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Partial characterization of the antigen recognized by a monoclonal antibody to Ascaris suum ovary extracts
Veterinary Parasitology 146 (2007) 281–287 www.elsevier.com/locate/vetpar
Partial characterization of the antigen recognized by a monoclonal antibody to Ascaris suum ovary extracts T. Inoue *, M. Takashima, S. Murakami, T. Watanabe Laboratory of Animal Health, Tokyo University of Agriculture, 1737 Funako, Atsugi City, Kanagawa Prefecture 243-0034, Japan Received 27 September 2006; received in revised form 22 February 2007; accepted 22 February 2007
Abstract A monoclonal antibody produced against ovary extracts from the worm Ascaris suum showed immunoreactivity against granules in the rachis and oocytes, the inner layer of the eggshell and the middle layer of some egg, but not against either ovary wall or uterus wall. Furthermore, the same antigens were detected on the body surface of migrated larva in guinea pig lung, whereas none were detected in adult male worm or adult female worm, except for the female reproductive organs. The ovary extracts were passed through an affinity column and the eluted fractions analyzed by SDS-PAGE, Western blotting and native-PAGE. Western blotting after SDS-PAGE detected chemiluminescence primarily as three bands of about 70, 78 and 90 kDa. However, Western blotting after native-PAGE of the partially purified ovary extracts demonstrated only one band at a position of about 230 kDa. LC-nanoESI-MS/ MS analysis of protein band gel slices from silver-stained SDS-PAGE revealed one peptide sequence ‘‘ILVGLIGTNR’’, that matched only the hypothetical protein F14D2.8 of Caenorhabditis elegans (gi/7499081). # 2007 Elsevier B.V. All rights reserved. Keywords: Ascaris suum; Ovary extracts; Mab; Immunohistochemistry; Western blotting; LC-nanoESI-MS/MS analysis
1. Introduction A number of methods have been tried to detect antibody to Ascaris suum in pig serum for serological diagnosis of infection of pigs (Taffs, 1964; Eriksen et al., 1980, 1992; Yoshihara et al., 1987, 1993; Saeki et al., 1991; Roepstorff, 1998; Santra et al., 2001; Frontera et al., 2004). In these methods, the antigens used for detection of antibody were adult worm extract (Yoshihara et al., 1987; Frontera et al., 2004), larval worm extract (Frontera et al., 2004), adult body fluid (Eriksen et al., 1992; Yoshihara et al., 1993; Frontera et al., 2004), excretory–secretory products from adult worm (Santra et al., 2001), excretory–secretory * Corresponding author. Tel.: +81 46 270 6595; fax: +81 46 270 6597. E-mail address: [email protected] (T. Inoue). 0304-4017/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2007.02.028
products from larva (Roepstorff, 1998; Frontera et al., 2004) and eggs (Eriksen et al., 1980) of the nematodes. In our previous study (Takashima et al., 2003) ovary extracts of A. suum reacted positively with sera from conventional pigs with or without hepatic milk spots and contained rich antigenic constituents. However, the ovary extracts consisted of numerous unknown antigens. Thus, we have further analyzed the characteristics of these antigenic components of the ovary for application to serodiagnosis and vaccination against ascariosis, and for research of their biological activities. We describe here the production of a monoclonal antibody to the aforementioned ovary extracts, examination of the distribution of the antigen recognized by the Mab in A. suum worm body, and peptide analysis of the antigen by LC-nanoESI-MS/MS method and matching by an NCB Inr database search.
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2. Materials and methods 2.1. Experimental animals All mice were handled and maintained according to the Guidelines for Animal Experiments of Tokyo University of Agriculture. All mice were housed under specific-pathogen free conditions in an air-conditioned room for experimental animals and had free access to a commercial feed and water. 2.2. Preparation of crude ovary extracts from A. suum Ovary extracts were prepared, with slight modification, according to the method described in our previous paper (Takashima et al., 2003). Adult female worms were obtained from the slaughterhouse at Atsugi City, Kanagawa prefecture. Fresh nematodes were transported to our laboratory at the University and the ovaries removed. A 1 g amount of ovary tissue was then suspended in 5 ml of 0.15 M phosphate-buffered saline (PBS) (pH 7.4) and homogenized using a supersonic vibrator (UD201, Tomy Seiko, Tokyo, Japan) for 10 min by short bursts (30 s). The homogenized suspension was centrifuged at 7000 g for 20 min and the supernatant then mixed with an equal volume of saturated ammonium sulfate solution. After 3–4 h the mixture was again centrifuged at 7000 g for 20 min and the obtained precipitate washed 3 times in PBS. The washed precipitate was then resuspended in a small volume of water and dialyzed against PBS (pH 7.4) overnight at 4 8C. The next day the supernatant was obtained by centrifugation at 7000 g for 20 min. 2.3. Monoclonal antibody to ovary extracts Murine monoclonal antibodies (Mabs) were produced as described elsewhere (Shinagawa et al., 1989; Yamamoto, 1991; Iwata et al., 2000). Five-week-old BALB/cA Jcl mice (purchased from Funabashi Farm and maintained at our laboratory) were inoculated subcutaneously with 0.2 ml of an emulsion of the above described ovary extract (500 mg/ml) and complete Freund’s adjuvant. The inoculations were performed subcutaneously with an emulsion of the crude ovary extract and incomplete Freund’s adjuvant at 2 and 4 weeks after the first immunization. A booster inoculation of ovary extract (30 mg/mouse) without adjuvant was injected intraperitoneally 1 week after the third immunization. The spleen was harvested 3–4 days after the booster injection.
Fusion of spleen cells from immunized mice with prepared P3NS-1/1Ag4.1 myeloma cells (purchased from the Cell Bank Riken Bioresource Center, Japan) was performed by addition of 50% polyethylene glycol 1500 (Roche Applied Science, Germany) in RPMI 1640. Fused cells were suspended in growth medium containing 1% HAT (Gibco, U.S.A.) and dispensed into 96-well cell culture plates. The plates were incubated at 37 8C at an atmosphere of 5% CO2 concentration in a CO2 incubator for 10 through 14 days until hybridoma colonies were observed, changing a half volume of the growth medium every 3 days. At the end of incubation, the supernatants of clones were screened by enzymelinked immunosorbent assay (ELISA) as described below. The hybridomas producing desired antibodies were cloned twice by the limiting dilution method, incubating with feeder cells prepared from the spleens of BALB/cA Jcl mice. The presence of a Mab in the supernatants of clones was confirmed by ELISA at the cloning stages. The Mab isotype was determined using the ImmunoPure Monoclonal Antibody Isotyping Kit (HRP/ABTS, Pierce, U.S.A.). Mabs from the selected clones were produced in pristine-(2,6,10,14-tetramethylpentadecane, Sigma, U.S.A.) primed BALB/cA Jcl mice, via inoculation of 1 106 hybridoma cells into the peritoneal cavity. Ascites fluid was collected when an adequate volume had accumulated and the supernatant of the fluid then obtained by centrifugation at 2000 g for 10 min. 2.4. Protein determination Protein concentrations were estimated by the method of Lowry et al. (1951) with bovine serum albumin as the standard. Ultra-violet absorbance at 280 nm (Johnstone and Thorpe, 1987) was also applied to estimate protein concentration. 2.5. ELISA Hybridoma supernatants were screened with ELISA as described by Ali et al. (2004) with slight modification. Multi-well plates (Sumitomo Bakelite Co., Japan) were coated with 10 mg/ml of the crude ovary extract in 0.05 M carbonate-bicarbonate buffer (pH 9.5), kept at 4 8C overnight and washed twice with PBS supplemented with 0.05% (v/v) Tween-20 (PBST). The plates were blocked with 0.1% (v/v) bovine serum albumin in PBST (BSA-PBST, 150 ml/well) at 37 8C for 2 h and washed twice with PBST. The supernatants and diluted positive and negative mouse sera were dispensed into the wells of plates at 100 ml/well and then allowed to
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stand at 37 8C for 1 h. After three washings, horse radish peroxidase-conjugated goat anti-mouse IgG (affinity purified antibody to mouse IgG (H + L), KPL, U.S.A.) was diluted 1:2000 in BSA-PBST, dispensed into the wells (150 ml/well), incubated at 37 8C for 1 h and washed three times. The enzyme reaction was initiated by adding 100 ml of substrate solution, containing 0.3% 2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (Wako Chemicals, Japan) in 0.003% (v/v) H2O2–0.1 M citrate buffer (pH 4.0). After incubation at room temperature for 15 min, the reaction was stopped by the addition of 50 ml of 1.25% sodium fluoride. Absorbance was measured at 405 nm, using a microtiter plate reader (MTP-32, Corona Electric, Japan). 2.6. Immunohistochemistry Fresh A. suum worms obtained from the slaughterhouse and the lungs of Hartley guinea pigs experimentally infected with A. suum (originally used for another study) were fixed in 20% neutral-buffered formalin and embedded in paraffin wax. Sections 4– 5 mm thick were stained with hematoxylin and eosin to reconfirm morphologically the presence of the uterus and the ovary in the adult worms. Other sections were stained by the streptavidin-biotin-peroxidase conjugate (SAB) method (Murakami et al., 1998). The Mab produced against A. suum ovary extracts was used at an optimal dilution as the primary antibody. Controls for the immunoreactions were specimens with normal mouse serum (Dako Cytomation Inc., U.S.A.) in place of the primary antibody. The antigenantibody reaction was detected using the Histofine SAB-PO(M) kit (Nichirei Co., Japan) according to the manufacturer’s instructions. The development of signal was performed using the AEC substrate kit (Nichirei Co., Japan) according to the manufacturer’s instructions. The sections were counterstained with hematoxylin. 2.7. Affinity chromatography Affinity chromatography was performed using a HiTrap NHS-activated HP 1-ml column (GE Healthcare, Amersham Biosciences AB, Sweden) according to the manufacturer’s instructions. The precipitate of 50% saturated ammonium sulfate from the ascites fluid was used as the coupling ligand. The crude ovary extract described above was centrifuged at 14,000 g for 20 min and filtered through a 0.45 m disposable syringe filter (DISMIC-25CS type, Toyo
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Roshi Kaisha Ltd., Japan) before application to the column. 2.8. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and native-PAGE Equal volumes of the sample preparations and a solution buffer composed of 2% SDS, 6% 2-mercaptoethanol (2-ME), 10% glycerol and 50 mM Tris–HCl buffer (pH 6.8) were mixed and boiled at 100 8C for 3 min. The electrophoresis was performed in an AE-6530MWmPAGE apparatus (ATTO Co., Japan) according to the manufacturer’s instructions. The concentrations of polyacrylamide in the stacking gel and the separating gel were 4.5 and 14.5%, respectively. The electrophoresis was run at 40 mA of constant current until the tracking dye reached near the bottom of the separating gel. Detection of separated materials was performed with Silver Stain ‘‘DAIICHI’’ (Daiichi Pure Chemicals Co., Japan). The Prestained SDS-PAGE Standards, Low Range (BioRad Laboratories, U.S.A.) which consisted of phosphorylase (106.9 kDa), bovine serum albumin (93.6 kDa), ovalbumin (52.3 kDa), carbonic anhydrase (37.2 kDa), soybean trypsin inhibitor (28.2 kDa) and lysozyme (18.8 kDa) were used as the standard proteins. The native-PAGE was carried out under nondenaturing conditions (without SDS and 2-ME) (Gallaghar and Smith, 1991). The mixture of equal volumes of the sample preparation and solution buffer without SDS and 2-ME were not boiled. Molecular Weight Marker ‘‘DAIICHI’’ I (DAIICHI PURE CHEMICALS, CO, LTD, Japan) consisting of thyroglobulin (669.0 kDa), ferritin (443.0 kDa), lactate dehydrogenase (133.9 kDa), albumin (66.3 kDa) and trypsin inhibitor (20.1 kDa) was used as standard protein for native SDS-PAGE. 2.9. Western blocking After electrophoresis, proteins were transferred to a PVDF sheet using a semidry transfer apparatus (BC BIO CRAFT Model 310, BIOCRAFT CO., Japan) at 120 mA of constant current for 30 min. Any antigens on the transfer sheet were detected using the ECL Western Blotting Analysis System (GE Healthcare, Amersham Biosciences AB, Sweden) according to the manufacturer’s instructions. Chemiluminescence was recorded using an ECL mini-camera (GE Healthcare, Amersham Biosciences AB, Sweden) loaded with FUJIFILM instant pack films FP-3000B (Fuji Photo Film Co., Japan).
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2.10. Sequence analysis
3.2. Immunohistochemistry
Peptide sequencing of the protein recognized by the Mab was performed by LC-nanoESI-MS/MS method and the obtained sequences were searched with an NCB Inr database (Barrett et al., 2005; Lasonder et al., 2002). Further de Novo sequencing was performed and sequences obtained were also searched with the data base. These analyses were performed by Geneworld Co., Japan.
In the ovary the positive immunoreaction with the Mab was found on granules in oocytes and the rachis (Fig. 1). However no reacted antigen was found at the outer muscle layer and epithelial cells (Fig. 1). In the uterus the strong reactions were observed at the inner layer of the eggshell and the weak reaction at the middle layer of some egg (Fig. 2). However no stained antigen was detected on the outer layer of the eggshell, at the egg cytoplasm, on the muscle layer or on the epithelium of the uterus (Fig. 2). On the larva in the bronchiole of guinea pig lung, the body surface exhibited the strong reaction (Fig. 3). In addition, the weak reaction was found on membrane structure in the larval body (Fig. 3).
3. Results 3.1. Mab The Mab produced and used in this study belonged to the IgG1 subclass and k light chain type which were determined using the ImmunoPure Monoclonal Antibody Isotyping Kit (HRP/ABTS, Pierce, U.S.A.).
3.3. Electrophoresis The crude ovary extract treated as described above was passed twice through the affinity column, and then
Fig. 1. The immunohistochemical findings of the ovary of Ascaris suum. Paraffin sections 4–5 mm thick were stained using the SAB method. (a) An SAB-stained section reacted with Mab against ovary extract as the primary antibody and (b) hematoxylin-stained serial section reacted with normal mouse serum in place of the Mab. NU: nuclei; OE: ovary epithelium; OO: oocyte; RA: rachis. Bar indicates 10 mm.
Fig. 2. The immunohistochemical findings of the uterus of A. suum. See the legend of Fig. 1. EG: egg; ML: muscle layer; UE: uterus epithelium. Bar indicates 25 mm.
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Fig. 3. The immunohistochemical findings of the migrated larva in the bronchiole of guinea pig lung. See the legend of Fig. 1. EO: eosinophile; LA: larva; MA: macrophage. Bar indicates 10 mm.
analyzed by SDS-PAGE, Western blotting and nativePAGE along with the original crude ovary extract (Fig. 4). Western blotting after SDS-PAGE showed the chemiluminescence in both the crude ovary extracts and the fraction eluted from the affinity column mainly as three bands at about 70, 78 and 90 kDa. Moreover three faint bands were also observed in the eluted fraction at about 35, 39 and 45 kDa. In SDS-PAGE the partially purified ovary extracts exhibited a number of stained bands at positions of faster mobility than that of about 90 kDa and good elimination of the larger molecules than 70 kDa from the crude extract by the affinity chromatography. On the other hand Western blotting after native-PAGE of the partially purified ovary extracts demonstrated only one band at a position of about 230 kDa. 3.4. Sequence analysis of the antigen The sample for LC-nanoESI-MS/MS analysis was excised from a silver-stained gel and pooled (Fig. 4). One peptide sequence of the many including de novo sequences obtained was ‘‘ILVGLIGTNR’’ which matched only the hypothetical protein F14D2.8 of Caenorhabditis elegans (gi/7499081). 4. Discussion Fig. 4. Electrophoresis of the ovary extracts from A. suum. (a) Markers, (b) Western blotting after SDS-PAGE of the crude ovary extracts, (c) silver-stained SDS-PAGE of the crude ovary extracts, (d) Western blotting after SDS-PAGE of the partially purified ovary extracts, (e) silver-stained SDS-PAGE of the partially purified ovary extracts, (f) markers for native-PAGE and (g) Western blotting after native-PAGE of the same partially purified ovary extracts as used for SDS-PAGE. Arrows show the sites excised for LC-nanoESI-MS/MS analysis.
This paper describes analysis of the properties of an antigen in the ovary of A. suum, using a Mab, that reacts positively with sera from conventional pigs (Takashima et al., 2003) and peptide sequence analysis by LCnanoESI-MS/MS method and an NCB Inr database search (Lasonder et al., 2002; Barrett et al., 2005). The immunohistochemical study showed that the antigen recognized by the Mab was present on granules
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inside oocytes and the rachis of the ovary, on the inner layer and the middle layer of eggshell of the eggs in the uterus and on the body surface and membrane structures in the body of the migrated larva in guinea pig lung. The antigen was not found in adult male worm and adult female worm excluding the female reproductive organs. Thus, expression of this antigen may be related to the development of oocytes through larvae. Further immunohistochemical analysis of L1 and L2 stage larvae is necessary for confirmation of this possibility. On the other hand, there were many granules reacted with the Mab in the oocytes and the rachis. Bird (1971) described that refringent granules were numerous in the oocytes and absent in the rachis. However the reacted granules were observed in the rachis. Thus the positively reacted granules appeared to be different from the refringent granules. Or the reacted granules in the rachis are thought to be precursors of refringent granules. In addition, the membrane structures reacted with the Mab were observed in the body of the larva in the lung of guinea pig. This structure is thought to be a part of the female reproductive organ, because the female reproductive system of the Nematode arises from genital primordia in the larval stages and sexual differentiation may first occur as early as the second stage larva (Bird, 1971). Western blotting showed chemiluminescence was detected in both the crude ovary extracts and the fraction that passed through the affinity column as three bands at about 70, 78 and 90 kDa. Moreover three faint bands were also observed in the eluted fraction at about 35, 39 and 45 kDa. Dubinsky et al. (1982) reported that SDS-PAGE of the supernatant of homogenate from ovary displayed proteins of 72 and 75 kDa. However, in the present study Western blotting after native-PAGE of partially purified ovary extracts treated by affinity chromatography demonstrated one band at a position of about 230 kDa. Moreover SDS-PAGE showed that the partially purified ovary extracts exhibited a number of stained bands at positions of faster mobility than that of about 70 kDa. This discrepancy between the SDSPAGE and native-PAGE results is likely due to the possible subunit structure of the antigen in the ovary extracts. Kennedy and Qureshi (1986) described that the excretory–secretory products from larvae were affected by reducing conditions. Furthermore, the antigen recognized by the Mab appeared to be equivalent to the hypothetical protein F14D2.8 of C. elegans (gi/7499081). This protein is composed of 1466 amino acids and is calculated to be 170,252 Da with a calculated pI value of 6.01. This protein is also thought to be a membrane protein and to
have two transmembrane helices (the 444th through 466th amino acids from the N-terminal and 470th through 492nd) (SOSUI, http://sosui.proteome.bio.tuat.ac.jp/cgi-bin/sosui.cgi?/). The other F14D2.8 of C. elegans (gi/71986492), which consists of 277 amino acids has been deposited in the NCBI protein database. NCBI BLASTP analysis indicated that the latter F14D2.8 matched with the 8th amino acid through the 340th one of the former F14D2.8. The sequence ‘‘ILVGLIGTNR’’ obtained here was identical with the 501st through the 510th amino acids of the former F14D2.8. This sequence was derived from the three bands on SDS-PAGE of about 70, 78 and 90 kDa recognized by the Mab. However no protein of A. suum did not match the sequences obtained by both LCnanoESL-MS/MS method and de novo sequencing. Analysis of cDNA from the ovary will clarify the precise sequence of this antigen. Acknowledgements The authors thank Professor Dr. Hiroyuki Iwata, Department of Veterinary Medicine, Faculty of Agriculture, Yamaguchi University for his advice on Western blotting and Professor Dr. Soichi Imai, Faculty of Veterinary Medicine, Nippon Veterinary and Life Science University for his advice on the structure of A. suum. References Ali, H.A., Sawada, T., Hatakeyama, H., Ohtsuki, N., Itoh, O., 2004. Characterization of a 39 kDa capsular protein of avian Pasteurella multocida using monoclonal antibodies. Vet. Microbiol. 100, 43– 53. Barrett, J., Brophy, P.M., Hamilton, J.V., 2005. Analyzing proteomic data. Int. J. Parasitol. 34, 543–553. Bird, A.F. (Ed.), 1971. The Structure of Nematodes. Academic Press, New York and London, 318 pp. Dubinsky, P., Rybos, M., Turcekova, L., 1982. Electrophoretic characteristic of the proteins in growing oocytes of Ascaris suum. Helmintologia 19, 289–297. Eriksen, L., Andersen, S., Nielsen, K., Pedersen, A., Nielsen, J., 1980. Experimental Ascaris suum infection in pigs. Serological response, eosinophilia, in peripheral blood, occurrence of white spots in the liver and worm recovery from the intestine. Nord. Vet. Med. 32, 233–242. Eriksen, L., Lind, P., Nansen, P., Roepstorff, A., Urban, J., 1992. Resistance to Ascaris suum in parasite naı¨ve and naturally exposed growers, finishers and sows. Vet. Parasitol. 41, 137–149. Frontera, E., Roepstorff, A., Serrano, F.J., Gazques, A., Reina, D., Navarrete, I., 2004. Presence of immunoglobulins and antigens in serum, lung and small intestine in Ascaris suum infected and immunised pigs. Vet. Parasitol. 119, 59–71. Gallaghar, S.R., Smith, J.A., 1991. Nondenaturing discontinuous gel electrophoresis. In: Coligan, J.E., Kruisbeek, A.M., Margulies,
T. Inoue et al. / Veterinary Parasitology 146 (2007) 281–287 D.H., Shevach, E.M., Strober, W. (Eds.), Current Protocols in Immunology, vol. 1. John Wiley & Sons, New York, Chichester, Brisbane, Toronto and Singapore, p. 8.4.9. Iwata, H., Murase, K., Inoue, T., 2000. Monoclonal antibody against bovine a1-acid glycoprotein. J. Vet. Med. Sci. 62, 1099–1100. Johnstone, A., Thorpe, R., 1987. Ultra-violet absorption. In: Johnstone, A., Thorpe, R. (Eds.), Immunochemistry in Practice. 2nd ed. Blackwell Scientific Publications, Oxford, London, Edinburgh, Boston, Palo Alto, Melbourne, pp. 1–3. Kennedy, M.W., Qureshi, F., 1986. Stage-specific secreted antigens of the parasitic larval stages of the nematode Ascaris. Immunology 58, 515–522. Lasonder, E., Ishihama, Y., Andersen, J.S., Vermunt, A.M.W., Pain, A., Sauerwein, R.W., Eling, W.M.C., Hall, N., Waters, A.P., Stunnenberg, H.G., Mann, M., 2002. Analysis of the Plasmodium falciparum proteome by high-accuracy mass spectrometry. Nature 419, 537–542. Lowry, L.O., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 192, 265–275. Murakami, S., Azuma, R., Koeda, T., Oomi, H., Watanabe, T., Fujiwara, H., 1998. Immunohistochemical detection for Actinomyces sp. in swine tonsillar abscess and granulomatous mastitis. Mycopathologia 141, 15–19. Roepstorff, A., 1998. Natural Ascaris suum infections in swine diagnosed by coprological and serological (ELISA) methods. Parasitol. Res. 84, 537–543. Saeki, H., Suzuki, S., Sei, A., Takahashi, T., Ishii, T., 1991. Application of ELISA for field survey of milk spots caused by
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Ascaris suum infection in porkers. Jpn. J. Parasitol. 40, 374– 382. Santra, A., Bhattacharya, T., Chowdhury, A., Ghosh, A., Ghosh, N., Chatterjee, B.P., Mazunder, D.N.G., 2001. Serodiagnosis of ascariasis with specific IgG4 antibody and its use in an epidemiological study. Trans. R. Soc. Trop. Med. Hyg. 95, 289–292. Shinagawa, E., Matsushita, K., Inoue, T., Adachi, O., Ameyama, M., 1989. Monoclonal antibody recognizing the quinoprotein subunit of alcohol dehydrogenase complex from Gluconobacter species. Agric. Biol. Chem. 53, 2011–2012. Taffs, L.F., 1964. Immunological studies on experimental infection of pigs with Ascaris suum Goerge. III. The antibody response and acquired immunity. J. Helmintol. 38, 129–150. Takashima, M., Ohmi, H., Watanabe, T., Okamoto, K., Kanoe, M., Nagai, S., 2003. Attempts to separate female Ascaris suum antigen and to investigate its partial characterization. Vet. J. 165, 164–168. Yamamoto, W.M., 1991. Production of monoclonal antibodies. In: Coligan, J.E., Kruisbeek, A.M., Margulies, D.H., Shevach, E.M., Strober, W. (Eds.), Current Protocols in Immunology, vol. 1. John Wiley & Sons, New York, Chichester, Brisbane, Toronto and Singapore, pp. 2.5.1–12.5.17. Yoshihara, S., Nakagawa, M., Sudo, H., 1987. Detection of complement fixation antibody against Ascaris suum antigen in pigs with white spots in the liver. Jpn. J. Vet. Sci. 49, 561–945. Yoshihara, S., Oya, T., Furuya, T., Goto, N., 1993. Use of body fluid of female Ascaris suum as antigen in the enzyme-linked immunosorbent assay (ELISA) for diagnosis of swine ascariosis. J. Helmintol. 67, 279–286.
Partial characterization of the antigen recognized by a monoclonal antibody to Ascaris suum ovary extracts T. Inoue *, M. Takashima, S. Murakami, T. Watanabe Laboratory of Animal Health, Tokyo University of Agriculture, 1737 Funako, Atsugi City, Kanagawa Prefecture 243-0034, Japan Received 27 September 2006; received in revised form 22 February 2007; accepted 22 February 2007
Abstract A monoclonal antibody produced against ovary extracts from the worm Ascaris suum showed immunoreactivity against granules in the rachis and oocytes, the inner layer of the eggshell and the middle layer of some egg, but not against either ovary wall or uterus wall. Furthermore, the same antigens were detected on the body surface of migrated larva in guinea pig lung, whereas none were detected in adult male worm or adult female worm, except for the female reproductive organs. The ovary extracts were passed through an affinity column and the eluted fractions analyzed by SDS-PAGE, Western blotting and native-PAGE. Western blotting after SDS-PAGE detected chemiluminescence primarily as three bands of about 70, 78 and 90 kDa. However, Western blotting after native-PAGE of the partially purified ovary extracts demonstrated only one band at a position of about 230 kDa. LC-nanoESI-MS/ MS analysis of protein band gel slices from silver-stained SDS-PAGE revealed one peptide sequence ‘‘ILVGLIGTNR’’, that matched only the hypothetical protein F14D2.8 of Caenorhabditis elegans (gi/7499081). # 2007 Elsevier B.V. All rights reserved. Keywords: Ascaris suum; Ovary extracts; Mab; Immunohistochemistry; Western blotting; LC-nanoESI-MS/MS analysis
1. Introduction A number of methods have been tried to detect antibody to Ascaris suum in pig serum for serological diagnosis of infection of pigs (Taffs, 1964; Eriksen et al., 1980, 1992; Yoshihara et al., 1987, 1993; Saeki et al., 1991; Roepstorff, 1998; Santra et al., 2001; Frontera et al., 2004). In these methods, the antigens used for detection of antibody were adult worm extract (Yoshihara et al., 1987; Frontera et al., 2004), larval worm extract (Frontera et al., 2004), adult body fluid (Eriksen et al., 1992; Yoshihara et al., 1993; Frontera et al., 2004), excretory–secretory products from adult worm (Santra et al., 2001), excretory–secretory * Corresponding author. Tel.: +81 46 270 6595; fax: +81 46 270 6597. E-mail address: [email protected] (T. Inoue). 0304-4017/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2007.02.028
products from larva (Roepstorff, 1998; Frontera et al., 2004) and eggs (Eriksen et al., 1980) of the nematodes. In our previous study (Takashima et al., 2003) ovary extracts of A. suum reacted positively with sera from conventional pigs with or without hepatic milk spots and contained rich antigenic constituents. However, the ovary extracts consisted of numerous unknown antigens. Thus, we have further analyzed the characteristics of these antigenic components of the ovary for application to serodiagnosis and vaccination against ascariosis, and for research of their biological activities. We describe here the production of a monoclonal antibody to the aforementioned ovary extracts, examination of the distribution of the antigen recognized by the Mab in A. suum worm body, and peptide analysis of the antigen by LC-nanoESI-MS/MS method and matching by an NCB Inr database search.
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2. Materials and methods 2.1. Experimental animals All mice were handled and maintained according to the Guidelines for Animal Experiments of Tokyo University of Agriculture. All mice were housed under specific-pathogen free conditions in an air-conditioned room for experimental animals and had free access to a commercial feed and water. 2.2. Preparation of crude ovary extracts from A. suum Ovary extracts were prepared, with slight modification, according to the method described in our previous paper (Takashima et al., 2003). Adult female worms were obtained from the slaughterhouse at Atsugi City, Kanagawa prefecture. Fresh nematodes were transported to our laboratory at the University and the ovaries removed. A 1 g amount of ovary tissue was then suspended in 5 ml of 0.15 M phosphate-buffered saline (PBS) (pH 7.4) and homogenized using a supersonic vibrator (UD201, Tomy Seiko, Tokyo, Japan) for 10 min by short bursts (30 s). The homogenized suspension was centrifuged at 7000 g for 20 min and the supernatant then mixed with an equal volume of saturated ammonium sulfate solution. After 3–4 h the mixture was again centrifuged at 7000 g for 20 min and the obtained precipitate washed 3 times in PBS. The washed precipitate was then resuspended in a small volume of water and dialyzed against PBS (pH 7.4) overnight at 4 8C. The next day the supernatant was obtained by centrifugation at 7000 g for 20 min. 2.3. Monoclonal antibody to ovary extracts Murine monoclonal antibodies (Mabs) were produced as described elsewhere (Shinagawa et al., 1989; Yamamoto, 1991; Iwata et al., 2000). Five-week-old BALB/cA Jcl mice (purchased from Funabashi Farm and maintained at our laboratory) were inoculated subcutaneously with 0.2 ml of an emulsion of the above described ovary extract (500 mg/ml) and complete Freund’s adjuvant. The inoculations were performed subcutaneously with an emulsion of the crude ovary extract and incomplete Freund’s adjuvant at 2 and 4 weeks after the first immunization. A booster inoculation of ovary extract (30 mg/mouse) without adjuvant was injected intraperitoneally 1 week after the third immunization. The spleen was harvested 3–4 days after the booster injection.
Fusion of spleen cells from immunized mice with prepared P3NS-1/1Ag4.1 myeloma cells (purchased from the Cell Bank Riken Bioresource Center, Japan) was performed by addition of 50% polyethylene glycol 1500 (Roche Applied Science, Germany) in RPMI 1640. Fused cells were suspended in growth medium containing 1% HAT (Gibco, U.S.A.) and dispensed into 96-well cell culture plates. The plates were incubated at 37 8C at an atmosphere of 5% CO2 concentration in a CO2 incubator for 10 through 14 days until hybridoma colonies were observed, changing a half volume of the growth medium every 3 days. At the end of incubation, the supernatants of clones were screened by enzymelinked immunosorbent assay (ELISA) as described below. The hybridomas producing desired antibodies were cloned twice by the limiting dilution method, incubating with feeder cells prepared from the spleens of BALB/cA Jcl mice. The presence of a Mab in the supernatants of clones was confirmed by ELISA at the cloning stages. The Mab isotype was determined using the ImmunoPure Monoclonal Antibody Isotyping Kit (HRP/ABTS, Pierce, U.S.A.). Mabs from the selected clones were produced in pristine-(2,6,10,14-tetramethylpentadecane, Sigma, U.S.A.) primed BALB/cA Jcl mice, via inoculation of 1 106 hybridoma cells into the peritoneal cavity. Ascites fluid was collected when an adequate volume had accumulated and the supernatant of the fluid then obtained by centrifugation at 2000 g for 10 min. 2.4. Protein determination Protein concentrations were estimated by the method of Lowry et al. (1951) with bovine serum albumin as the standard. Ultra-violet absorbance at 280 nm (Johnstone and Thorpe, 1987) was also applied to estimate protein concentration. 2.5. ELISA Hybridoma supernatants were screened with ELISA as described by Ali et al. (2004) with slight modification. Multi-well plates (Sumitomo Bakelite Co., Japan) were coated with 10 mg/ml of the crude ovary extract in 0.05 M carbonate-bicarbonate buffer (pH 9.5), kept at 4 8C overnight and washed twice with PBS supplemented with 0.05% (v/v) Tween-20 (PBST). The plates were blocked with 0.1% (v/v) bovine serum albumin in PBST (BSA-PBST, 150 ml/well) at 37 8C for 2 h and washed twice with PBST. The supernatants and diluted positive and negative mouse sera were dispensed into the wells of plates at 100 ml/well and then allowed to
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stand at 37 8C for 1 h. After three washings, horse radish peroxidase-conjugated goat anti-mouse IgG (affinity purified antibody to mouse IgG (H + L), KPL, U.S.A.) was diluted 1:2000 in BSA-PBST, dispensed into the wells (150 ml/well), incubated at 37 8C for 1 h and washed three times. The enzyme reaction was initiated by adding 100 ml of substrate solution, containing 0.3% 2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (Wako Chemicals, Japan) in 0.003% (v/v) H2O2–0.1 M citrate buffer (pH 4.0). After incubation at room temperature for 15 min, the reaction was stopped by the addition of 50 ml of 1.25% sodium fluoride. Absorbance was measured at 405 nm, using a microtiter plate reader (MTP-32, Corona Electric, Japan). 2.6. Immunohistochemistry Fresh A. suum worms obtained from the slaughterhouse and the lungs of Hartley guinea pigs experimentally infected with A. suum (originally used for another study) were fixed in 20% neutral-buffered formalin and embedded in paraffin wax. Sections 4– 5 mm thick were stained with hematoxylin and eosin to reconfirm morphologically the presence of the uterus and the ovary in the adult worms. Other sections were stained by the streptavidin-biotin-peroxidase conjugate (SAB) method (Murakami et al., 1998). The Mab produced against A. suum ovary extracts was used at an optimal dilution as the primary antibody. Controls for the immunoreactions were specimens with normal mouse serum (Dako Cytomation Inc., U.S.A.) in place of the primary antibody. The antigenantibody reaction was detected using the Histofine SAB-PO(M) kit (Nichirei Co., Japan) according to the manufacturer’s instructions. The development of signal was performed using the AEC substrate kit (Nichirei Co., Japan) according to the manufacturer’s instructions. The sections were counterstained with hematoxylin. 2.7. Affinity chromatography Affinity chromatography was performed using a HiTrap NHS-activated HP 1-ml column (GE Healthcare, Amersham Biosciences AB, Sweden) according to the manufacturer’s instructions. The precipitate of 50% saturated ammonium sulfate from the ascites fluid was used as the coupling ligand. The crude ovary extract described above was centrifuged at 14,000 g for 20 min and filtered through a 0.45 m disposable syringe filter (DISMIC-25CS type, Toyo
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Roshi Kaisha Ltd., Japan) before application to the column. 2.8. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and native-PAGE Equal volumes of the sample preparations and a solution buffer composed of 2% SDS, 6% 2-mercaptoethanol (2-ME), 10% glycerol and 50 mM Tris–HCl buffer (pH 6.8) were mixed and boiled at 100 8C for 3 min. The electrophoresis was performed in an AE-6530MWmPAGE apparatus (ATTO Co., Japan) according to the manufacturer’s instructions. The concentrations of polyacrylamide in the stacking gel and the separating gel were 4.5 and 14.5%, respectively. The electrophoresis was run at 40 mA of constant current until the tracking dye reached near the bottom of the separating gel. Detection of separated materials was performed with Silver Stain ‘‘DAIICHI’’ (Daiichi Pure Chemicals Co., Japan). The Prestained SDS-PAGE Standards, Low Range (BioRad Laboratories, U.S.A.) which consisted of phosphorylase (106.9 kDa), bovine serum albumin (93.6 kDa), ovalbumin (52.3 kDa), carbonic anhydrase (37.2 kDa), soybean trypsin inhibitor (28.2 kDa) and lysozyme (18.8 kDa) were used as the standard proteins. The native-PAGE was carried out under nondenaturing conditions (without SDS and 2-ME) (Gallaghar and Smith, 1991). The mixture of equal volumes of the sample preparation and solution buffer without SDS and 2-ME were not boiled. Molecular Weight Marker ‘‘DAIICHI’’ I (DAIICHI PURE CHEMICALS, CO, LTD, Japan) consisting of thyroglobulin (669.0 kDa), ferritin (443.0 kDa), lactate dehydrogenase (133.9 kDa), albumin (66.3 kDa) and trypsin inhibitor (20.1 kDa) was used as standard protein for native SDS-PAGE. 2.9. Western blocking After electrophoresis, proteins were transferred to a PVDF sheet using a semidry transfer apparatus (BC BIO CRAFT Model 310, BIOCRAFT CO., Japan) at 120 mA of constant current for 30 min. Any antigens on the transfer sheet were detected using the ECL Western Blotting Analysis System (GE Healthcare, Amersham Biosciences AB, Sweden) according to the manufacturer’s instructions. Chemiluminescence was recorded using an ECL mini-camera (GE Healthcare, Amersham Biosciences AB, Sweden) loaded with FUJIFILM instant pack films FP-3000B (Fuji Photo Film Co., Japan).
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2.10. Sequence analysis
3.2. Immunohistochemistry
Peptide sequencing of the protein recognized by the Mab was performed by LC-nanoESI-MS/MS method and the obtained sequences were searched with an NCB Inr database (Barrett et al., 2005; Lasonder et al., 2002). Further de Novo sequencing was performed and sequences obtained were also searched with the data base. These analyses were performed by Geneworld Co., Japan.
In the ovary the positive immunoreaction with the Mab was found on granules in oocytes and the rachis (Fig. 1). However no reacted antigen was found at the outer muscle layer and epithelial cells (Fig. 1). In the uterus the strong reactions were observed at the inner layer of the eggshell and the weak reaction at the middle layer of some egg (Fig. 2). However no stained antigen was detected on the outer layer of the eggshell, at the egg cytoplasm, on the muscle layer or on the epithelium of the uterus (Fig. 2). On the larva in the bronchiole of guinea pig lung, the body surface exhibited the strong reaction (Fig. 3). In addition, the weak reaction was found on membrane structure in the larval body (Fig. 3).
3. Results 3.1. Mab The Mab produced and used in this study belonged to the IgG1 subclass and k light chain type which were determined using the ImmunoPure Monoclonal Antibody Isotyping Kit (HRP/ABTS, Pierce, U.S.A.).
3.3. Electrophoresis The crude ovary extract treated as described above was passed twice through the affinity column, and then
Fig. 1. The immunohistochemical findings of the ovary of Ascaris suum. Paraffin sections 4–5 mm thick were stained using the SAB method. (a) An SAB-stained section reacted with Mab against ovary extract as the primary antibody and (b) hematoxylin-stained serial section reacted with normal mouse serum in place of the Mab. NU: nuclei; OE: ovary epithelium; OO: oocyte; RA: rachis. Bar indicates 10 mm.
Fig. 2. The immunohistochemical findings of the uterus of A. suum. See the legend of Fig. 1. EG: egg; ML: muscle layer; UE: uterus epithelium. Bar indicates 25 mm.
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Fig. 3. The immunohistochemical findings of the migrated larva in the bronchiole of guinea pig lung. See the legend of Fig. 1. EO: eosinophile; LA: larva; MA: macrophage. Bar indicates 10 mm.
analyzed by SDS-PAGE, Western blotting and nativePAGE along with the original crude ovary extract (Fig. 4). Western blotting after SDS-PAGE showed the chemiluminescence in both the crude ovary extracts and the fraction eluted from the affinity column mainly as three bands at about 70, 78 and 90 kDa. Moreover three faint bands were also observed in the eluted fraction at about 35, 39 and 45 kDa. In SDS-PAGE the partially purified ovary extracts exhibited a number of stained bands at positions of faster mobility than that of about 90 kDa and good elimination of the larger molecules than 70 kDa from the crude extract by the affinity chromatography. On the other hand Western blotting after native-PAGE of the partially purified ovary extracts demonstrated only one band at a position of about 230 kDa. 3.4. Sequence analysis of the antigen The sample for LC-nanoESI-MS/MS analysis was excised from a silver-stained gel and pooled (Fig. 4). One peptide sequence of the many including de novo sequences obtained was ‘‘ILVGLIGTNR’’ which matched only the hypothetical protein F14D2.8 of Caenorhabditis elegans (gi/7499081). 4. Discussion Fig. 4. Electrophoresis of the ovary extracts from A. suum. (a) Markers, (b) Western blotting after SDS-PAGE of the crude ovary extracts, (c) silver-stained SDS-PAGE of the crude ovary extracts, (d) Western blotting after SDS-PAGE of the partially purified ovary extracts, (e) silver-stained SDS-PAGE of the partially purified ovary extracts, (f) markers for native-PAGE and (g) Western blotting after native-PAGE of the same partially purified ovary extracts as used for SDS-PAGE. Arrows show the sites excised for LC-nanoESI-MS/MS analysis.
This paper describes analysis of the properties of an antigen in the ovary of A. suum, using a Mab, that reacts positively with sera from conventional pigs (Takashima et al., 2003) and peptide sequence analysis by LCnanoESI-MS/MS method and an NCB Inr database search (Lasonder et al., 2002; Barrett et al., 2005). The immunohistochemical study showed that the antigen recognized by the Mab was present on granules
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inside oocytes and the rachis of the ovary, on the inner layer and the middle layer of eggshell of the eggs in the uterus and on the body surface and membrane structures in the body of the migrated larva in guinea pig lung. The antigen was not found in adult male worm and adult female worm excluding the female reproductive organs. Thus, expression of this antigen may be related to the development of oocytes through larvae. Further immunohistochemical analysis of L1 and L2 stage larvae is necessary for confirmation of this possibility. On the other hand, there were many granules reacted with the Mab in the oocytes and the rachis. Bird (1971) described that refringent granules were numerous in the oocytes and absent in the rachis. However the reacted granules were observed in the rachis. Thus the positively reacted granules appeared to be different from the refringent granules. Or the reacted granules in the rachis are thought to be precursors of refringent granules. In addition, the membrane structures reacted with the Mab were observed in the body of the larva in the lung of guinea pig. This structure is thought to be a part of the female reproductive organ, because the female reproductive system of the Nematode arises from genital primordia in the larval stages and sexual differentiation may first occur as early as the second stage larva (Bird, 1971). Western blotting showed chemiluminescence was detected in both the crude ovary extracts and the fraction that passed through the affinity column as three bands at about 70, 78 and 90 kDa. Moreover three faint bands were also observed in the eluted fraction at about 35, 39 and 45 kDa. Dubinsky et al. (1982) reported that SDS-PAGE of the supernatant of homogenate from ovary displayed proteins of 72 and 75 kDa. However, in the present study Western blotting after native-PAGE of partially purified ovary extracts treated by affinity chromatography demonstrated one band at a position of about 230 kDa. Moreover SDS-PAGE showed that the partially purified ovary extracts exhibited a number of stained bands at positions of faster mobility than that of about 70 kDa. This discrepancy between the SDSPAGE and native-PAGE results is likely due to the possible subunit structure of the antigen in the ovary extracts. Kennedy and Qureshi (1986) described that the excretory–secretory products from larvae were affected by reducing conditions. Furthermore, the antigen recognized by the Mab appeared to be equivalent to the hypothetical protein F14D2.8 of C. elegans (gi/7499081). This protein is composed of 1466 amino acids and is calculated to be 170,252 Da with a calculated pI value of 6.01. This protein is also thought to be a membrane protein and to
have two transmembrane helices (the 444th through 466th amino acids from the N-terminal and 470th through 492nd) (SOSUI, http://sosui.proteome.bio.tuat.ac.jp/cgi-bin/sosui.cgi?/). The other F14D2.8 of C. elegans (gi/71986492), which consists of 277 amino acids has been deposited in the NCBI protein database. NCBI BLASTP analysis indicated that the latter F14D2.8 matched with the 8th amino acid through the 340th one of the former F14D2.8. The sequence ‘‘ILVGLIGTNR’’ obtained here was identical with the 501st through the 510th amino acids of the former F14D2.8. This sequence was derived from the three bands on SDS-PAGE of about 70, 78 and 90 kDa recognized by the Mab. However no protein of A. suum did not match the sequences obtained by both LCnanoESL-MS/MS method and de novo sequencing. Analysis of cDNA from the ovary will clarify the precise sequence of this antigen. Acknowledgements The authors thank Professor Dr. Hiroyuki Iwata, Department of Veterinary Medicine, Faculty of Agriculture, Yamaguchi University for his advice on Western blotting and Professor Dr. Soichi Imai, Faculty of Veterinary Medicine, Nippon Veterinary and Life Science University for his advice on the structure of A. suum. References Ali, H.A., Sawada, T., Hatakeyama, H., Ohtsuki, N., Itoh, O., 2004. Characterization of a 39 kDa capsular protein of avian Pasteurella multocida using monoclonal antibodies. Vet. Microbiol. 100, 43– 53. Barrett, J., Brophy, P.M., Hamilton, J.V., 2005. Analyzing proteomic data. Int. J. Parasitol. 34, 543–553. Bird, A.F. (Ed.), 1971. The Structure of Nematodes. Academic Press, New York and London, 318 pp. Dubinsky, P., Rybos, M., Turcekova, L., 1982. Electrophoretic characteristic of the proteins in growing oocytes of Ascaris suum. Helmintologia 19, 289–297. Eriksen, L., Andersen, S., Nielsen, K., Pedersen, A., Nielsen, J., 1980. Experimental Ascaris suum infection in pigs. Serological response, eosinophilia, in peripheral blood, occurrence of white spots in the liver and worm recovery from the intestine. Nord. Vet. Med. 32, 233–242. Eriksen, L., Lind, P., Nansen, P., Roepstorff, A., Urban, J., 1992. Resistance to Ascaris suum in parasite naı¨ve and naturally exposed growers, finishers and sows. Vet. Parasitol. 41, 137–149. Frontera, E., Roepstorff, A., Serrano, F.J., Gazques, A., Reina, D., Navarrete, I., 2004. Presence of immunoglobulins and antigens in serum, lung and small intestine in Ascaris suum infected and immunised pigs. Vet. Parasitol. 119, 59–71. Gallaghar, S.R., Smith, J.A., 1991. Nondenaturing discontinuous gel electrophoresis. In: Coligan, J.E., Kruisbeek, A.M., Margulies,
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