Schistosoma mansoni: Description of a Potentially Useful Monoclonal Antibody that Recognizes Soluble Egg Antigen (SEA)

Schistosoma mansoni; Monoclonal antibodies; Soluble egg antigen (SEA); Carbohydrate epitopes

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Schistosoma mansoni: DESCRIPTION OF A POTENTIALLY USEFUL MONOCLONAL ANTIBODY THAT RECOGNIZES SOLUBLE EGG ANTIGEN (SEA)

Maria Cristina NAKHLE(¹) & Célia Regina Whitaker CARNEIRO(²)

KEYWORDS: Schistosoma mansoni; Monoclonal antibodies; Soluble egg antigen (SEA); Carbohydrate epitopes.

Many research groups have obtained monoclonal antibodies (MAbs) that react with SEA derived from Schistosoma mansoni. Most of them recognize carbohydrate epitopes that are shared by different developmental stages of the parasite^{5, 6, 9}. Some of these reagents were shown to be useful to quantitate egg circulating antigens and can be considered a good assessment of infected individuals’ egg load. As a consequence, these assays are a potential diagnostic parameter for morbidity⁷. Also, they can be used to study the fate of the antigen in the host¹.

One of us have previously reported the production of an IgM MAb (3C6) that reacted with schistosomula, eggs and the inner layer of the gut from adult worms of S. mansoni³. It has been raised by immortalization of spleen cells of a seven-week-infected mouse. As a consequence, it represents an antibody produced under normal circumstances of infection, and the recognized epitope deserves investigation. The hybridoma 3C6 was recently re-cloned by limiting dilution generating a stable cell line called 3C6H6. Gel filtration-purified MAb was obtained from ascitic fluid and used in all experiments.

Immunoperoxidase reactions over liver tissue sections of infected hamsters showed a strong reactivity with egg antigens both inside and outside the egg shell (Fig. 1). When tested on adult worm sections MAb 3C6H6 bound to the digestive tract and tegument as previously shown³. ELISAs using purified SEA² and adult worm antigen (AWA)⁴ showed a saturable reaction with SEA, and almost no recognition of AWA (Fig. 2).

Fig. 1 - Immunoperoxidase of MAb 3C6H6 (5 µg/ml) on an infected hamster liver tissue section showing recognition of SEA diffusing from the egg shell. The aecond biotinylated antibody as well as avidin-biotin-peroxidase complex were from Vector Labs. (Vecstatin ABC kit). Diaminobenzydine diluted in PBS containing 0.005% H₂O₂ (v/v) was used as substrate, and hematoxylin as counterstain (400x magnification)

Fig. 2 - ELISA of MAb 3C6H6 tested in serial dilutions on solid phase coated with SEA (10 µg/ml)(t) and AWA (10 µg/ml)( ). 5mM (O) and 20 mM (x) NalO₄^- treated SEA were also tested. Second antibody was a goat anti-mouse IgM from Sigma, and the substrate employed was orto-phenylenediamine in citrate-phosphate buffer containing 0.03% H₂O₂ (v/v).

In order to partially characterize the chemical nature of the recognized epitope, SEA was treated by the sugar oxidant sodium periodate in different concentrations, according to WOODWARD, 1985⁸. Figure 3 depicts an immunoblotting assay of MAb 3C6H6 recognition of SEA transferred to nitrocellulose sheets and treated or not with NaIO₄. These results were confirmed in ELISA, as shown in figure 2.

Fig. 3 - Immunoblotting of MAb 3C6H6 (10µg/ml) on SEA (10µg/slot) submitted to SDS-PAGE in a 10% gel using reducing conditions and transferred to nitrocellulose membrane. Before incubation with MAb 3C6H6, membrane strips were treated or not (lane 1) with NalO₄ using the following concentrations: 5 mM (lane 2) and 20 mM (lane 3). The second antibody and the substrate employed were goat antimouse IgM alkaline-phosphatase conjugate and BCIP/NBT (from Sigma), respectively.

Taken together, these data demonstrate that the epitope recognized by MAb 3C6H6 presents a glycidic nature, and can be similar to one of the already described MAbs^{5, 6, 9}. This reagent shows its usefulness in the recognition of antigen deposits trapped in glomeruli of infected experimental animals (DE BRITO, T. et al.; manuscript in preparation). Also, its previously shown reactivity with adult worms digestive tract and schistosomula opens the possibility of studies on protection immunity and/or its potential utilization on detection of circulating antigens.

Recebido para publicação em 04/09/1997

Aceito para publicação em 07/11/1997

• 1. BOGERS, J. J. P. M.; NIBBELING, H. A. M.; DEELDER, A. M. & VAN MARCK, E. A. E. Immunohistochemical and ultrastructural localization of Schistosoma mansoni soluble egg antigens processed by the infected hosts. Parasitology, 112: 537-543, 1996.
• 2. BOROS, D. I. & WARREN, K. S. Delayed hypersensitivity-type granuloma formation and dermal reaction induced and elicited by a soluble factor isolated from Schistosoma mansoni eggs. J. exp. Med., 132: 488-507, 1970.
• 3. CARNEIRO, C. R. W. & LOPES, J. D. Surface antigen detected by a Schistosoma mansoni monoclonal antibody in worm extracts and kidney deposits of infected mice and hamsters. Infect. Immun., 52: 230-235, 1986.
• 4. DEELDER, A. M.; KLAPPE, H. T. M.; VAN DER AARDWEG, G. J. M. & VAN MEERBEKE, E. H. E. M. Schistosoma mansoni: demonstration of two circulating antigens in infected hamsters. Exp. Parasit., 40: 189-197, 1976.
• 5. KÖSTER, B. & STRAND, M. Schistosoma mansoni: immunolocalization of two different fucose-containing carbohydrate epitopes. Parasitology, 108: 433-446, 1994.
• 6. NOUR EL DIN, M. S.; KORNELIS, D.; VAN ZEYL, R. J. M. & DEELDER, A. M. Immunological characterization of two monoclonal antibodies reactive with repetitive carbohydrate epitopes of circulating Schistosoma mansoni egg antigen. Amer. J. trop. Med. Hyg., 50: 487-498, 1994.
• 7. NOUR EL DIN, M. S. A.; NIBBELING, R.; ROTMANS, P. et al. Quantitative determination of circulating soluble egg antigen in urine and serum of Schistosoma mansoni-infected individuals using a combined two-site enzyme-linked immunosorbent assay. Amer. J. trop. Med. Hyg., 50: 585-594, 1994.
• 8. WOODWARD, M. P.; YOUNG, W. W. & BLOODGOOD, R. A. Detection of monoclonal antibodies specific for carbohydrate epitopes using periodate oxidation. J. immunol. Meth., 78: 143-153, 1985.
• 9. YI, X.; OMER-ALI, P.; KELLY, C.; SIMPSON, A. J. G. & SMITHERS, S. R. IgM antibodies recognizing carbohydrate epitopes shared between schistosomula and miracidia of Schistosoma mansoni that block in vitro killing. J. Immunol., 137: 3946-3954, 1986.

(1

) Instituto de Medicina Tropical de São Paulo, São Paulo, SP, Brasil

(2

) Departamento de Microbiologia, Imunologia e Parasitologia da Universidade Federal de São Paulo/Escola Paulista de Medicina (UNIFESP/EPM), São Paulo, SP, Brasil.

Correspondence to: Célia Regina Whitaker Carneiro, Universidade Federal de São Paulo (UNIFESP/EPM), Disciplina de Imunologia. Rua Botucatu, 862, 4º andar, 04023-900 São Paulo, SP, Brasil. Phone: [phone]. Fax: [phone]. e-mail: [e-mail]

Publication Dates

• Publication in this collection
  09 Oct 1998
• Date of issue
  Nov 1997

History

• Received
  04 Sept 1997
• Accepted
  07 Nov 1997