasis patients respectively by double antibody sandwich ELISA. Either 12D5 or 21B7 McAbs was used as antibody and protein A was conjugated with colloid gold as the detection marker. A special pad for GICA was designed according to the reaction procedure, and CAg were detected by GICA in the sera of rats infected with A. cantonensis and angiostrongyliasis patients respectively. 12D5 McAb was identified as IgG1 and 21B7 McAb was IgM. Results from Western blotting showed that two McAbs could be used to identified 55 KD protein of adult worms of A. cantonensis. The detection rates of CAg in the sera of infected rats was 100% (48/48) and the detection rates of CAg in the sera of angiostrongyliasis patients was 100% (32/32). No cross-reaction to sera of patients with other infection of parasites, such as clonorchiasis, fasciolopsiasis, ancylostomiasis, ancylostomiasis, anisakiasis as well as schistosomiasis wee seen and normal sera did not react with 12D5 and 21B7 McAbs. Results from sandwich ELISA and GICA with 12D5 and 21B7 McAbs showed high specificity and acting as detecting CAg of A. cantonensis in sera of infected animals and patients. We noticed that GICA with 12D5 and 21B7 was not only rapid and simple that without requirement of special instrument, but also rather sensitive and specific for the detection of current infection with A. cantonensis.

A Case of Optic Nerve Compression Caused by Angiostrongylus cantonensis

A 47-year-old man presented with right-side torso pain and numbness and intermittent fever and was diagnosed with pulmonary infection. However, he reported blurred vision in his right eye during therapy. Further questioning of the patient about his behavior uncovered that he ate inadequately cooked Pomacea canaliculata twice within 54 days. This piece of information, along with the fact that it coincided with an outbreak of angiostrongyliasis in Beijing, 1 brought to our attention the possibility of this disease. Because of this, some related examinations were performed. An investigation based on circulating antigens (CAg) of Angiostrongylus cantonensis was positive, orbital computed tomography (CT) scan showed that there was a soft tissue lesion (6.7 × 2.6 mm in size) at the lower part of the right optic nerve sheath ( Figure 1 ), and ocular magnetic resonance imaging (MRI) showed abnormal enhancement of the right ocular nerve sheath ( Figure 2 ). Unfortunately, an orbital CT scan showed that the soft tissue lesion (7 × 2.2 mm in size) remained unchanged ( Figure 3 ) at the 3-month follow-up. Several treatments have been used in ocular angiostrongyliasis, including surgical removal. 2 In this case, the orbital CT dynamic detections showed that the granuloma was still, indicating that the larva was dead—the patient was therefore not treated with albendazole. Furthermore, the granuloma could not be removed by surgery because of the particular location of infection—this led to an unfortunate lack of visual acuity improvement after treatment.