OBJECTIVETo clone the coding gene of the stage-specific antigen cC1 from Cysticercus cellulosae and express high levels of soluble cC1 in E.coli.

METHODSThe cC1 gene was amplified from Cysticercus cellulosae by RT-PCR and cloned into pMD18-T vector, followed by subcloning into the prokaryotic expression plasmid pET28a. The recombinant plasmid was transformed into E.coli BL21(DE3) and the expression conditions were optimized. The expressed product was purified by Ni(+)-affinity chromatography, analyzed by high-performance liquid chromatography (HPLC), and identified with SDS-PAGE and Western blotting.

RESULTSThe fragment length of the amplification product by RT-PCR was 1056 bp. Comparison of the amplified gene sequence with the cC1 gene in Genbank identified a samesense point mutation at 423 position in the gene cloned into the expression plasmids. After a 6-h induction with 0.05 mmol/L IPTG at 37 degrees celsius;, the expression of the 40 kd soluble fusion protein exceeded 60% of the total bacterial protein, and the fusion protein was recognized by Cysticercus-infected human sera. The purity of the fusion protein was about 94% after purification by affinity chromatography.

CONCLUSIONThe stage-specific antigen cC1 from Cysticercus cellulosae has been successfully cloned and the soluble protein efficiently expressed in E.coli, which provides the basis for its further study and application.

Animals ; Antigens, Helminth ; biosynthesis ; genetics ; immunology ; Cloning, Molecular ; Cysticercus ; immunology ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; Humans ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Solubility ; Swine ; Taenia solium ; immunology

Seroprevalence of the IgG antibodies for Clonorchis sinensis, Paragonimus westermani, Taenia solium metacestode (cysticercus), and Spirometra erinacei plerocercoid (sparganum) was measured using enzyme-linked immunosorbent assay (ELISA) in sera of patients in Korea from 1993 to 2006. A total of 74,448 specimens referred nationwide from 121 hospitals revealed an IgG positive rate of 7.6% for the 4 parasites. The IgG positive rate (18.7%) for the 4 parasites in 1993 decreased gradually to 6.6% in 2006. Individual positive rate decreased from 5.2% (1993) to 1.6% (2006) for C. sinensis, from 2.8% (1993) to 1.1% (2006) for P. westermani, from 8.3% (1993) to 2.2% (2006) for cysticercus, and from 2.6% (1993) to 1.6% (2006) for sparganum. The positive rate was highest (21.2%) in the group of patients who ranged in age from 50-59 yr old, and in the group that was referred from the Seoul area (55.9%). In conclusion, our results suggest that tissue invading parasitic infections should always be included in differential diagnosis for patients with eosinophilia associated lesions of the central nervous system, liver, and lungs in Korea.
Adolescent ; Adult ; Age Factors ; Aged ; Aged, 80 and over ; Animals ; Antibodies, Helminth/*blood ; Child ; Child, Preschool ; Clonorchiasis/diagnosis/*epidemiology ; Clonorchis sinensis/immunology/isolation & purification ; Cysticercosis/diagnosis/*epidemiology ; Cysticercus/immunology/isolation & purification ; Diagnosis, Differential ; Enzyme-Linked Immunosorbent Assay ; Eosinophilia/immunology ; Female ; Humans ; Immunoglobulin G/blood ; Infant ; Male ; Middle Aged ; Paragonimiasis/diagnosis/*epidemiology ; Paragonimus westermani/immunology/isolation & purification ; Republic of Korea/epidemiology ; Seroepidemiologic Studies ; Sparganosis/diagnosis/*epidemiology ; Sparganum/immunology/isolation & purification

To obtain the recombinant 18 kD protein with high purity and normal bioactivity of Cysticercus cellulosae (rCE18), E. coli cells with the rCE18 were disrupted ultra-sonically, and the inclusion bodies were washed with a solution containing 0.2% deoxycholic acid sodium (DOC)and 2% DOC, respectively. Then they were denatured with 0.9% sodium lauroyl sarcosine (SKL) followed by dialysis and gel filtration to refold and purify the target protein. At the same time, this method was compared with GST-FF affinity chromatography and recovering from SDS-PAGE gel. Biological activity of purified rCE18 was analyzed with indirect ELISA, and the purity of the products was identified using SDS-PAGE. The purity of refolded inclusion bodies exceeded 60% and the total recovery of activated protein rCE18 was about 41.3%. The specificity of rCE18 reached up to 97.2% using indirect ELISA. An effective way for purifying and refolding rCE18 expressed in E. coli as inclusion bodies was established, rCE18 with higher purity and activity was obtained, which has the potential for developing diagnosis methods of porcine cysticercosis.
Animals ; Antigens, Helminth ; biosynthesis ; genetics ; immunology ; isolation & purification ; Chromatography, Gel ; Cysticercus ; genetics ; immunology ; metabolism ; Escherichia coli ; genetics ; metabolism ; Inclusion Bodies ; metabolism ; Protein Renaturation ; Recombinant Proteins ; genetics ; immunology ; isolation & purification

Prevalence survey of neurocysticercosis was made in a mixed epilepsy patients of Changmi Club in Korea. From February 1987 to July 1990, a total of 2,667 randomly selected patients at 27 local centers was tested for their serum levels of anti-Cysticercus antibody (IgG) by enzyme-linked immunosorbent assay. Positive rate of the antibody was 4.0% in the examined patients. The standardized antibody positive rate by provincial population was 3.1%. The rate was the highest in patients living in Cheju Do (8.4%). The patient age brackets of 0 approximately 9 years and over 50-year showed higher positive rates of the antibody. In 750 normal persons who checked up routine physical examination, the antibody positive rate was 2.1% (standardized rate was 1.8%). These seroepidemiological data disclosed for the first time the prevalence of cysticercosis in epileptic patients and in population.
Adolescent ; Adult ; Age Factors ; Aged ; Animals ; Antibodies, Helminth/*blood ; Child ; Child, Preschool ; Cysticercosis/complications/*epidemiology ; Cysticercus/*immunology ; Epilepsy/*complications ; Female ; Humans ; Infant ; Infant, Newborn ; Korea/epidemiology ; Male ; Middle Aged ; Sex Factors

We studied the serological reaction between various antigenic components from Cysticercus cellulosae and IgG antibodies in sera of cysticercosis, sparganosis, hydatidosis patients and normal humans by ELISA and EITB. In serological tests by ELISA, we recognized cross reaction of Cysticercus antigenic components with IgG antibodies in heterologous sera such as sparganosis and hydatidosis patients or normal humans. The crude antigenic components of Cysticercus showed lower ELISA sensitivity in homologous sera from cysticercosis patients than heterologous sera from hydatidosis patients. A total of 31 polypeptide bands with 260 KDa-22 KDa molecular weights were detected by SDS-PAGE, and 11 of them showed strong intensity. Total 22 components of them were recognized by IgG antibodies in cysticercosis patients sera. However, 12 of them were recognized also by normal human sera, 11 were by sparganosis sera, and 21 were by hydatidosis patients sera. The crude antigenic components of 104 KDa, 82 KDa, 72 KDa, 59 KDa and 34 KDa molecular weights were nonspecific ones, which cross-reacted with sera of either cysticercosis, sparganosis, hydatidosis patients or normal humans.
parasitology-helminth-cestoda ; Spirometra erinacei ; Taenia solium ; cysticercus ; sparganum ; immunology ; antigen

The applicability of indirect immunofluorescent antibody test (IFAT) was compared with enzyme-linked immunosorbent assay (ELISA) in sera from 163 cases of confirmed neurocysticercosis, 101 other neurologic and parasitic diseases and 100 normal controls. As antigen, frozen sections of a Taenia solium metacestode from a human brain was used in IFAT and cystic fluid was used in ELISA. For the detection of specific IgG antibody, IFAT was equally sensitive (89. 6%) and specific (85. l%) as ELISA. The antibody titers by IFAT were correspondingly increased with mean absorbance of ELISA. The corresponding rate of positivity in the two techniques was 90.8%. Except for the difficulty in detecting antibodies in cerebrospinal fluid (CSF), IFAT was concluded to be very useful for the serodiagnosis of human neurocysticercosis.
parasitology-helminth-cestoda ; Taenia solium ; cysticercus ; enzyme-linked immunosorbent assay ; cerebrospinal fluid ; brain ; IgG ; immunofluorescence ; immunology

To determine the source of Cysticercus-specific IgG antibody in cerebro-spinal fluid(CSF), paired samples of serum and CSF were collected from confirmed neurocysticercosis, other neurologic diseases and normal control. The antibody levels in serum and CSF were measured by enzyme-linked immunosorbent assay (ELISA). With the measurement of total protein, albumin and IgG concentration in serum and CSF, the contribution of IgG in CSF were calculated in transudation, exudation and intracranial synthesis using the formula of Tourtellotte and Ma (1978). Mean concentrations of total protein, albumin, IgG and proportional IgG levels in CSF by transudation, exudation and intracranial synthesis were elevated in neurocysticercosis. But only the intracranial synthesis of IgG showed a statistically significant correlation with the specific IgG antibody levels in CSF. In CSF from lateral ventricle in the 4th ventricular neurocysticercosis, the protein concentrations were normal and the specific antibody levels were negative. However, in consecutively secured lumbar CSF from the same patients, the former were increased and the latter were positive. These results indicated that, in neurocysticercosis, the specific IgG antibody in CSF was a local product of intracranial synthesis.
parasitology-helminth-cestoda ; Taenia solium ; cysticercus ; enzyme-linked immunosorbent assay ; cerebrospinal fluid ; brain ; IgG ; immunology

/A total of 69 patients of confirmed neurocysticercosis was followed serologically by ELISA up to 22 months after praziquantel treatment. The intervals and numbers of follow-up were variable by patients. Serially collected samples of serum and CSF were examined simultaneously for their specific IgG antibody levels by ELISA, using cystic fluid, saline extracts of bladder wall and scolex as antigen. Within 4 months after praziquantel treatment, the antibody levels were elevated temporarily in both serum and CSF in most patients. In some cases antibody levels exhibited steady declining tendency after the treatment. Concomitant administration of dexamethasone appeared to suppress the elevation of antibody levels. The rate of mean absorbance of antibody changed more in serum than in CSF. The rate of elevation was greater in antibodies to parenchymal antigens than that to cystic fluid, but absolute difference of antibody levels was greater in anitbody to cystic fluid. Previously negative samples for IgG antibody may become positive after praziquantel treatment, which could be used as a complementary tool(provocation test) in serodiagnosis. One month was considered to be sufficient interval for the follow-up test for that purpose. In the follow-up of up to 22 months, only few cases of chronic neurocysticercosis showed declining tendency of IgG antibody levels below negative range. During acute encephalitic attacks in chronic patients, IgG antibody to parenchymal antigen were elevated in CSF temporarily. These results indicated that serologic follow-up of every year was recommendable to differentiate the cured patients from chronic patients with slowly calcifying lesions.
parasitology-helminth-cestoda ; Taenia solium ; cysticercus ; brain ; immunology ; praziquantel-chemotherapy ; praziquantel

The applicability of micro-ELISA was evaluatd in human neuro-cysticercosis using paired samples of serum and CSF. A total of 355 cases who were mostly neurologic patients was subjected. Cystic fluid of C. cellulosae was used as antigen in protein concentration of 2.5 micro-g/ml. Serum was diluted to 1:100 and CSF was undiluted in the assay for the specific IgG antibody level. The differential criterion of the positive reaction was the abs. of 0.18 in both samples. The results were summarized as follows: The overall sensitivity of the micro-ELISA in 71 confirmed neurocysticercosis was 90.1%; the sensitivity by serum was 77.5% and that by CSF was 83.1%. CSF was a more sensitive and valuable material. Most of the false negative cases of neuro-cysticercosis showed far lower level of abs. rather than marginal. The overall specificity of the micro-ELISA in 52 confirmed other neurologic diseases was 88.5% ; the specificities by serum and by CSF were 94.2% respectively. Cases of other neurologic diseases did not show false positive reactions in both samples. When serum was assayed, taeniasis(2/18), sparganosis(2/20), paragonimiasis(1/56), clonorchiasis(1/15) and fascioliasis(1/1) cases showed cross reactions. When CSF was assayed, 2 of 10 neuro-sparganosis showed cross reactions while none of 9 neuro-paragonimiasis showed it. Out of 71 confirmed neuro-cysticercosis cases, 6 and 11 showed cross reactions by serum and CSF to crude extract antigen of sparganum; but no case did show it to crude extract antigen of Paragonimus westermani. Ventricular CSF showed low or negative levels of IgG antibody than lumbar CSF unless the lesion was at the lateral ventricle itself. Out of 4 racemose cysticercosis cases, 3 showed positive reaction in serum while all of 3 examined CSF were positive. The above results indicated that the serological test for detecting the specific IgG antibody by micro-ELISA using paired samples of serum and CSF was very helpful for clinical differentiation of neuro-cysticercosis from neurologic diseases of other causes.
parasitology-helminth-cestoda ; immunology ; Taenia solium ; cysticercus ; enzyme-linked-immunosorbent assay ; serum ; cerebrospinal fluid ; IgG

Seven cases of surgically proven sparganosis were serologically tested by means of micro ELISA for their specific IgG antibody levels. For that purpose, crude saline extract of spargana from snake, Natrix tigrina lateralis was prepared and used as antigen. The sparganosis sera were also tested with Paragonimus and Cysticercus antigens to observe the cross reactivity. A total of 71 sera from normal control, ectopic and pulmonary paragonimiasis, clonorchiasis, cysticercosis and Taenia saginata cases were also included. Except for one case of old calcified infection, all of 6 human sparganosis showed higher serum levels of specific IgG antibody when the differential point of positive reaction was set at the absorbance value of 0.25 (the sensitivity being 85.7%). In control and other helminthic infections, all except 3 cases of T. saginata infection showed negative reaction to sparganum antigen (the specificity being 95.7%). None of sparganosis cases showed cross reactivity to Paragonimus and Cysticercus antigens. Undiluted cerebrospinal fluid also showed high levels of antibody when central nervous system was invaded. The serologic diagnosis by means of micro-ELISA could be a useful tool in epidemiological study of human sparganosis in susceptible population, as well as in individual diagnosis.
parasitology-helminth-cestoda ; sparganosis ; sparganum ; Paragonimus ; Cysticercus ; ELISA ; immunology ; serology ; human