[Screening and identification of therapeutic effect evaluation antigens of angiostrongyliasis].
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To identify antigens which may help evaluate the therapeutic effect of angiostrongyliasis from adult worm antigen of Angiostrongylus cantonensis.The adult worm antigens of A. cantonensis were analyzed by Western blotting with the sera of rats infected with A. cantonensis before and after treatment. The sera of rats were tested by enzyme-linked immunosorbent assay (ELISA).The antigens with relative molecular mass between 38,000 and 78,000 reacted not only with the sera of rats before treatment, but also with that after treatment. The antigens with M(r) between 190,000 and 17,000 reacted with the sera of rats before treatment but not with that after treatment; those with M(r) between 32,000 and 24,000 antigens strongly reacted with the former, but the reaction became much weakened with the latter. The AC32-IgG antibody appeared earlier than the AC-IgG, and disappeared rapidly after treatment. Six of the 10 treated rats became negative for AC-IgG as found by ELISA.The antigens of adult worm antigen of A. cantonensis with M(r) of 190,000, 32,000, 24,000, 17,000 and 16,000 may serve as candidate antigens for therapeutic effect evaluation of angiostrongyliasis.Keywords:
Angiostrongyliasis
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To develop and identify monoclonal antibodies (McAbs) against adult worm of Angiostrongylus cantonensis and observe its applicability.BALB/c mice were immunized with soluble antigen of adult worms of A. cantonensis. The spleen cells of immunized mice were fused with myeloma cell, and the hybridoma secreting high titer of McAbs with high specificity was screened. By using the McAbs, serum of angiostrongyliasis patient and sera of the rats infected with A. cantonensis were detected by Western blotting and double antibody sandwich ELISA respectively.Three McAbs were established (2A2, 3F1, 4H2), which all showed no cross reaction with antigens of Schistosoma japonicum, Paragonimus westermani, Cysticercus cellulosae and Trichinella spiralis. Western blotting analysis demonstrated that the three McAbs recognized a Mr 15,000 soluble antigen of adult worm of A. cantonensis and recognized the Mr 24,000 and Mr 15,000 circulating antigens from the serum of angiostrongyliasis patient. The double antibody sandwich ELISA detection showed a positive rate of 76.5%.Three hybridoma cell lines against adult worm of A. cantonensis have been established which secret high titer of McAbs with high specificity and seem promising in detecting the circulating antigen of the angiostrongyliasis patient.
Angiostrongyliasis
Trichinella spiralis
Paragonimus westermani
Antibody titer
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Two monoclonal antibodies, which recognize the epitope on an antigen with a molecular weight of 204 kD from the fifth-stage worm of Angiostrongylus cantonensis, were previously prepared and used to detect circulating antigens in patients with eosinophilic meningitis or meningoencephalitis and in mice experimentally infected with this parasite by a double-antibody, sandwich enzyme-linked immunosorbent assay (ELISA). The levels of this circulating antigen in experimentally infected mice were significantly higher three weeks after infection. The ELISA values in the detection of circulating antigens in cerebrospinal fluid (CSF) from patients were markedly higher than those in serum. Immunodiagnosis of patients with angiostrongyliasis by this technique proved to be highly specific for circulating antigens in serum and CSF specimens; however, the sensitivity in CSF was significantly higher than in serum.
Angiostrongyliasis
Trichinella spiralis
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To analyze the difference among antigens of Angiostrongylus cantonensis in different developmental stages and identify dominant diagnostic antigen for angiostrongyliasis.Antigens of A. cantonensis in different developmental stages were analyzed by SDS-PAGE and immunoblot.The protein bands of all developmental stages were similar on SDS-PAGE. The Mr 40000, 50000, 66000 and 80000 antigens reacted not only with the sera of rats infected by A. cantonensis but also with the sera of normal rats. The Mr 104000 antigen could be discerned by sera of rats infected with A. cantonensis for 2 weeks. The Mr 32000 antigen could be recognized by sera of rats 2 weeks after infection, and the reaction became stronger with the infection continued.The Mr 40000, 50000, 66000 and 80000 antigens might result in the unspecific reaction in the immunodiagnosis of angiostrongyliasis using the crude antigen of A. cantonensis. The Mr 104000 of larva, Mr 33000 of adult females and Mr 32000 of the worms might be used as candidate antigens in early diagnosis and epidemiological survey of angiostrongyliasis.
Angiostrongyliasis
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The enzyme-linked immunosorbent assay (ELISA) was employed for the sero-diagnosis of Angiostrongylus cantonensis infections in rats and man. Metabolic and 'purified' antigens of adult or juvenile A. cantonensis were evaluated by ELISA and compared against Toxocara cant's antigen for sensitivity and specificity. Sera and cerebrospinal fluid (CSF) from both rats and man gave higher ELISA values against antigens of A. cantonensis than those obtained from negative control samples. The results showed that cross reaction occurred between T. canis and A. cantonensis. Of four A. cantonensis antigens tested, a Sephacryl S-300 "purified" fraction was shown to be superior in the ELISA to determine angiostrongyliasis.
Angiostrongyliasis
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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
Angiostrongyliasis
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To identify antigens which may help evaluate the therapeutic effect of angiostrongyliasis from adult worm antigen of Angiostrongylus cantonensis.The adult worm antigens of A. cantonensis were analyzed by Western blotting with the sera of rats infected with A. cantonensis before and after treatment. The sera of rats were tested by enzyme-linked immunosorbent assay (ELISA).The antigens with relative molecular mass between 38,000 and 78,000 reacted not only with the sera of rats before treatment, but also with that after treatment. The antigens with M(r) between 190,000 and 17,000 reacted with the sera of rats before treatment but not with that after treatment; those with M(r) between 32,000 and 24,000 antigens strongly reacted with the former, but the reaction became much weakened with the latter. The AC32-IgG antibody appeared earlier than the AC-IgG, and disappeared rapidly after treatment. Six of the 10 treated rats became negative for AC-IgG as found by ELISA.The antigens of adult worm antigen of A. cantonensis with M(r) of 190,000, 32,000, 24,000, 17,000 and 16,000 may serve as candidate antigens for therapeutic effect evaluation of angiostrongyliasis.
Angiostrongyliasis
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Objective To establish a method for the isolation of stage-1 larvae of A. cantonsis,and to evaluate its diagnostic value for the treatment of angiostrongyliasis. Method Three different purification procedures,namely Baermann funnel method,sucrose density centrifugation method and modified Baermann funnel combined with trypsin digestion,were compared. Stage-1 larval antigens and adult worm antigens were analyzed by SDS-PAGE,Western blotting and enzyme-linked immunosorbent assay (ELISA). Result The separation efficiency of the three methods was 50.1%,33.6% and 19.7%,respectively. Highly purified larvae with 90% of viability were obtained by the modified Baermann funnel combined with trypsin digestion method. 99% of larvae obtained by the Baermann funnel method were viable,but the purity was low. Most of the larvae obtained by sucrose centrifugation method were inactive. SDS-PAGE analysis showed that the total number of protein bands of stage-1 larvae was less than that of antigens prepared from adult worm. Western blotting analysis showed that the 104 000 Mr stage-1 larval antigen strongly reacted with the immune sera from rats infected with A. cantonensis for 2 weeks. Both 31 000 Mr and 32 000 Mr antigens were found to react with the immune sera of rats infected with A. cantonensis for 6 weeks. The 32 000 Mr antigen also reacted with the sera of patients with angiostrongyliasis,and immuno-reactivity to normal sera was not observed. Both stage-1 larval antigens and the adult antigens showed the same level of reactivity to sera of patients with angiostrongyliasis and rats infected with A. cantonensis by the ELISA method. Conclusion Viable and highly purified larvae can be obtained by the modified Baermann funnel combined with trypsin digestion method. The 31 000,32 000 and 104 000 Mr stage-1 larval antigens can be used in serological diagnosis of angiostrongyliasis.
Angiostrongyliasis
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The possibility of cross-reactivity was previously investigated by indirect ELISA with sera from Angiostrongylus cantonensis infections, normal controls and A. costaricensis antigen. 5 microg/ml of crude antigen from both sexes of each species reacted with diluted serum samples (1:800) of each of 20 cases of angiostrongyliasis and normal controls, and further with anti-human IgG conjugate at 1:1,000. The mean absorbance values were evaluated as follows; normal controls showed a value of 0.033 using A. costaricensis antigen lower than (0.085) A. costaricensis antigen. Both mean values of angiostrongyliasis cases were rather close (0.491) using A. costaricensis antigen and the other antigen (0.518). The present study continued with a crude antigen of 13 A. costaricensis females and males. Serum samples were analyzed; 27 sera of angiostrongyliasis, 30 negative controls and 193 cases of other parasitic infections (91 cases of nematodiasis; 45 cases of cestodiasis; 47 cases of trematodiasis and 10 cases of HIV) and 7 cases of other brain infections. This antigen was evaluated for ELISA with a concentration of 5 microg/ml, serum dilution 1:400 and anti-human IgG conjugate at 1:2,000. The test gave sensitivity and specificity at cut-off value 0.261; 92.59% and 73% respectively. The antigen was cross-reactive with 30 cases from 9 out of 10 different kinds of nematodiasis (gnathostomiasis, strongyloidiasis, ascariasis, hookworm infections, trichinosis, toxocariasis, trichuriasis, onchocercosis and Wuchereria bancrofti infections. Five cases from 3 of 6 kinds of cestodiasis (neurocysticercosis, echinococcosis and Hymenolepis nana infections) and 18 cases of 4 out of 5 kinds of trematodiasis (Paragonimus heterotremus infections, opisthorchiasis, schistosomiasis and fascioliasis). One case of other brain infections was observed. The crude antigen of A. costaricensis showed a high percentage sensitivity with serum antibodies of angiostrongyliasis cases. Low specificity of the test was observed by reactions of those serum antibodies with various kinds of antigenic molecules. This study provides baseline data for further immunodiagnosis of human angiostrongyliasis.
Angiostrongyliasis
Ancylostomiasis
Trichuriasis
Strongyloidiasis
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Objective To analyze the urea-soluble antigen of Angiostrongylus cantonensis adult worm, and identify and evaluate the dominant diagnostic antigen for angiostrongyliasis. Method The urea-soluble antigens (U-Ag) were obtained from the urea soluble fraction of the homogenized A.cantonensis adult worm. The urea-soluble antigens were analyzed and compared with water-soluble antigens (W-Ag) by SDS-PAGE, Western blotting and enzyme-linked immunosorbent assay (ELISA). Result The number of protein bands of U-Ag was less than W-Ag on SDS-PAGE. Besides the bands observed in the Western blotting of U-Ag were fewer than that of W-Ag. A strong band was detected at 32 000 Mr of U-Ag with sera obtained from patients with angiostrongyliasis, rats infected with A.cantonensis and rabbits vaccinated with W-Ag by Western blotting. The U-Ag had the same positive ratio as the W-Ag in detection of sera of patients suspected with angiostrongyliasis and rats infected with A.cantonensis by ELISA. At the same time, the singal in Western blotting was lower than that in ELISA in the detection of sera of normal rats, blood donors and patients without angiostrongyliasis. Conclusion There is a 32 000 Mr antigen or more than one 32 000 Mr antigens in the urea-soluble antigens of A.cantonensis adult worm that can be detected by the sera of angiostrongyliasis patients, infected rats of immunized rabbits. Urea-soluble antigens have not only the same sensitivity but the higher specificity than water-soluble antigens in the immunodiagnosis of angiostrongyliasis.
Angiostrongyliasis
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To evaluate the specificity and sensitivity of immunodiagnosis with the antigen with molecular mass of 32 000 of Angiostrongyliasis cantonensis(AC32).The major antigenic protein AC32 was purified from the antigens of A.cantonensis adult worm by electroelution from SDS-polyacrylamide gels. AC32-enzyme linked immunosorbent assay (ELISA) and adult worm antigen (AWA)-ELISA were used to detect the specific IgG in the sera of normal rats (n=5) and rats with angiostrongyliasis (n=61), sera of healthy individuals (n=50) and patients (n=50) with angiostrongyliasis, schistosomiasis and other parasitosis.The positivity rate in AC32-ELISA of the sera from rats with angiostrongyliasis was 100%; without false positive results or cross reaction between AC32 and the sera of the normal control and patients infected with parasites other than A.cantonensis.AC32 of A.cantonensis is a valuable candidate antigen for immunodiagnosis of angiostrongyliasis with high sensitivity and specificity.
Angiostrongyliasis
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