Journal of Immunological Methods, 131 (1990) 159-163 Elsevier
159
JIM05631
Sandwich ELISA assay for quantitative measurement of SP-40,40 in seminal plasma and serum N a m - H o Choi 1, Takashi T o b e 1, K a t s u t o s h i H a r a 1, H i d e k i Y o s h i d a 2 a n d M o t o w o T o m i t a 1 I Department of Physiological Chemistry, School of Pharmaceutical Sciences, and • Department of Urology, School of Medicine, Showa University, 1-5-8, Hatanodai, Shinagawa-ku, Tokyo 142, Japan (Received 21 February 1990, revised received 2 April 1990, accepted 3 April 1990)
SP-40,40 was purified from human plasma by PEG fractionation, DEAE-Sephacel, Phenyl-Toyopearl 650M, Bio-Gel A-0.5m and hydroxylapatite chromatographies. Three monoclonal antibodies (IF12, IID9 and IVF4) to this protein were prepared: IF12 and IID9 were specific for the fl subunit and IVF4 for the a subunit. The concentrations of SP-40,40 in seminal plasmas and sera were determined using a sandwich ELISA method. The results showed that the average concentrations of SP-40,40 were 438 _ 285/~g/ml in seminal plasmas and 111 + 50 g g / m l in sera of normal donors. SP-40,40 concentrations in seminal plasmas of Klinefelter and excretory azoospermia patients were similar to those of normal donors. However, those of oligozoospermia and idiopathic azoospermia patients were about half the normal value. Key words: SP-40,40; Monoclonal antibody; ELISA; Seminal plasma
Introduction
SP-40,40 was first found in a soluble membrane attack complex (SMAC, SC5b-9) of complement as a protein of MW 80,000 consisting of two subunits (a chain and fl chain) each with a MW of 40,000 (Murphy et al., 1988). The amino acid sequence of SP-40,40 deduced from the nucleotide sequence of its cDNA bore a high homology to rat sulfated glycoprotein-2 (SGP-2), which is a major secreted product of Sertoli cells in testes (Jenne et
Correspondence to: N.-H. Choi, Department of Physiological Chemistry, School of Pharmaceutical Sciences, Showa University, 1-5-8, Hatanodai, Shinagawa-ku, Tokyo 142, Japan. Abbreviations: ELISA, enzyme-linked immunosorbent assay; PEG, polyethylene glycol; DEAE, diethylaminoethyl; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; BSA, bovine serum albumin; PBS, phosphatebuffered saline.
al., 1989; Kirszbaum et al., 1989), to quail T64 protein, which is induced in the cells transformed by Rous sarcoma virus (Michel et al., 1989), and to ram clusterin, which seems to be a cell adhesion molecule (Cheng et al., 1988). It has been suggested that SGP-2 plays a critical role in spermatogenesis (Groswold et al., 1986; Collard et al., 1987) and SP-40,40 is also present in human seminal plasma. Recently, we and others reported that SP-40,40 had a modulatory or an inhibitory action on the formation of membrane attack complex (MAC, C5b-9) of complement (Choi et al., 1989; Jenne et al., 1989; Murphy et al., 1989). In this paper, we describe the quantitation of SP-40,40 in seminal plasma and serum by a sandwich ELISA method using two monoclonal antibodies. Isolation of SP-40,40 on a preparative scale is also described, because, although two methods have been reported for the isolation of SP-40,40, one method requires the monoclonal antibody to SP-40,40 (Murphy et al., 1988) and
0022-1759/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
160 the other method is not suitable for large scale preparation of SP-40,40 (Choi et al., 1989).
Materials and methods
Materials PEG 4000 was purchased from Wako Pure Chemicals. DEAE-Sephacel was obtained from Pharmacia Fine Chemicals and Phenyl-Toyopearl 650M was from Tosoh. Bio-Gel A-0.5m was purchased from Bio-Rad and hydroxylapatite was from Pentax. SDS-PA GE and Western blot analysis SDS-PAGE was performed by the method of Laemmli (1970). After electrophoresis, proteins were transferred to a nitrocellulose filter and blocked with gelatin. The filter was treated with anti-SP-40,40 antibody and developed using a Vectastain kit (Vector) as described previously (Choi et al., 1989). A26o
a)
Preparation of monoclonal antibodies Monoclonal antibodies against SP-40,40 were prepared according to the method of Galfr~ et al. (1981). Antibody producing hybridoma cells were injected intraperitoneally into BALB/c mice which had already been injected with pristan. Monoclonal antibodies were purified from the ascites fluids by ammonium sulfate precipitation and DEAE-Sephacel chromatography. IgG subclass and light chain of monoclonal antibodies were determined with a monoclonal antibody isotyping kit (Pharmingen). Sandwich ELISA for quantitation of SP-40,40 Sandwich ELISA was performed according to the method of Lems-Van Kan et al. (1983) with minor modifications. The wells of plastic plates (96 fiat-bottomed wells, Nunc) were coated with anti-SP-40,40 monoclonal antibody IVF4 (100 /tl of 20 /Lg/ml in PBS) as capturing antibody at room temperature for 2 h. The wells were filled with 1% BSA in PBS and kept at 4 ° C overnight to
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159
JIM05631
Sandwich ELISA assay for quantitative measurement of SP-40,40 in seminal plasma and serum N a m - H o Choi 1, Takashi T o b e 1, K a t s u t o s h i H a r a 1, H i d e k i Y o s h i d a 2 a n d M o t o w o T o m i t a 1 I Department of Physiological Chemistry, School of Pharmaceutical Sciences, and • Department of Urology, School of Medicine, Showa University, 1-5-8, Hatanodai, Shinagawa-ku, Tokyo 142, Japan (Received 21 February 1990, revised received 2 April 1990, accepted 3 April 1990)
SP-40,40 was purified from human plasma by PEG fractionation, DEAE-Sephacel, Phenyl-Toyopearl 650M, Bio-Gel A-0.5m and hydroxylapatite chromatographies. Three monoclonal antibodies (IF12, IID9 and IVF4) to this protein were prepared: IF12 and IID9 were specific for the fl subunit and IVF4 for the a subunit. The concentrations of SP-40,40 in seminal plasmas and sera were determined using a sandwich ELISA method. The results showed that the average concentrations of SP-40,40 were 438 _ 285/~g/ml in seminal plasmas and 111 + 50 g g / m l in sera of normal donors. SP-40,40 concentrations in seminal plasmas of Klinefelter and excretory azoospermia patients were similar to those of normal donors. However, those of oligozoospermia and idiopathic azoospermia patients were about half the normal value. Key words: SP-40,40; Monoclonal antibody; ELISA; Seminal plasma
Introduction
SP-40,40 was first found in a soluble membrane attack complex (SMAC, SC5b-9) of complement as a protein of MW 80,000 consisting of two subunits (a chain and fl chain) each with a MW of 40,000 (Murphy et al., 1988). The amino acid sequence of SP-40,40 deduced from the nucleotide sequence of its cDNA bore a high homology to rat sulfated glycoprotein-2 (SGP-2), which is a major secreted product of Sertoli cells in testes (Jenne et
Correspondence to: N.-H. Choi, Department of Physiological Chemistry, School of Pharmaceutical Sciences, Showa University, 1-5-8, Hatanodai, Shinagawa-ku, Tokyo 142, Japan. Abbreviations: ELISA, enzyme-linked immunosorbent assay; PEG, polyethylene glycol; DEAE, diethylaminoethyl; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; BSA, bovine serum albumin; PBS, phosphatebuffered saline.
al., 1989; Kirszbaum et al., 1989), to quail T64 protein, which is induced in the cells transformed by Rous sarcoma virus (Michel et al., 1989), and to ram clusterin, which seems to be a cell adhesion molecule (Cheng et al., 1988). It has been suggested that SGP-2 plays a critical role in spermatogenesis (Groswold et al., 1986; Collard et al., 1987) and SP-40,40 is also present in human seminal plasma. Recently, we and others reported that SP-40,40 had a modulatory or an inhibitory action on the formation of membrane attack complex (MAC, C5b-9) of complement (Choi et al., 1989; Jenne et al., 1989; Murphy et al., 1989). In this paper, we describe the quantitation of SP-40,40 in seminal plasma and serum by a sandwich ELISA method using two monoclonal antibodies. Isolation of SP-40,40 on a preparative scale is also described, because, although two methods have been reported for the isolation of SP-40,40, one method requires the monoclonal antibody to SP-40,40 (Murphy et al., 1988) and
0022-1759/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
160 the other method is not suitable for large scale preparation of SP-40,40 (Choi et al., 1989).
Materials and methods
Materials PEG 4000 was purchased from Wako Pure Chemicals. DEAE-Sephacel was obtained from Pharmacia Fine Chemicals and Phenyl-Toyopearl 650M was from Tosoh. Bio-Gel A-0.5m was purchased from Bio-Rad and hydroxylapatite was from Pentax. SDS-PA GE and Western blot analysis SDS-PAGE was performed by the method of Laemmli (1970). After electrophoresis, proteins were transferred to a nitrocellulose filter and blocked with gelatin. The filter was treated with anti-SP-40,40 antibody and developed using a Vectastain kit (Vector) as described previously (Choi et al., 1989). A26o
a)
Preparation of monoclonal antibodies Monoclonal antibodies against SP-40,40 were prepared according to the method of Galfr~ et al. (1981). Antibody producing hybridoma cells were injected intraperitoneally into BALB/c mice which had already been injected with pristan. Monoclonal antibodies were purified from the ascites fluids by ammonium sulfate precipitation and DEAE-Sephacel chromatography. IgG subclass and light chain of monoclonal antibodies were determined with a monoclonal antibody isotyping kit (Pharmingen). Sandwich ELISA for quantitation of SP-40,40 Sandwich ELISA was performed according to the method of Lems-Van Kan et al. (1983) with minor modifications. The wells of plastic plates (96 fiat-bottomed wells, Nunc) were coated with anti-SP-40,40 monoclonal antibody IVF4 (100 /tl of 20 /Lg/ml in PBS) as capturing antibody at room temperature for 2 h. The wells were filled with 1% BSA in PBS and kept at 4 ° C overnight to
b)
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F r a c t i o n Number
A280
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