Changes in serum immunoglobulin M (IgM) concentrations during early development of chum salmon (Oncorhynchus keta) as determined by sensitive elisa technique

Camp. Biochem.

Physiol. Vol. 106A. No. I, pp. 69-74, 1993 0

Printed in Great Britain

0300-9629/93 66.00 + 0.00 1993 PergamonPressLtd

CHANGES IN SERUM IMMUNOGLOBULIN M (IgM) CONCENTRATIONS DURING EARLY DEVELOPMENT OF CHUM SALMON (ONCORHYNCHUS KETA) AS DETERMINED BY SENSITIVE ELISA TECHNIQUE MASAKINAGAE,* HIROTOSHIFUDA,? AKIHIKO HARA,t HIROSHIKAWAMURA~and KOHEIYAMAUCHI*§ *Department of Biology and Aquaculture, Faculty of Fisheries, Hokkaido University, Hakodate,

041, Japan (Tel. 81-138-405545; Fax 81-138-435105); tNanae Fish Culture Experimental Station, Faculty of Fisheries, Hokkaido University, Kameda, 041-I 1, Japan (Tel. 81-138-652344; Fax 81-138-652239); SMakkari Branch, Hokkaido Fish Hatchery, Abuta, 048-16, Japan (Tel. 81-136-453473; Fax 81-136453473)

(Received 23 October 1992; accepted 27 November 1993) Abstract-l.

developed

A specific and sensitive sandwich enzyme-linked immunosorbent assay for the measurement

of low levels of serum

immunoglobulin

M (IgM)

(ELISA) was of chum salmon

Oncorhynchusketa. 2. In this assay, 5~1 serum was enough

to measure the concentration of IgM and the minimum detectable concentration of serum IgM was about 5 ng/ml. 3. Coefficients of variation within and between assays ranged from 2.90 to 9.61%. 4. IgM concentrations remained at low level (< 300 ng/ml) until 40 days after hatching and then increased rapidly at the period of emergence (48 days after hatching).

channel catfish, Ictafurus punctatus (Ourth, 1986) and masu salmon, Oncorhynchus masou (Fuda et al., 1991). Adult fish were used in these studies, and the high serum IgM concentrations permitted analysis by single radial immunodiffusion @RID). Studies on serum IgM concentration during early developmental stages are scarce. At this stage, it is estimated that serum IgM concentration is low and is not detectable by SRID. Therefore, a more sensitive assay system is needed for ontogenic research on IgM. Enzymelinked immunosorbent assay (ELISA), first reported by Engvall and Perlann (1971), is a precise and sensitive assay method for the quantitative determination of rabbit IgG and based on the same principle as radioimmunoassay. Furthermore, this method avoids the use of radioisotopes and excessive use of chemicals. The present paper describes the establishment of ELISA for chum salmon IgM and the measurement of serum IgM concentrations from 21 to 67 days after hatching.

Immunoglobulin M (IgM) is a humoral immune factor in fish as well as a lysozyme, chitinase, interferon, etc. It is assumed that the essential function of IgM is defence against foreign antigens, for example, infectious agents. Many studies on fish immunobiology have dealt with the phylogeny of immunoglobulin structure and function coupled with investigations into the factors affecting the immune response after antigenic stimulation (Ingram, 1980). However, there have been only few studies of serum IgM concentration in fish from the point of view of physiology and fish pathology. The measurement of serum IgM concentration is required to obtain basic information on the immune system as well as for epidemiologic research. Concentrations of IgM have been measured in serum from the nurse shark, Ginglymosstoma cirratum(Fidler et al., 1969), paddlefish, PoIyodon spathula (Acton et al., 1971b; Legler et al., 1971), gar, Lepisosteus osseus (Acton et al., 1971a), carp, Cyprinus carpio (Richter et al., 1973; Vilain et al., 1984), brown trout, Sulmo trutta (Ingram and Alexander, 1981), coho salmon, Oncorhynchus kisutch (Voss et al., 1980), chum salmon, Oncorhynchus keta (Kobayashi et al., 1982), goldfish, Carassius auratus and tenth, Tinca tinca (Vilain and Wetzel, 1984), yellowtail, Seriolu quinqueradiuta (Matsubara et al., 1985), rainbow trout, Oncorhynchus mykiss (Olesen and Jsrgensen, 1986),

MATERIALSANDMETHODS Animals

Mature chum salmon (Oncorhynchus keta) were caught in the Torisaki River, southern Hokkaido, Japan. One batch of eggs was fertilized artificially and kept in freshwater at 7.8”C in darkness at Hokkaido Fish Hatchery, Makkari branch, Hokkaido. Fish were fed commercial diets just after emergence. Blood was collected from hatched larvae and fry by severing their caudal peduncle. Blood samples were allowed to

§Author to whom all correspondence should be addressed. 69

MASAKI NAGAE et

70

stand at room temperature for several hours and serum was separated by centrifugation at 3000rpm for 10 min and stored at -40°C until use for ELISA. PuriJication of serum IgM Purification of chum salmon IgM was carried out according to Kobayashi et al. (1982) and Fuda et al. (1991). Adult chum salmon serum was precipitated by addition of ammonium sulfate at 50% saturation. After centrifugation at 10,000 rpm for 10min the precipitate was collected, dissolved in and dialysed against 0.015 M Tris-HCl buffer, pH 8.0. The dialysate was fractionated on a DEAE cellulose (DE-52, Whatman, Kent, U.K.) column equilibrated with Tris-HCl buffer using a stepwise elution with the same buffer containing NaCl at 0.05, 0.1, 0.15, 0.2 and 0.4 M. The fraction eluted by 0.1 M NaCl was reprecipitated with 50% ammonium sulfate, and collected by centrifugation at 10,000 rpm for 10 min. The precipitate was dissolved in 0.02 M Tris-HCl buffer, pH 8.0, containing 2% NaCl and 0.1% NaN,. It was then gel-filtered through a Scpharose 6B (Pharmacia, Uppsala, Sweden) column using the same buffer. Two distinct peaks were obtained. The first peak was collected, concentrated by lyophilization and was again chromatographed on Sepharose 6B. A single and symmetrical peak was obtained and collected as the purified chum salmon IgM. Preparation

of antisera

Polyvalent antiserum to chum salmon serum was carried out by immunizing rabbit as described previously (Hara, 1976). Specific antiserum to chum salmon IgM was obtained from rabbit immunized with 2 ml solution containing 200 pg of purified IgM mixed with an equal volume of Freund’s complete adjuvant. The rabbit received four injections at about 7 day intervals. Double antibody sandwich ELISA The procedure for labeling antibody (Fab’ fragment) with enzyme was performed according to the method of Ishikawa (1983). Preparation from IgM

of the Fragments

antigen binding (Fab’)

Antiserum to purified chum salmon IgM (28 ml) was precipitated by addition of ammonium sulfate at 35% saturation. The precipitate collected after centrifugation at 10,000 rpm for 10 min was dissolved in and dialysed against 0.0175 M phosphate buffer, pH 6.3. The dialysate was fractionated on DEAE cellulose (DE-52 Whatman) column (2.8 x 5.2cm) equilibrated with 0.0175 M phosphate buffer. The fraction containing high amount of IgG was used for horseradish peroxidase (HRP) labeling. The IgG dialysed against 0.1 M acetate buffer, pH 4.5 was incubated with 1 mg pepsin (Sigma, St Louis, MO) with gentle stirring for 20 hr at 37°C. The reaction was stopped by adding about 30mg Tris-HCI salt.

al.

The mixture was gel filtered through a Sephadex G-200 (Pharmacia) column (2.5 x 73 cm) equilibrated with 0.01 M PBS (pH 7.0). The fraction containing F(ab’), fragment was concentrated to 15 mg/ml, and 48 mg F(ab’), were reduced with 96 mM mercaptoethylamine (Nacarai, Kyoto, Japan) for 90min at 37°C. After this incubation, the Fab fragment was isolated on a Sephadex G-75 column using 0.01 M PBS (pH 6.5) containing 5 mM ethylenediaminetetraacetic acid (EDTA). Introduction

of maleimide groups into HRP

Twenty mg of HRP (Toyobo, Osaka, Japan) dissolved in 3.0 ml PBS (pH 7.0) was incubated with 8 mg of N-Hydroxy-succinimide ester of CHM (Zieben them., Tokyo, Japan) dissolved in 300 pi of N, N-dimethylformamide for 60min at 37 C with mild stirring. The precipitate formed was removed by centrifugation and the supernatant was gel filtered through a Sephadex G-25 using 0.01 M PBS (pH 7.0). Fractions containing protein were pooled and concentrated by ultrafiltration. Conjugation

of maleimide -peroxidase

and Fab ’

Ten mg of maleimide-peroxidase in 1 ml 0.01 M PBS (pH 7.0) was incubated with 10 mg Fab’ in 1 ml 0.01 M PBS containing 5 mM EDTA (pH 6.15) for 20 hr at 4°C. The mixture was gel filtered through a Sephadex G-200 (2.5 x 73 cm) using 0.01 M PBS (pH 7.0). Each fraction was measured at absorbance 280 nm for protein and 403 nm for enzyme activity. The enzyme-antibody conjugates were stored at -20°C until use. ELISA

assay procedure

Assays were carried out in 96-well polystyrene ELISA microtiter plates (Titertex, Horsham, PA). Antibody coating. The microtiter plates were coated with rabbit anti-chum salmon IgM IgG which was fractionated by DE-52 at a concentration of 40 pg/ml in 0.01 M PBS. A volume of 150 ~1 was dispensed into each well and incubated for 4 hr at 4 C. Blocking. After one washing with 200 ~1 of 0.01 M PBS + 0.1% Tween 20 per well and two washings with 200 ~1 of PBS + 1% BSA, 0.01% thimerosol was added to each well and incubated for 2 hr at room temperature. Incubation of samples and standards. After washings as described above, 100 ~1 of sample and standard were placed into the appropriate wells in the microtiterplates and incubated at room temperature. Incubation with peroxidase labeled antibody. After washings as described above, each well received 150 ~1 of peroxidase labeled antibody 1: 1600 in PBS-BSA, followed by incubation for 12 hr at room temperature. Enzymatic color reaction. The plates were washed as described above and 150 p I 0 -phenylenediamine (3 mg/ml 0.1 M citric acid-phosphate buffer (pH 5.0) containing 0.02% HzOz were added to each well for

IgM in early development

Fig. 1. Immunoelectrophoresis a-S: rabbit antiserum against

of chum

salmon

71

of purified chum salmon IgM. S: chum salmon serum; M: purified chum salmon serum protein; a-M: specific antiserum to chum salmon

color reaction. The reaction was stopped after 30 mitt at room temperature by adding 100 ~1 of 4 N HCI. The absorbance at 492 nm was measured using an ELISA plate reader (Bio-Rad 2550, Richmond, CA).

enzymatic

IgM; IgM.

Conjugation of maieimide -peroxidase and Fab ’ The elution pattern of the Iabeled antibody is shown in Fig. 2. The first peak was collected as the peroxidase labeled antibody. Concentration of antibody solution for coating the microtiter plate

RESULTS

Purification of chum salmon IgM The purified IgM revealed a single precipitin line when reacted against a polyvalent antiserum to chum salmon serum proteins as well as when it was precipitated by the specific antiserum to chum salmon IgM (Fig. 1). Conversely, the antiserum raised against the purified IgM preparation yielded a single precipitin line with the chum salmon whole serum, suggesting that the IgM preparation was immunologically pure.

An appropriate concentration of antibody was incubated with 100 ~1 of solution containing 10, 20, 40 and 80 pg of purified IgG/ml for 8 hr. The incubation time for the first reaction was set for 4 hr at room temperature while the second reaction was set for 12 hr at room temperature. A satisfactory calibration curve was obtained for antibody concentrations ranging from 20 to 80 pg/ml. The concentration of 40 pg antibody/ml was chosen for coating the plates to obtain a maximum absorbance value. Effect of coating time The microtiter plates were coated with purified IgG at a concentration of 40 pg/ml for 2, 4, 8 and 12 hr at room temperature. Three different concentrations of IgM (40, I60 and 640 ng/ml) were used as samples. The first and second reaction were done as described above. As shown in Fig. 3, binding of antigen on the wells proceeded rapidly and reached a plateau by 4 hr regardless of IgM concentration.

o’3 1o’3

Eflect of b&king

Fraction Number

Fig. 2. Gel filtration of peroxidase labeled Fab’ on Sephadex G-200 (2.5 x 73 cm) equilibrated with 0.01 M PBS (PH 7.0). Each

fraction was measured at absorbance at 280nm protein (0) and 403 nm for enzyme activity (e),

for

time

The coated microtiter plates were blocked by 0.01 M PBS-l% BSA for 2,4, 8 and 12 hr. The first and second reaction were done as described above. A satisfactory calibration curve was obtained regardless of blocking time. Therefore, plates were blocked for 2 hr.

MASAKI NAGAE et

;

r;

;

al.

0

1;

’

4

4

. . . . ..

..I

.

.

.

. . . ..I

10

1

.

.

.

. . . ..I

.

lo3

lo2

-

.

.--I

IO’

IgM (nglml)

Incubation time (hr)

Fig. 3. Effect of time on coating the microtiter plate with antibody. IgM concentration is represented by 0,40 ng/ml; 0, 160 ng/ml and 0, 640 ng/ml.

Eflect of incubation time The incubation time necessary for the first reaction was examined with various incubation periods from 2 to 12 hr. The period for the second reaction was set at 12 hr. The first reaction proceeded rapidly and reached a plateau within 8 hr regardless of IgM concentration (Fig. 4A). The incubation time necessary for the second reaction was examined between 2-24 hr. The period for the first reaction was set at 8 hr. The second reaction also proceeded rapidly for the first 4 hr (Fig. 4B) and reached a plateau within 12 hr regardless of IgM concentration. Based from Figs 4A, B, the optimum incubation time for the first

Fig. 5. Standard curve of chum salmon IgM.

reaction was determined second reaction. Under curves of chum salmon moidal curve (Fig. 5).

to be 8 and 12 hr for the these conditions, standard IgM showed a typical sig-

Precision tests of the assay Precision tests were performed using three sera with various IgM concentrations. The results within and between assay variations are described in Table 1. Coefficients of variations ranged from 2.90 to 7.87% within assay and from 7.30 to 9.61% between assays. Quantitation

of chum salmon IgM

IgM concentrations were measured in samples of serum pooled from about 100 fishes from 21 to 67 days after hatching. A concentration of 153 ng IgM/ml was detected 21 days after hatching and remained at constant until 33 days. The concentration increased gradually to about 4pg/ml at 48 days (emergence) and then increased rapidly to about 22 pg/ml at 67 days after hatching (Fig. 6). DISCUSSION

0

Until now, ELISA has been used to measure IgM concentrations of yellowtail, Seriola quinqueradiata (Matsubara et al., 1985). In this case, however, serum IgM levels below 1 mg/ml were not measured. The aim of the present study is to develop a sensitive ELISA for measurement of small amounts of IgM in developing larva of chum salmon, since the volume of serum that can be collected from one larva is very small (0.5 ml). The minimum detectable concentration of IgM using this ELISA was 5 ng/ml. More-

Incubation time (hr)

Table I. Precision

tests of the assays

Mean IgM

0 024

12

concentration

24

Incubation time (hr)

Fig. 4A. Effect of incubation time on the first reaction. IgM concentration is represented by 0, 40 ng/ml; 0, 160 ng/ml and 0, 64Ong/ml. B. Effect of incubation time on the second reaction. IgM concentration is represented by 0, 4Ong/mI; 0, 160ng/ml and 0, 640ng/ml.

Variation Within

(naiml) assay

Between assay

Number of determinations

CV(%)

48.5 210.3 911.0

IO IO IO

2.90 4.74 7.87

45.6 221.3 1162.5

5 5 5

7.59 7.01 9.54

IgM in early development

of chum

73

salmon

emergence, alevins are known to start feeding after completion of yolk absorption. This event requires many morphological and physiological changes. Kaeriyama et al. (1986) reported that survival rate of chum salmon may decline at emergence. Therefore, it is possible that newly produced IgM at the time of emergency plays a role in the survival of larva.

s

ra 10

0 0

-

20

30

40

50

60

70

Days after hatching

Fig. 6. Changes in serum IgM concentrations of chum salmon. l represents IgM concentration.

over, coefficients of variation within and between assays were under 10%. Therefore, the ELISA developed in the present study is precise and highly sensitive. The serum IgM concentrations increased gradually from 33 days after hatching by the period of emergence (48 days after hatching) in the chum salmon. The presence of maternal Ig in fish eggs and fetus has been noted in the Brook trout Salvelinus fontinalis (Wolf et al., 1963), Surfperch Cymatogaster aggreta (Hogarth, 1976) carp Cyprinus carpio (Van Loon et al., 1981) plaice Pfeuronectes patessa (Bly et al., 1986), guppy Poecilia retioculata (Takahashi and Kawahara. 1987) tilapia Oreochromis aureus (Mor and Avtalion, 1990) and chum salmon Oncorhynchus keta (Fuda et al., 1992). Fielder et al. (1969) identified two kinds of IgM (7s and 19s IgM) in nurse shark Ginglymosstoma cirratum, and suggested that 19s IgM present at birth may be of maternal origin. Fuda et al. (1992) observed a peculiar IgM-related component (IgM-like protein) in chum salmon eggs. Therefore, it is most likely that serum IgM observed prior to emergence in the chum salmon is maternal in origin. The molecular weight of this IgM-like protein was 495 kD, whereas that of serum IgM was 750 kD. However, the polyclonal anti-IgM antibody recognized both IgM and IgM-like protein, suggesting that IgM-like protein was antigenically identical with serum IgM. The concentrations of serum IgM increased dramatically just after emergence. According to immunofluorescent staining observations, the appearance of membrane-bound IgM at 45 days after hatching coincided with the onset of feeding in the Atlantic salmo Salmo salar (Ellis, 1977). Furthermore, in the rainbow trout Oncorhynchus mykiss, IgM-positive cells appeared in the spleen and thymus at 30 days after hatching (Razquin et al., 1990). These results indicate that IgM may be produced around the time of emergence in salmonids. Therefore, the dramatic increase in IgM just after emergence observed in the present study may be due to the de novo production. However, the function of the newly produced IgM at the time of emergence are unknown. At the period of

Acknowledgements-We would like to thank to Professor Hiroya Takahashi, Hokkaido University, for his critical advice during this study. Thanks are also due to Dr Reynaldo Patiiio, Texas Tech University, for reading the manuscript. This study was supported in part by a grant-inaid for Scientific Research from the Ministry of Education. Science and Culture, Japan.

REFERENCES

Acton R. T., Weinheimer P. F., Dupree H. K.. Evans E. E. and Bennett J. C. (1971a) Phylogeny ofimmunoglobulins. Characterization of a 14s immunoglobulin from the gar, lepisosteus osseas. Biochemistry 10, 20282036 Acton R. T., Weinheimer P. F., Dupree H. K., Russell T. R., Wolcott M., Evans E. E.. Schrohenloher R. E. and Bennett J. C. (1971 b) Isolation and characterization of the immune macroglobulin from the paddlefish, Polyodon spathula. J. biol. Chem. 246, 6760 6769. Bly J. E., Grimm A. S. and Moriss I. G. (1986) Transfer of passive immunity from mother to young in a teleost fish: haemagglutinating activity in the serum and eggs of plaice, Pleuronectes platessa L. Camp. Biochem. Physiol. 84A, 3099313. Ellis A. E. (1977) Ontogeny of the immune response in Salmo salar. Histogenesis of the lymphoid organs and appearance of membrane immunoglobulin and mixed leucocyte reactivity. In Deaelopmental Immunobiolog~ (Edited by Solomon J. B. and Horton J. D.), pp. 225-23 I. Elsevier/North Holland Biomedical Press, Amsterdam. Engvall E. and Perlmann P. (197 I) Enzyme-linked immunosorbent assay (ELISA) Quantitative assay of immunoglobulin G. Immunochemistry 8, 871-874. Fidler J. E., Clem L. W. and Small Jr P. A. (1969) Immunoglobulin synthesis in neonatal nurse sharks (Ginglymostoma cirratum). Comp. Biochem. Plr>asiol. 31, 3655371. Fuda H., Soyano K., Yamazaki F. and Hara A. (1991) Serum immunoglobulin M (IgM) during early development of masu salmon (Oncorhynchus masou). Comp. Biochem. Physiol. WA, 637643. Fuda H.. Hara A., Yamazaki F. and Kobayashi K. (1992) A peculiar immunoglobulin M (IBM) identified in eggs of chum salmon (Oncorhynchus keta). Dec. romp. Immunol. (in press). Hara A. (1976) Iron-binding activity of female-specific serum proteins of rainbow trout (Salmo gairdneri) and chum salmon (Oncorhynchus keta). Biochem. hiophys. Acta 427, 549-557. Hogarth P. J. (1976) The Institute of Biolog.v’.r Studies in Biology No.75. Viviparity. Edward Arnord, London. Ingram G. A. (1980) Substances involved in the natural resistance of fish to infection-A review. J. Fish Biol. 16, 23-60. Ingram G. A. and Alexander J. B. (1981) A comparison of the immune response of the brown trout (Salmo trurta) to a protein and carbohydrate antigen. In Aspects of Der’elopmental and Comparative Immunology I (Edited by Solomon, J. B.), pp; 471472. Pergamon Press, Oxford. Ishikawa E., Imaaawa, M.. Hashida S.. Yoshitake S. Hamaguchi Y. and Ueno T. (1983) Enzyme-labeling of antibodies and their fragments for enzyme immunoassay

74

MASAKI NAGAE et al

and immunohistochemical staining. J. Immunoassay 4, 209-327 Kaeriyama M. (1986) Ecological study on early life of the chum salmon, Oncorhyuchus Keta (Walbaum). Scienrifc Reports of The Hokkaido Salmon Halchery 40, 31-92 Kobayashi K., Hara A., Takano K. and Hirai H. (1982) Studies on subunit components of immunoglobulin M from a bony fish, the chum salmon (Oncorhynchus keta). Mol. Immunol. 19, 95-103. Legler D. W., Weinheimer P. F., Acton R. T., Dupree H. K. and Russell T. R. (1971) Humoral immune factors in the paddlefish, Polyodon spathula. Comp. Biochem. Physiol. 38B, 523-521. Matsubara A., Mihara S. and Kusuda R. (1985) Quantitation of yellowtail immunoglobulin by enzyme-linked immunosorbent assay (ELISA). Bull. Japan. Sac. Sci. Fish. 51, 921-925. Mor A. and Avtalion R. R. (1990) Transfer of antibody activity from immunized mother to embryo in tilapias. J. Fish Biol. 31, 249-255 Olesen N. J. and Jorgensen P. E. V. (1986) Quantification of serum immunoglobulin in rainbow trout Salmo gairdneri under various environmental conditions. Dis. Aquat. Org. 1, 183-189. Ourth D. D. (1986) Purification and quantitation of channel catfish (Ictalurus puncratus) Immunoglobulin M. J. appl. Ichrhyol. 3, 140-143. Razquin B. E., Castillo A., Lopez-Fierro P., Alvarez F., Zapata A. and Villena A. J. (1990) Ontogeny of IgM-pro-

ducing cells in the lymphoid organs of rainbow trout, Salmo gairdneri Richardson: an immuno- and enzymehistochemical study. J. Fish Biol. 36, 159-173. Richter R.. Frenzel E.-M.. Hadae D.. Koooerschhieer G. and Ambrosius H. (1973) Structural and immunochemical studies on the immunoglobulin of carp (Cyprinus carpio L.) 1. Analysis at the total molecule. Acta Biol. Med. Ger. 30, 735-749. Takahashi Y. and Kawahara E. (1987) Maternal immunity in newborn fry of the ovoviparous guppy. Bull. Japan Sac. Sci. Fish. 53, 721-125. Van Loon J. J. A., Van Oosterom R. and Van Muiswinkel W. B. (1981) Development of the immune system in carp (Cyprinus carpio). In Aspects Of Developmental and Cam parafitle Immunology I (Edited by Solomon J. B.). pp. 469470. Pergamon Press, Oxford. Vilain C., Wetzel M.-C., Du Pasquier L. and Charlemagne J. (1984) Structural and functional analysis of spontaneous anti-nitrophenyl antibodies in three cyprinid fish species: carp (Cyprinus carpio), goldfish (Carassius aurafus) and tenth (Tinca tinca). Dw. camp. Immunol. 8, 61 I-622. Voss E. W. J.. Groberg W. J. J. and Fryer J. L. (1980) Metabolism of coho samon Ig. catabolic rate of coho salmon tetrameric Ig in serum. Mol. Immunol. 17, 445452. Wolf K., Quinby M. C. and Bradford A. D. (1963) Eggassociated transmission of IPN virus of trouts. Virolog) 21, 317-321. I

.-