Development of an Indirect Enzyme-Linked Immunoabsorbent Assay Using a Monoclonal Antibody to Identify Ictalurus Sp. Fillets

Aquaculture Research, 2005, 36, 1279^1284 doi:10.1111/j.1365-2109.2005.01329.x

Development of an indirect enzyme-linked immunoabsorbent assay using a monoclonal antibody to identify Ictalurus sp. ﬁllets

Shawn T McNulty & Phillip H Klesius USDA, ARS Aquatic Animal Health Research Laboratory,Auburn, AL, USA

Correspondence: S T McNulty, USDA, ARS Aquatic Animal Health Research Laboratory, PO Box 952, Auburn, AL 36831 USA. E-mail: [email protected]

Abstract crease in the supply of a lower cost substitute caused the pond-bank prices of channel cat¢sh to drop sub- The deceptive marketing of imported basa, Pangasius stantially.The US cat¢sh industry lobbied for a provi- bocourti (Sauvage), ¢llets as cat¢sh has resulted in sion in the 2001 farm bill that permited only ¢sh of serious economic losses to the channel cat¢sh, Icta- the Ictaluridae family to be labelled as ‘cat¢sh’. How- lurus punctatus (Ra¢nesque), industry in the US. The ever,a spokesman for the Foodand Drug Administra- similarappearance of channel cat¢sh and basa ¢llets tion (FDA) stated that the FDA does not have the created a need for a rapid method to di¡erentiate un- money to enforce the law, especially since there is cooked, cooked and/or marinated channel cat¢sh no easy way to di¡erentiate between species (Mercer ¢llets from basa ¢llets and other ¢sh products. A 2002). Previously monoclonal antibodies (MAbs) monoclonal antibody (MAb) speci¢c for a 36.8 kDa have been used to distinguish between di¡erent spe- channel cat¢sh ¢llet protein was produced and char- cies of seafood. An, Klein, Kao, Marshall, Otwell and acterized by an indirect enzyme-linked immunoab- Wei (1990) developed an MAb that could detect the sorbent assay (ELISA) and Western blotting. This presence of rock shrimp, Sicyonia brevirostris (Stimp- MAb was used to develop an indirect ELISA speci¢c son), as low as 4.3 ng sample 1 on an average in a for a ¢llet protein unique to ¢sh of the genus Icta- sample mixture containing various seafood or meat lurus. Using this ELISA, 100% of raw and cooked samples. Huang, Marshall and Wei (1995) developed channel cat¢sh ¢llets were correctly identi¢ed and two murine MAbs that could not only identify red di¡erentiated from other ¢sh in a single-blind study. snapper, Lutjanus campechanus (Poey), from 24 var- These results show that the indirect ELISA using ious seafood extracts but could also di¡erentiate red MAbs speci¢c for unique Ictalurus sp. ¢llet proteins snapper from vermilion snapper, Rhomboplites auror- is a rapid and sensitive method for the identi¢cation ubens (Cuvier), lane snapper, L. synagris (Linnaeus), of raw and cooked cat¢sh ¢llets. mutton snapper, L. analis (Cuvier) and yellowtail snapper, Ocyurus chrysurus (Bloch). The purpose of Keywords: channel cat¢sh, basa, Vietnamese im- this study was to determine if an MAb against a ports, monoclonal antibody channel cat¢sh protein could be developed, and to of- fer a rapid method to identify channel cat¢sh ¢llets Introduction and di¡erentiate them from other ¢sh. The importationand marketing of basa (Pangasius bocourti)aschannelcat¢sh(Ictalurus punctatus)¢llets have caused economic losses to the US channel cat- Materials and methods ¢sh industry (Quagrainie & Engle 2002). In 2001, Antigen preparation imported Vietnamese ¢sh ¢llets captured 12% of the US frozen ¢llet market from the sale of 17 million Previously frozen ¢sh ¢llets were purchased from pounds of ¢llets (The Cat¢sh Journal 2002). This in- several local seafood stores in Auburn, AL, USA. Each

r 2005 Blackwell Publishing Ltd 1279 Identi¢cation of cat¢sh ¢llets S T McNulty & P H Klesius Aquaculture Research, 2005, 36, 1279^1284

¢sh species sample contained at least ¢ve ¢sh. Blue cat¢sh, I. furcatus (Valenciennes); hybrid cat¢sh, I. punctatus I. furcatus;basa,P. bocourti;wildstriped bass, Morone saxatilis (Walbaum); red snapper L. campechanus; Chilean sea bass, Dissostichus eleginoides (Smitt); £ounder, Paralichthys dentatus L.; Nile tilapia, Oreochromis niloticus L.; sword¢sh Xiphias gladius L and pre-seasoned lemon-herb channel cat¢sh, I. punctatus, ¢llets were purchased. Channel cat¢sh (NWAC-103) from USDA-ARS stocks were used as the authentic cat¢sh sample. The ¢llets were then cut into small pieces, placed in whirlpak bags and stored at 20 1C until needed. Fish tissue samples were placed in 50 mL centrifuge tubes and spun at 3500 g for 15min. The supernatant was collected and the tissue was discarded. The supernatant (0.33 mg mL 1) was loaded into a readymade isoelectric focusing (IEF) gel (BioRad, Hercules, CA, USA) to compare the protein pro¢les of all ¢sh tested (data not shown). The isoelectric points of the proteins were determined using pI standards (BioRad) (Fig. 1). Channel cat¢sh proteins were then eluted from the gel and were used to immunize three BALB/c mice.

Isoelectric focusing electrophoresis and Figure 1 Isoelectric focusing pro¢les of water-soluble determination of protein pI values proteins from channel cat¢sh and basa. The arrow indi- cates the proteins used to immunize BALB/c mice. Lane An IEF Ready Gel (BioRad) with a pH gradient of 1, channel cat¢sh extract; lane 2, basa extract. 3^10 was used to compare the protein pro¢les of the ¢sh.The pI values of the proteins were determined by bands were then eluted using an elution bu¡er (3.8 g comparing the bands of the ¢sh proteins with those Tris, 13.6 g glycine, 1.4 g SDS, 1L distilled water) by of known IEF standards (BioRad). Protein standards running a Centriluter micro-electroeluter (Amicon, containing phycocyanin (three bands), pI 4.45, 4.65, CT, USA) at 3 W and 7 mA for 1.5 h. Bicinchoninic 4.75; b-lactoglobulin B, pI 5.1; bovine carbonic anhy- acid (BCA) protein analysis was performed on eluted drase, pI 6.0; human carbonic anhydrase, pI 6.5; samples to determine the ¢nal protein concentration. equine myoglobin (two bands), 6.8, 7.0; human hae- moglobin A, pI 7.1; human hemoglobin C, pI 7.5; le nt i l lectin (three bands), pI 7.8,8.0,8.2 and cytochrome c, Monoclonal antibody production pI 9.6 were included.The pI of the proteins was calcu- lated using Quantity One Version 4.1 software from The eluted proteins were suspended at a ratio of 1:1 BioRad. with Freund’s complete adjuvant (Sigma, St Louis, MO, USA), and 200 mL(33mg protein mouse 1)was administered to three mice by intraperitoneal and Identi¢cation and puri¢cation of unique subcutaneous injection. This was repeated ¢ve times channel cat¢sh proteins at 21-day intervals with the same dosage; however, The channel cat¢sh proteins used to immunize mice for the subsequent injections, the immunogen was were eluted from the IEF gel. The gel was run at100 V mixed 1:1 (v/v) with Freund’s incomplete adjuvant for 1h, 250 V for 1h and 500 V for 30 min. The gels (F-5506, Sigma). Mouse serum from tail bleedings were then stained with IEF Gel Staining Solution was screened for the presence of anti-channel cat¢sh (BioRad). New gels were run following the same pro- ¢llet antibodies by enzyme-linked immunoabsorbent cedure but were not stained. The areas that corre- assay (ELISA). The supernatant was collected from sponded to the desired bands were cut out. The frozen ¢llets for all species as above and was used to

1280 r 2005 Blackwell Publishing Ltd, Aquaculture Research, 36, 1279^1284 Aquaculture Research, 2005, 36, 1279^1284 Identi¢cation of cat¢sh ¢llets S T McNulty & P H Klesius coat microtitre plates (Becton-Dickinson, Lincoln raw and cooked ¢sh ¢llets. Randomly selected ¢llet Park, NJ, USA) with 100 mLofa100mg mL 1 protein samples were coded and randomized to mask the solution of ¢llet supernatant in sodium carbonate buf- identity of the ¢llet ¢sh and the proteins were ex- fer (pH 9.6) for 1h at room temperature. Plates were tracted by a second technician. The ELISA procedure washed ¢ve times with phosphate-bu¡ered saline was then performed with the coded samples. The re- (PBS) with 0.05% Tween-20 (PBST) (pH 7.2). Plates activity of the MAb with protein extracts from raw were blocked with 1% non-fat-dry milk in PBST for channel cat¢sh, basa, blue cat¢sh, hybrid cat¢sh, 30 min at room temperature, washed as above and wild striped bass, red snapper, Chilean sea bass, 50 mLwell 1 of the mouse serum was added to the £ounder, Nile tilapia, sword¢sh and a pre-seasoned plate and incubated at room temperature for 30 min. lemon-herb channel cat¢sh ¢llet was determined. A Plates were washed. Peroxidase-conjugated rabbit 25 g sample of each ¢sh ¢llet was placed in a 50 mL anti-mouse IgG (BioRad) was diluted 1:5000 in PBST; centrifuge tube and centrifuged at 3500 g for15min. 10 0 mL was added to each well and incubated at room The supernatant was collected and used for analysis. temperature for15min and washed as above. Fifty mL Microtitre plates were coated with100 mgof¢lletpro- of 3,30,5,5 0-tetramethylbenzidine (TMB) (Pierce Bio- tein in 100 mL in sodium carbonate bu¡er for 1h at technology,Rockford, IL, USA) was added to each well room temperature. The wells were blocked with 1% and incubated at room temperature for15min (Shelby, non-fat dry milk for 30 min, and a 1:32000 dilution Shoemaker & Klesius 2002).The reaction was stopped of ascites-produced MAb was added to each well. For with 50 mLof3MH2SO4, and the optical density was the blind study of cooked ¢sh, ¢llets were baked at read at 450 nm with an ELISA reader.The mouse that 170 1C for 20 min. A 25 g sample of the cooked ¢llet had the highest titres against the channel cat¢sh ¢llet was placed in a 50 mL centrifuge tube and 5 mL of extract and low titres against a basa ¢llet extract was water was added. The tubes were centrifuged at selected for hybridoma production (Harlow & Lane 3500 g for15min. Again,100 mLwas analysed by ELI- 1988). Hybridomas were screened for channel cat¢sh SA. These were the only modi¢cations made to the speci¢cityas above. Clone1A10was chosen for utiliza- previously described protocol. tion and further characterization.

Dot-blot Molecular size and isotype determination The Bio-Dot SF micro¢ltration apparatus (BioRad) Channel cat¢sh proteins were separated by sodium do- was used to demonstrate the reactivity of the MAb decyl sulphate polyacrylamide gel electrophoresis with the target protein on a nitrocellulose membrane. (Laemmli1970) and transferred to nitrocellulose mem- Channel cat¢sh and basa ¢llet proteins were applied branes byWestern blot as described byTowbin, Straehe- to the membrane at a rate of 500 mLofa0.01mg mL 1 lin and Gordon (1979). Nitrocellulose membranes were solution. The diluted proteins were allowed to absorb blocked with1% non-fat dry milk in PBST for 30 min at to the membrane for15min and then ¢ltered through room temperature.The membranes were washed three the membrane by gentle vacuum. Wells were then times for 5 min each in PBST.The membranes were in- washed three times with 500 mL of PBST, blocked cubated in supernatant from the anti-channel cat¢sh with Superblock (BioRad) for 15min and the mem- MAb for 30 min at room temperature. The membranes branes were washed as above. The anti-cat¢sh MAb were washed as above and a 1:5000 dilution of peroxi- supernatant was added to each well and incubated dase-conjugated rabbit anti-mouse in PBST was added for 10min. The supernatant was removed and the to the membrane. The blot was washed, and bound membrane was washed. A 1:5000 peroxidase-conju- antibody was detected with 4-chloronapthol. Molecu- gated rabbit anti-mouse antiserum (Pierce) was lar weights were determined by comparing the protein added to each well for10min.The reaction was visua- bands with Precision Plus protein standards (BioRad). lized by the addition of 4-chloronapthol to each well. The isotype of clone 1A10 was determined using goat anti-mouse isotype antibodies (Sigma). Statistical analysis

Data for this experiment were statistically analysed Single-blind study by one-way analysis of variance. Duncan multiple A single-blind test using indirect ELISAwas used to range tests were used to determine signi¢cant di¡er- determine the speci¢cityof the MAb for proteins from ences (Po0.05) in optical density.

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Results and discussion Fish ¢llet proteins resolved by isoelectric focusing in- dicated two proteins unique to ¢sh from the genus Ic- talurus with isoelectric points of 5.92 and 5.79 (Fig.1). These two unique proteins were used to produce hybridomas that secreted anti-channel cat¢sh protein antibodies. During the screening process, nine of the original 703 seeded cultures were found to produce antibodies that showed four to eight times higher reactivity for channel cat¢sh protein than for the basa proteins as determined by ELISA. Clone 1A10 was chosen for utilization and characterization in

ELISA.The MAb belonged to the IgG1isotype as determined by the isotyping kit. Western blotting of the clone 1A10 showed only one positive reaction area, which corresponded to a 36.8 kDa channel cat¢sh protein (Fig. 2). There was no reaction with basa proteins. To determine whether any cross-reactivity occurred, the MAb was tested byWestern blot and ELISA against seven unre- lated ¢sh species, two other ictalurids (blue and hybrid cat¢sh) and pre-seasoned channel cat¢sh (lemon-herb, purchased frozen). The MAb showed si- milar reactivity with the blue and hybrid cat¢sh ¢llet Figure 2 Western blot of the anti-cat¢sh antibody. The proteins, as well as the lemon-herb cat¢sh ¢llet pro- molecular weight standard (Std.) is a precision plus pre- teins, indicating that this MAb was highly speci¢c for stained molecular weight standard (BioRad). Channel cat- ¢sh (CCF) and basa extracts were prepared as previously ¢llet proteins from ¢sh of the genus Ictalurus (Fig. 3). described.

Raw samples 0.9 * * Cooked samples 0.8 * * 0.7

0.6

0.5

0.4 ** ** Optical density (450 nm) Optical density (450 0.3 **

0.2 **

0.1

0 Channel Blue Hybrid Lemon- Basa Flounder Red Sea bass Striped Swordfish Tilapia catfish catfish catfish herb snapper bass catfish

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No cross-reactivity was observed with proteins from lose membrane. Virtually no cross-reactivity oc- other ¢sh species. curred between the channel cat¢sh and basa ¢llets. Proteins from raw Ictalurus sp. ¢llets were equally reactive with the MAb and were correctly identi¢ed from eleven various ¢sh ¢llet protein samples in the Conclusion blind study using raw ¢llets.When the resultant optical densities from the reactivity between the anti-cat- The US cat¢sh industry experienced a dramatic de- ¢sh antibodyand proteins from raw and cooked ¢llets cline in ¢sh prices due to the in£ow of cheaper sub- were analysed by Duncans multiple range test, all op- stitutes from foreign markets, with a signi¢cant per- tical density readings for ictalurid ¢llet proteins were centage of the US market being captured beginning in 2001. To combat this problem, regulations have signi¢cantly higher (Po0.05) than for all other ¢sh proteins tested (Fig. 3, Table 1). The reactivity of the been promulgated which allow only ¢sh of the Icta- MAb for raw Ictalurus ¢llet proteins was seven times luridae family to be labelled as ‘cat¢sh’. However, no higher than that of the other ¢sh samples.The colour rapid, reliable and economical test for species identi- development was so intense that visual inspection ¢cation was available. Therefore, we developed an as- could be used for identi¢cation (data not shown). say that could rapidly and e⁄ciently identify The absorbance levels for the cooked Ictalurus ¢llets Ictalurus sp. ¢llets and di¡erentiate these ¢llets were two times higher than for all other ¢sh ¢llets from other ¢sh products. To this end, we developed a tested. MAb that identi¢es Ictalurus sp. based on a unique To demonstrate the reactivity of the MAb, a dot- 36.8 kDa protein. The anti-channel cat¢sh MAb cor- blot was performed. Proteins from a channel cat¢sh rectly di¡erentiated proteins from both raw and ¢llet and a basa ¢llet were absorbed onto a nitrocellu- cooked Ictalurus sp. ¢llets from ¢llet proteins from other ¢sh species tested. The anti-channel cat¢sh MAb can be used at ports of entry,supermarkets and Table 1 Reactivity of the 1A10 MAb with the extracts of restaurants to ensure proper labelling of ¢llets. raw and cooked ¢sh ¢llets in the blind study using ELISA

Optical density Acknowledgments Unknown sample Raw samples Cooked samples The authors would like to thank Dr Richard Shelby,

Ã Channel catfish 0.725 0.056a 0.290 0.008x Mr Roger Bridgman and the sta¡ at the Auburn Uni- Blue catfish 0.748 0.042a 0.308 0.034x versity Hybridoma Laboratory for their technical as- Hybrid catfish 0.796 0.061a 0.351 0.024x sistance. Also, the authors would like to thank Drs a y Lemon-herb catfish 0.757 0.104 0.187 0.015 Les Torrans and Kevin Huggins for reviewing this cd z Basa 0.124 0.005 0.080 0.011 manuscript. Use of trade name or commercial pro- Flounder 0.099 0.009d 0.088 0.044z Red snapper 0.104 0.009d 0.074 0.008z ducts is solely for the purpose of providing speci¢c in- Sea bass 0.093 0.009d 0.066 0.011z formation and does not imply endorsement by USDA. Striped bass 0.097 0.011d 0.065 0.014z Swordfish 0.103 0.010d 0.059 0.005z Tilapia 0.190 0.009b 0.073 0.011z References ÃDenotes signi¢cant di¡erence in Duncan’s multiple range test grouping. AnH.,KleinP.A.,KaoK.J.,MarshallM.R.,OtwellW.S.&Wei MAb, monoclonal antibody; ELISA, enzyme-linked immunosor- C.I. (1990) Development of a monoclonal antibody for rock bent assay. shrimp identi¢cation using ELISA. Journal of Agriculture and Food Chemistry 38, 2094^2100. Harlow E. & Lane D. (1988) Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, Figure 3 Reactivity of the monoclonal antibody to chan- USA. nel cat¢sh ¢llet with the extracts of raw and cooked ¢sh Huang T.S., Marshall M.R. & Wei C.I. (1995) Identi¢cation of ¢llets in a blind study using indirect ELISA. ÃSigni¢cantly red snapper using electrophoretic techniques. Journal of di¡erent (Po0.05) between raw samples as determined by Food Science 60,279^283. Duncan multiple range test. ÃÃSigni¢cantly di¡erent Laemmli U.K. (1970) Cleavage of structural proteins during (Po0.05) between cooked samples as determined by Dun- assembly of the head of bacteriophage T4. Nature 277, can multiple range test. 680^685.

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Mc Call M. (ed.) (2002) New bill signed; contains cat¢sh la- Shelby R.A., Shoemaker C.A. & Klesius P.H. (2002) Detection beling provision. Cat¢shJournal16, 1^2. of humoral response to Streptococcus iniae infection Mc Call M. (ed.) (2003) Commerce department sets higher of Nile tilapia, Oreochromis niloticus, by a monoclonal tari¡s onVietnam ¢sh imports. Cat¢shJournal17, 1, 16. antibody-based ELISA. Journal of Applied Aquaculture 12, Mercer D. (2002) Southern cat¢sh farmers watch Vietna- 23^31. mese ¢sh imports for false cat¢sh label. Arkansas Demo- Towbin H., Straehelin T. & Gordon J. (1979) Electrophoretic crat-Gazette156,6A. transfer of proteins from polyacrylamide gels to nitrocel- Quagrainie K.K. & Engle C.R. (2002) Analysis of cat¢sh pri- lulose sheets: procedure and some applications. Proceed- cing and market dynamics: the role of imported cat¢sh. ings of the National Academy of Science 76, 4350^4354. Journal of theWorld Aquaculture Society 33,389.

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