Mouse strain difference in visceral larva migrans of Toxocara canis

The mode of larval migration (visceral larva migrans) in Toxocara canis infection was compared for BALB/c, C57BL/6, C3H/He, DBA/2, NC and BALB/c nude mice following oral infection with 400 eggs. The mean recovery of larvae from the liver on day 2 post infection (PI) was not different in terms of the strain, age or sex of the mice. The number of larvae recovered from the liver decreased in all strains on days 6, 12 and 21 PI, but the mean for BALB/c and (NC X BALB/c) F1 mice was significantly higher than that for C567BL/6, NC and BALB/c nude mice, unless the total number of larvae in the carcasses on day 21 PI was the same among those strains including athymic nude mice. The mean recovery of larvae from the liver on day 6 PI increased with age in both NC and BALB/c mice, although no sex difference was observed. From these results, it is emphasized that the age and strain of animals should be properly selected for animal experimentation with T. canis infection.     

Visceral larva migrans: examinations by means of enzyme-linked immunosorbent assay of human sera for antibodies to excretory-secretory antigens of the second-stage larvae of Toxocara canis

An enzyme-linked immunosorbent assay is described for the detection of serum antibodies to visceral larva migrans (Toxocariasis). Excretory-secretory antigens of the second-stage larvae of Toxocara canis were used as antigen to coat the polystyrene plates. With sera from patients high antibody titers were observed in both ocular and visceral disorders. Cross-reactions due to other parasitic infections could be excluded, including other migrating larval infections such as ascariasis, trichinellosis, strongyloidiasis, filariasis, and anisakiasis. In a small seroepidemiologic survey of healthy primary schoolchildren, a remarkably high percentage (7.1) reacted positively to this method. These children showed eosinophilia as compared to the seronegative group. The data were compared with those observed in other countries and the results prompt reconsideration of the significance of T. canis for public health.     

Do Toxocara canis larval antigens used in enzyme-linked immunosorbent assay for visceral larva migrans cross-react with AB isohemagglutinins and give false positive results?

A variety of helminth parasites have A and B blood group antigens on their surface. These antigens may cross-react with elevated concentrations of A and B isohemagglutinins in some patients and give false-positive results in the serologic diagnosis of visceral larva migrans caused by T. canis. To clarify this point, serum from patients with visceral larva migrans and elevated T. canis antibody titers as determined by ELISA were absorbed with AB blood cells and retested by ELISA without a demonstrable decline in T. canis antibody titers. Similarly, absorption with T. canis embryonated egg antigens of serum containing elevated levels of anti-A or anti-B isohemagglutinins failed to decrease the isohemagglutinin titer. This indicates that the ELISA using T. canis embryonated egg antigen does not give false positive results with sera containing high concentrations of anti-A or anti-B isohemagglutinins.     

Ocular larva migrans caused by Toxocara cati in Mongolian gerbils and a comparison of ophthalmologic findings with those produced by T. canis

To elucidate the pathogenic potential of Toxocara cari, we observed the ophthalmologic changes of the fundi in Mongolian gerbils, Meriones unguiculatus, after oral inoculation of 17 embryonated eggs/g body weight. Ophthalmic conditions in 8 T. cati-infected gerbils were monitored using an ophthalmoscope from day 0 to day 156 and were compared with those of 57 T. canis-infected gerbils. The results showed that T. cati larvae migrated into the eye of the gerbil and then elicited ophthalmic changes, including retinal (25%) and vitreous (50%) hemorrhaging, vasculitis (37.5%), and exudative lesions (25%). Lesions were less prevalent, however, in T. cati-infected than in T. canis-infected gerbils. Unlike in T. canis-infected gerbils, the hemorrhagic lesions did not reappear in T. cati-infected gerbils after they were absorbed. These findings suggested that T. cati larvae are a potentially hazardous pathogen for ocular toxocariasis and that Mongolian gerbils infected with T. cati may be a useful model for the study of human ocular toxocariasis caused by T. cati. This is the first study to report that T. cati larvae can induce ophthalmic lesions in the retina of gerbils.     

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.     

静注人免疫球蛋白(pH4)中抗-HBs效价双抗原夹心ELISA的验证

目的用双抗原夹心ELISA检测静注人免疫球蛋白(p H4)(human immunoglobulin for intravenous injection,IVIG)中抗-乙型肝炎病毒表面抗体(HBs)效价并进行方法验证。方法依据2003年版《药品生产验证指南》,对用双抗原夹心ELISA进行专属性、重复性、中间精密度、线性、准确度、范围、耐用性的验证;并与放射免疫方法测定抗-HBs效价的结果进行比对。结果专属性验证中,6次独立试验的回收率在83.7%~101.1%区间内;重复性试验中10、40、80、160 IU/L 4个浓度的3次重复检测结果的RSD均<20%;中间精密度验证结果显示,两位分析人员不同时间各测定6次,检测结果的RSD=5.2%,均<20%。F检验显示,人员和时间两因素在α=0.05水平上对检测结果均无统计学意义(P=0.41>0.05,F=2.19);线性验证中,抗-HBs免疫球蛋白国家标准品和IVIG成品各重复3次的相关系数r分别为0.984 3、0.990 7、0.982 2、0.991 7、0.988 3、0.992 5,均>0.980 0;在准确度验证中,3次重复检测结果的回收率为84.10%~114.10%;耐用性结果显示,在试验加入终止液后0、3、6、9、12 min的测定结果均与0 min无统计学意义(Dunnett t test,α=0.05,P>0.05,t(3)=1.350.05,t=2.27)。结论经验证,用双抗原夹心ELISA检测IVIG中抗-HBs效价,其专属性、重复性、中间精密度、线性、准确性、范围和耐用性均符合要求,该方法准确可靠、简便快速,可替代放射免疫法(radioimmunoassay,RIA)用于IVIG中抗-HBs效价的测定。     

Quantification of intact 146S foot-and-mouth disease antigen for vaccine production by a double antibody sandwich ELISA using monoclonal antibodies

A double antibody sandwich (DAS) enzyme-linked immunosorbent assay (ELISA) was developed to quantify 146S antigen of foot-and-mouth disease virus (FMDV) strain A10 Holland grown in suspension cultures of surviving bovine tongue epithelium. When virus harvests were incubated with trypsin--which affects VP1, the most immunogenic structural protein of FMDV--the concentration of 146S antigen as determined by ELISA was reduced by greater than 90%. Therefore, the test detected essentially only those virus particles with intact VP1. When the test was compared with the sucrose density gradient method, concentrations of 146S antigen correlated well (r = 0.87). The rate of variation in both tests was the same. In contrast to the sucrose density gradient method, the DAS-ELISA can simultaneously quantify 146S antigen in many samples, and also indicates when VP1 of 146S particles has disintegrated by the action of proteases.     

A case of relapsing cutaneous larva migrans transmitted by a cheetah (Acinonyx jubatus)

A case of relapsing "creeping eruption" connected with parasites of the cheetah is reported in the man. The parasitological examination, carried out in two cheetahs liable of this syndrome, has shown two different helmintic species: Ancylostoma caninum and Ancylostoma tubaeforme.     

hGH单克隆抗体的筛选及双抗体夹心ELISA检测方法的建立

采用杂交瘤法制备单克隆抗体,并用辛酸-硫酸铵法纯化单抗,通过ELISA方法和Western blotting测定抗体的效价与特异性,并进行抗体类型、相对亲和力测定;应用纯化的单抗建立hGH双抗体夹心ELISA检测方法。筛选出两株可以稳定分泌抗hGH单抗的杂交瘤细胞株,分别命名为3E11、2G9,抗体类型均为IgG1,抗体滴度均可达10-10,特异性好,相对亲和力高,以筛选到的两株单抗建立的双抗夹心ELISA法线性范围为0.09～1.5625ng/mL,R2>0.9,灵敏度为0.09ng/mL。筛选出高效抗hGH的单抗,并建立了hGH双抗体夹心ELISA检测方法。     

Characterization of murine monoclonal antibodies to human interferon-gamma (IFN-gamma) and their application for sandwich enzyme-linked immunosorbent assay (ELISA)

The three murine monoclonal antibodies (MAb), D1G2, D9D10, and D13C8, are specific for human interferon-gamma (IFN-gamma), but not human IFN-alpha and IFN-beta. They react weakly with heat-treated IFN-gamma. The three antibodies recognize different epitopes of the IFN-gamma molecule, as evaluated by antibody-binding inhibition experiments. We have used these three monoclonal antibodies to construct a sandwich enzyme-linked immunosorbent assay (ELISA). The best result was obtained when we used D1G2 or D9D10 MAb as a solid-phase immunosorbent and D1G2 or D9D10 MAb as a tracer. When we measured IFN-gamma in sera by a combination of D1G2 (a solid-phase) and D1G2 (a tracer), a result similar to the one by a combination of D9D10 (a solid-phase) and D1G2 (a tracer), was obtained. This may suggest that human IFN-gamma exists in oligomeric form. Recombinant human IFN-gamma expressed in E. coli is detectable at a concentration of 1 ng/ml in this sandwich ELISA. This assay can be employed for the analysis of the structural characteristics of the human IFN-gamma molecule as well as measurement of IFN-gamma in human sera and tissue culture fluids.     

IL-5 drives eosinophils from bone marrow to blood and tissues in a guinea-pig model of visceral larva migrans syndrome

This study was undertaken to evaluate the role of IL-5 in eosinophil migration and in the maintenance of eosinophilia in a guinea-pig model of visceral larva migrans syndrome. The results show that the infection of animals with Toxocara canis induced an early increase in serum IL-5 levels that might be essential for eosinophil differentiation and proliferation and for the development of eosinophilia. When infected guinea-pigs were treated with mAb anti-IL-5 (TRFK-5) given at the same time or 1 or 3 days after infection, there was a high percentage of reduction of eosinophil counts 18 days after infection. However, when the mAb was administered during the peak of eosinophilia, there was high inhibition in blood, no inhibition in bronchoalveolar lavage fluid (BALF) or peritoneum and an increase in eosinophil numbers in bone marrow. Thus, a basic level of IL-5 may be essential to drive eosinophils from bone marrow to blood and tissues, and for the maintenance of eosinophilia in infected animals. We may also conclude that when eosinophils have already migrated to the lungs, TRFK-5 has no power to inhibit eosinophilia, which is also under control of local lung cells producing IL-5. In this way, only one later TRFK-5 treatment may not be sufficient to modify the lung parenchyma microenvironment, since T. canis antigens had already stimulated some cell populations to produce IL-5.     

Characterization of O-GlcNAcylation in starfish (Asterina pectinifera) development from fertilization to bipinnaria larva

Though O-linked β-N-acetylglucosaminylation (O-GlcNAcylation) of nucleocytoplasmic proteins has been found in many multicellular organisms, its presence or absence in Echinodermata is unknown. Here we report the occurrence of O-GlcNAcylation in starfish (Asterina pectinifera) oocytes and the apparent O-GlcNAcylation pattern in starfish early development. O-GlcNAcylation might participate in the regulation of starfish development at the mid-blastula stage and thereafter.     

Visceral larval migrans (Human toxocariasis) cause of hypereosinophilia and visceral granulomas in adults]

A 24-year-old woman 2-3 months after a normal parturation presented geophagy. Due to hypermenorrhea she consulted a gynecologist and in a hemogram a 57% (6,893 x mm3) hypereosinophilia was detected. A chest TAC showed bilateral pulmonary nodules. The following tests resulted positive: ELISA IgG for toxocariasis 1:1000, isohemagglutinins anti A 1:2048 and anti B 1:512. The patient was treated with albendazole and prednisone during 10 days. One month after treatment eosinophilia decreased to 2.590 x mm3 and ELISA IgG for toxocariasis descended to 1:128. Different aspects of human toxocariasis are commented. When hypereosinophia is observed in adult patients, toxocariasis must be checked.     

Development and application of a monoclonal antibody-based sandwich ELISA for quantification of Japanese medaka (Oryzias latipes) vitellogenin

Vitellogenin (Vtg) was purified from ascitic fluid of a 17beta-estradiol (E2)-treated female Japanese medaka by anion-exchange chromatography. The molecular mass of medaka Vtg by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), corresponding to the Vtg monomer, was 200 kDa. BALB/c mice were immunized with purified-Vtg and two hybridoma clones producing specific antibodies against medaka Vtg were selected. The specificity of these monoclonal antibodies (mAbs) was evaluated by Western blot analysis of the plasma proteins separated on SDS-PAGE, and no cross-reactivity was observed with plasma proteins from control males. A sandwich enzyme-linked immunosorbent assay (ELISA) for quantification of medaka Vtg was developed using these mAbs. The assay range was between 1 and 100 ng/ml, and the intra- and inter-assay variations determined from plasma samples were within 7.7 and 8.5%, respectively. Recovery of medaka Vtg added to plasma was 92-111%. In a plasma dilution test, plots of Vtg concentration gave a straight line. After exposure of male medaka to E2 (10 ng/l), Vtg appeared in liver and plasma on the first day and reached a maximum on the 3rd to 5th day. The sandwich ELISA could be useful for the detection of estrogenic properties, and the medaka Vtg bioassay could be a very sensitive and good tool for screening of endocrine disrupting compounds.     

Enzyme-linked immunosorbent assay (ELISA) using a glycolipid antigen for the serodiagnosis of melioidosis

Abstract The serodiagnosis of melioidosis is commonly performed with tests using protein or polysaccharide as antigen. However, due to the low sensitivity, specificity and difficulty in the preparation of the antigens, more simple, precise and reproducible diagnostic tests were required. A purified glycolipid antigen (GL) which is a specific lipid component of Burkholderia pseudomallei has been used in an ELISA. With this antigen, specific immunoglobulin G (IgG) was detected in 49 out of 50 melioidosis sera. IgG was also detected in 2 out of 185 (Japanese) and 16 out of 181 (Vietnamese) control sera. Thus, the sensitivity was 98.0%, and specificity was 98.9% and 91.1% in the Japanese and Vietnamese sera, respectively. When the ELISA and indirect haemagglutination (IHA) tests were combined, a sensitivity of 100% and specificity of 97.8% were achieved. The advantages of the glycolipid antigen are ease of preparation, stability, high sensitivity and specificity.     

Prevalence of Toxocara canis, Toxascaris leonina and Dirofilaria immitis in dogs in Chuncheon, Korea (2004)

The intestines and hearts of dogs were examined for Toxocara canis, Toxascaris leonina, and Dirofilaria immitis, after necropsy between June 26 and September 29, 2004 in Chuncheon, Korea. Of the 662 dogs examined, 6 were infected with T. canis (0.9%), 86 with T. leonina (13.0%). Fifty dogs were infected with D. immitis among 500 dogs examined (10.0%). Five were co-infected with T. canis and T. leonina, and three were co-infected with T. leonina and D. immitis. The cumulative positive infection rate for three species was 134/662 (20.2%). Considering previously reported seropositive rates of T. canis excretory-secretory antigen, i.e., 5% in the adult population in Korea, the possibility of toxocariasis caused by T. leonina should be reevaluated.