HYBRIDOMA Volume 11, Number 3, 1992
Mary
Ann
Liebert, Inc., Publishers
Monoclonal Antibodies Specific to Sailfish Serum Albumin: Development of an Assay for the Identification of Fish Species in the Field EDMUND A.
ROSSI, SCOT R. SHEPARD, JAMES C. POYER, and JAMES X. HARTMANN
Department of Biological Sciences, Florida Atlantic University,
Boca Raton, Florida 33431
ABSTRACT
Balb/c mice were immunized with albumin purified from sailfish ÍIstiophorus albicansl serum. Hybridomas were produced and screened by ELISA for reactivity with the purified albumins of sailfish, blue marlin (Makaira niqricans) and white marlin (Tetrapturus albidus). Monoclonal antibodies (MAbs) from 16 different clones exhibited Thirteen of the MAbs showed activity against sailfish albumin. Three MAbs exhibited cross-reactivity with the marlin species. distinct specificity for sailfish albumin. One of these species specific MAbs (M2D1) was conjugated to horseradish peroxidase (HRP) in order to construct an ELISA for identification of sailfish from The ELISA for sailfish correctly identified eight sailfish serum. from 26 billfish serum samples. The MAb-peroxidase conjugate was highly specific toward sailfish in that no reaction against heterologous species
was
detected.
INTRODUCTION
Billfishes,
members of the family Istiophoridae, are among the marine fishes and are highly prized by sportfishermen and commercial fisheries alike. The survival of billfish stocks may depend largely on the ability to accurately identify these species in the field (1). Three closely related billfishes, sailfish, blue marlin and white marlin, cannot be easily distinguished after commercial processing (2). Since commercial long-lining activities account for 90% of the billfish catch (3), and the processed carcasses cannot be accurately identified, catch statistics are often inaccurate. Accurate statistics are necessary for the proper management of the billfish fishery. The electrophoretic techniques currently used for species identification of fish tissues (4,5,6,7) cannot be used in the field. Agencies, such as the National Marine Fisheries Services need a field-portable billfish identification kit to enforce current legislation regarding the capture and transport of billfishes and to improve the reliability of catch statistics.
largest
One approach to provide fisheries biologists and legal authorities with a means of positively identifying processed billfish carcasses is the production of field-portable immunoassay kits. Serum albumin, a readily purified, highly immunogenic protein was considered for use in species identification. As one of the most rapidly evolving proteins, albumin has been used in numerous taxonomic, systematic and evolutionary studies (8,9). Schill and Dorazio (10) employed polyclonal anti-sera against albumin to discriminate between stocks of striped bass (Morone saxatilis). This communication describes the production of SFA-specific
333
monoclonal antibodies (MAbs) that have been used in an immunoassay to distinguish the sera of sailfish from that of blue and white marlin. MATERIALS and METHODS
Albumin Purification Albumin from sailfish (SFA), blue marlin (BMA) and white marlin (WMA) were purified from serum following the methods of Hoch and Chanutin (11) with adjustments recommended by Ray Simon (12). Serum was diluted 1:2 with distilled water, and caprylic acid was added to 0.2 M. The solution was incubated for 90 min at 63°C, and An equal then clarified by centrifugation at 10,000 X g (4°C) volume of saturated ammonium sulfate was added to the supernatant while maintaining pH 7.4. After one hour at 4°C, the solution was centrifuged at 10,000 X g. The supernatant was dialyzed extensively against distilled water and then lyophilized. .
Immunization Schedule
Hybridomas were generated according to the techniques of Goldsby (13) and Harlow and Lane (14). Female Balb/c mice were immunized intraperitoneally with 100 µg of purified SFA emulsified in 500 µ Dulbecco's phosphate buffered saline (Grand Island Biological Co., Grand Island, NY) and 500 µ Freund's complete adjuvant (Sigma Chemical Co., St. Louis, MO). On days 14 and 28 post immunization, mice were boosted with SFA emulsified in Freund's Six weeks after incomplete adjuvant, as described above. immunization, sera were assayed for anti-SFA activity by ELISA. When an anti-SFA antibody titre greater than 5000 was achieved, the mouse was given a final boost via a tail vein with 50µg of purified SFA in 80µ1 PBS. Fusion Three days after the final boost, the activated splenocytes fused to X-63 myelomas using procedures outlined by Harlow and Lane (14). The resulting hybridomas were suspended in 100 ml of HY/HAT (Sigma) medium supplemented with 20% fetal bovine serum The P-388 (FBS)/(Sigma) and 5% macrophage conditioned medium. macrophage tumor line was grown to confluency in HY medium supplemented with 2% FBS, then activated with lipopolysaccharide to The hybridoma supply the cytokine-rich conditioned medium. suspension was plated at 100 µ /well into five 96 well tissue culture plates (Corning Inc., Corning, NY). Additionally five plates of a 1:2 dilution and ten plates of a 1:4 dilution of the were
original suspension
were
prepared.
Post Fusion We used the Bio-Rad Clone Selector Mouse Monoclonal
Antibody (Bio-Rad Laboratories, Richmond, Ca) to screen for anti-SFA activity 10 days post fusion. Corning high binding ELISA plates were coated with one microgram of SFA per well. The ELISA plates were read with a Bio-Tek microplate auto-reader set at 405 nm (Bio-Tek Instruments, Winooski, VT). Samples that tested positive for anti-SFA activity (O.D. > 0.1) were screened for crossreactivity using ELISA plates coated with BMA and WMA. Samples that were specific for SFA were cloned by limiting dilution at 0.3 cells/well. Clone M2D1 was selected for further study. Screening
Kit
Purification and Isotyping of Anti-SFA MAb MAbs from the sailfish-specific clone M2D1 that had been grown cell culture and as an ascites tumor in Balb/C mice were Further precipitated with 50% saturated ammonium sulfate. purification was accomplished using a goat anti-mouse IgG agarose The column was (GAMA) affinity chromâtography column (Sigma). equilibrated with 0.01 M sodium phosphate, 0.5 M NaCl buffer pH 7.5 and the antibody eluted with 0.1 M glycine, 0.15 M NaCl buffer pH 2.5. The isotype of the M2D1 MAb was resolved using the Immunotype Mouse MAb Isotyping Kit (Sigma).
in
334
Conjugation of SFA-MAb to HRP
Immuno-affinity purified M2D1 MAbs were conjugated to Immunopure Activated Peroxidase (Pierce Chemical Co., Rockford, IL) according to the manufacturer's protocol. The conjugate was termed
M2D1-HRP.
Assay of Billfish Sera Sera of ten blue marlin, eight white marlin, and eight sailfish (maintained at -20°C) were diluted 1:5000 in Tris-buffered saline pH 7.4 (TBS) and 100 µ aliquote of each was adsorbed to
three wells of an ELISA plate for one hour. All procedures were conducted at room temperature. The plate was blocked for 30 min with a 1% BSA solution and then washed 3 times with 0.05% (v/v) Tween-20 in TBS (TTBS). The M2D1-HRP conjugate was diluted 1:2000 in antibody buffer (0.5% BSA in TTBS) and 100 µ were incubated in the antigen-coated wells for one hour. After washing 5 times with of peroxidase substrate TTBS, 100 µ (3,3',5,5' tetramethyl benzidine) solution (Pierce) were added to each well. After five minutes, the reaction was stopped with IM H3PO„ and the plate was read spectrophotometrically at 450 nm.
RESULTS and DISCUSSION
Albumin Purification Billfish serum yielded of albumin using the method. purification 0.5
mg/ml
Hydroxyapatite-HPLC of the chemically
analysis purified albumin revealed one major and two minor peaks (Fig. 1). The major peak was identified as albumin by comparison of its retention time with that of a bovine serum albumin standard. Albumin has several characteristics that make it
practical
as
identification
a
species
marker
in
immunoassays: ample quantities of antigen can be readily
obtained, it is heat stabile, well characterized, and is present in billfish red muscle
FIGURE 1. Hydroxyapatite-HPLC of Purified Saüfish Albumin. The major peak was identified as serum albumin by comparison to bovine serum albumin.
(15). Hartmann and coworkers (16) attempted sailfish identification by employing polyclonal antisera against sarcoplasmic proteins. This method proved to be inadequate due to cross-reaction with other billfish species. When the antiserum was purified using an affinity chromatography column constructed of blue and white marlin sarcoplasmic proteins, reactivity with sailfish proteins was lost. extracts
Hybridoma Production Of the 16 hybridoma-containing wells that demonstrated antithree did not exhibit cross-reactivity with BMA or WMA (Table 1). One of the three, 21G12, that appeared to consist of a single colony was chosen for its high level of reactivity to SFA. Limiting dilution of 21G12 yielded clone M2D1. The MAbs produced by M2D1 were used in the following experiments. Table 1 shows several MAbs with strong reactivity to both SFA and marlin albumins. These MAbs demonstrate the existence of common epitopes on the albumins of these closely related species. MAbs such as 12A4 are being tested as capture antibodies to be used in conjunction with the species specific MAbs of M2D1 in a sandwich ELISA (17). This format can be used for the identification of sailfish from tissue samples or other biological fluids with low concentrations of albumin. SFA
activity,
335
TABLE 1
Reactivity of Supernatant Fluids from Wells Containing Hybridomas that Produced Antibodies Against Sailfish Albumin. Well
Sailfish Albumin
Blue & White
Well
Sailfish Albumin
Blue & White Marlin Albumin
17A5
0.151
0.055
0.351
0.337
Marlin Albumin 22H1
0.157
0.156
0.117
0.000
8B11
0.139
0.191
21G1
0.207
0.010
23G12
0.227
0.301
21G12
0.355
0.000
3E1
0.374
0.274
21H1
0.386
0.305
18A6
0.150
0.053
21H2
0.278
0.201
18G7
0.339
0.183
21H3
0.123
0.088
18G8
0.292
0.121
2C3
0.120
0.000
Values are optical densities from an alkaline phosphatase-based ELISA. Only wells with antibodies that showed a value of >0.100 versus sailfish albumin were indicated in the table.
Purification. Isotyping and Conjugation of M2D1 Mabs
Affinity purified MAbs from clone M2D1, at a protein concentration of 6 mg/ml, maintained anti-SFA activity (O.D. > 0.100) up to a dilution of 1:50,000. The M2D1 MAbs were shown to be of the mouse IgGl isotype. The M2D1-HRP conjugate retained its activity toward SFA at a dilution of 1:2000. Assay of Billfish Sera The M2D1-HRP conjugate accurately identified the sera of eight sailfish from 26 billfish samples (Figure 2). Furthermore, the conjugate did not show any reactivity with the sera of eight white marlin or that of ten blue marlin. Serum samples were tested in triplicate, with O.D. (450nm) readings ranging from 0.245 to 0.847 for the sailfish samples. The replicate readings for each sample were consistent and differed by no more than 0.040 O.D. units. The largest value recorded for any marlin serum sample replicate was 0.008. In this assay, optimal results were obtained when the serum samples were diluted 1:5000 (data not shown). At dilutions of less than 1:1000, O.D. readings were markedly reduced and inconsistent due to antigen excess. Only 26 samples were available for testing at the time of this study because of the rarity of these species and the tag and release practices of most sportiishermen. All samples were obtained from the Western Atlantic Ocean. The M2D1-HRP conjugate accurately distinguished between sailfish or non-sailfish for all available samples. However, it has been determined if not the will conjugate identify sailfish from O.D. 450 nm oceans. other The conjugate, as well as other MAbs derived from this study, may allow the determination of the number of species or stocks of sailfish that exist. This has been a fisheries of concern since the biologists existence of locationspecific stocks selected through evolution may be ABCDEFOH I JKLMNOPQRSTUVWXYZ essential in maintaining Serum Sample certain local fisheries. The current immunoassay could be used FIGURE 2. Reactivity of M2D1-HRP Conjugate with Billfish Sera by field biologists to in an ELISA. A-J are blue marlin sera; K-R are sailfish sera; S-Z are identify processed sail— white marlin sera. O.D. is the average of triplicate readings.
_
336
fish
carcasses that were caught in the Western Atlantic Ocean. Up 80 samples could be tested in less than two hours. Modifications of this assay can be made to reduce the assay time to approximately 30 minutes. As discussed above, capture antibodies could be employed to facilitate the assay and allow the use of tissues such as red muscle. Adaptation of the assay to a paper format would further reduce the assay time. These modifications are being investigated by this laboratory.
to
ACKNOWLEDGMENTS
We are grateful to Dr. E. Prince for providing the majority of the billfish samples. Dr. R. Waldner for his assistance throughout the project and Dr. D. Binninger for his critical review. We are grateful to Dr. M. Flajnik for the X-63 and P-388 cell lines. This work was supported by The Billfish Foundation through The International Commission for the Conservation of Atlantic Tunas. Mr. Rossi was awarded the Andrew J. Boehm Grant from The American Fishing Tackle Manufacturers Association in the amount of $5000.
REFERENCES
1.
Conser,
R.J. 1985. The status of world billfish stocks. In: World
Angling and Resources and Challenges. R.H. Stroud, ed. Proc. First World Angling Conf., Cap d'Agde, France. Inter. Game Fish Assoc,
Lauderdale, FL. 291-308. Prince, E. Personal communication. NMFS Southeast Region. Virginia Key, FL. 3. King, D.M. 1989. Economic trends affecting commercial billfish fisheries. In: Planning the Future of Billfishes, part 1, R.H. Stroud, ed. National Coalition for the Marine Conservation Inc., Savannah, GA. 89-102. 4. Lundstrom, R.C. 1980. Fish species identification by thin layer polyacrylamide gel isoelectric focusing: Collaborative study. Ft.
2.
J.A.O.A.C. 53: 7-9.
Lundstrom, R.C. 1981. Fish species identification by isoelectric focusing: Sarcoplasmic protein polymorphism in monkfish Lophius
5.
americanuBl. J.A.O.A.C. 64: 38-43. 6. Lundstrom, R.C. 1981. agarose gel isoelectric J.A.O.A.C. 64: 38-43. 7. Lundstrom, R.C. 1983.
Rapid fish species identification by focusing of sarcoplasmic proteins.
Identification of Pacific rockfish (Sebastesl species by isoelectric focusing. J.A.O.A.C. 66: 974-980. 8. Lowenstein, J.M. 1985. Molecular approaches to the identification of species. Amer. Sci. 73: 541-547. 9. Sarich, V.M. 1969. Pinniped origins and the rate of evolution of carnivore albumins. Syst. Zool. 18: 286-295. 10. Schill, W.B. and R.M. Dorazio. 1990. Immunological discrim¬ ination of Atlantic striped bass stocks. Trans. Am. Fish. Soc. 119: 77-85. 11. Hoch,
H.
and
A.
Chanutin.
1954.
Albumin
from
plasma: preparation and electrophoretic properties. Biophys. 51: 271-276.
12. Simon, R. Personal Kearnesville, WV.
Communication.
Fish
heated human Arch. Biochem.
Health
Laboratory.
13. Goldsby, R.A. 1989. A practical guide to making hybridomas. In: Nucleic acid and monoclonal antibody probes. Applications in diagnostic microbiology. B. Swaminathan and G.Prakash, eds. Mercel Dekker Inc., New York, NY. 367-381. 14. Harlow,E. and D. Lane. 1988. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, USA. 53-312. 15. Hartmann, J.X., R.E. Waldner, J.C. Poyer and E.A. Rossi. 1990. Albumin as a biomarker for serological identification of billfish (Istiophoridae) carcasses. Proc. 41st Ann. Tuna Conf. C.H. Boggs (ed). S.W. Fisheries Center NMFS-NOAA Administrative report 11-9007. p. 8. 16. Hartmann, J.X. and R.E. Waldner. 1990. Isolation of biomarker proteins from blue marlin, white marlin, and sailfish from the Western Atlantic Ocean. In: Planning the Future of Billfish, Part 2, R.H. Stroud, ed. National Coalition for Marine Conservation Inc., Savannah, GA. 231-237.
337
17. Abbas, . ., . . Lichtman and J.S. Molecular Immunology. W.B. Saunders Co.
Pober. 1991. Cellular and PA. 60-62.
Philadelphia
Address
reprint requests
to:
James X. Hartmann
Department of Biological Sciences Florida Atlantic Boca
Phone Number:
Received for publication: 12/9/91 after revision: 1/28/92
Accepted
338
University
Raton, FL 33431
(407) 367-3334
Mary
Ann
Liebert, Inc., Publishers
Monoclonal Antibodies Specific to Sailfish Serum Albumin: Development of an Assay for the Identification of Fish Species in the Field EDMUND A.
ROSSI, SCOT R. SHEPARD, JAMES C. POYER, and JAMES X. HARTMANN
Department of Biological Sciences, Florida Atlantic University,
Boca Raton, Florida 33431
ABSTRACT
Balb/c mice were immunized with albumin purified from sailfish ÍIstiophorus albicansl serum. Hybridomas were produced and screened by ELISA for reactivity with the purified albumins of sailfish, blue marlin (Makaira niqricans) and white marlin (Tetrapturus albidus). Monoclonal antibodies (MAbs) from 16 different clones exhibited Thirteen of the MAbs showed activity against sailfish albumin. Three MAbs exhibited cross-reactivity with the marlin species. distinct specificity for sailfish albumin. One of these species specific MAbs (M2D1) was conjugated to horseradish peroxidase (HRP) in order to construct an ELISA for identification of sailfish from The ELISA for sailfish correctly identified eight sailfish serum. from 26 billfish serum samples. The MAb-peroxidase conjugate was highly specific toward sailfish in that no reaction against heterologous species
was
detected.
INTRODUCTION
Billfishes,
members of the family Istiophoridae, are among the marine fishes and are highly prized by sportfishermen and commercial fisheries alike. The survival of billfish stocks may depend largely on the ability to accurately identify these species in the field (1). Three closely related billfishes, sailfish, blue marlin and white marlin, cannot be easily distinguished after commercial processing (2). Since commercial long-lining activities account for 90% of the billfish catch (3), and the processed carcasses cannot be accurately identified, catch statistics are often inaccurate. Accurate statistics are necessary for the proper management of the billfish fishery. The electrophoretic techniques currently used for species identification of fish tissues (4,5,6,7) cannot be used in the field. Agencies, such as the National Marine Fisheries Services need a field-portable billfish identification kit to enforce current legislation regarding the capture and transport of billfishes and to improve the reliability of catch statistics.
largest
One approach to provide fisheries biologists and legal authorities with a means of positively identifying processed billfish carcasses is the production of field-portable immunoassay kits. Serum albumin, a readily purified, highly immunogenic protein was considered for use in species identification. As one of the most rapidly evolving proteins, albumin has been used in numerous taxonomic, systematic and evolutionary studies (8,9). Schill and Dorazio (10) employed polyclonal anti-sera against albumin to discriminate between stocks of striped bass (Morone saxatilis). This communication describes the production of SFA-specific
333
monoclonal antibodies (MAbs) that have been used in an immunoassay to distinguish the sera of sailfish from that of blue and white marlin. MATERIALS and METHODS
Albumin Purification Albumin from sailfish (SFA), blue marlin (BMA) and white marlin (WMA) were purified from serum following the methods of Hoch and Chanutin (11) with adjustments recommended by Ray Simon (12). Serum was diluted 1:2 with distilled water, and caprylic acid was added to 0.2 M. The solution was incubated for 90 min at 63°C, and An equal then clarified by centrifugation at 10,000 X g (4°C) volume of saturated ammonium sulfate was added to the supernatant while maintaining pH 7.4. After one hour at 4°C, the solution was centrifuged at 10,000 X g. The supernatant was dialyzed extensively against distilled water and then lyophilized. .
Immunization Schedule
Hybridomas were generated according to the techniques of Goldsby (13) and Harlow and Lane (14). Female Balb/c mice were immunized intraperitoneally with 100 µg of purified SFA emulsified in 500 µ Dulbecco's phosphate buffered saline (Grand Island Biological Co., Grand Island, NY) and 500 µ Freund's complete adjuvant (Sigma Chemical Co., St. Louis, MO). On days 14 and 28 post immunization, mice were boosted with SFA emulsified in Freund's Six weeks after incomplete adjuvant, as described above. immunization, sera were assayed for anti-SFA activity by ELISA. When an anti-SFA antibody titre greater than 5000 was achieved, the mouse was given a final boost via a tail vein with 50µg of purified SFA in 80µ1 PBS. Fusion Three days after the final boost, the activated splenocytes fused to X-63 myelomas using procedures outlined by Harlow and Lane (14). The resulting hybridomas were suspended in 100 ml of HY/HAT (Sigma) medium supplemented with 20% fetal bovine serum The P-388 (FBS)/(Sigma) and 5% macrophage conditioned medium. macrophage tumor line was grown to confluency in HY medium supplemented with 2% FBS, then activated with lipopolysaccharide to The hybridoma supply the cytokine-rich conditioned medium. suspension was plated at 100 µ /well into five 96 well tissue culture plates (Corning Inc., Corning, NY). Additionally five plates of a 1:2 dilution and ten plates of a 1:4 dilution of the were
original suspension
were
prepared.
Post Fusion We used the Bio-Rad Clone Selector Mouse Monoclonal
Antibody (Bio-Rad Laboratories, Richmond, Ca) to screen for anti-SFA activity 10 days post fusion. Corning high binding ELISA plates were coated with one microgram of SFA per well. The ELISA plates were read with a Bio-Tek microplate auto-reader set at 405 nm (Bio-Tek Instruments, Winooski, VT). Samples that tested positive for anti-SFA activity (O.D. > 0.1) were screened for crossreactivity using ELISA plates coated with BMA and WMA. Samples that were specific for SFA were cloned by limiting dilution at 0.3 cells/well. Clone M2D1 was selected for further study. Screening
Kit
Purification and Isotyping of Anti-SFA MAb MAbs from the sailfish-specific clone M2D1 that had been grown cell culture and as an ascites tumor in Balb/C mice were Further precipitated with 50% saturated ammonium sulfate. purification was accomplished using a goat anti-mouse IgG agarose The column was (GAMA) affinity chromâtography column (Sigma). equilibrated with 0.01 M sodium phosphate, 0.5 M NaCl buffer pH 7.5 and the antibody eluted with 0.1 M glycine, 0.15 M NaCl buffer pH 2.5. The isotype of the M2D1 MAb was resolved using the Immunotype Mouse MAb Isotyping Kit (Sigma).
in
334
Conjugation of SFA-MAb to HRP
Immuno-affinity purified M2D1 MAbs were conjugated to Immunopure Activated Peroxidase (Pierce Chemical Co., Rockford, IL) according to the manufacturer's protocol. The conjugate was termed
M2D1-HRP.
Assay of Billfish Sera Sera of ten blue marlin, eight white marlin, and eight sailfish (maintained at -20°C) were diluted 1:5000 in Tris-buffered saline pH 7.4 (TBS) and 100 µ aliquote of each was adsorbed to
three wells of an ELISA plate for one hour. All procedures were conducted at room temperature. The plate was blocked for 30 min with a 1% BSA solution and then washed 3 times with 0.05% (v/v) Tween-20 in TBS (TTBS). The M2D1-HRP conjugate was diluted 1:2000 in antibody buffer (0.5% BSA in TTBS) and 100 µ were incubated in the antigen-coated wells for one hour. After washing 5 times with of peroxidase substrate TTBS, 100 µ (3,3',5,5' tetramethyl benzidine) solution (Pierce) were added to each well. After five minutes, the reaction was stopped with IM H3PO„ and the plate was read spectrophotometrically at 450 nm.
RESULTS and DISCUSSION
Albumin Purification Billfish serum yielded of albumin using the method. purification 0.5
mg/ml
Hydroxyapatite-HPLC of the chemically
analysis purified albumin revealed one major and two minor peaks (Fig. 1). The major peak was identified as albumin by comparison of its retention time with that of a bovine serum albumin standard. Albumin has several characteristics that make it
practical
as
identification
a
species
marker
in
immunoassays: ample quantities of antigen can be readily
obtained, it is heat stabile, well characterized, and is present in billfish red muscle
FIGURE 1. Hydroxyapatite-HPLC of Purified Saüfish Albumin. The major peak was identified as serum albumin by comparison to bovine serum albumin.
(15). Hartmann and coworkers (16) attempted sailfish identification by employing polyclonal antisera against sarcoplasmic proteins. This method proved to be inadequate due to cross-reaction with other billfish species. When the antiserum was purified using an affinity chromatography column constructed of blue and white marlin sarcoplasmic proteins, reactivity with sailfish proteins was lost. extracts
Hybridoma Production Of the 16 hybridoma-containing wells that demonstrated antithree did not exhibit cross-reactivity with BMA or WMA (Table 1). One of the three, 21G12, that appeared to consist of a single colony was chosen for its high level of reactivity to SFA. Limiting dilution of 21G12 yielded clone M2D1. The MAbs produced by M2D1 were used in the following experiments. Table 1 shows several MAbs with strong reactivity to both SFA and marlin albumins. These MAbs demonstrate the existence of common epitopes on the albumins of these closely related species. MAbs such as 12A4 are being tested as capture antibodies to be used in conjunction with the species specific MAbs of M2D1 in a sandwich ELISA (17). This format can be used for the identification of sailfish from tissue samples or other biological fluids with low concentrations of albumin. SFA
activity,
335
TABLE 1
Reactivity of Supernatant Fluids from Wells Containing Hybridomas that Produced Antibodies Against Sailfish Albumin. Well
Sailfish Albumin
Blue & White
Well
Sailfish Albumin
Blue & White Marlin Albumin
17A5
0.151
0.055
0.351
0.337
Marlin Albumin 22H1
0.157
0.156
0.117
0.000
8B11
0.139
0.191
21G1
0.207
0.010
23G12
0.227
0.301
21G12
0.355
0.000
3E1
0.374
0.274
21H1
0.386
0.305
18A6
0.150
0.053
21H2
0.278
0.201
18G7
0.339
0.183
21H3
0.123
0.088
18G8
0.292
0.121
2C3
0.120
0.000
Values are optical densities from an alkaline phosphatase-based ELISA. Only wells with antibodies that showed a value of >0.100 versus sailfish albumin were indicated in the table.
Purification. Isotyping and Conjugation of M2D1 Mabs
Affinity purified MAbs from clone M2D1, at a protein concentration of 6 mg/ml, maintained anti-SFA activity (O.D. > 0.100) up to a dilution of 1:50,000. The M2D1 MAbs were shown to be of the mouse IgGl isotype. The M2D1-HRP conjugate retained its activity toward SFA at a dilution of 1:2000. Assay of Billfish Sera The M2D1-HRP conjugate accurately identified the sera of eight sailfish from 26 billfish samples (Figure 2). Furthermore, the conjugate did not show any reactivity with the sera of eight white marlin or that of ten blue marlin. Serum samples were tested in triplicate, with O.D. (450nm) readings ranging from 0.245 to 0.847 for the sailfish samples. The replicate readings for each sample were consistent and differed by no more than 0.040 O.D. units. The largest value recorded for any marlin serum sample replicate was 0.008. In this assay, optimal results were obtained when the serum samples were diluted 1:5000 (data not shown). At dilutions of less than 1:1000, O.D. readings were markedly reduced and inconsistent due to antigen excess. Only 26 samples were available for testing at the time of this study because of the rarity of these species and the tag and release practices of most sportiishermen. All samples were obtained from the Western Atlantic Ocean. The M2D1-HRP conjugate accurately distinguished between sailfish or non-sailfish for all available samples. However, it has been determined if not the will conjugate identify sailfish from O.D. 450 nm oceans. other The conjugate, as well as other MAbs derived from this study, may allow the determination of the number of species or stocks of sailfish that exist. This has been a fisheries of concern since the biologists existence of locationspecific stocks selected through evolution may be ABCDEFOH I JKLMNOPQRSTUVWXYZ essential in maintaining Serum Sample certain local fisheries. The current immunoassay could be used FIGURE 2. Reactivity of M2D1-HRP Conjugate with Billfish Sera by field biologists to in an ELISA. A-J are blue marlin sera; K-R are sailfish sera; S-Z are identify processed sail— white marlin sera. O.D. is the average of triplicate readings.
_
336
fish
carcasses that were caught in the Western Atlantic Ocean. Up 80 samples could be tested in less than two hours. Modifications of this assay can be made to reduce the assay time to approximately 30 minutes. As discussed above, capture antibodies could be employed to facilitate the assay and allow the use of tissues such as red muscle. Adaptation of the assay to a paper format would further reduce the assay time. These modifications are being investigated by this laboratory.
to
ACKNOWLEDGMENTS
We are grateful to Dr. E. Prince for providing the majority of the billfish samples. Dr. R. Waldner for his assistance throughout the project and Dr. D. Binninger for his critical review. We are grateful to Dr. M. Flajnik for the X-63 and P-388 cell lines. This work was supported by The Billfish Foundation through The International Commission for the Conservation of Atlantic Tunas. Mr. Rossi was awarded the Andrew J. Boehm Grant from The American Fishing Tackle Manufacturers Association in the amount of $5000.
REFERENCES
1.
Conser,
R.J. 1985. The status of world billfish stocks. In: World
Angling and Resources and Challenges. R.H. Stroud, ed. Proc. First World Angling Conf., Cap d'Agde, France. Inter. Game Fish Assoc,
Lauderdale, FL. 291-308. Prince, E. Personal communication. NMFS Southeast Region. Virginia Key, FL. 3. King, D.M. 1989. Economic trends affecting commercial billfish fisheries. In: Planning the Future of Billfishes, part 1, R.H. Stroud, ed. National Coalition for the Marine Conservation Inc., Savannah, GA. 89-102. 4. Lundstrom, R.C. 1980. Fish species identification by thin layer polyacrylamide gel isoelectric focusing: Collaborative study. Ft.
2.
J.A.O.A.C. 53: 7-9.
Lundstrom, R.C. 1981. Fish species identification by isoelectric focusing: Sarcoplasmic protein polymorphism in monkfish Lophius
5.
americanuBl. J.A.O.A.C. 64: 38-43. 6. Lundstrom, R.C. 1981. agarose gel isoelectric J.A.O.A.C. 64: 38-43. 7. Lundstrom, R.C. 1983.
Rapid fish species identification by focusing of sarcoplasmic proteins.
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