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Characterization of IgM of Indian major carps and their cross-reactivity with anti-fish IgM antibodies
Fish & Shellfish Immunology 26 (2009) 275–278
Contents lists available at ScienceDirect
Fish & Shellfish Immunology journal homepage: www.elsevier.com/locate/fsi
Characterization of IgM of Indian major carps and their cross-reactivity with anti-fish IgM antibodies Manas Ranjan Bag, M. Makesh*, K.V. Rajendran, S.C. Mukherjee Central Institute of Fisheries Education, Versova, Andheri West, Mumbai 400061, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 August 2008 Received in revised form 3 November 2008 Accepted 12 November 2008 Available online 27 November 2008
Indian major carps (IMC), rohu (Labeo rohita), catla (Catla catla) and mrigal (Cirrhinus mrigala) were immunized with bovine serum albumin and the serum immunoglobulin M (IgM) was purified by affinity chromatography. The heavy and light chain of IgM of all the three species of IMC were about 88 and 26 kDa, respectively. Anti-fish IgM antibody against all the three species were raised in mice and the reaction of anti-fish IgM antibodies with IgM of all the three species of IMC were studied by Western blot. The anti-fish IgM antibodies reacted strongly with the heavy chain of the same species against which it was raised while the reactions with the heavy chain of other species were milder indicating some degree of epitope sharing among the heavy chains of IgM of IMCs. However, there was no cross-reaction with the light chain of any of the IgM. Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Indian major carps Labeo rohita Catla catla Cirrhinus mrigala Immunoglobulin IgM
1. Introduction Humoral adaptive immunity in fish is mediated by immunoglobulins (Ig), and IgM class is the primary immunoglobulin in most teleost fish. IgM is tetrameric in teleosts and pentameric in elasmobranchs [1]. The majority of carp immunoglobulins have tetrameric structures with a molecular weight of about 760 kDa. Each monomer of the tetrameric Ig contains two heavy (H) chains of molecular weight 70 kDa and two light (L) chains of 25 kDa [2]. Although the H and L chains of different species possess different amino acid sequences, all the H and L chains within an antibody molecule are identical to one another [3]. Cross-reaction of anti-fish antibodies with the immunoglobulins of other species from the same family or different family provides information on their phylogenetic relationship and the level of sensitivity and specificity of anti-fish immunoglobulins. Piscine species of the same taxonomic order were expected to react with anti-Ig sera of each other, confirming similarity in the structure of their antibodies [4]. Anti-fish antibodies against IgM of one species cross-reacting with IgM of other species can be used as a probe to detect and quantify the antibodies in these species. Indian major carps (IMC), consisting of Labeo rohita, Catla catla and Cirrhinus mrigala, are the most widely cultured fish in the
* Corresponding author. Tel.: þ91 22 2636 1446; fax: þ91 22 2636 1573. E-mail address: [email protected] (M. Makesh). 1050-4648/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2008.11.009
Indian subcontinent. Despite their high commercial value, studies on immunoglobulins of Indian major carps are scanty and include isolation and characterization of the immunoglobulins of Labeo rohita [5]. The present study was designed to characterize the immunoglobulins of all the three Indian major carps and detect their cross-reactivity with the anti-fish IgM antibodies. 2. Materials and methods 2.1. Immunization of Indian major carps Thirty each of apparently healthy rohu (Labeo rohita) and catla (Catla catla) weighing about 100–200 g and mrigal (Cirrhinus mrigala) weighing about 50–70 g were obtained from a commercial farm near Mumbai, India. Each species was kept separately in a 1000 l FRP tank with continuous aeration. The fish were fed with pellet feed prepared at the nutrition laboratory of the Central Institute of Fisheries Education, Mumbai, twice daily. About 20% of the water was siphoned out from the bottom and replaced with fresh water daily. The water temperature ranged from 25 to 30 C throughout the experiment. The fish were immunized intraperitoneally with bovine serum albumin (BSA) (Bangalore Genei, India) (200 mg fish1) in Freund’s complete adjuvant (Bangalore Genei, India). The fish were boosted twice at 14 days interval with a similar dose of antigen in Freund’s incomplete adjuvant (Bangalore Genei, India). Two weeks after the second booster the fish were anaesthetized and bled from the caudal vein. The blood was
276
M.R. Bag et al. / Fish & Shellfish Immunology 26 (2009) 275–278
allowed to clot in a 2 ml microcentrifuge tube. After clotting, the blood was stored at 4 C for 4 h. The tubes were centrifuged at 2000 g for 5 min and the clear serum was stored at 80 C. 2.2. Estimation of total protein content of serum The protein concentration of the serum samples were estimated by the Biuret and BCG dye binding method [6], using total protein estimation kit (Qualigens, India). 2.3. Purification of fish immunoglobulin The Ig was purified from 2 ml each of catla, rohu and mrigal sera separately. The serum was clarified, diluted 1:3 with Tris buffered saline (TBS: 0.02 M Tris–HCl; 0.15 M NaCl, pH 7.4) and filtered through a 0.45 mm syringe filter (Millipore, India). The Ig was purified by affinity chromatography using a 5 ml immunoaffinity column (Bangalore Genei, India) containing BSA crosslinked to agarose beads. Before loading the samples, the column was washed with five-bed volumes (25 ml) of washing buffer (TBS) and then stripped with four-bed volumes (20 ml) of elution buffer (0.1 M glycine NaOH, pH 11.0) followed by washing with ten-bed volumes (50 ml) of TBS. The serum was loaded to the column and allowed to pass through the column very slowly. The flow-through was repeatedly added to the column for efficient binding of the Igs to the column. The column was washed with TBS till the absorbance of the flow-through at 280 nm wavelength, was same as that of TBS. The bound Ig in the column was eluted with 10 ml of elution buffer (0.1 M Glycine NaOH, pH 11.0) [7] and the flow-through was collected in 1.5 ml aliquots. The pH of the collected samples was neutralized immediately with 2 M Tris–HCl, pH 2.5. The column was washed with 50 ml washing buffer and stored in 2 ml of washing buffer with 0.05% of sodium azide at 4 C. The absorbance of the fractions was measured in a spectrophotometer at 280 nm. The fractions having absorbance above 0.03 for rohu and catla; and above 0.02 for mrigal were pooled. The pooled fractions were dialyzed against PBS in a cellulose membrane dialysis tubing (Sigma, USA), and concentrated in centriconÒ (Millipore, India) centrifugal filter device with 10 kDa cut off. The protein concentrations of purified samples were estimated by Bradford [8] method, using BSA as the standard. 2.4. Production of anti-fish IgM antibody in mice Each of the purified IgM of catla, rohu and mrigal was emulsified in Freund’s complete adjuvant separately and injected intraperitoneally into two Balb/c mice (40 mg of IgM mice1). The animals were boosted on 14th and 28th day of immunization, intraperitoneally with the same dose of respective IgM emulsified in Freund’s incomplete adjuvant. The mice were bled on the 42nd day of immunization by puncturing the orbital plexus using a capillary tube after anaesthetizing. The blood was allowed to clot at 4 C for 4 h, centrifuged at 2000 g for 5 min and the serum was separated and stored at 80 C. 2.5. Immune response in fish and mice The immune response in the fish and mice was confirmed by Western blotting. BSA (2 mg/ml) was electrophoresed in four lanes along with a protein marker on a 10% polyacrylamide gel. The gel was run initially at 60 V till the tracking dye reached the separating gel and thereafter the voltage was increased to 110 till the dye reached the bottom of the gel. The gel was removed and the lanes pertaining to the marker and one lane of BSA were cut and stained with 0.25% Coomassie Brilliant Blue R-250. The protein bands in the
rest of the unstained gel were transferred to a PVDF membrane using an electroblotter (Technosource, India) at 350 mA constant current for 1.5 h. The membrane was cut into three pieces each pertaining to one lane of BSA. Each membrane was incubated with 1:1000 dilution of one of the IMC serum for 1 h at room temperature (RT). The membranes were washed three times in PBS and incubated with the respective mice anti-fish IgM diluted 1:1000 for 1 h at RT. The membranes were washed three times and incubated with rabbit anti-mice conjugate (1:1000 dilution) (Bangalore Genei, India) for 1 h at RT. The membranes were washed five times and incubated in substrate solution (10 mg diaminobenzidine in 10 ml of PBS containing 10 ml of hydrogen peroxide) for about 10 min in dark. The membranes were washed in tap water and air dried. 2.6. Characterization of fish immunoglobulin 2.6.1. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) SDS-PAGE was carried out according to the method of Bowtell and Sambrook [9] on a 7.5 cm 8.5 cm polyacrylamide gel of 1 mm thickness in a vertical gel electrophoresis apparatus (Technosource, India) in order to determine the types and molecular weight of heavy chains and light chains present in Ig molecules of IMC. Twenty ml of purified Ig of IMC (0.6 mg/ml) and 5 ml of molecular weight markers were electrophoresed on a 10% polyacrylamide gel. The gel was initially run at 60 V till the tracking dye reached the separating gel and thereafter the voltage was increased to 110 till the dye reached the bottom of the gel. The gel was stained with 0.25% Coomassie Brilliant Blue R-250, destained and analyzed using Dolphin-Scan Gel Image System (Wealtec Corp., USA). 2.6.2. Cross-reaction of mice anti-IMC sera SDS-PAGE of three sets of purified IgM of rohu, catla and mrigal was carried out along with molecular weight markers. Initially the gel was electrophoresed at 60 V till the tracking dye reached the separating gel and the voltage was increased to 100 V till the tracking dye reached the bottom of the gel. The lane pertaining to molecular weight markers was cut, stained with Coomassie brilliant blue and destained. Protein bands in the unstained gel were transferred to PVDF membrane by electroblotting. Blotting was carried out in an electroblotting apparatus (Technosource, India) at a constant current of 350 mA for 1.5 h. After the transfer, the PVDF membrane was cut into three pieces each having a lane of rohu, catla and mrigal IgM. The membranes were washed with methanol for 3 s, dried at RT and blocked with blocking buffer (1% BSA in PBS) for 1 h at RT. The membranes were incubated with one of the mice anti-IMC IgM serum (1:1000) for 1 h at RT, washed three times in washing buffer (0.25% BSA in PBS) and incubated with rabbit antimice conjugate (1:1000 in washing buffer) (Bangalore Genei, India) for 1 h at RT. The membranes were further washed five times and incubated with substrate solution (10 mg of diaminobenzidine in 10 ml of PBS containing 10 ml of hydrogen peroxide) for about 10 min. The membranes were washed with tap water and air-dried. After the final washing the membranes were observed for any reactions.
Table 1 Total protein and IgM content of immunized IMC sera. Species
Total serum protein (mg ml1)
Total IgM (mg ml1)
IgM as % of total serum protein
Rohu Catla Mrigal
28.94 28.66 26.45
0.330 0.315 0.255
1.14 1.10 0.96
M.R. Bag et al. / Fish & Shellfish Immunology 26 (2009) 275–278
L1
L2
L3
L4
L5
97.4 kDa
277
IgM purified from 1 ml of rohu, catla and mrigal serum and their percentage in total serum proteins are given in Table 1. 3.3. Immune response in fish and mice
66.0 kDa
Western blotting of BSA with immunized fish serum and mice anti-fish IgM serum gave a specific band at 66 kDa pertaining to BSA for all the three species of IMC indicating specific immune response in fish against BSA and in mice against fish IgM (Fig. 1).
43.0 kDa
3.4. Characterization of fish immunoglobulin 29.0 kDa
3.4.1. SDS-PAGE of purified IMC immunoglobulin The affinity purified immunoglobulin of IMC in SDS-PAGE revealed two bands (Fig. 2). For rohu, catla and mrigal the heavy chain bands were estimated to be about 88 kDa and the light chain bands were of molecular weight of about 26 kDa.
20.1 kDa
3.4.2. Cross-reaction of mice anti-IMC IgM sera The cross-reactivity and specificity of mice anti-IMC IgM sera was studied by Western blot technique. The mice anti-rohu IgM sera reacted strongly with heavy chain of rohu IgM, while its reaction with heavy chain of catla and mrigal IgM was milder. The mice anti-rohu IgM sera also reacted with the light chain of rohu IgM but not with that of catla and mrigal. The mice anti-catla IgM sera gave a similar reaction as that of mice anti-rohu IgM sera. The mice anti-mrigal IgM sera reacted strongly against the heavy chain mrigal IgM, while its reaction with the heavy chain of catla and rohu IgM was weak. The mice anti-mrigal IgM sera did not react with the light chain of any of the three species (Fig. 3).
Fig. 1. Western blot showing the Immune response in fish and mice. L1 – BSA. L2 – Molecular weight marker. L3 – BSA Western blotted with rohu serum. L4 – BSA Western blotted with catla serum. L5 – BSA Western blotted with mrigal serum.
3. Results 3.1. Serum protein content The total serum protein content of immunized rohu, catla and mrigal are given in Table 1. 3.2. Purification of immunoglobulin During each purification process, a single peak was obtained in the elution with 0.1 M glycine NaOH buffer, pH 11.0. The quantity of
L1
97.4 kDa
L2
4. Discussion The BSA specific serum IgM concentration of IMC ranged from 0.255 to 0.33 mg/ml of serum. The BSA specific IgM concentration reported for rohu weighing 500 g is 1.41 mg/ml of serum [5] and for Asiatic catfish weighing 100–125 g it is 0.15 mg/ml of serum [7].
L3
L4
H chain (88 kDa)
66.0 kDa
43.0 kDa
29.0 kDa L chain (26 kDa)
20.1 kDa
Fig. 2. SDS-PAGE for purified IgM of IMC. L1 – Molecular weight marker. L2 – Rohu IgM. L3 – Catla IgM. L4 – Mrigal IgM.
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M.R. Bag et al. / Fish & Shellfish Immunology 26 (2009) 275–278
L1
L2
L3
L4
97.4 kDa
L5
L6
L7
L8
L9
L10 H chain (88 kDa)
66.0 kDa
43.0 kDa
29.0 kDa
L chain (26 kDa)
20.1 kDa
14.3 kDa Fig. 3. Western blot of purified IgM with mice anti-IMC IgM sera. L1 – Marker. L2, L3 and L4 – Rohu, catla and mrigal IgM, respectively, immunoblotted with mice anti-mrigal IgM sera. L5, L6 and L7 – Rohu, catla and mrigal IgM, respectively, immunoblotted with mice anti-catla IgM sera. L8, L9 and L10 – Rohu, catla and mrigal IgM, respectively, immunoblotted with mice anti-rohu IgM sera.
The serum Ig level increases with the increase in the age of the fish [10] and the serum Ig level in this study is in proportion with the report for rohu, taking the size of the fish into consideration. The SDS-PAGE of rohu, catla and mrigal Ig showed two bands, indicating the presence of a single heavy chain and a single light chain. Single heavy and light chains were also reported in Japanese eel [11]; chum salmon [12]; Atlantic salmon [13]; sea bass [14]; gilthead sea bream [15] and Atlantic cod [16]. The heavy chains of rohu, catla and mrigal were estimated to be about 88 kDa and the light chains were about 26 kDa. The heavy chain and light chain reported earlier for rohu are 85 and 23 kDa, respectively [5]. In general, the molecular weight of H and L chains varies from 60–77 to 23–26 kDa, respectively for most of the osteichthyan fishes but the higher molecular weight H chains, as found in Ig of IMCs, has also been observed in some other species such as cod (81 kDa) [16]; tilapia (90 kDa) [17] and barramundi (86 kDa) [18]. The bony fish Ig has been proved to be tetrameric [(H2L2)4] in nature by electron microscopic studies [19–21]. Assuming the tetrameric structure of IMC immunoglobulins, an 88 kDa heavy chain and a 26 kDa light chain would give a theoretical value of 912 kDa for Ig of IMC. A non-denaturing PAGE also gave a molecular weight for rohu IgM as 908 kDa (results not shown). However, the molecular weight of IgM of rohu reported earlier is 850 kDa [5]. The anti-IMC IgM sera reacted strongly with the heavy chain of their respective IgM while their reaction to IgM of other species was comparatively milder indicating that there is epitope sharing to some extent among the heavy chains of IgM of all the three species of IMC. The results of cross-reactions in the present investigation support the hypothesis that the fish species in the same taxonomic order are expected to react with anti-Ig sera of each other [4], as the IMC belongs to same family Cyprinidae. Similar patterns of cross-reaction have been reported earlier. However, contrary to the findings of Israelsson et al. [4] that the light chain might be more conserved than the heavy chain, the present study reveals no cross-reaction between the light chains. Since there is sharing of epitopes among the heavy chains of IgM of all the three species of IMC, monoclonal antibodies produced against any of the common epitopes should be useful in detecting and quantifying the immune response in these species.
References [1] Voss EW, Sigel MM. Valence and temporal change in affinity of purified 7S and 18S nurse shark anti-2, 4-dinitrophenyl antibodies. J Immunol 1972;109:665–73.
[2] Vesely T, Reschova S, Pokorova D, Hulova J, Nevorankova Z. Production of monoclonal antibodies against immunoglobulin heavy chain in common carp (Cyprinus carpio L.). Veterinarni Medicina 2006;51:296–302. [3] Iwama G, Nakanishi T. The fish immune system: organism, pathogen, and environment. San Diego, USA: Academic Press; 1996. p. 159–242. [4] Israelsson O, Petersson A, Bengten E, Wiersma EJ, Andersson J, Gezelius G, et al. Immunoglobulin concentration in Atlantic cod, Gadus morhua L., serum and cross-reactivity between anti-cod-antibodies and immunoglobulins from other species. J Fish Biol 1991;39:265–78. [5] Suresh Babu PP, Shankar KM, Honnananda BR, Vijaya Kumaraswamy HV, Prasanna Shama K, Suryanarayana VVS, et al. Isolation and characterization of immunoglobulin of the Indian major carp, rohu [Labeo rohita (Ham.)]. Fish Shellfish Immunol 2008;24:779–83. [6] Reinhold JG. Manual determination of serum total protein, albumin and globulin fractions by biuret method. In: Reiner M, editor. Standard method of clinical chemistry. New York: Academic Press; 1953. p. 88. [7] Swain T, Mohanty J, Sahu AK. One step purification and partial charaterisation of serum immunoglobulin from Asiatic catfish (Clarius batrachus L.). Fish Shellfish Immunol 2004;17:397–401. [8] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem 1976;72:248–54. [9] Bowtell D, Sambrook J. DNA microarrays: a molecular cloning manual. New York: Cold Spring Harbor Laboratory Press; 2002. [10] Zilberg D, Klesius PH. Quantification of immunoglobulin in the serum and mucus of channel catfish at different ages and following infection with Edwardsiella ictaluri. Vet Immunol Immunopathol 1997;58:171–80. [11] Uchida D, Hirose H, Chang PK, Aranishi F, Hirayabu E, Mano N, et al. Characterization of Japanese eel immunoglobulin M and its level in serum. Comp Biochem Physiol 2000;127B:525–32. [12] Kobayashi K, Hara A, Takano K, Hirai H. Studies on subunit components of immunoglobulin M from a bony fish, the chum salmon (Oncorhynchus keta). Mol Immunol 1982;19:95–103. [13] Havarstein LS, Aasjord PM, Ness S, Endresen C. Purification and partial characterization of an IgM-like serum immunoglobulin from Atlantic salmon (Salmo salar). Dev Comp Immunol 1988;12:773–85. [14] Scapigliati G, Romano N, Picchietti S, Mazzini M, Mastrolia L, Scalia D, et al. Monoclonal antibodies against sea bass, Dicentrachus labrax (L.) immunoglobulins: immunolocalisation of immunoglobulin-bearing cells and applicability in immunoassays. Fish Shellfish Immunol 1996;6:383–401. [15] Palenzuela O, Sitja`-Bobadilla A, A´lvarez-Pellitero P. Isolation and partial characterization of serum immunoglobulins from sea bass (Dicentrarchus labrax L.) and gilthead sea bream (Sparus aurata L. Fish Shellfish Immunol 1996;6:81–94. [16] Pilstro¨m L, Petersson A. Isolation and partial characterization of immunoglobulin from cod (Gadus morhua L.). Dev Comp Immunol 1991;15:143–52. [17] Rajavarthini PB, Arunkumar R, Michael RD. Partial characterization of serum immunoglobulins of Oreochromis mosambicus. Ind J Exp Biol 2000;38:549–53. [18] Crosbie PBB, Nowak BF. Production of polyclonal antisera against barramundi (Lates calcarifer Bloch) serum immunoglobulin derived from affinity columns containing mannan-binding protein or staphylococcal protein A. Aquaculture 2002;211:49–63. [19] Shelton E, Smith M. The ultrastructure of carp (Cyprinus carpio) immunoglobulin: a tetrameric macroglobulin. J Mol Biol 1970;54:615–6. [20] Warr GW. Immunoglobulin of the toadfish, Spheroides glaber. Comp Biochem Physiol 1983;76B:507–14. [21] Whittington RJ. Purification and partial characterization of serum immunoglobulin of the European perch (Perca fluviatilis L.). Fish Shellfish Immunol 1993;3:331–43.
Contents lists available at ScienceDirect
Fish & Shellfish Immunology journal homepage: www.elsevier.com/locate/fsi
Characterization of IgM of Indian major carps and their cross-reactivity with anti-fish IgM antibodies Manas Ranjan Bag, M. Makesh*, K.V. Rajendran, S.C. Mukherjee Central Institute of Fisheries Education, Versova, Andheri West, Mumbai 400061, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 August 2008 Received in revised form 3 November 2008 Accepted 12 November 2008 Available online 27 November 2008
Indian major carps (IMC), rohu (Labeo rohita), catla (Catla catla) and mrigal (Cirrhinus mrigala) were immunized with bovine serum albumin and the serum immunoglobulin M (IgM) was purified by affinity chromatography. The heavy and light chain of IgM of all the three species of IMC were about 88 and 26 kDa, respectively. Anti-fish IgM antibody against all the three species were raised in mice and the reaction of anti-fish IgM antibodies with IgM of all the three species of IMC were studied by Western blot. The anti-fish IgM antibodies reacted strongly with the heavy chain of the same species against which it was raised while the reactions with the heavy chain of other species were milder indicating some degree of epitope sharing among the heavy chains of IgM of IMCs. However, there was no cross-reaction with the light chain of any of the IgM. Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Indian major carps Labeo rohita Catla catla Cirrhinus mrigala Immunoglobulin IgM
1. Introduction Humoral adaptive immunity in fish is mediated by immunoglobulins (Ig), and IgM class is the primary immunoglobulin in most teleost fish. IgM is tetrameric in teleosts and pentameric in elasmobranchs [1]. The majority of carp immunoglobulins have tetrameric structures with a molecular weight of about 760 kDa. Each monomer of the tetrameric Ig contains two heavy (H) chains of molecular weight 70 kDa and two light (L) chains of 25 kDa [2]. Although the H and L chains of different species possess different amino acid sequences, all the H and L chains within an antibody molecule are identical to one another [3]. Cross-reaction of anti-fish antibodies with the immunoglobulins of other species from the same family or different family provides information on their phylogenetic relationship and the level of sensitivity and specificity of anti-fish immunoglobulins. Piscine species of the same taxonomic order were expected to react with anti-Ig sera of each other, confirming similarity in the structure of their antibodies [4]. Anti-fish antibodies against IgM of one species cross-reacting with IgM of other species can be used as a probe to detect and quantify the antibodies in these species. Indian major carps (IMC), consisting of Labeo rohita, Catla catla and Cirrhinus mrigala, are the most widely cultured fish in the
* Corresponding author. Tel.: þ91 22 2636 1446; fax: þ91 22 2636 1573. E-mail address: [email protected] (M. Makesh). 1050-4648/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2008.11.009
Indian subcontinent. Despite their high commercial value, studies on immunoglobulins of Indian major carps are scanty and include isolation and characterization of the immunoglobulins of Labeo rohita [5]. The present study was designed to characterize the immunoglobulins of all the three Indian major carps and detect their cross-reactivity with the anti-fish IgM antibodies. 2. Materials and methods 2.1. Immunization of Indian major carps Thirty each of apparently healthy rohu (Labeo rohita) and catla (Catla catla) weighing about 100–200 g and mrigal (Cirrhinus mrigala) weighing about 50–70 g were obtained from a commercial farm near Mumbai, India. Each species was kept separately in a 1000 l FRP tank with continuous aeration. The fish were fed with pellet feed prepared at the nutrition laboratory of the Central Institute of Fisheries Education, Mumbai, twice daily. About 20% of the water was siphoned out from the bottom and replaced with fresh water daily. The water temperature ranged from 25 to 30 C throughout the experiment. The fish were immunized intraperitoneally with bovine serum albumin (BSA) (Bangalore Genei, India) (200 mg fish1) in Freund’s complete adjuvant (Bangalore Genei, India). The fish were boosted twice at 14 days interval with a similar dose of antigen in Freund’s incomplete adjuvant (Bangalore Genei, India). Two weeks after the second booster the fish were anaesthetized and bled from the caudal vein. The blood was
276
M.R. Bag et al. / Fish & Shellfish Immunology 26 (2009) 275–278
allowed to clot in a 2 ml microcentrifuge tube. After clotting, the blood was stored at 4 C for 4 h. The tubes were centrifuged at 2000 g for 5 min and the clear serum was stored at 80 C. 2.2. Estimation of total protein content of serum The protein concentration of the serum samples were estimated by the Biuret and BCG dye binding method [6], using total protein estimation kit (Qualigens, India). 2.3. Purification of fish immunoglobulin The Ig was purified from 2 ml each of catla, rohu and mrigal sera separately. The serum was clarified, diluted 1:3 with Tris buffered saline (TBS: 0.02 M Tris–HCl; 0.15 M NaCl, pH 7.4) and filtered through a 0.45 mm syringe filter (Millipore, India). The Ig was purified by affinity chromatography using a 5 ml immunoaffinity column (Bangalore Genei, India) containing BSA crosslinked to agarose beads. Before loading the samples, the column was washed with five-bed volumes (25 ml) of washing buffer (TBS) and then stripped with four-bed volumes (20 ml) of elution buffer (0.1 M glycine NaOH, pH 11.0) followed by washing with ten-bed volumes (50 ml) of TBS. The serum was loaded to the column and allowed to pass through the column very slowly. The flow-through was repeatedly added to the column for efficient binding of the Igs to the column. The column was washed with TBS till the absorbance of the flow-through at 280 nm wavelength, was same as that of TBS. The bound Ig in the column was eluted with 10 ml of elution buffer (0.1 M Glycine NaOH, pH 11.0) [7] and the flow-through was collected in 1.5 ml aliquots. The pH of the collected samples was neutralized immediately with 2 M Tris–HCl, pH 2.5. The column was washed with 50 ml washing buffer and stored in 2 ml of washing buffer with 0.05% of sodium azide at 4 C. The absorbance of the fractions was measured in a spectrophotometer at 280 nm. The fractions having absorbance above 0.03 for rohu and catla; and above 0.02 for mrigal were pooled. The pooled fractions were dialyzed against PBS in a cellulose membrane dialysis tubing (Sigma, USA), and concentrated in centriconÒ (Millipore, India) centrifugal filter device with 10 kDa cut off. The protein concentrations of purified samples were estimated by Bradford [8] method, using BSA as the standard. 2.4. Production of anti-fish IgM antibody in mice Each of the purified IgM of catla, rohu and mrigal was emulsified in Freund’s complete adjuvant separately and injected intraperitoneally into two Balb/c mice (40 mg of IgM mice1). The animals were boosted on 14th and 28th day of immunization, intraperitoneally with the same dose of respective IgM emulsified in Freund’s incomplete adjuvant. The mice were bled on the 42nd day of immunization by puncturing the orbital plexus using a capillary tube after anaesthetizing. The blood was allowed to clot at 4 C for 4 h, centrifuged at 2000 g for 5 min and the serum was separated and stored at 80 C. 2.5. Immune response in fish and mice The immune response in the fish and mice was confirmed by Western blotting. BSA (2 mg/ml) was electrophoresed in four lanes along with a protein marker on a 10% polyacrylamide gel. The gel was run initially at 60 V till the tracking dye reached the separating gel and thereafter the voltage was increased to 110 till the dye reached the bottom of the gel. The gel was removed and the lanes pertaining to the marker and one lane of BSA were cut and stained with 0.25% Coomassie Brilliant Blue R-250. The protein bands in the
rest of the unstained gel were transferred to a PVDF membrane using an electroblotter (Technosource, India) at 350 mA constant current for 1.5 h. The membrane was cut into three pieces each pertaining to one lane of BSA. Each membrane was incubated with 1:1000 dilution of one of the IMC serum for 1 h at room temperature (RT). The membranes were washed three times in PBS and incubated with the respective mice anti-fish IgM diluted 1:1000 for 1 h at RT. The membranes were washed three times and incubated with rabbit anti-mice conjugate (1:1000 dilution) (Bangalore Genei, India) for 1 h at RT. The membranes were washed five times and incubated in substrate solution (10 mg diaminobenzidine in 10 ml of PBS containing 10 ml of hydrogen peroxide) for about 10 min in dark. The membranes were washed in tap water and air dried. 2.6. Characterization of fish immunoglobulin 2.6.1. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) SDS-PAGE was carried out according to the method of Bowtell and Sambrook [9] on a 7.5 cm 8.5 cm polyacrylamide gel of 1 mm thickness in a vertical gel electrophoresis apparatus (Technosource, India) in order to determine the types and molecular weight of heavy chains and light chains present in Ig molecules of IMC. Twenty ml of purified Ig of IMC (0.6 mg/ml) and 5 ml of molecular weight markers were electrophoresed on a 10% polyacrylamide gel. The gel was initially run at 60 V till the tracking dye reached the separating gel and thereafter the voltage was increased to 110 till the dye reached the bottom of the gel. The gel was stained with 0.25% Coomassie Brilliant Blue R-250, destained and analyzed using Dolphin-Scan Gel Image System (Wealtec Corp., USA). 2.6.2. Cross-reaction of mice anti-IMC sera SDS-PAGE of three sets of purified IgM of rohu, catla and mrigal was carried out along with molecular weight markers. Initially the gel was electrophoresed at 60 V till the tracking dye reached the separating gel and the voltage was increased to 100 V till the tracking dye reached the bottom of the gel. The lane pertaining to molecular weight markers was cut, stained with Coomassie brilliant blue and destained. Protein bands in the unstained gel were transferred to PVDF membrane by electroblotting. Blotting was carried out in an electroblotting apparatus (Technosource, India) at a constant current of 350 mA for 1.5 h. After the transfer, the PVDF membrane was cut into three pieces each having a lane of rohu, catla and mrigal IgM. The membranes were washed with methanol for 3 s, dried at RT and blocked with blocking buffer (1% BSA in PBS) for 1 h at RT. The membranes were incubated with one of the mice anti-IMC IgM serum (1:1000) for 1 h at RT, washed three times in washing buffer (0.25% BSA in PBS) and incubated with rabbit antimice conjugate (1:1000 in washing buffer) (Bangalore Genei, India) for 1 h at RT. The membranes were further washed five times and incubated with substrate solution (10 mg of diaminobenzidine in 10 ml of PBS containing 10 ml of hydrogen peroxide) for about 10 min. The membranes were washed with tap water and air-dried. After the final washing the membranes were observed for any reactions.
Table 1 Total protein and IgM content of immunized IMC sera. Species
Total serum protein (mg ml1)
Total IgM (mg ml1)
IgM as % of total serum protein
Rohu Catla Mrigal
28.94 28.66 26.45
0.330 0.315 0.255
1.14 1.10 0.96
M.R. Bag et al. / Fish & Shellfish Immunology 26 (2009) 275–278
L1
L2
L3
L4
L5
97.4 kDa
277
IgM purified from 1 ml of rohu, catla and mrigal serum and their percentage in total serum proteins are given in Table 1. 3.3. Immune response in fish and mice
66.0 kDa
Western blotting of BSA with immunized fish serum and mice anti-fish IgM serum gave a specific band at 66 kDa pertaining to BSA for all the three species of IMC indicating specific immune response in fish against BSA and in mice against fish IgM (Fig. 1).
43.0 kDa
3.4. Characterization of fish immunoglobulin 29.0 kDa
3.4.1. SDS-PAGE of purified IMC immunoglobulin The affinity purified immunoglobulin of IMC in SDS-PAGE revealed two bands (Fig. 2). For rohu, catla and mrigal the heavy chain bands were estimated to be about 88 kDa and the light chain bands were of molecular weight of about 26 kDa.
20.1 kDa
3.4.2. Cross-reaction of mice anti-IMC IgM sera The cross-reactivity and specificity of mice anti-IMC IgM sera was studied by Western blot technique. The mice anti-rohu IgM sera reacted strongly with heavy chain of rohu IgM, while its reaction with heavy chain of catla and mrigal IgM was milder. The mice anti-rohu IgM sera also reacted with the light chain of rohu IgM but not with that of catla and mrigal. The mice anti-catla IgM sera gave a similar reaction as that of mice anti-rohu IgM sera. The mice anti-mrigal IgM sera reacted strongly against the heavy chain mrigal IgM, while its reaction with the heavy chain of catla and rohu IgM was weak. The mice anti-mrigal IgM sera did not react with the light chain of any of the three species (Fig. 3).
Fig. 1. Western blot showing the Immune response in fish and mice. L1 – BSA. L2 – Molecular weight marker. L3 – BSA Western blotted with rohu serum. L4 – BSA Western blotted with catla serum. L5 – BSA Western blotted with mrigal serum.
3. Results 3.1. Serum protein content The total serum protein content of immunized rohu, catla and mrigal are given in Table 1. 3.2. Purification of immunoglobulin During each purification process, a single peak was obtained in the elution with 0.1 M glycine NaOH buffer, pH 11.0. The quantity of
L1
97.4 kDa
L2
4. Discussion The BSA specific serum IgM concentration of IMC ranged from 0.255 to 0.33 mg/ml of serum. The BSA specific IgM concentration reported for rohu weighing 500 g is 1.41 mg/ml of serum [5] and for Asiatic catfish weighing 100–125 g it is 0.15 mg/ml of serum [7].
L3
L4
H chain (88 kDa)
66.0 kDa
43.0 kDa
29.0 kDa L chain (26 kDa)
20.1 kDa
Fig. 2. SDS-PAGE for purified IgM of IMC. L1 – Molecular weight marker. L2 – Rohu IgM. L3 – Catla IgM. L4 – Mrigal IgM.
278
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L1
L2
L3
L4
97.4 kDa
L5
L6
L7
L8
L9
L10 H chain (88 kDa)
66.0 kDa
43.0 kDa
29.0 kDa
L chain (26 kDa)
20.1 kDa
14.3 kDa Fig. 3. Western blot of purified IgM with mice anti-IMC IgM sera. L1 – Marker. L2, L3 and L4 – Rohu, catla and mrigal IgM, respectively, immunoblotted with mice anti-mrigal IgM sera. L5, L6 and L7 – Rohu, catla and mrigal IgM, respectively, immunoblotted with mice anti-catla IgM sera. L8, L9 and L10 – Rohu, catla and mrigal IgM, respectively, immunoblotted with mice anti-rohu IgM sera.
The serum Ig level increases with the increase in the age of the fish [10] and the serum Ig level in this study is in proportion with the report for rohu, taking the size of the fish into consideration. The SDS-PAGE of rohu, catla and mrigal Ig showed two bands, indicating the presence of a single heavy chain and a single light chain. Single heavy and light chains were also reported in Japanese eel [11]; chum salmon [12]; Atlantic salmon [13]; sea bass [14]; gilthead sea bream [15] and Atlantic cod [16]. The heavy chains of rohu, catla and mrigal were estimated to be about 88 kDa and the light chains were about 26 kDa. The heavy chain and light chain reported earlier for rohu are 85 and 23 kDa, respectively [5]. In general, the molecular weight of H and L chains varies from 60–77 to 23–26 kDa, respectively for most of the osteichthyan fishes but the higher molecular weight H chains, as found in Ig of IMCs, has also been observed in some other species such as cod (81 kDa) [16]; tilapia (90 kDa) [17] and barramundi (86 kDa) [18]. The bony fish Ig has been proved to be tetrameric [(H2L2)4] in nature by electron microscopic studies [19–21]. Assuming the tetrameric structure of IMC immunoglobulins, an 88 kDa heavy chain and a 26 kDa light chain would give a theoretical value of 912 kDa for Ig of IMC. A non-denaturing PAGE also gave a molecular weight for rohu IgM as 908 kDa (results not shown). However, the molecular weight of IgM of rohu reported earlier is 850 kDa [5]. The anti-IMC IgM sera reacted strongly with the heavy chain of their respective IgM while their reaction to IgM of other species was comparatively milder indicating that there is epitope sharing to some extent among the heavy chains of IgM of all the three species of IMC. The results of cross-reactions in the present investigation support the hypothesis that the fish species in the same taxonomic order are expected to react with anti-Ig sera of each other [4], as the IMC belongs to same family Cyprinidae. Similar patterns of cross-reaction have been reported earlier. However, contrary to the findings of Israelsson et al. [4] that the light chain might be more conserved than the heavy chain, the present study reveals no cross-reaction between the light chains. Since there is sharing of epitopes among the heavy chains of IgM of all the three species of IMC, monoclonal antibodies produced against any of the common epitopes should be useful in detecting and quantifying the immune response in these species.
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