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Development of an ELISA for Chum Salmon (Oncorhynchus keta) Growth Hormone
Comp. Biochem. Physiol. Vol. 117B, No. 3, pp. 387–392, 1997 Copyright 1997 Elsevier Science Inc.
ISSN 0305-0491/97/$17.00 PII S0305-0491(97)00134-X
Development of an ELISA for Chum Salmon (Oncorhynchus keta) Growth Hormone Haruhisa Fukada,1,3 Naoshi Hiramatsu,1,3 Koichiro Gen,2 and Akihiko Hara3 1
Department of Biology and Aquaculture, Faculty of Fisheries, Hokkaido University, Hakodate, Hokkaido, 041, Japan; 2 Institute of Endocrinology, Gunma University, Maebashi, Gunma, 371, Japan; and 3 Nanae Fish Culture Experimental Station, Faculty of Fisheries, Hokkaido University, Nanae, Kameda-gun, Hokkaido, 041-11, Japan ABSTRACT. A specific and sensitive enzyme-linked immunosorbent assay (ELISA) was developed for the measurement of low levels of serum growth hormone (GH) in chum salmon (Oncorhynchus keta). The antiserum to GH (a-rsGH) was obtained from a rabbit immunized with recombinant chum salmon GH. The noncompetitive ELISA was performed by a sandwich method using a-rsGH rabbit IgG as the first antibody, its biotinylated Fab′ fragment as the second antibody, and the avidin-biotin reaction for signal amplification. This assay could be run in 3 days and routinely detected GH at concentrations as low as 0.5 ng/ml. The development of an ELISA for GH made possible quantification of serum GH levels. In this assay system, parallel dilution curves were obtained using purified chum salmon GH and GH’s from several species of the genus Oncorhynchus. comp biochem physiol 117B;3:387–392, 1997. 1997 Elsevier Science Inc. KEY WORDS. ELISA, avidin-biotin, growth hormone, chum salmon, teleost, fish
INTRODUCTION Growth hormone (GH) stimulates somatic growth indirectly (4) and enhances adaptation for salt water, especially in salmonid species (20,26). There are reports of purification of GH from several fish species including tilapia, Oreochromis mossambica (7), chum salmon, Oncorhynchus keta (10,13,24), chinook salmon, Oncorhynchus tshawytscha (16), sturgeon, Acipenser guldenstadti (6), carp, Cyprinus carpio (3), and Japanese eel, Anguilla japonica (12). To explore GH physiology, it is necessary to develop a specific assay system for the hormone. Development of an assay for GH has been attempted in various fishes using radioimmunoassay (RIA) (2,3,11,17,25) and an enzyme immunoassay (22). RIA was used successfully to measure plasma GH in various fishes. There is one report of an enzyme-linked immunosorbent assay (ELISA) system for fish GH that can measure plasma GH levels using a monoclonal antibody (5). In general, ELISA has several advantages over RIA including safety, simplicity, and speed. In the present study, a sensitive, noncompetitive sandwich ELISA was developed for chum salmon GH, one using a polyclonal antibody to recombinant chum salmon GH as the first antibody, its biotinylated Fab′ fragment as the second antibody, and the avidin-biotin reaction for signal amplification.
Address reprint requests to: A. Hara, Nanae Fish Culture Experimental Station, Faculty of Fisheries, Hokkaido University, Nanae, Kameda-gun, Hokkaido, 041-11, Japan. Tel. 81-138-65-2344; Fax 81-138-65-2239. Received 13 December 1996; accepted 18 December 1996.
MATERIALS AND METHODS Fish and Samples Mature chum salmon were caught from the Yurappu River in southern Hokkaido. Pink salmon (O. gorbuscha), steelhead trout (O. mykiss), masu salmon (O. masou), brown trout (Salmo trutta), white-spotted charr (Salvelinus leucomaenis), and Sakhalin taimen (Hucho perryi) were obtained in 1993 and 1994 from broodstocks reared in freshwater ponds at the Nanae Fish Culture Experimental Station, Faculty of Fisheries, Hokkaido University. Blood samples was taken by syringe from the caudal vessels of each fish. The samples were allowed to stand at 4°C for several hours and serum was separated by centrifugation at 16,000 g for 10 min and stored at 230°C until use. Pituitaries were collected and stored at 230°C until use. For measurement of GH using the ELISA, pituitary extracts and diluted serum were prepared. Pituitary glands were homogenized in 0.01 M phosphate buffer in isotonic saline, pH 7.0 (PBS) and homogenates were centrifuged at 16,000 g for 20 min at 4°C as discussed below. The supernatant was used as the pituitary extract. Serum was diluted with phosphate buffer including 0.2 M NaCl, 1% bovine serum albumin (BSA) and 0.05% normal rabbit serum (NRS) (PBS-BSA) and used in the ELISA at a total volume of 100 µ l of solution per well. Purification of Chum Salmon GH For GH purification, chum salmon pituitary glands were homogenized in 0.01M PBS with a ground glass homogenizer
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and centrifuged at 12,000 g for 20 min at 4°C. The supernatant was precipitated by addition of ammonium sulfate at 35% saturation. The precipitate was collected after centrifugation at 28,000 g for 30 min and dissolved in and dialyzed against 0.02 M Tris-HCl buffer, pH 8.0. The dialysate was applied to a Mono-Q column (FPLC system, Pharmacia, Uppsala, Sweden) and eluted stepwise with Tris-HCl buffer containing NaCl at 0.1 or 1 M. Thereafter, the fraction containing GH was subjected to gel filtration on a Superose 12 column equilibrated with 0.02 M Tris-HCl buffer including 0.2% NaCl (pH 8.0). The presence of chum salmon GH (sGH) in the fractions was assessed by Western blotting using a rabbit antiserum against recombinant chum salmon GH (rsGH). Antiserum An antiserum against rsGH (a-rsGH) was raised in a rabbit by intradermal injection with rsGH (1 mg) emulsified in an equal volume of complete Freund’s adjuvant. Injections were done six times at 1-week intervals. The specificity of this a-rsGH was demonstrated previously by Bjo¨rnsson et al. (1) as follows, cross-reactivities with chum salmon gonadotropins I and II are 0.03% and 0.02%, respectively. Electrophoresis Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out using a Tris-glycine buffer system (15). Gels were stained for protein with 0.1% Coomassie Brilliant Blue R 250 in a solution of 40% ethanol/10% acetic acid/50% distilled water (CBB). The molecular weights of proteins banded in the gel were estimated using the following molecular mass markers (Pharmacia, Uppsala, Sweden): α-lactalbumin (14.4 kDa), trypsin inhibitor (20.1 kDa), carbonic anhydrase (30 kDa), ovalbumin (43 kDa), albumin (67 kDa), and phosphorylase b (94 kDa). Western blotting was carried out according to our previous report (14) with the primary antiserum used at a 1:1000 dilution. Biotin Labeling of Fab′ Fab′ was purified from a-rsGH according to our previous report (19). Biotinylated Fab′ was prepared using a biotin labeling kit (Boehringer Mannheime Biochem.). A 22.5 µl mixture, containing 10 µ l biotin-7-NHS (D-biotinylatedo-aminocaproic acid-N-hydroxysuccinimide ester) solution (20 mg/ml) and 90 µ l DMSO (dimethylsulfoxide), was added to 1 mg of Fab′ in solution (1 mg/ml PBS) with gentle stirring for 2 hr at room temperature. Remaining nonreacted biotin-7-NHS was separated by dialysis of the solution against PBS overnight at 4°C.
ANTIBODY COATING. The immunoglobulin G (IgG) solution was purified from a-rsGH rabbit serum by chromatography on DE-52 (19). Wells were coated and incubated for 4 hr at 24°C with 150 µl IgG solution at a concentration of 40 µ g/ml in PBS. BLOCKING. After three consecutive washes with 200 µ l of PBS-1% Tween buffer (PBS-T), and followed by wash with the same volume of PBS per well, the plates were incubated with 200 µl of PBS-1%BSA per well for 4 hr at 24°C or overnight at 4°C. INCUBATION OF SAMPLES AND STANDARDS (STEP 1).
After three washes with PBS-T and one with PBS, 100 µ l of sample or standard, diluted serially with PBS-BSA, was added into each well of the microtiter plates and incubated for 8 hr at 24°C. The standard solutions (rsGH 0.098 ng/ ml to 200 ng/ml) were prepared by dissolving a known amount of rsGH in PBS-BSA. INCUBATION WITH BIOTINYLATED a-rsGH (STEP 2). After washing as described above for step 1, each well received 100 µ l of labeled antibody diluted 1 : 400 in 0.02 M phosphate buffer, pH 7.0 containing 0.1% BSA, 0.1% Tween and 0.002% thimerosal (PB-BSA-T), followed by incubation for 16 hr at 4°C. INCUBATION WITH STREPTAVIDIN-HORSERADISH PEROX-
After the plates were again washed as described above, each well received 100 µ l of streptavidin-HRPO conjugate (streptavidin coupled to peroxidase, Boehringer Mannheim Biochem) diluted 1:2000 with PB-BSA-T, followed by incubation for 1.5 hr at 24°C. IDASE (HRPO) CONJUGATE (STEP 3).
ENZYMATIC COLOR REACTION. The plates were washed as described above and color was developed at room temperature for 15 min in the dark by adding 150 µl of o-phenylenediamine (3 mg/ml 0.1 M citric acid-phosphate buffer pH 5.0 containing 0.02% H2O2 ) to each well. The reaction was stopped by adding 150 µl of 4N HCl per well. The absorbance at 492 nm was measured using an ELISA plate reader (Bio-Rad 2550, Richmond, CA).
Expression of the Results The parallelism between the regression curves was tested by analysis of covariance according to Man˜ano´s et al. (18). RESULTS Purification of sGH The purified sGH appeared as a single band of 23 kDa after SDS-PAGE. This band stained specifically with polyvalent a-rsGH in Western blotting (Fig. 1).
ELISA Assay Procedure The ELISA was carried out in 96-well polystyrene ELISA microtiter plates (ICN Biomedicals, Horsham, PA) according to the method of Guesdon et al. (8).
Effect of Concentration of NaCl and NRS NaCl at four different concentrations (0.1, 0.2, 0.3, and 0.4 M) was mixed in 0.01 M Phosphate Buffer-1%BSA for use
Salmon Growth Hormone ELISA
FIG. 1. 12.5% SDS-PAGE (A) and Western blotting pattern using a-rsGH (B) of pituitary extracts (lane 1) and purified GH (lane 2). Samples were reduced with 2-mercaptoethanol.
as the dilution buffer in step 1. Color development serially decreased with increasing NaCl concentration. At 0.1 M NaCl, diluted serum did not run parallel with the standard curve in the ELISA. At 0.2 M NaCl, good color development was obtained and serially diluted serum run parallel to the standard curve (Fig. 2A). Four different concentrations of normal rabbit serum (NRS) (1%, 0.5%, 0.1% and 0.05%) were mixed with PBS-1%BSA containing 0.2M NaCl. The best standard curve and serum dilution series were obtained when PBS-1%BSA containing 0.05% NRS was used (see Fig. 2B). Consequently the standard ELISA procedure used dilution buffer containing 0.2M NaCl and 0.05% NRS. Standard Curve and Precision of the ELISA Under routine conditions (coating: 4 hr at 24°C, step 1: 8 hr at 24°C, step 2: 16 hr at 4°C, step 3: 1.5 hr at 24°C),
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FIG. 2. Typical standard curve in the ELISA using 0.2 M NaCl (A) and 0.05% NRS (B) in the dilution buffer at step 1. Parallelism between regression curves was assessed by the F-test ( P , 0.05). d, standard curve; s, masu salmon serum.
standard curves of rsGH (range 0.098–50 ng/ml) showed a typical sigmoidal curve (Fig. 3). Precision tests were performed using five samples with various rsGH concentrations (200, 100, 50, 25, and 12.5 ng/ml). The between assay coefficient of variation was 6.90% (n 5 15) and the within assay coefficient of variation was 5.54% (n 5 15). Cross-Reactivity with Pituitary and Serum GH from Other Salmonids Serum and pituitary extracts from other salmonid species were recognized in the ELISA. Serial dilutions of pituitary extracts showed a sigmoidal curve, but the degree of immunoreactivity was different between genera (Fig. 4B). As to
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Measurement of Serum GH Levels in Mature Chum Salmon Serum GH levels were measured in mature chum salmon captured on their spawning migration in the Yurappu river. Serum GH levels of male chum salmon were 6.39 6 0.69 ng/ml (n 5 5). Serum GH levels were higher in female chum salmon (12.42 6 1.89 ng/ml, n 5 5) than in males. DISCUSSION
FIG. 3. Routine standard curve of rsGH in the ELISA (range
0.098–50 ng/ml).
serum, serially diluted samples from masu salmon (genus Oncorhynchus) ran parallel to the standard curve. Serum from species of other genera (Salmo, Hucho, and Salvelinus) was recognized by this ELISA but did not run parallel with the standards when serially diluted (Fig. 4C). Serially diluted serum from four species of the genus Oncorhynchus ran parallel to the standard curve. The purified sGH also ran parallel to rsGH in the ELISA standard curve, but it showed low immunoreactivity as compared to rsGH (Fig. 5).
The a-rsGH used in this ELISA is the same antibody used in a radioimmunoassay for salmon GH in a previous study (1). The reported cross-reactivities with chum salmon gonadotropins I and II were 0.03% and 0.02%. After immunostaining pink salmon pituitary sections with a-rsGH, prolactin-producing cells in the rostral pars distalis, identified in salmonids on the basis of their morphology in relation to specific immunostaining characteristics (10), were not stained at all (data not shown). Furthermore, results of Western blotting demonstrated the high degree of specificity of the a-rsGH. Thus, the a-rsGH is specific enough to use for ELISA of GH. The purification of sGH was performed using an FPLC system. The purified sGH appeared as a single band at a position corresponding to 23 kDa in SDS-PAGE and the band reacted positively and specifically in Western blots using a-rsGH. This estimated molecular weight for GH is similar to that reported for other teleost GHs in previous studies [see review (5)]. A specific and sensitive ELISA for chum salmon GH was developed utilizing a noncompetitive sandwich method with the avidin-biotin system in polystyrene microtiter plates as the solid phase (8,23). Owing to use of the non-
FIG. 4. Serial dilution of rsGH standards in the ELISA (A) corresponding and serial dilutions of pituitary extracts (B) and serum (C) from masu salmon (d), brown trout (m), white-spotted charr (■) and Sakhalin taimen (✚).
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FIG. 5. Parallelism with the
rsGH standard curve in the ELISA of serum from fish of the genus Oncorhynchus (masu, pink, and chum salmon, and steelhead trout) and relative immunoreactivity of rsGH and purified chum salmon GH (sGH) in the ELISA. Parallelism between regression curves was assessed by the F-test ( P , 0.05).
competitive sandwich ELISA method, the confounding reaction of endogenous peroxidase can be avoided. To determine optimal ELISA conditions, various experiments were performed in this study. A concentration of 40 µ g/ml of purified IgG was chosen for coating the plates to insure maximum sensitivity. Overnight incubation at 4°C was selected as a long blocking time at 4°C contributes to reduction of nonspecific binding. The optimal incubation time, temperature, and dilution buffer for the assay were also investigated. In the step 1 reaction, both room temperature (24°C) and low temperature (4°C) were tested. Incubation at 4°C required a long time compared to incubation at 24°C for optimal reaction. In the step 2 reaction, the same conditions were tested. Low temperature reduced nonspecificity compared with room temperature. Finally, reaction temperatures of 24°C for step 1 and 4°C for step 2 were chosen. Four concentrations of NaCl (0.1, 0.2, 0.3, and 0.4 M) and NRS (1%, 0.5%, 0.1%, and 0.05%) were tested in the dilution buffer for step 1. Low concentrations of NaCl enhanced the color reaction, but nonspecific reactivity also increased. While very high concentrations of NaCl reduced nonspecific reactions, they also reduced color development (9). Therefore, 0.2 M was chosen as the optimum NaCl concentration. The level of 0.05% NRS was chosen as it provided the best color reaction and standard curve. Cross-reactivity in the ELISA of purified sGH and serum from other salmonids (masu salmon, steelhead trout, pink salmon, brown trout, white-spotted charr, and Sakhalin taimen) were verified. Dilutions of serum from fish of the genera Salmo, Hucho, and Salvelinus were not parallel to the standard curve, but were recognized by this assay system. However, the ELISA should be able to measure GH levels in these species if their GHs are first purified. Dilutions of serum from fish of the genus Oncorhynchus were parallel with standard curve. Purified sGH ran parallel to the standard curve in the ELISA, but its immunoreactivity was lower than that of rsGH. These results correspond with those shown previously for immunoreactivity of rsGH ver-
sus sGH (21). Our study supports the idea of that the main difference between sGH and rsGH is the higher solubility of rsGH (22). In a previous study (2), plasma GH levels in chum salmon were reported to be 21.7 6 3.6 ng/ml (males) or 31.8 6 9.8 ng/ml (females). Serum GH levels in mature chum salmon were 6.39 6 0.69 ng/ml (males) and 12.42 6 1.89 ng/ml (females) when measured using our ELISA. We suspect that the reason for this discrepancy is due to a difference between the GHs used as standard. However, female chum salmon GH levels were higher than male GH levels in both reports. In conclusion, we developed a sensitive and specific ELISA for chum salmon GH in this study. This ELISA should be useful for further physiological and endocrinological studies of the biology of GH in this and related salmonid species. We thank Professor Minero Saneyoshi, Department of Biological Sciences, Nishi-Tokyo University, and Dr. Seiga Itoh, Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., for the gift of rsGH and Professor Craig V. Sullivan, Department of Zoology, North Carolina State University, for critically reading the manuscript. Thanks are also due to Mr. Julio Roberto Watanabe, Mr. Munetaka Shimizu, and Mr. Shinyo Iwasaki for their help, and to Mr. Shizuo Kimura, Ms. Makiko Kitamura, Ms. Hiroe Kumakura, and Ms. Miho Watanabe for maintenance of fish and technical assistance during this study. This research was supported by a Grand-in-Aid for Scientific Research from the Japanese Ministry of Education, Science and Culture to A. Hara.
References 1. Bjo¨rnsson, B.T.; Taranger, G.L.; Hansen, T.; Stefansson, S.O.; Haux, C. The interrelation between photoperiod, growth hormone, and sexual maturation of adult Atlantic salmon (Salmo salar). Gen. Comp. Endocrinol. 93:70–81; 1994. 2. Bolton, J.P.; Takahashi, A.; Kawauchi, H.; Kubota, J.; Hirano, T. Development and validation of salmon growth hormone radioimmunoassay. Gen. Comp. Endocrinol. 62:230–238; 1986. 3. Cook, A.F.; Wilson, S.W.; Peter, R.E. Development and vali-
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16. Le Bail, P.Y.; Boulard, G.; Zygmunt, M. Purification of chinook salmon (Oncorhynchus tshawytscha) GH for receptor study. Fish Physiol. Biochem. 7:243–251;1989. 17. Le Bail, P.Y.; Sumpter, J.P.; Carragher, J.F.; Mourot, B.; Niu, P.D.; Weil, C. Development and validation of a highly sensitive radioimmunoassay to chinook salmon (Oncorhynchus tshawystscha) growth hormone. Gen. Comp. Endocrinol. 83: 75–85;1991. 18. Man˜ano´s, E.; Nu´n˜ez, J.; Zauny, S.; Carrillo, M.; Le Menn, F. Sea bass (Dicentrachus labrax L.) vitellogenin. II-Validation of an enzyme-linked immunosorbent assay (ELISA). Comp. Biochem. Physiol. 107B:217–223;1994. 19. Nagae, M.; Fuda, H.; Hara, A.; Kawamura, H.; Yamauchi, K. Changes in serum immunoglobulin M (IgM) concentrations during early development of chum salmon (Oncorhynchus keta) as determined by sensitive ELISA technique. Comp. Biochem. Physiol. 106A:69–74;1993. 20. Prunet, P.; Bouef, G.; Bolton, J.P.; Young, G. Smoltification and seawater adaptation in Atlantic salmon (Salmo salar): Plasma prolactin, growth hormone and thyroid hormones. Gen. Comp. Endocrinol. 74:355–366;1989. 21. Stefansson, S.O.; Bjo¨rnsson, B.T.; Hansen, T.; Haux, C.; Taranger, G.L.; Saunders, R.L. Growth, parr-smolt transformation, and changes in growth hormone of Atlantic salmon (Salmo salar) reared under different photoperiods. Can. J. Fish. Aquat. Sci. 48:2100–2108;1991. 22. Takahashi, A.; Kawazoe, I.; Kawauchi, H. Competitive enzyme immunoassay for chum salmon growth hormone. Nippon Suisan Gakkaishi 57:267–272;1991. 23. Vilja, P.; Krohn, K.; Tuohimaa, P. A rapid and sensitive noncompetitive avidin-biotin assay for lactoferrin. J. Immunol. Methods 76:73–83;1985. 24. Wagner, G.F.; Fargher, R.C.; Brown, J.C.; McKeown, B.A. Further characterization of growth hormone from the chum salmon (Oncorhynchus keta). Gen. Comp. Endocrinol. 60:27– 34;1985. 25. Wagner, G.F.; McKeown, B.A. Development of a salmon growth hormone radioimmunoassay. Gen. Comp. Endocrinol. 60:27–34;1986. 26. Young, G.; Bjo¨rnsson, B.T.; Prunet, P.; Lin, R.J.; Bern, H.A. Smoltification and seawater adaptation in coho salmon (Oncorhynchus mykiss): Plasma prolactin, growth hormone, thyroid hormones and cortisol. Gen. Comp. Endocrinol. 74:335– 345;1989.
ISSN 0305-0491/97/$17.00 PII S0305-0491(97)00134-X
Development of an ELISA for Chum Salmon (Oncorhynchus keta) Growth Hormone Haruhisa Fukada,1,3 Naoshi Hiramatsu,1,3 Koichiro Gen,2 and Akihiko Hara3 1
Department of Biology and Aquaculture, Faculty of Fisheries, Hokkaido University, Hakodate, Hokkaido, 041, Japan; 2 Institute of Endocrinology, Gunma University, Maebashi, Gunma, 371, Japan; and 3 Nanae Fish Culture Experimental Station, Faculty of Fisheries, Hokkaido University, Nanae, Kameda-gun, Hokkaido, 041-11, Japan ABSTRACT. A specific and sensitive enzyme-linked immunosorbent assay (ELISA) was developed for the measurement of low levels of serum growth hormone (GH) in chum salmon (Oncorhynchus keta). The antiserum to GH (a-rsGH) was obtained from a rabbit immunized with recombinant chum salmon GH. The noncompetitive ELISA was performed by a sandwich method using a-rsGH rabbit IgG as the first antibody, its biotinylated Fab′ fragment as the second antibody, and the avidin-biotin reaction for signal amplification. This assay could be run in 3 days and routinely detected GH at concentrations as low as 0.5 ng/ml. The development of an ELISA for GH made possible quantification of serum GH levels. In this assay system, parallel dilution curves were obtained using purified chum salmon GH and GH’s from several species of the genus Oncorhynchus. comp biochem physiol 117B;3:387–392, 1997. 1997 Elsevier Science Inc. KEY WORDS. ELISA, avidin-biotin, growth hormone, chum salmon, teleost, fish
INTRODUCTION Growth hormone (GH) stimulates somatic growth indirectly (4) and enhances adaptation for salt water, especially in salmonid species (20,26). There are reports of purification of GH from several fish species including tilapia, Oreochromis mossambica (7), chum salmon, Oncorhynchus keta (10,13,24), chinook salmon, Oncorhynchus tshawytscha (16), sturgeon, Acipenser guldenstadti (6), carp, Cyprinus carpio (3), and Japanese eel, Anguilla japonica (12). To explore GH physiology, it is necessary to develop a specific assay system for the hormone. Development of an assay for GH has been attempted in various fishes using radioimmunoassay (RIA) (2,3,11,17,25) and an enzyme immunoassay (22). RIA was used successfully to measure plasma GH in various fishes. There is one report of an enzyme-linked immunosorbent assay (ELISA) system for fish GH that can measure plasma GH levels using a monoclonal antibody (5). In general, ELISA has several advantages over RIA including safety, simplicity, and speed. In the present study, a sensitive, noncompetitive sandwich ELISA was developed for chum salmon GH, one using a polyclonal antibody to recombinant chum salmon GH as the first antibody, its biotinylated Fab′ fragment as the second antibody, and the avidin-biotin reaction for signal amplification.
Address reprint requests to: A. Hara, Nanae Fish Culture Experimental Station, Faculty of Fisheries, Hokkaido University, Nanae, Kameda-gun, Hokkaido, 041-11, Japan. Tel. 81-138-65-2344; Fax 81-138-65-2239. Received 13 December 1996; accepted 18 December 1996.
MATERIALS AND METHODS Fish and Samples Mature chum salmon were caught from the Yurappu River in southern Hokkaido. Pink salmon (O. gorbuscha), steelhead trout (O. mykiss), masu salmon (O. masou), brown trout (Salmo trutta), white-spotted charr (Salvelinus leucomaenis), and Sakhalin taimen (Hucho perryi) were obtained in 1993 and 1994 from broodstocks reared in freshwater ponds at the Nanae Fish Culture Experimental Station, Faculty of Fisheries, Hokkaido University. Blood samples was taken by syringe from the caudal vessels of each fish. The samples were allowed to stand at 4°C for several hours and serum was separated by centrifugation at 16,000 g for 10 min and stored at 230°C until use. Pituitaries were collected and stored at 230°C until use. For measurement of GH using the ELISA, pituitary extracts and diluted serum were prepared. Pituitary glands were homogenized in 0.01 M phosphate buffer in isotonic saline, pH 7.0 (PBS) and homogenates were centrifuged at 16,000 g for 20 min at 4°C as discussed below. The supernatant was used as the pituitary extract. Serum was diluted with phosphate buffer including 0.2 M NaCl, 1% bovine serum albumin (BSA) and 0.05% normal rabbit serum (NRS) (PBS-BSA) and used in the ELISA at a total volume of 100 µ l of solution per well. Purification of Chum Salmon GH For GH purification, chum salmon pituitary glands were homogenized in 0.01M PBS with a ground glass homogenizer
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and centrifuged at 12,000 g for 20 min at 4°C. The supernatant was precipitated by addition of ammonium sulfate at 35% saturation. The precipitate was collected after centrifugation at 28,000 g for 30 min and dissolved in and dialyzed against 0.02 M Tris-HCl buffer, pH 8.0. The dialysate was applied to a Mono-Q column (FPLC system, Pharmacia, Uppsala, Sweden) and eluted stepwise with Tris-HCl buffer containing NaCl at 0.1 or 1 M. Thereafter, the fraction containing GH was subjected to gel filtration on a Superose 12 column equilibrated with 0.02 M Tris-HCl buffer including 0.2% NaCl (pH 8.0). The presence of chum salmon GH (sGH) in the fractions was assessed by Western blotting using a rabbit antiserum against recombinant chum salmon GH (rsGH). Antiserum An antiserum against rsGH (a-rsGH) was raised in a rabbit by intradermal injection with rsGH (1 mg) emulsified in an equal volume of complete Freund’s adjuvant. Injections were done six times at 1-week intervals. The specificity of this a-rsGH was demonstrated previously by Bjo¨rnsson et al. (1) as follows, cross-reactivities with chum salmon gonadotropins I and II are 0.03% and 0.02%, respectively. Electrophoresis Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out using a Tris-glycine buffer system (15). Gels were stained for protein with 0.1% Coomassie Brilliant Blue R 250 in a solution of 40% ethanol/10% acetic acid/50% distilled water (CBB). The molecular weights of proteins banded in the gel were estimated using the following molecular mass markers (Pharmacia, Uppsala, Sweden): α-lactalbumin (14.4 kDa), trypsin inhibitor (20.1 kDa), carbonic anhydrase (30 kDa), ovalbumin (43 kDa), albumin (67 kDa), and phosphorylase b (94 kDa). Western blotting was carried out according to our previous report (14) with the primary antiserum used at a 1:1000 dilution. Biotin Labeling of Fab′ Fab′ was purified from a-rsGH according to our previous report (19). Biotinylated Fab′ was prepared using a biotin labeling kit (Boehringer Mannheime Biochem.). A 22.5 µl mixture, containing 10 µ l biotin-7-NHS (D-biotinylatedo-aminocaproic acid-N-hydroxysuccinimide ester) solution (20 mg/ml) and 90 µ l DMSO (dimethylsulfoxide), was added to 1 mg of Fab′ in solution (1 mg/ml PBS) with gentle stirring for 2 hr at room temperature. Remaining nonreacted biotin-7-NHS was separated by dialysis of the solution against PBS overnight at 4°C.
ANTIBODY COATING. The immunoglobulin G (IgG) solution was purified from a-rsGH rabbit serum by chromatography on DE-52 (19). Wells were coated and incubated for 4 hr at 24°C with 150 µl IgG solution at a concentration of 40 µ g/ml in PBS. BLOCKING. After three consecutive washes with 200 µ l of PBS-1% Tween buffer (PBS-T), and followed by wash with the same volume of PBS per well, the plates were incubated with 200 µl of PBS-1%BSA per well for 4 hr at 24°C or overnight at 4°C. INCUBATION OF SAMPLES AND STANDARDS (STEP 1).
After three washes with PBS-T and one with PBS, 100 µ l of sample or standard, diluted serially with PBS-BSA, was added into each well of the microtiter plates and incubated for 8 hr at 24°C. The standard solutions (rsGH 0.098 ng/ ml to 200 ng/ml) were prepared by dissolving a known amount of rsGH in PBS-BSA. INCUBATION WITH BIOTINYLATED a-rsGH (STEP 2). After washing as described above for step 1, each well received 100 µ l of labeled antibody diluted 1 : 400 in 0.02 M phosphate buffer, pH 7.0 containing 0.1% BSA, 0.1% Tween and 0.002% thimerosal (PB-BSA-T), followed by incubation for 16 hr at 4°C. INCUBATION WITH STREPTAVIDIN-HORSERADISH PEROX-
After the plates were again washed as described above, each well received 100 µ l of streptavidin-HRPO conjugate (streptavidin coupled to peroxidase, Boehringer Mannheim Biochem) diluted 1:2000 with PB-BSA-T, followed by incubation for 1.5 hr at 24°C. IDASE (HRPO) CONJUGATE (STEP 3).
ENZYMATIC COLOR REACTION. The plates were washed as described above and color was developed at room temperature for 15 min in the dark by adding 150 µl of o-phenylenediamine (3 mg/ml 0.1 M citric acid-phosphate buffer pH 5.0 containing 0.02% H2O2 ) to each well. The reaction was stopped by adding 150 µl of 4N HCl per well. The absorbance at 492 nm was measured using an ELISA plate reader (Bio-Rad 2550, Richmond, CA).
Expression of the Results The parallelism between the regression curves was tested by analysis of covariance according to Man˜ano´s et al. (18). RESULTS Purification of sGH The purified sGH appeared as a single band of 23 kDa after SDS-PAGE. This band stained specifically with polyvalent a-rsGH in Western blotting (Fig. 1).
ELISA Assay Procedure The ELISA was carried out in 96-well polystyrene ELISA microtiter plates (ICN Biomedicals, Horsham, PA) according to the method of Guesdon et al. (8).
Effect of Concentration of NaCl and NRS NaCl at four different concentrations (0.1, 0.2, 0.3, and 0.4 M) was mixed in 0.01 M Phosphate Buffer-1%BSA for use
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FIG. 1. 12.5% SDS-PAGE (A) and Western blotting pattern using a-rsGH (B) of pituitary extracts (lane 1) and purified GH (lane 2). Samples were reduced with 2-mercaptoethanol.
as the dilution buffer in step 1. Color development serially decreased with increasing NaCl concentration. At 0.1 M NaCl, diluted serum did not run parallel with the standard curve in the ELISA. At 0.2 M NaCl, good color development was obtained and serially diluted serum run parallel to the standard curve (Fig. 2A). Four different concentrations of normal rabbit serum (NRS) (1%, 0.5%, 0.1% and 0.05%) were mixed with PBS-1%BSA containing 0.2M NaCl. The best standard curve and serum dilution series were obtained when PBS-1%BSA containing 0.05% NRS was used (see Fig. 2B). Consequently the standard ELISA procedure used dilution buffer containing 0.2M NaCl and 0.05% NRS. Standard Curve and Precision of the ELISA Under routine conditions (coating: 4 hr at 24°C, step 1: 8 hr at 24°C, step 2: 16 hr at 4°C, step 3: 1.5 hr at 24°C),
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FIG. 2. Typical standard curve in the ELISA using 0.2 M NaCl (A) and 0.05% NRS (B) in the dilution buffer at step 1. Parallelism between regression curves was assessed by the F-test ( P , 0.05). d, standard curve; s, masu salmon serum.
standard curves of rsGH (range 0.098–50 ng/ml) showed a typical sigmoidal curve (Fig. 3). Precision tests were performed using five samples with various rsGH concentrations (200, 100, 50, 25, and 12.5 ng/ml). The between assay coefficient of variation was 6.90% (n 5 15) and the within assay coefficient of variation was 5.54% (n 5 15). Cross-Reactivity with Pituitary and Serum GH from Other Salmonids Serum and pituitary extracts from other salmonid species were recognized in the ELISA. Serial dilutions of pituitary extracts showed a sigmoidal curve, but the degree of immunoreactivity was different between genera (Fig. 4B). As to
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Measurement of Serum GH Levels in Mature Chum Salmon Serum GH levels were measured in mature chum salmon captured on their spawning migration in the Yurappu river. Serum GH levels of male chum salmon were 6.39 6 0.69 ng/ml (n 5 5). Serum GH levels were higher in female chum salmon (12.42 6 1.89 ng/ml, n 5 5) than in males. DISCUSSION
FIG. 3. Routine standard curve of rsGH in the ELISA (range
0.098–50 ng/ml).
serum, serially diluted samples from masu salmon (genus Oncorhynchus) ran parallel to the standard curve. Serum from species of other genera (Salmo, Hucho, and Salvelinus) was recognized by this ELISA but did not run parallel with the standards when serially diluted (Fig. 4C). Serially diluted serum from four species of the genus Oncorhynchus ran parallel to the standard curve. The purified sGH also ran parallel to rsGH in the ELISA standard curve, but it showed low immunoreactivity as compared to rsGH (Fig. 5).
The a-rsGH used in this ELISA is the same antibody used in a radioimmunoassay for salmon GH in a previous study (1). The reported cross-reactivities with chum salmon gonadotropins I and II were 0.03% and 0.02%. After immunostaining pink salmon pituitary sections with a-rsGH, prolactin-producing cells in the rostral pars distalis, identified in salmonids on the basis of their morphology in relation to specific immunostaining characteristics (10), were not stained at all (data not shown). Furthermore, results of Western blotting demonstrated the high degree of specificity of the a-rsGH. Thus, the a-rsGH is specific enough to use for ELISA of GH. The purification of sGH was performed using an FPLC system. The purified sGH appeared as a single band at a position corresponding to 23 kDa in SDS-PAGE and the band reacted positively and specifically in Western blots using a-rsGH. This estimated molecular weight for GH is similar to that reported for other teleost GHs in previous studies [see review (5)]. A specific and sensitive ELISA for chum salmon GH was developed utilizing a noncompetitive sandwich method with the avidin-biotin system in polystyrene microtiter plates as the solid phase (8,23). Owing to use of the non-
FIG. 4. Serial dilution of rsGH standards in the ELISA (A) corresponding and serial dilutions of pituitary extracts (B) and serum (C) from masu salmon (d), brown trout (m), white-spotted charr (■) and Sakhalin taimen (✚).
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FIG. 5. Parallelism with the
rsGH standard curve in the ELISA of serum from fish of the genus Oncorhynchus (masu, pink, and chum salmon, and steelhead trout) and relative immunoreactivity of rsGH and purified chum salmon GH (sGH) in the ELISA. Parallelism between regression curves was assessed by the F-test ( P , 0.05).
competitive sandwich ELISA method, the confounding reaction of endogenous peroxidase can be avoided. To determine optimal ELISA conditions, various experiments were performed in this study. A concentration of 40 µ g/ml of purified IgG was chosen for coating the plates to insure maximum sensitivity. Overnight incubation at 4°C was selected as a long blocking time at 4°C contributes to reduction of nonspecific binding. The optimal incubation time, temperature, and dilution buffer for the assay were also investigated. In the step 1 reaction, both room temperature (24°C) and low temperature (4°C) were tested. Incubation at 4°C required a long time compared to incubation at 24°C for optimal reaction. In the step 2 reaction, the same conditions were tested. Low temperature reduced nonspecificity compared with room temperature. Finally, reaction temperatures of 24°C for step 1 and 4°C for step 2 were chosen. Four concentrations of NaCl (0.1, 0.2, 0.3, and 0.4 M) and NRS (1%, 0.5%, 0.1%, and 0.05%) were tested in the dilution buffer for step 1. Low concentrations of NaCl enhanced the color reaction, but nonspecific reactivity also increased. While very high concentrations of NaCl reduced nonspecific reactions, they also reduced color development (9). Therefore, 0.2 M was chosen as the optimum NaCl concentration. The level of 0.05% NRS was chosen as it provided the best color reaction and standard curve. Cross-reactivity in the ELISA of purified sGH and serum from other salmonids (masu salmon, steelhead trout, pink salmon, brown trout, white-spotted charr, and Sakhalin taimen) were verified. Dilutions of serum from fish of the genera Salmo, Hucho, and Salvelinus were not parallel to the standard curve, but were recognized by this assay system. However, the ELISA should be able to measure GH levels in these species if their GHs are first purified. Dilutions of serum from fish of the genus Oncorhynchus were parallel with standard curve. Purified sGH ran parallel to the standard curve in the ELISA, but its immunoreactivity was lower than that of rsGH. These results correspond with those shown previously for immunoreactivity of rsGH ver-
sus sGH (21). Our study supports the idea of that the main difference between sGH and rsGH is the higher solubility of rsGH (22). In a previous study (2), plasma GH levels in chum salmon were reported to be 21.7 6 3.6 ng/ml (males) or 31.8 6 9.8 ng/ml (females). Serum GH levels in mature chum salmon were 6.39 6 0.69 ng/ml (males) and 12.42 6 1.89 ng/ml (females) when measured using our ELISA. We suspect that the reason for this discrepancy is due to a difference between the GHs used as standard. However, female chum salmon GH levels were higher than male GH levels in both reports. In conclusion, we developed a sensitive and specific ELISA for chum salmon GH in this study. This ELISA should be useful for further physiological and endocrinological studies of the biology of GH in this and related salmonid species. We thank Professor Minero Saneyoshi, Department of Biological Sciences, Nishi-Tokyo University, and Dr. Seiga Itoh, Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., for the gift of rsGH and Professor Craig V. Sullivan, Department of Zoology, North Carolina State University, for critically reading the manuscript. Thanks are also due to Mr. Julio Roberto Watanabe, Mr. Munetaka Shimizu, and Mr. Shinyo Iwasaki for their help, and to Mr. Shizuo Kimura, Ms. Makiko Kitamura, Ms. Hiroe Kumakura, and Ms. Miho Watanabe for maintenance of fish and technical assistance during this study. This research was supported by a Grand-in-Aid for Scientific Research from the Japanese Ministry of Education, Science and Culture to A. Hara.
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