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Journal of Veterinary Diagnostic Investigation Vol. 19 Issue 5, 465-470
Copyright © 2007 by the American Association of Veterinary Laboratory Diagnosticians
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Development and validation of monoclonal and polyclonal antibodies for the detection of immunoglobulin G of bottlenose dolphins (Tursiops truncatus)

Hendrik Nollens1, Linda G. Green, Diane Duke, Michael T. Walsh, Beth Chittick, Scott Gearhart, Paul A. Klein and Elliott R. Jacobson

Correspondence: 1 Corresponding Author: Hendrik Nollens, Marine Mammal Health Program, SACS, College of Veterinary Medicine, University of Florida, PO Box 100126, Gainesville, FL 32610, e-mail: NollensH{at}vetmed.ufl.edu


   Abstract
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Antibodies directed against species-specific immunoglobulin G (IgG) have a broad range of applications in serologic and immunologic research and in the development of clinical assays. Validated anti-IgG antibodies for marine mammal species are in short supply. The objective of this study was to produce and validate antibodies with specificity for IgG of the common bottlenose dolphin (Tursiops truncatus). Bottlenose dolphin IgG was purified using protein G. Two mouse monoclonal antibodies and a rabbit polyclonal antibody were developed from mice and rabbits immunized with bottlenose dolphin IgG. The specificity of the monoclonal antibodies and the polyclonal antibody for bottlenose dolphin IgG was first verified by Western blot analysis and enzyme-linked immunosorbent assay (ELISA). For further validation, both monoclonal antibodies and the polyclonal antibody were incorporated in an indirect ELISA for the detection of the immune response of bottlenose dolphins to a vaccine antigen. Three bottlenose dolphins were immunized with a commercial Erysipelothrix rhusiopathiae vaccine, and serial blood samples were collected from all dolphins for measurement of levels of circulating antibodies. Seroconversion was observed in all 3 dolphins by use of both monoclonal antibodies and the polyclonal antibody. Circulating antibodies were detectable as early as 6 days after immunization in 1 dolphin. Peak antibody levels were detected 14 days after the immunization. The ability to detect seroconversion in all 3 immunized bottlenose dolphins firmly establishes the specificity of the monoclonal antibodies and the polyclonal antibody for IgG of the common bottlenose dolphin.

Key Words: Antibody • dolphin • immunoglobulin • monoclonal • polyclonal • serology • Tursiops


   Introduction
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The order of cetaceans comprises the group of marine mammals that consists of all whales, dolphins, and porpoises. Although some cetacean species are commonly kept in captivity, cetacean veterinary medicine is a relatively new science. Among other disciplines, the development of serodiagnostic tests for cetaceans is still in its infancy. Cetacean antibody responses are traditionally measured by use of serum neutralization, precipitation, and agglutination assays.16 The most commonly used assay to measure pathogen-specific antibodies is the serum neutralization (SN) test.4,5,16 SN tests are both highly sensitive and specific and are commonly considered the gold standard serologic assay. However, SN tests are labor intensive and may require waiting up to 5 days to obtain results. They require live virus and cell cultures and can therefore only be performed in specialized laboratories. Other assays, such as radioimmunoassays and precipitation and agglutination assays have been used to determine the prevalence of antibodies to pathogens such as Toxoplasma gondii3 and Brucella spp.15 While these assays are less expensive, they are also labor-intensive and are considered less sensitive than SN tests.

Of all serologic assays, the enzyme-linked immunosorbent assay (ELISA) has emerged as the preferred assay system for determining the presence of specific antibodies in plasma and serum of many species, including cetaceans.2,3,11–15,17 While competitive ELISAs can be applied to multiple host species, species-specific indirect ELISAs (iELISA) are often considered the serodiagnostic method of choice.1,6,12 They are rapid and inexpensive. They do not require live, infectious virus and are easily applied to large numbers of samples and multiple antigens. However, iELISAs require the availability of species-specific anti-immunoglobulin monoclonal antibodies (MAb) or polyclonal antibodies (polyAb). These MAb and polyAb directed against species-specific IgG have a broad range of applications including the development of serologic assays for measuring exposure to infectious agents, immunological research, and establishing diagnoses of immune dysfunction or failure of passive transfer of antibodies.

The relative shortage of validated species-specific anti-immunoglobulin antibodies has been one of the greatest impediments to the use of immunodiagnostic tests in cetacean medicine.1,12 A limited number of MAbs and polyAbs with specificity for bottlenose dolphin IgG have been developed using different protocols.a,2 However, these antibodies are not readily available, and their validity has not always been firmly established. The objectives of the study reported here were 1) to develop mouse MAb and rabbit polyAb with specificity for IgG of bottlenose dolphins and 2) to validate the specificity of these antibodies by demonstrating their ability to detect seroconversion of bottlenose dolphins in response to a vaccine antigen.


   Materials and Methods
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Animals, Immunizations, and Sample Collection
Three 1-year-old, male, captive-bred bottlenose dolphins (Tursiops truncatus; FTt0102, FTt0104, and FTt0105) housed at an oceanarium were enrolled in an ongoing vaccination program from 8 to 14 November, 2002. All 3 dolphins received a single immunization with a commercial Erysipelothrix rhusiopathiae bacterinb for swine (ER Bac Plus). Immunizations were given in the dorsal musculature lateral and cranial of the dorsal fin. After each immunization, all animals were monitored for adverse reactions for 30 min. An initial blood sample was collected from the ventral tail vein from each dolphin immediately before the first immunization. Four subsequent blood samples were collected at approximately 1-week intervals. In total, 11 samples were collected from the 3 bottlenose dolphins. Blood samples (6 ml) were collected into lithium heparin tubes and were immediately placed into a portable cooler for transportation to the laboratory. Plasma was obtained by low speed centrifugation (300 x g for 10 min) and frozen at –70°C until purification of IgG and ELISA analysis. All sample collection protocols were approved by the University of Florida Institutional Animal Care and Use Committee (IACUC# C408).

Purification of Bottlenose Dolphin Igg
Bottlenose dolphin IgG was purified from a 12.65-ml pool of plasma from the 3 dolphins. IgG was purified from the pooled plasma by affinity chromatography using a 5-ml Hi-Trap Protein G column,c following the manufacturer's instructions. The pooled dolphin plasma was diluted 1:6 in 0.01 M phosphate-buffered saline containing 0.02% sodium-azide (PBS/Az) and was loaded onto the column. The diluted plasma was allowed to filter through the protein G matrix 3 times at a flow rate of 3.0 ml/min. Five-milliliter fractions were collected throughout the third chromatographic period, and the protein concentration of each fraction was measured spectrophotometrically. The leading and trailing portions of each peak were pooled separately for evaluation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). All fractions were concentrated separately using an Ultrafree centrifugal filter device.d The original whole dolphin plasma, the peak representing the IgG-rich fraction, and the column flow-through were subjected to SDS-PAGE (45 min at 200 V) under denaturing conditions, on a precast 10% (wt/vol) polyacrylamide Nupage Novex BisTris gele with morpholinepropanesulfonic acid running buffer. The gel was stained with Simply Blue Safestaine to assess the purity of the eluates (Fig. 1).


Figure 01
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  		Figure 1 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis with Coomassie blue staining of products from the bottlenose dolphin IgG purification steps. The predominant bands in the protein G-purified fraction (lanes 5 and 6) are consistent with the dolphin 55 kDa {gamma} heavy chain and 25 Da {kappa} and {lambda} light chains. Lane 1: benchmark protein ladder (MW). Lanes 2–4: whole bottlenose dolphin plasma (1:100, 1:150, and 1:250 in PBS/Az). Lanes 5–6: 1 µg and 0.5 µg protein G-purified fraction. Lanes 7–8: protein G purification flow-through at 1:150 and 1:250 (in PBS/Az).

 
Production of Polyab
The purified dolphin antibody fraction was delivered to a private biological company,f and polyclonal antiserum was produced in rabbit SB-A9713 with a standard 70-day protocol. Plasma samples were obtained from the rabbit before immunization (pre-immune plasma). The rabbit was immunized 4 times with 200 µg of affinity purified bottlenose dolphin immunoglobulins with complete Freund's adjuvant (first immunization) or incomplete Freund's adjuvant (subsequent immunizations). Following a final intraperitoneal immunization (without adjuvant), the rabbit was anesthetized and exsanguinated. The polyclonal serum SB-A9713 was separated and stored at –70°C. Serum anti-dolphin IgG titers were evaluated in an indirect ELISA system, and the specificity of the polyAb for was evaluated via Western blot analysis (Fig. 2).


Figure 02
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  		Figure 2 Western immunoblotting of whole bottlenose dolphin plasma (1:250 in PBS/Az) using the polyAb and MAbs. The predominant bands detected are consistent with the dolphin 55 kDa {gamma} heavy chain and 25 Da {kappa} and {lambda} light chains. Lane 1: prestained molecular mass standards SeeBlue Plus2 (MW). Lanes 2–8 were probed with 1:80,000 polyAb SB-A9713 (lane 2), 1:80,000 normal rabbit serum control (lane 3), 0.05 µg/ml MAb HL1912 (lane 4), 0.025 µg/ml MAb HL1912 (lane 5), 1 x PBS/Az control (lane 6), 5 µg/ml mAb HL1914 (lane 7), 1 x PBS/Az control (lane 8). All antibodies were diluted to the desired concentration in 1 x PBS/Az. Conjugates were goat anti-rabbit Ig polyAb (1:2,000 in PBS/Az, lanes 2x3) and streptavidin-alkaline phosphatase (1:2,000 in PBS/Az, lanes 4–8), respectively.

 
Production of Mab
Mouse MAbs against bottlenose dolphin immunoglobulins were produced by use of a standard hybridoma protocol used by the Hybridoma Core Laboratory at the University of Florida Interdisciplinary Center for Biotechnology Research.7–10 In brief, an 8-week-old female Balb/c mouse was immunized subcutaneously with 30 µg of affinity purified dolphin immunoglobulins with the RIBI adjuvant system.g Three and 9 weeks later, the inoculation was repeated with 30 µg of immunoglobulin and RIBI adjuvant. One week after the second immunization, blood was collected from the tail, and serum anti-dolphin IgG titers were evaluated in an indirect ELISA system. One month after the third subcutaneous injection, 15 µg of immunoglobulin was administered intraperitoneally. Four days later, the spleen was harvested for hybridoma production. Mouse spleen cells were fused with mouse myeloma cells (Sp2/0) in the presence of polyethylene glycolh and added to 96-well culture plates. Eight 96-well culture plates were seeded. The supernatants from the resulting sodium hypoxanthine, aminopterin, and thymidine (HAT)-resistant hybridoma cells were screened 10 days later for the presence of antibodies specific to dolphin IgG. Hybridomas from positive wells (OD405 3–20 times higher than background) were transferred to 24-well plates and grown to confluency. The supernatants were then screened and mouse antibody isotype was determined via ELISA with antibodies specific for anti-mouse IgG whole molecule, anti-mouse {gamma} chain, or anti-mouse µ chain. Two supernatants that consistently scored positively were selected for cloning by limiting dilution to yield MAb-producing hybridomas (HL1912 and HL1914). The hybridomas were grown in controlled process serum replacement medium (BD Cell MAb Medium Quantum Yieldi). Monoclonal antibodies were purified from the supernatant by use of protein G affinity chromatography.j The IgG isotype of MAbs HL1912 and HL1914 was determined with a MAb isotyping kitk following the manufacturer's instructions.

Biotinylation of Mabs
The protein concentration of the purified MAbs HL1912 and HL1914 was measured spectrophotometrically at 280 nm. Five milligrams of biotin (sulfosuccinimidyl 6-(biotinamido)hexanoate)l was dissolved in ddH2O. A volume of 31 µl of 5 mg/ml biotin solution was mixed with 1.3 mg of each of the MAbs, and the solution was allowed to stand for 2 hr at room temperature. The mixture was then dialyzed for 48 hr against PBS/Az.

Western Immunoblotting
The specificity of polyAb SB-A9713 and MAbs HL1912 and HL1914 was evaluated by Western immunoblot (Fig. 2). Dolphin plasma at 1:250 dilution (in PBS/Az) was separated electrophoretically under reducing conditions on a precast 10% (wt/vol) polyacrylamide Nupage Novex BisTris gele with morpholinepropanesulfonic acid running buffer. The reduced serum immunoglobulins were electrophoretically transferred to a nitrocellulose sheete with a Novex Western transfer apparatus. Blotting time was 60 min at a constant voltage of 30 V. The nitrocellulose sheet was placed in 5% nonfat dry milk dissolved in PBS/Az to block overnight at room temperature with gentle agitation. The blocked nitrocellulose sheets were washed 3 times for 5 min each in PBS/Az containing 0.05% Tween 20 (PBS-T). Lanes were incubated with SB-A9713 (1:80,000), biotinylated HL1912 (0.05 µg/ml and 0.025 µg/ml) or biotinylated HL1914 (5 µg/ml) (Fig. 2), diluted in PBS/Az. A negative control lane (normal rabbit serum at 1:80,000 or PBS/Az) was included for each of the 3 antibodies. The nitrocellulose sheets were incubated with gentle rocking at room temperature for 1 hr, after which the blot was washed 4 times for 5 min each with PBS/Az containing 0.05% Tween 20. Goat anti-rabbit Ig polyAb (1:2,000 in PBS/Az) and streptavidin-alkaline phosphatasee (1:2,000 in PBS/Az) were used to develop the polyAb and MAb lanes, respectively. The blot was incubated with gentle rocking for 60 min, and after 4 more washes the blot was incubated with substrate buffer (0.1 M Tris HCl, 1 mM MgCl2, pH 8.8) containing 4.4 µl/ml nitroblue tetrazolium chloridem and 3.3 µl/ml 5-bromo-4-chloro-3-inolylphosphate p-toluidine salt.l

Validation of The Mabs and Polyab
An iELISA was developed for the evaluation and validation of the MAb and polyAb. Proprietary ERBac Plus bacterin 65 kDa protein antigen (Ery p65) was kindly provided by Pfizer Animal Health.b A capture monoclonal antibody (ERHU MAb), specific for the 65 kDa ERBac Plus protein antigen was provided by the USDA Animal and Plant Health Inspection Service.n Wells of a high protein binding microplateo were coated with ERHU MAb diluted 1:800 in PBS and were left to adsorb overnight at 4°C. After this and each subsequent step, all wells were washed 3 times with PBS/Az and 0.05% PBS-T using an automated microplate washer. After washing, all wells were blocked with 1% bovine serum albumin (BSA)h in PBS/Az, after which the Ery p65 antigen was applied (1:600 in PBS). The dolphin plasma samples were diluted 1:1,000 in 1% BSA and applied in 3-fold to each of 2 wells. One percent BSA was applied to 6 wells to serve as duplicate negative control wells for the respective antibodies. PolyAb SB-A9713 (1:40,000 in 1% BSA), MAb HL1912 or HL1914 (5 µg/ml, diluted in 1% BSA) were applied as the secondary reagent for the detection of bound dolphin antibodies. Alkaline phosphatase-labeled goat anti-rabbit IgGi (1:1,000 in 1% BSA) was used as tertiary reagent for the detection of bound polyAb, whereas alkaline phosphatase-labeled streptavidinp (1:2,000 in 1% BSA) was used to detect the presence of bound MAbs HL1912 and HL1914. Each step of the ELISA was left to incubate with gentle agitation for 1 hr at approximately 22°C. Finally, 100 µ1 of P-nitrophenyl phosphatei substrate at 1 mg/ml was added. The optical density at 405 nm (OD405) was recorded 30 min after addition of the substrate using a Spectramax 250 microplate reader.q For analysis, the average OD405 of the negative controls were subtracted from the average OD405 readings of all other wells of the corresponding antibody (corrected OD405).


   Results
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Purification of Bottlenose Dolphin Igg
The protein G affinity purification of the 12.65 ml of pooled dolphin plasma yielded 3.35 mg of Tursiops IgG. Electrophoretic separation of the purified fraction using a phastgel yielded predominant bands in the protein G–purified fraction that were consistent with the dolphin 55 kDa {gamma} heavy chain and 25 kDa {kappa} and {lambda} light chains (Fig. 1).

Mabs and Polyab
Primary screening of the fusion products by ELISA revealed 38 hybridomas reactive with dolphin IgG. Ten hybridomas were chosen for further screening via Western blot (Fig. 2). Six of these 10 hybridomas had specificity for the heavy or light chain region of bottlenose dolphin IgG. Hybridomas 1E5 and 4E5 were chosen for cloning on the basis of their rapid cell growth, their IgG isotype, and their respective reactivities with bottlenose dolphin IgG heavy and light chain. PolyAb SB-A9713 was reactive with both the bottlenose dolphin heavy and light chain.

Validation of The Mabs and Polyab
An antibody response to the Erysipelothrix rhusiopathiae bacterin was detected in all 3 dolphins using all 3 antibodies (Fig. 3A–C). Circulating anti-Ery p65 antibodies were first detectable in 1 dolphin (FTt0105) 6 days after immunization, and all 3 dolphins uniformly developed anti-Ery p65 antibodies through day 14. At day 14, the strongest antibody increase was measured using MAb HL1912 (respectively, a 2.6-, 7.0-, and 5.2-fold increase in comparison to the initial bleed for FTt0102, FTt0104, and FTt0105). The intensities of the dolphin antibody response measured using MAbs HL1912 and HL1914 appeared similar to each other but distinct from the intensity of the response measured using polyAb SB-A9713 (Fig. 3A–C).


Figure 03
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  		Figure 3 The IgG antibody responses of three 1-year-old bottlenose dolphins after inoculation with an Erysipelothrix rhusiopathiae vaccine antigen. Antibody levels (corrected OD405) were measured by ELISA and were corrected for interplate variation. Seroconversion was detected in all 3 dolphins using A, MAb HL1912; B, MAb HL1914; and C, polyAb SB-A9713.

 

   Discussion
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The aim of this study was to develop a validated set of species-specific anti-IgG antibodies for use in bottlenose dolphins. Monoclonal antibodies are highly valued because they are consistent and specific reagents. They are exceptionally useful for developing serodiagnostic tests to determine exposure of animals to pathogens. These diagnostic tests can be applied in a clinical diagnostic setting, but also in retrospective sero-epidemiological surveys of captive and wild populations and prospective monitoring of disease agents of concern. However, these antibodies also have multiple applications in cetacean immunological research, including quantitative IgG assays.

In this study, 1 polyAb and 2 MAbs with specificity for bottlenose dolphin IgG were developed, and their reactivity with bottlenose dolphin antibodies was firmly established by showing the ability of the MAb and polyAb to detect seroconversion of 3 bottlenose dolphins in response to an immunogen. The range of species in which these antibodies can be applied remains to be determined. However, their ability to detect a band on Western blot analysis and to detect seroconversion in an ELISA format does demonstrate that both MAbs and the polyAb can be applied to detect bottlenose dolphin IgG in both Western and ELISA formats. Compared with HL1912 and SB-A9713, the MAb HL1914 did generate a high background signal in the iELISA (initial OD405 = 0.900–1.080). However, HL1914 was retained in this study because of its unique apparent specificity for the immunoglobulin light chain(s) on Western blot. This high background signal in the iELISA may be due to cross-reactivity with the {kappa} and {lambda} light chains of IgA, D, E, and M molecules, which are not detected using MAb HL1912. HL1914 may still be a useful antibody when used under different assay conditions in other ELISA or Western blot applications.

The main impediment in developing anticetacean IgG antibodies is the validation of the resultant antibodies. The specificity of antibodies can be evaluated in the laboratory using assays such as the ELISA and Western immunoblot. However, the usefulness of the antibodies can only be unequivocally established by demonstrating their ability to detect seroconversion. However, access to marine mammals as study subjects is limited. Free-ranging marine mammals are protected by the legislature, and managers of captive populations are generally hesitant to participate in experimental research. As a result, challenge studies and injections with immunogens are not feasible in cetacean species. The ability of the previously existing anti-dolphin IgG antibodies to detect seroconversion has not yet been unequivocally demonstrated. This study made use of a unique collaboration with an oceanarium in which, first, dolphins were serologically monitored to help evaluate the usefulness of a vaccination program and, second, anti-dolphin IgG antibodies were developed. Thus, this study not only provided useful clinical information on the antibody response of bottlenose dolphins to the antigen, but successfully validated these antibodies for future use in serologic and immunologic assays.

This is also the first published account in which the timing and intensity of the humoral antibody response of bottlenose dolphins to a single-point exposure was recorded. In terrestrial mammals, no antibody is detectable for several days after first exposure to an antigen.14 When antibodies do appear, their level climbs to a peak by 10 to 14 days before declining and disappearing within a few weeks. In the dolphins in this study, circulating antibodies were first detectable 6 days after immunization, and antibody levels appeared to approach peak levels 14 days after immunization. Therefore, the temporal characteristics of the IgG response of bottlenose dolphins appear to be very similar to those of the antibody responses of terrestrial mammals.


   Acknowledgments
 
The authors wish to thank the animal care staff at SeaWorld of Orlando for their assistance with animal handling and sample collection. We thank Pfizer Animal Health for providing the proprietary vaccine antigen. This work was funded by a faculty seed grant from the College of Veterinary Medicine of the University of Florida.


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From the Hendrik H. Nollens, Marine Mammal Health Program, and the Department of Small Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL 32610Hybridoma Core Laboratory, Interdisciplinary Center for Biotechnology Research, University of Florida, Gainesville, FL 32610(Green, Duke); SeaWorld Orlando, 7007 SeaWorld Drive, Orlando, Florida 32821(Walsh, Chittick, Gearhart), and the Department of Pathology, Immunology and Laboratory Medicine, College of Medicine, University of Florida, PO Box 100275, Gainesville, FL 32610(Klein). Back

a. Bethyl Laboratories, Montgomery, TX. Back

b. Pfizer Animal Health, Pizer Inc., New York, NY. Back

c. GE Healthcare Bio-sciences Corp., Piscataway, NJ. Back

d. Millipore Corp., Billerica, MA. Back

e. Invitrogen Corp., Carlsbad, CA. Back

f. Strategic Biosolutions, Newark, DE. Back

g. Roche Diagnostics, Indianapolis, IN. Back

h. BD Biossciences, San Jose, CA. Back

i. Protein G sepharose fast flow, Pfizer-Pharmacia, Pizer Inc., New York, NY. Back

j. Code RPN (Amersham), GE Healthcare Bio-sciences Corp., Piscataway, NJ. Back

k. Ultraspec II, LKB Biochrom Ltd., Cambridge, UK. Back

l. Immunoselect, Gibco BRL, Gaithersburg, MD. Back

m. United States Department of Agriculture, Animal and Plant Health Inspection Service, Center for Veterinary Biologics, Ames, IA. Back

n. Nunc Maxisorp, Fisher Scientific, Pittsburgh, PA. Back

o. Zymed Laboratories, San Francisco, CA. Back

p. Molecular Devices, Sunnyvale, CA. Back


   References
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 Abstract
 Introduction
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 Results
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 References
 

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H. H. Nollens, C. Ruiz, M. T. Walsh, F. M. D. Gulland, G. Bossart, E. D. Jensen, J. F. McBain, and J. F. X. Wellehan
Cross-Reactivity between Immunoglobulin G Antibodies of Whales and Dolphins Correlates with Evolutionary Distance
Clin. Vaccine Immunol., October 1, 2008; 15(10): 1547 - 1554.
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