Measurement of 150 kDa protein of <i>Taenia solium</i> metacestodes by antibody-sandwich ELISA in cerebrospinal fluid of neurocysticercosis patients

, Cho, S Y; , Kong, Y; , Kim, S I; , Kang, S Y

doi:1992.30.4.299

Korean J Parasito > Volume 30(4):1992 > Article

Original Article


Korean J Parasitol. 1992 Dec;30(4):299-307. English. Published online Mar 20, 1994. http://dx.doi.org/10.3347/kjp.1992.30.4.299
Copyright © 1992 by The Korean Society for Parasitology


Measurement of 150 kDa protein of Taenia solium metacestodes by antibody-sandwich ELISA in cerebrospinal fluid of neurocysticercosis patients
S Y Cho,Y Kong,S I Kim,^** and S Y Kang
Department of Parasitology, College of Medicine, Chung-Ang University, Seoul 156-756, Korea.


Abstract
An antigenic protein in cystic fluid of Taenia solium metacestodes (CF) of 150 kDa was measured by antibody-sandwich ELISA in serum and cerebrospinal fluid (CSF) of neurocysticercosis patients. Capture antibodies were rabbit antisera against CF (RACF) and a monoclonal antibody (MAb) against 150 kDa protein in CF. Lower limit of antibody-sandwich ELISA was 8 ng/ml of the protein. Except CF, no tested helminths extracts reacted. Levels of the protein in 351 sera from 255 patients (55 surgery confirmed and 202 antibody and CT/MRI confirmed) were below sensitivity of the assay. Of 276 CSF from 212 patients, 31 samples (11.2%) showed positive findings. This assay, therefore, was not sensitive enough to be a diagnostic. Instead, the 150 kDa protein appeared in CSF in such situations as in 2 days after praziquantel treatment, or as in a patient infected with a racemose cysticercus with degenerated cyst wall. Of cases whose follow-up CSF were assayed, 2 cases showed that the protein appeared intermittently. These results suggest strongly that appearance of free 150 kDa protein is associated with cyst wall rupture. In CSF which contained the 150 kDa protein over 61 ng/ml, the protein was recognized in SDS-PAGE before and after immunoprecipitation.

Figures


	Fig. 1 Relation between 150 kDa protein concentration in Cf and absorbance by antibody-sandwich ELISA. The concentration was calculated as 70% of whole proteins in CF (6.0 mg/ml) as measured by densitometry (Cho et al., 1988). The standard curve was linear in protein range of 8~60 ng/ml. This range was considered to be the best measurement interval form the 150 kDa protein.


	Fig. 2 SDS-PAGE findings of CSF and surgically collected cystic fluid of NCC patients. The samples were electrophoresed on 10~15% reducing gel with the supply of 30 mA constant current. Mr: Molecular mass in kDa, Lanes 1 and 5 : Surgically collected cystic fluid, Lanes 2,3,4,6,7,8,9, and 10: NCC patients CSF, CF: Cystic fluid collected from a pig, C: Band C protein (150 kDa protein).

Fig. 3
SDS-PAGE and immunoblot findings of CSF from NCC patients after the immunoprecipitation. The proteins were separate using 10~17.5% pradient gel. Mr: Molecular mass in kDa, N: CSF from other mcurologic patient, 1~7: CSF collected each NCC patient, CF: Cystic fluid collected from a pig. (A): MAb bound antigenic subunits of molecular mass of 10 kDa were recognized at the bottom of each lane (arrow head). (B): When this immune complex was immunoblotted with RACF, 10 kDa bands of 150 kDa protein were reacted (arrow head). Heavy and light chains of rabbit IgG (in Pansorbin) were non-specifically reacted with RACF.

Tables


	Table 1 Number of NCC cases and their samples assayed in this study


	Table 2 Range of 150 kDa protein concentration (ng/ml) in CSF of NCC patients


	Table 3 Antibody and 150 kDa protein levels in serum and CSF of a NCC patient who was treated with proziquantel since Now. 11, 1987


	Table 4 Antibody and 150 kDa protein levels in serum and CSF of a NCC patient with hydrocephalus


	Table 5 Antibody and 150 kDa protein levels in serum and CSF of another NCC patient with hydrocephalus and multiple low densities

References


1.	Brandt JR, Geerts S, De Deken R, Kumar V, Ceulemans F, Brijs L, Falla N. A monoclonal antibody-based ELISA for the detection of circulating excretory-secretory antigens in Taenia saginata cysticercosis. Int J Parasitol 1992;22(4):471–477.

2.	Chang KH, Kim WS, Cho SY, Han MC, Kim CW. Comparative evaluation of brain CT and ELISA in the diagnosis of neurocysticercosis. AJNR Am J Neuroradiol 1988;9(1):125–130.

3.	Cho SY, Kang SY, Kim SI. Analysis of antigen specificity using monoclonal and polyclonal antibodies to Cysticercus cellulosae by enzyme-linked immunoelectrotransfer blot technique. Korean J Parasitol 1987;25(2):159–167.

4.	Cho SY, Kim SI, Kang SY, Choi DY, Suk JS, Choi KS, Ha YS, Chung CS, Myung HJ. Evaluation of enzyme-linked immunosorbent assay in serological diagnosis of human neurocysticercosis using paired samples of serum and cerebrospinal fluid. Korean J Parasitol 1986;24(1):25–41.

5.	Cho SY, Kim SI, Kang SY, Kong Y. Biochemical properties of a purified protein in cystic fluid of Taenia solium metacestodes. Korean J Parasitol 1988;26(2):87–94.

6.	Choi CS, et al. Chung-Ang J Med 1990;15:319–327.

7.	Choromanski L, Estrada JJ, Kuhn RE. Detection of antigens of larval Taenia solium in the cerebrospinal fluid of patients with the use of HPLC and ELISA. J Parasitol 1990;76(1):69–73.

8.	Correa D, Sandoval MA, Harrison LJ, Parkhouse RM, Plancarte A, Meza-Lucas A, Flisser A. Human neurocysticercosis: comparison of enzyme immunoassay capture techniques based on monoclonal and polyclonal antibodies for the detection of parasite products in cerebrospinal fluid. Trans R Soc Trop Med Hyg 1989;83(6):814–816.

9.	Estrada JJ, Estrada JA, Kuhn RE. Identification of Taenia solium antigens in cerebrospinal fluid and larval antigens from patients with neurocysticercosis. Am J Trop Med Hyg 1989;41(1):50–55.

*10.*	Estrada JJ, Kuhn RE. Immunochemical detection of antigens of larval Taenia solium and anti-larval antibodies in the cerebrospinal fluid of patients with neurocysticercosis. J Neurol Sci 1985;71(1):39–48.

*11.*	Cho SY, Kim SI, Kang SY. Serologic follow-up study in neurocysticercosis patients by ELISA after praziquantel treatment. Korean J Parasitol 1986;24(2):159–170.

*12.*	Kong Y, Cho SY, Kim SI, Kang SY. Immunoelectrophoretic analysis of major component proteins in cystic fluid of Taenia solium metacestodes. Korean J Parasitol 1992;30(3):209–218.

*13.*	Laemmli UK. Nature 1970;227:681–685.

*14.*	Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193(1):265–275.

*15.*	Nash TE, Neva FA. Recent advances in the diagnosis and treatment of cerebral cysticercosis. N Engl J Med 1984;311(23):1492–1496.

*16.*	Tellez-Giron E, Ramos MC, Dufour L, Alvarez P, Montante M. Detection of Cysticercus cellulosae antigens in cerebrospinal fluid by dot enzyme-linked immunosorbent assay (Dot-ELISA) and standard ELISA. Am J Trop Med Hyg 1987;37(1):169–173.

*17.*	Tsang VC, Brand JA, Boyer AE. An enzyme-linked immunoelectrotransfer blot assay and glycoprotein antigens for diagnosing human cysticercosis (Taenia solium). J Infect Dis 1989;159(1):50–59.

*18.*	Wilson M, Bryan RT, Fried JA, Ware DA, Schantz PM, Pilcher JB, Tsang VC. Clinical evaluation of the cysticercosis enzyme-linked immunoelectrotransfer blot in patients with neurocysticercosis. J Infect Dis 1991;164(5):1007–1009.