Developmentaland ComparativeImmunology,Vol. 15, pp. 143-152, 1991 Printed in the USA. All rights reserved.

0145-305X/91 $3.00 + .00 Copyright © 1991 Pergamon Press plc

ISOLATION AND PARTIAL CHARACTERIZATION OF IMMUNOGLOBULIN FROM COD (GADUS MORHUA L.) Lars Pilstr6m* and Annika Petersson Department of Immunology, BMC, Uppsala University, Box 582, S-751 23 Uppsala, Sweden

(SubmittedJanuary 1990;Accepted February 1990) mAbstract--Serum immunoglobulins (CS-Ig) from cod (Gadus morhua L.) were prepared by precipitation with ammonium sulphate and molecular sieving. The molecular weight estimated from molecular sieving and electrophoresis was 851 kD for the whole molecule and 81 and 27.5 kD for the two subunits. This suggests a tetrameric structure of the molecule. The isoelectric point of CS-Ig was determined to approximately pH 5.0. The amino acid composition and absorbancy at 280 nm are very similar to published data of IgM from other f'Lsh species as well as from several mammals. CS-Ig has a natural binding capacity to a number of antigens used for immunization. Assays of antibody activity of fractionated cod serum indicates that CS-Ig does not exist as a monomeric molecule. As the characteristics of CS-Ig are very similar to those found in other fish species, we believe that CS-Ig is an IgMlike molecule.

DKeywords--Cod; Gadus morhua; Immunoglobulin properties; Serum. Introduction

The increasingly polluted marine environment has been shown to affect the defense mechanisms in cod (1). The immune system of cod, however, is not studied and for this r eas on we have begun to study the humoral component. Fish immunoglobulins studied so far (2-4) are mostly similar to the mammalian IgM class although the pentameric structure is not general in teleost fish, but rather, tetramers occur. In some

species, monomeric IgM has also been found (5,6). Immunization of fish results in prod u c t i o n of a n t i b o d i e s with specific binding capacity to the antigen (7-9), but there are also 'natural' antibodies against several commonly used antigens like mammalian blood cells and haptens like DNP or TNP (10,11).

Materials and Methods

Fish and Serum Preparation Cod were caught in the fjord Gullmaren at the Swedish west coast in 1986-1988 by trawling and netting. Blood was collected by making an incision in the bulbous arteriosus of cod anesthetized with 0.05% tricaine methane-sulfonate (MS-222) in sea water, allowed to clot on ice and in a refrigerator for 12-18 h before removal of serum. Serum was centrifuged at 2000 g for 5 min at room temperature and stored either frozen (at - 7 0 ° C) or in a refrigerator. Precipitates are formed in the sera upon storage and it seems that the degree of preci pi t at i on increases after freezing and thawing. Thus, experiments have been performed with sera which have been frozen as well as with unfrozen sera to which sodium azide was added.

Preparation of Cod Immunoglobulin * Author to whom correspondence should be addressed.

Serum was centrifuged at 27,000 g for 30 min at 4-8°C and the precipitate re143

144

moved. Saturated ammonium sulphate (adjusted to pH 7.2-7.4) was added slowly until the serum reached 40-50% saturation with ammonium sulphate and the preparation was centrifuged as described above. The precipitate was dissolved in equal volume of distilled water and phosphate-buffered saline (PBS) described by Hudson and Hay (12). The immunoglobulin fraction was isolated from the dissolved precipitate by gel chromatography on a Sephacryl S-300 HR column (5.0 x 75 cm) and a Sephacryl S-400HR column (5.0 × 40 cm) in series. The columns were equilibrated and eluted with PBS containing 0.2% azide.

L. PilstrOm and A. Petersson

was added and the sample boiled before application to the gel. The HMW- and L M W - M a r k e r Kit (Pharmacia Fine Chemicals, Uppsala) were used as molecular size markers. The gels were run and stained with silver nitrate according to the manufacturer's instructions. For immunostaining, the gels, run as above, were diffusion blotted onto nitrocellulose (Schleicher & Schuell: BA 85) filters and incubated with rabbit polyclonal anti-cod immunoglobulin. The specific binding of antibodies was detected with goat-anti-rabbit-IgG coupled to alkaline phosphatase (Sigma) and visualized after incubation with 5-bromo4-chloro-3-indolyl phosphate (BCIP) and nitroblue tetrazolium (NBT) according to Blake et al. (13).

Test of Purity of Preparations and Estimation of Molecular Weight Isoelectric Point For e s t i m a t i o n of the m o l e c u l a r weight, a Sephacryl S-300HR column (2.6 × 90 cm) was equilibrated with PBS and the void and total volumes were measured with raw dextran (gift from Pharmacia AB, Uppsala, Sweden) and acetone, respectively. It was then run twice with known reference proteins (from Gel Filtration Calibration Kit, Pharmacia Fine Chemicals, Uppsala) and CS-Ig. The flow rate was 60 mL h in all cases. The experiment was repeated in the same way with another, similar column. The obtained preparation of CS-Ig was checked for purity by polyacrylamide gel electrophoresis (PAGE) in a Pharmacia Phast-Gel apparatus under native and reduced conditions using either 10-15% gradient gels for reduced conditions or 4-15% gels (Experimental gel from Pharmacia AB) for native conditions. The sample containing c. 0.2 mg p r o t e i n / m L was m i x e d with e q u a l volume of sample buffer (10 mM trisHCI pH 8,0 1 mM EDTA and 0.01% bromophenol blue), for reduced conditions 2.5% SDS and 5% 13-mercaptoethanol

The isoelectric point of CS-Ig was determined by isoelectric focusing of a sample containing c. 2 mg protein/mL on a thin 1% agarose IEF gel with Pharmalyte 4-6.5, 0.12% sorbitol (w/v) and pl calibration proteins (Pharmacia LKB). The gel was run 4000 Vh (1000V, 15W and 4h) fixed in 5% sulphosalicylic acid/10% trichloroacetic acid, washed overnight, and stained with 0.2% Coomassie Brilliant Blue.

Amino Acid and Protein Analyses For the determination of amino acids, the CS-Ig preparation was rerun on a S-300 and a S-400 column and the purity checked by SDS-PAGE under native and reduced conditions. A sample (35 ~g/100 IxL) of CS-Ig was hydrolyzed at l l0°C for 24 h with 5 mL 6 M HC1 containing 5 p~moles of norleucine and 2 mg/mL of reagent grade phenol in thoroughly evacuated and sealed Pyrex tubes. Half-cystine and methionine were determined as cysteic acid and methionine sulfone, re-

Immunoglobulin from cod

spectively, with a separate sample hydrolyzed in the same way following oxidation with performic acid according to Moore (14). The hydrolysates were analyzed with the LKB Model 4151 Alpha Plus amino acid analyzer using the ninhydrin reagent and hydrolysate program essentially as described in the apparatus manual. The threonine and serine values were corrected 4% and 10%, respectively, for destruction during hydrolysis. The amino acid composition of CS-Ig was compared with those of paddlefishIg (Polyodon spathula), Ig from mammalian species and with unrelated proteins by calculating SAQ as described by Marchalonis (3). In short, the mole percentage of each amino acid of one protein is s u b t r a c t e d from the corresponding figure of the other protein, the difference is squared and the sum estimated giving SAQ. Data for the proteins used in the comparisons were obtained and/or recalculated from Acton et al. (15), Jeske & Capra (16), and from the E M B L and GenBank computer data bases. CS-Ig easily forms aggregates even in high salt solutions, which makes estimations of protein content by measurement of absorbance at 280 nm unreliable unless the absorbance at 310 nm is measured and subtracted from the value obtained at 280 nm. Analysis of protein content was performed with a modified L o w r y method (17) of three different preparations of CS-Ig of known absorbance and with BSA as standard.

Immunization Immunization of mice. For the development of an ELISA for CS-Ig, an antiserum was produced by i.p. immunization of three CBA mice each with 25 p,g/100 IxL CS-Ig (the purest preparation available at that time) in equal volume of Freund's complete adjuvant (FCA). Before and 10 days after the immunization

145

the animals were bled from their tails and the serum tested in E L I S A (see below). The mice were then bled three times at weekly intervals and a booster injection of 20 ~g/100 fxL CS-Ig in Freund's incomplete adjuvant (FIA) was given 48 days after the primary immunization. Three days after the booster, one mouse was killed for preparation of hybridoma cell lines producing monoclonal antibodies (see below). The other two mice were exsanguinated 10 days after the booster in order to obtain a pooled antiserum with high titer.

Immunization of rabbits. Two rabbits were bled from their ear veins in order to obtain preimmune serum and each was then immunized by s.c. injections of 1 mg/0.5 mL CS-Ig (crude preparation from S-300 column) in the same volume of FCA. Three months later, the animals were boosted with the same dose of antigen in FlA. One week after the booster and then three times every second day, blood was drawn from the middle artery of the ear and serum was prepared from the blood. This serum has been used for Western Blots and as secondary antibody in ELISA's.

Enzyme-Linked Immunosorbent Assays ELISA for anti-CS-lg antibodies. Wells of microtiter plates were coated for 3 h at 37°C with 50 IxL of 5 Ixg/mL CS-Ig in phosphate-buffered saline (PBS) containing 0.2% sodium azide. In some experiments, 50 mM carbonate buffer, pH 9.6 was used instead of PBS but any difference in binding could not be demonstrated. The antigen solution was discharged and the residual binding sites were blocked at 4°C overnight with 100 ~L of either 1% ovalbumin or 2% milk casein in PBS with azide. After washing the plates in PBS, rabbit-anti2CS-Ig serum or monoclonal mouse-anti-CS-Ig antibodies diluted in PBS containing

146

L. Pilstr0m and A. Petersson

0.05% Tween-20 and 0.02% azide (PBST) were added to the wells and incubated for 2 h at 37°C. Fifty ~L alkaline phosphatase conjugated Protein A (coupled as described by Heyman et al. (18)) or alkaline phosphatase conjugated goatanti-mouse-IgG (Sigma) appropriately diluted with PBS-T were added to each well and the plates were incubated for 2 h at 37°C. The color reaction was started by addition of I00 txL 1 mg/mL p-nitrophenylphosphate in 0.85 M diethanolamine buffer, pH 9.8, 0.5 mM MgC12 and measured in a Titertec multiscan photometer at 405 nm. The plates were washed three times with PBS between each addition.

ELISA for anti-TNP-antibodies. TNP was coupled to bovine serum albumin (BSA) by adding 5 mg 2,4,6-trinitroben-

zene sulfonic acid per mL to 10 mg/mL BSA in 0.28 M cacodylate buffer, pH 6.9. The sample was left at room temperature for 1 h and then 10 mg glycylglycine was added to each mL reaction mixture. The preparation was dialyzed against three changes of PBS during 3 days. The absorbance at 280 and 340 nm showed a total protein content o'f 5.25 mg/mL and an average of 16.3 TNP-residues per BSA molecule. For ELISA, this preparation was diluted with 50 mM carbonate buffer, pH 9.6, to 15 ~g/mL and 50 ~L per well of this solution was used for coating microtiter plates. After blocking with ovalbumin as for the CS-Ig-ELISA, the plates were used for detection and estimation of c o d - a n t i - T N P - a n t i b o d i e s . Fifty IxL of different dilutions (in PBS-T) of cod serum were added to each well

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Figure 1. Elution diagram of cod serum precipitated with 50% saturation of ammonium sulphate (pH 7.2-7.4) and redissolved in 0.5 x PBS. 5 mL sample was applied to a Sephacryl S-300 column (5.0 x 75 cm) coupled in series with a Sephacryl S-400 column (5.0 x 40 cm) and eluated with PBS at a flow rate of 120 mL h -1. The denoted fraction contained cod immunoglobulin,

Immunoglobulin from cod

147

and incubated overnight at 4°C. The monoclonal mouse-anti-CS-Ig antibody 2A2, (see below) or rabbit anti-CS-Ig serum was added and the plates were incubated for 3 h at 37°C. The monoclonal antibody was detected with goat-antimouse-IgG conjugated with alkaline phosphatase and rabbit antibodies detected with protein A conjugated with the same enzyme. Colour reaction was achieved by addition of p-nitrophenyl phosphate as above. The plates were washed three times with PBS-T between each addition.

s u s p e n d e d in D u l b e c c o ' s M o d i f i e d Eagle's Medium (DMEM) supplemented with 5% fetal calf serum and cultivated together with rat thymocytes as filler cells (20). HAT-selection was used for 10 days of culture when the supernatants were tested in CS-Ig-ELISA. Approximately 10 positive hybridoma cell lines were obtained, two of which have been cloned in order to acertain monoclonality. The antibodies produced by these cell lines, called 2A2 and 1F1, showed precipitation bands in Ouchterlony's immunodiffusion assay against goat-antimouse-IgG but not against goat-antim o u s e - I g M or IgA. The two monoclonals have been used in ELISA and Western Blot experiments.

Monoclonal Antibodies

Spleen cells from a mouse immunized with CS-Ig (see above) were prepared and fused with the nonsecreting fusion partner Sp 2/0 (19) at a ratio 10:1 by means of centrifugation and treatment with polyethyleneglycoll. The cells were

Western Blot

In order to test the binding specificity of the monoclonals, 0.5 mg/mL of CS-Ig

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Polyacrylamide gel electrophoresis and blotting of cod immunoglobulins. CS-Ig was run under native and reduced conditions in a Pharmacia Phast-Gel apparatus and stained with silver nitrate. For immunostaining, the gels were diffusion blotted, incubated with antibodies, and visualized with BCIP and NBT.

148

L. Pilstr6m and A. Petersson

Results and Discussion

was m i x e d w i t h e q u a l v o l u m e o f Laemmli sample buffer (125 mM trisHCI pH 6.8, 4% SDS, 20% glycerol, pyronin Y and 10% 13-mercaptoethanol), boiled and run on a 15% polyacrylamide gel in 0.2% SDS, 0.4 M tris-HC1, pH 8.7, and then blotted onto nitrocellulose filters using 500 Vh in a Bio-Rad transblot apparatus. A strip of the paper was then stained for protein with amidoblack. Immunoblotting was performed with the monoclonal antibodies as well as with polyclonal antibodies from rabbit and mice. The specific binding of antibodies was detected with goat-anti-rabbit-IgG or goat-anti-mouse-IgG coupled to alkaline phosphatase (Sigma) and visualized after incubation in BCIP and NBT according to Blake et al. (13).

Isolation and Purification of Cod hnmunoglobulin For isolation and purification of cod immunoglobulin cod serum was precipitated with saturated ammonium sulphate to 40-50% saturation. The dissolved precipitate was further separated by gelsieving chromatography on a Sephacryl S-300HR and a Sephacryl S-400HR columns coupled in series. Figure 1 shows an elution diagram of cod serum after precipitation with ammonium sulphate. The fraction with a molecular weight of approximately 800-900 kD contained cod serum immunoglobulins (CS-Ig) and was collected for further in-

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Immunoglobulin from cod

149

vestigation. After isolation, the preparation was analyzed for purity by polyacrylamide gel electrophoresis. The two bands in SDS-PAGE under reduced conditions are very similar to L- and Hchain of Ig-M. They appear strongly in SDS-PAGE of whole serum and this together with the gel-sieving experiments show that CS-Ig is a major serum protein. For immunostaining, the native CS-Ig appears as a single band and the reduced CS-Ig as two bands on the nitrocellulosepaper corresponding to the bands observed on the gels (Fig. 2).

Characterization Estimation of molecular weight of CS-Ig was performed using two different methods: molecular sieving on S-300 as well as electrophoresis. Both methods

gave an approximate value of 850 kD. Gel sieving was performed on two different calibrated columns and gave 843 and 867 kD, respectively. Native PAGE gave an average molecular weight of 851 _+ 21 kD (mean _+ S.D., range 821-882 kD). Estimations of molecular weights for the H- and L-chains in SDS-PAGE under reduced conditions gave 81.0 _+ 3.1 and 27.5 __+ 2.0 kD, respectively. Assuming a tetrameric structure of CS-Ig, these data will give a molecular weight of 868 kD for the native protein. Thus, it seems very likely that cod immunoglobulin has a tetrameric structure as in other teleost fish. The isoelectric point of CS-Ig was estimated to be approximately pH 5.0 (4.9-5.2) with isoelectric focusing (Fig. 3). This is in accordance with observations from other species of fish where immunoglobulins are distributed between pH 4-6.5 (21).

Table 1. Amino acid composition end Its statistical difference between Immunoglobulln from cod and some other species as well as some unrelated proteins. MOLE % of amino acids

Alanine Cysteine Aspartic acid & aspargine Glutaminic acid & glutamine Phenylalanine Glycine Histidine Isoleucine Lysine Leucine Methionine Proline Arginine Serine Threonine Valine Tyrosine S-&-Q

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V

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9.1 3.7 7.0 2.0 4.7 6.5 7.1 1.4 4.4 1.7 12.5 9.1 7.5 4.3 15.2

9.1 3.9 6.2 1.8 4.6 6.0 9.1 1.0 6.3 3.7 11.5 9.0 7.4 3.7 13.3

9.5 4.2 6.0 1.7 3.8 4.7 8.7 1.3 6.1 4.1 13.8 9.2 8.3 3.2 23.8

9.6 4.1 6.1 1.8 3.3 5.4 8.8 1.3 6.0 4.0 13.4 9.1 8.7 3.2 22.4

10.3 3.9 6.3 1.4 3.3 5.7 8.4 1.0 6.2 4.3 12.2 9.1 8.6 3.5 15.1

10.2 3.6 6.2 1.4 3.4 5.7 8.2 1.0 6.7 4.1 12.3 9.6 8.3 3.5 20.2

15.6 2.3 6.8 3.8 2.7 3.0 7.6 0.8 5.7 9.1 4.6 8.4 4.9 4.9 92.6

11.1 3.9 6.6 3.5 5.1 3.1 10.9 2.7 4.3 8.9 7.4 7.0 2.7 2.7 161.8

13.3 5.8 2.1 2.6 1.5 10.0 10.5 1.2 4.0 4.4 4.6 4.8 3.1 3.1 192.4

I. Cod immunoglobulin II. Paddlefish immunoglobulin III. Mouse IgM with kappa L-chain IV & V. Rabbit IgM with kappa and lambda L-chain, respectively. VI & VIL Human IgM with kappa and lambda L-chain, respectively. VIII & IX. Human MHC class 2 antigen, alpha and beta chain. X. Human serum albumin.

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150

The total amino acid composition of immunoglobulin from cod is very similar to that from p a d d l e f i s h (Polyodon spathula) (15) as well as IgM from mouse, rabbit, and human (Table 1). An approximate degree of relatedness can be obtained by calculation of the statistical parameter S-A-Q as described by Marchalonis (3). S-A-Q between cod immunoglobulin and the mentioned immunoglobulins and some unrelated proteins are shown in Table I. Among the immunoglobulins, the value is always less than 25 but when compared with unrelated proteins the value is close to or higher than 100. Protein analyses show that 1 mg/mL CS-Ig has (A2s0-A31o) = 1,115, which corresponds to figures for mammalian IgM (12).

Immunological Assays In order to study antibody activity in cod, immunochemical methods have been developed for detection and quantification of CS-Ig. Rabbits have been immunized with CS-Ig for production of polyclonal antibodies against CS-Ig. One of our purest preparations of CS-Ig was used for immunization of mice in order to obtain both immune serum and fusion partners for hybridization of cells for production of monoclonal antibodies. The sera were used for the development of enzyme-linked immunosorbent assays (ELISA), which gave a good signal to noise ratio with enzyme conjugates of either anti-rabbit- or antimouse-antibodies or protein A from Staphylococcus aureus. Thus, protein A does not bind to CS-Ig (Cf. review 22). The mouse-anti-CS-Ig ELISA was used for screening anti-CS-Ig-positive clones in fusion experiments. From the first fus i o n , t w o a n t i b o d y - s e c r e t i n g hybridomas have been cloned. Figure 4 shows a Western Blot of cod immunoglobulin. Anti-CS-Ig-serum from rabbit and mouse and the monoclonal anti-

L. Pilstr0m and A. Petersson

bodies 1F1 and 2A2 were used for detection of the cod immunoglobulin fragments. The monoclonal antibody 1F1 bind to the heavy chain and 2A2 to the light chain in Western Blot. Immunization of cod with different antigens (SRBC-TNP and oxazoloneOA) did not result in elevated amounts of specific antibodies in comparison to unimmunized cod (data not shown). Thus, it seems that the antibodies detected are natural antibodies against the different haptens and proteins and that the immunization for some reason did not result in production of specific antibodies. However, in order to detect if cod serum contain low molecular weight immunoglobulin as reported for some other fish species (6-8), cod serum was run on a gel-sieving column and the fractions analyzed for TNP-binding capacity with ELISA. As seen in Fig. 5, no other

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Figure 4. 'Western Blot' of cod i m m u n o g l o b u l i n f r a c t i o n after Sephacryl S-300 c h r o m a t o g r a p h y . A n t i - C S - I g - s e r u m f r o m rabbit and mouse and the m o n o c l o n a l a n t i b o d i e s 1F1 and 2A2 were used for detection of the cod i m m u n o g l o b u l i n f r a g m e n t s . ( F o r details, see M a t e r i a l and Methods.)

Immunoglobulin from cod

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Isolation and partial characterization of immunoglobulin from cod (Gadus morhua L.).

Serum immunoglobulins (CS-Ig) from cod (Gadus morhua L.) were prepared by precipitation with ammonium sulphate and molecular sieving. The molecular we...
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