Regulatory Peptides, 39 (1992) 201-214 © 1992 Elsevier Science Publishers B.V. All rights reserved 0167-0115/92/$05.00

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Immunodetection of secretogranin II in animal and human tissues by new monoclonal antibodies Micaela Pelagi a, Antonia Zanini b, Anna Gasparri c, Laura Ermellino e, Anna M. Giudici b, Stefano Ferrero d, Antonio G. Siccardi c and Roberto Buffa e aIstituto Scientifico Ospedale S. Raffaele, Milano (Italy), b Centro CNR Infrastrutture Cellulari, Dipartimento di Farmacologia, Universitgt di Milano, Milano (Italy), CDipartimento di Biologia e Genetica per le Scienze Mediehe, Milano (Italy), d III Cattedra di Anatomia Patologica, Universitd di Milano, Milano (Italy) and e Dipartimento di Patologia Umana ed Ereditaria dell'Universith di Pavia. Sezione di Anatomia Patologica II, Sede Ospedale Multizonale, Varese (Italk9

(Received 12 September 1991; revised version received 20 January 1992; accepted 4 March 1992)

Key words: Immunoblotting; Immunocytochemistry; Neuroendocrine tissue; Secretogranin II; Secretory product

Summary Secretogranin II (chromogranin C) is an acidic tyrosine-sulfated secretory protein, known to be a marker of neuroendocrine secretory products and of specific neuroendocrine tumours. In order to obtain anti-secretogranin II monoclonal antibodies for cell biology studies and, in particular, for clinical applications, we immunized mice with a secretogranin II-enriched fraction prepared from homogenates of bovine anterior pituitaries. Hybridoma supernatants obtained from the splenocytes of a hyperimmune mouse, screened with an enzyme-linked immunosorbent assay, were analyzed by both immunocytochemistry and two-dimensional immunoblotting. By using this experimental approach, we were able to identify two monoclonal antibodies (8G1 and 5A7) which recognize bovine secretogranin II. Both immunocytochemistry and immunoblotting revealed that one of them, the 5A7 antibody, cross-reacts with the human antigen. The distribution patterns of the immunoreactivity, obtained by immunocytochemistry with the 5A7 antibody in animal and human tissues, partially overlap those, obtained by using a polyclonal antiserum elicited against bovine secretogranin II, previously described. Moreover, the 5A7, but not the polyclonal antibody, reacts with some

Correspondence: M. Pelagi, Dipartimento di Biologia e Genetica per le Scienze Mediche, Via Viotti 3/5, 20133 Milano, Italy.

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duodeno-jejunal cells. In conclusion, both the 5A7 and 8G1 antibodies can be useful for cell biology studies. The 5A7 antibody can be used for the detection of secretogranin II in human tissues and should be of help in clinical and pathological practices.

Introduction

Secretogranin II (SglI) (previously also called chromogranin C, see Ref. 1 for nomenclature) is an acidic tyrosine-sulfated secretory protein present, like chromogranin A (CgA) and chromogranin B (CgB), in secretory granules of a variety of neuroendocrine tissues (for reviews see Refs. 2-5). The function of SgII is still unclear, although recent data on its primary sequence suggest that this protein, like CgA and CgB with which it shares biochemical and biophysical properties, might be the precursor of peptides with possible a hormonal or paracrine role [4,5]. Recently, SgII has been shown to be a useful marker for neoplasms derived from neuroendocrine cells. Its presence in pheochromocytomas [6], C-cell carcinomas [7], bronchial and intestinal carcinoids [8,9], insulinomas, Merkel cell carcinomas and neuroblastomas [ 10] has been detected by immunocytochemistry. However, SgII is not as widely reported in surgical pathology as CgA and CgB [5], possibly because only polyclonal antibodies against bovine or rat SgII have been obtained by different groups [ 11-14] and no monoclonal antibodies have been described. The present paper reports on the preparation of monoclonal antibodies against bovine SgII and their characterization by two-dimensional immunoblotting and immunohistochemistry.

Materials and Methods

Preparation of SglI-enriched fractions An SglI-enriched fraction was prepared from homogenates of bovine anterior pituitaries which were pulse-labeled with [35S]sulfate by means of ion-exchange chromatography on DEAE-Sephadex, as previously reported [ 11 ]. This fraction was used as an immunogen in mice. Anterior pituitary homogenates, used to set up an enzyme-linked immunosorbent assay (ELISA) and as antigen for immunoblotting, were prepared in 0.3 M sucrose containing 20 mM Tris-HC1 (pH 7.4) and 0.5 mM EGTA. An SgII-containing fraction was prepared from human pheochromocytoma by the heat-treatment procedure [6,12]. Pheochromocytoma tissue was frozen in liquid nitrogen immediately after surgical excision, homogenized in distilled water, boiled for 6 min and centrifuged at 120,000 g for 1 h. The supernatant was used as antigen for ELISA and immunoblotting.

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Two-dimensional polyacrylamide gel electrophoresis and immunoblotting Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) was performed according to O'Farrell et al. [ 15], with the modifications previously reported [ 16,17], using Mini Protean II Cells (Bio-Rad Laboratories, Richmond, CA). Immunoblotting was performed as previously described [17]. Proteins separated by 2D-PAGE and electrophoretically transferred to nitrocellulose membranes were visualized with Ponceau S. (Serva Feinbiochemica, Heidelberg) and incubated overnight at 4 °C in 8~o bovine serum albumin (BSA) in phosphate-buffered saline (PBS). Membranes were then incubated for 2 h with the first antibodies (anti-bovine SglIenriched fraction mouse antiserum diluted: 1:200; anti-purified bovine SglI rabbit antibodies, purified by affinity chromatography as reported [18], 2 mg/ml; undiluted hybridoma supernatants; ascite fluids, diluted 1:500). After several washings in PBS containing 0.3 ~o Triton X-100 the membranes were incubated for 1 h with horseradish peroxidase conjugated goat anti-mouse IgG and IgM antibodies (Jackson Immuno Research Laboratories, Inc., Avondale, PA), diluted 1:2000 or goat anti-rabbit IgG antibodies (Pasteur Institute, Paris, France), diluted 1:1000. Visualization of the reaction was performed using 3,3'-diaminobenzidine (DAB) or, in the case of anti-SgII rabbit antibodies, 4-chloro-l-naphthol.

Immunizations Balb/c mice, 10 weeks old, were immunized i.p. with 50 mg of the SgII-enriched fraction, prepared from bovine anterior pituitaries, in complete Freund's adjuvant. The second dose, in incomplete Freund's adjuvant, was given on day 15. Doses of 50 mg in PBS were subsequently given every 15 days thereafter.

Fusions and screening of hybridoma supernatants 3 days before killing, mice were boosted i.p. with 50 mg of the immunogen. Spleen cells were fused by standard methods, using 50~o polyethylene glycol (M r 155; Sigma) and the P3-X63 Ag8-NS1 cell line as fusion partner. Hybridomas producing anti-SglI antibodies were revealed by ELISA. 96-well microtitre plates (PBI) were coated with 0.3 mg/well of anterior pituitary homogenates or the SglI-containing fraction prepared from human pheochromocytoma in 50 mM carbonate buffer (pH 9.5) for 90 min at 37 °C. Non-specific binding of antibodies to the plates was then blocked by incubation with 3 ~o bovine serum albumin (B SA) in PB S overnight. The antibody binding assay was performed in three steps: (a) 100 ml of culture fluid was incubated in each well overnight at 4 °C and unbound immunoglobulin was then removed from the well by washing three times with PB S; (b) plates were incubated for 1 h at room temperature with peroxidase conjugated rabbit anti-mouse immunoglobulins diluted 1:1000; (c)after a final wash, a chromogenic substrate (o-phenylenediamine) was added and the reaction was blocked by addition of 2 M n 2 s o 4. Hybridomas producing antibodies with specificity for SglI were cloned by limiting dilution and expanded. Ascitic fluids were obtained from Balb/c mice primed with 2,6,10,11-tetramethyl-

204 pentadecane (Pristane, Aldrich Chem. Co., Steinheim, Germany) and inoculated with hybridoma cells. Anti-serotonin antibodies were purchased from Dakopatts (Glostrup, Denmark).

Immunohistochemistry Fresh samples from the pituitary, thyroid, parathyroid, adrenals, pancreas and gastrointestinal tract were taken from oxen, guinea-pigs, rats, rabbits, cats and dogs. Fragments from human adrenals, pancreas, thyroid, parathyroid and gastrointestinal mucosa were obtained from surgical specimens or during endoscopic examination; human pituitary, thymus and lung were collected at necroscopy. The following fixatives were used: PAF (neutral picric acid-formaldehyde [ 19]), AAF (70 ~o ethanol- 5 ~o acetic acid-4~o formaldehyde, modified from Bodian [20]), Bouin's solution and buffered formaldehyde (4~o in 0.1 M (pH 7.3) phosphate). After dewaxing, paraffin sections were immunostained with an avidin-biotin technique (ABC reagent from Vector, Burlingame, CA, USA). The primary reagents were applied overnight in 0.15 M Tris buffer (pH 7.3)-0.145 M saline (TBS). Peroxidase activity was detected with either DAB or 4 CI-1 naphthol (0.5 mM in TBS supplemented with 0.005 mM H202). Pertinent specificity tests (controls included the use of antibodies added with related and unrelated antigens and substitution of the primary reagent with non immune sera) were done according to Van Noorden [21].

Results

Anti-bovine SgII-enriched fraction antisera The antisera obtained by immunizing two mice with the SgII-enriched fraction prepared from bovine anterior pituitaries [ 11 ] were analyzed by direct-binding ELISA, using anterior pituitary homogenates as antigen. As shown in Fig. 1, a strong reaction, significantly different from the one obtained with the preimmune sera, was obtained with both antisera. Two-dimensional immunoblotting of anterior pituitary homogenates revealed that the two antisera (Fig. 2a) recognized a component corresponding to SgII, as demonstrated by comparison with the pattern obtained by using previously characterized polyclonal antibodies anti-purified bovine SgII raised in rabbit [11,18] (Fig. 2b). Other components (SgII breakdown products and, probably, CgB and its breakdown products) were also recognized. When a heat stable fraction obtained from human pheochromocytoma (Fig. 2c,d) was used as antigen, a component corresponding to SgII was recognized by both the polyclonal antiserum raised in mice and the polyclonal antibodies raised in rabbit. SgII breakdown components in the human pheochromocytoma were also recognized by both antibodies. These products were more abundant in the case of the mouse serum which also recognized putative CgB. At immunohistochemistry (not shown), the polyclonal antisera reacted with several endocrine/paracrine cells scattered in different tissues obtained from seven mammalian species. In PAF-fixed human tissues immunoreactivity was also found in non-

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Immunodetection of secretogranin II in animal and human tissues by new monoclonal antibodies.

Secretogranin II (chromogranin C) is an acidic tyrosine-sulfated secretory protein, known to be a marker of neuroendocrine secretory products and of s...
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