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Universal assay of vitellogenin as a biomarker for environmental estrogens.

Universal assay of vitellogenin as a biomarker for environmental estrogens. Universal Assay of Vitellogenin as a Biomarker for Environmental Estrogens A. Heppell,1 Nancy D. Denslow,2 Leroy C. Folmar,3 and Scott V. Sullivan' Craig 'Department of Zoology, North Carolina State University, Raleigh, North Carolina; 2Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida; 3U.S. Environmental Protection Agency, Gulf Breeze, Florida Vitellogenin (VTG), the serum phospholipoglycoprotein precursor to egg yolk, is potentially an ideal biomarker for environmental estrogens. This study was undertaken to develop antibodies against conserved regions on the VTG molecule that could form the basis for establishing bioassays to detect estrogen exposure in any oviparous vertebrate. We developed monoclonal antibodies (mAbs) generated against purified rainbow trout VTG purified from two phylogenetically distant vertebrates, mykiss) VTG and selected for the property of specifically recognizing (Oncorhynchus trout and striped bass (Morone saxatilis). Results of enzyme-linked immunosorbent assay and Western blotting indicated that these mAbs specifically recognize purified VTG and VTG or other estrogen-inducible proteins in plasma or serum from representative species of four vertebrate classes (fish, amphibians, reptiles, and birds). All of the mAbs generated were IgM class. A polyclonal antiserum was raised against a synthetic consensus peptide representing the conserved N-terminal amino acid sequence of VTG. The results of Western blotting indicate that this antiserum teleost fish of diverse families. It was used to detect VrG in Western blots of serum from specifically recognizes VTG in plasma or serum from with cancer and cholangio-carcinoma) collected from a contaminated industrial site outside of brown bullhead (Ameiurus nebulosus) (hepatocellular their normal vitellogenic season. Our results indicate that it is feasible to generate antibodies capable of recognizing VTG without regard to species and that development of a universal VTG assay is an achievable goal. - Environ Health Perspect 1 03(Suppl 7):9-15 (1995) Key words: vitellogenin, universal assay, environmental estrogens, xenobiotics, monoclonal, immunoassay, synthetic peptide, biomarker, polyclonal, hormone mimics effects on the reproduc- their use or release into the environment. and their potential Introduction their notoriously weak struc- tive biology of vertebrates. However, that the Vitellogenin (VTG) is potentially an Environmental estrogens, chemicals with ture-activity relationship requires of similar to estrogen, identity and potency of environmental ideal biomarker for the estrogenicity bioactivity endogenous The chemicals. It is a large serum phospholipo- are known to affect and sexual estrogens be defined by bioactivity. development pre- of vertebrates and are impli- current lack of effective bioassays is consid- glycoprotein that serves as the major maturation be a cause of the lack of cursor to the egg-yolk proteins of cated as cancer promoters (1). They ered to proximal of detergents, regulatory action regarding these com- oviparous vertebrates (3). VTG is synthe- indude breakdown products An effective needs to sized and secreted by the liver in response and a variety of chlo- pounds (2). bioassay pesticides, plasticizers, such chemicals be developed to assess the estrogenicity of to circulating estrogens in maturing rinated compounds. Ideally, before or their derivatives females and is normally undetectable would be evaluated for estrogenicity synthetic compounds (4,5) and in the plasma of immature animals males. The presence of this estrogen- in the of an ani- inducible protein serum on in the Environment, III: Global Health Implications This paper was presented at the Symposium Estrogens in DC. received: March 15, 1995; manuscript accepted: mal can be taken as evidence of exposure to held 9-11 January 1994 Washington, Manuscript April 4, 1995. or or endogenous exogenous estrogens assistance in either samples, fish, or both. The following peo- We thank several people for obtaining plasma estrogen mimics. This potential ofVTG as and animals: M. tuatara; P. Licht, snake; E. donated plasma from vitellogenic nonvitellogenic Brown, ple has been white T. F. and A. J. Hinshaw donated the a biomarker already explored and croaker; Siopes, Edens, Youngblood, chicken; Casillas, pollock A. Hara for the of antiserum to trout VTG and to L. Greene rainbow trout. We are grateful to gift homologous several fish for which both in using species, mAbs. We thank B. Parten for Protein sequencing was for the antihepatitis B-core protein microsequencing. have been devel- vivo and in vitro assays Core in the Center for Biotechnology Research at carried out in the Protein Chemistry Facility Interdisciplinary However, immunological Florida. C. Bidwell the for white vitellogenin cDNA. This work oped (6-10). the University of provided sequence sturgeon of an Electric Power Research Institute Graduate Research Fellowship was supported by the University Florida, and structural of VTG can properties vary North Carolina Center 13ARIG 0803 to and a cooperative agreement SAH), a Biotechnology grant (#91 CVS) (to greatly even among closely related species Environmental Protection and the of Florida to NDD). between the U.S. Agency University (#CR821437 which limits broad scale applica- (11-14), to Dr. V. of Zoology, Campus Box 7617, North Carolina Address correspondence Craig Sullivan, Department NC 27695. 515-7186. Fax: (919) 515-2698. E-mail: vertebrate taxa. State University, Raleigh, Telephone: (919) tion of these assays across [email protected] it has become apparent that Recently used: avidin-biotin complex; DAB, diaminobenzidene; ELISA, enzyme-linked immunosor- Abbreviations ABC, VTG is an ancient protein belonging to a FMOC, fluorenylmethoxycarbonyl; IgG, bent assay; E2, 17>-estradiol; Fab, antibody binding fragment; limpet hemocyanin; mAb, monoclonal antibody; Mr, multigene family that includes a variety of immunoglobulin G; IgM, immunoglobulin M; KLH, keyhole aromatic saline; relative mobility; OVA, ovalbumin; PAH, polynuclear hydrocarbons; PBS, phosphate-buffered vertebrate lipoproteins, including various fluoride immunoblot membranes; rpHPLC, reverse-phase PITC, phenylisothiocyanate; PVDF, polyvinilydene and invertebrate VTGs as well as the sodium dodecyl sulfate-polyacrylamide gel electrophore- high performance liquid chromatography; SDS-PAGE, trifluoroacetic human serum proteins, apolipoprotein TCSN, tissue culture supernatant; TFA, acid; VTG, vitellogenin. sis; Environmental Health Perspectives HEPPELL ETAL. B-100 and von Willebrand factor (15-18). of a 96-well plate that had been previously white croaker, Genyonemus lineatus; brown Coupled with prior evidence for conserva- coated with antigen. The second antibody trout, Salmo trutta; and pollock, Theragra tion of some functional and immunologi- was a goat antimouse IgM, p-chain specific as well as representative rep- chalcogramma) cal properties of the molecule (19-22), antibody (Sigma Chemical Co, St. Louis, tiles (tuatara, Sphenodon punctatus; and rat- these findings suggest that VTG may pos- MO) conjugated to horseradish peroxidase, tlesnake, Crotalus molossus) and a bird sess conserved epitopes that could be and antibody binding was visualized with (chicken, Gallus domesticus). Additional to antibodies and O-phenylenediamine/H202 in 0.1 M cit- screened by Western blot included exploited develop species immunoassays of a more universal nature. rate-phosphate buffer, pH 5.0. the bullfrog (Rana catesbeiana), the black This study was undertaken to develop Hybridoma colonies whose TCSN rat snake (Elaphe obsoleta), and the chicken. antibodies against conserved regions on the demonstrated immunoreactivity in the To initiate generation of polyclonal VTG molecule that could form the basis ELISA to VTG and E2-plasma from both antisera known conserved regions against for establishing bioassays to detect estrogen bass and trout but did not react to normal on the VTG molecule, we microsequenced exposure in any oviparous vertebrate. We male plasma were selected for cloning. amino acids in the N-terminal regions of a cho- VTGs purified from several phy- attempted to develop both monoclonal Rainbow trout and striped bass were number of antibodies generated against purified VTG sen as the for initial screenings logenetically diverse teleost fish (23). We species and polyclonal antisera raised against the because they represent distinct vertebrate obtained sequences for several teleost fish conserved N-terminal amino acid sequence lines that have evolved independently over induding the striped bass, brown bullhead pinfish (Lagodon of VTG (23). Using these two approaches a considerable period of time (-200x 106 (Ameiurus nebulosus), we demonstrated that it is feasible to gener- methods for their respec- rhomboides), yellow perch (Perca years), purifying flavescens), as as ate antibodies capable of recognizing VTG tive VTGs were well established (24,25), and medaka (Oryzias latipes), well without regard to species of origin and that and they were available because they were sequences deduced from the cDNA of heteroclitus) and development of a universal VTG assay is subjects of other endocrine investigations mummichog (Fundulus an achievable goal. in our laboratory. Positive colonies were white sturgeon (Acipenser transmontanus). to bass was used as the template subjected to limiting dilution produce The striped Methods monoclonal cell lines secreting mAbs with for comparison because it is the most highly immunized ip with characteristics. The mAbs from the in To determine an Balb/C mice were desired evolved fish our study. either 100 of rainbow trout monoclonal cultures were isotyped using optimal peptide segment for antibody tar- pg as we a consensus N-terminal (Oncorynchus mykiss) VTG purified commercial kits (Life Technologies, geting, analyzed Hara et al. or 100 of MD, or Sigma). with a protein structure program described by (24) Gaithersberg, sequence pg for a second which an antigenicity index striped bass (Morone saxatilis) VTG Western blots were used (28), provides as described by Tao et al. (25) series of mAb screens. Samples of purified at each position of the peptide. Using this purified or male from information we a consensus mixed 1:2 with Freund's complete adju- VTG, E2-plasma, plasma designed vant. Mice were boosted twice at 2- to 3- trout and bass were normalized for protein peptide fifteen amino acids long. with of mixed dode- The consensus peptide was assembled week intervals 50 pg antigen concentration, separated by sodium 1:2 with Freund's incomplete adjuvant. cyl sulfate 4 to 15% polyacrylamide gradi- on a Model 431A automated peptide syn- were on a thesizer Foster Four days before fusion, they again ent gel electrophoresis (SDS-PAGE) (Applied Biosystems, City, N- boosted with 100 pg of antigen in incom- PhastSystem electrophoresis apparatus CA) from the C-terminus towards the Monoclonal antibodies and then elec- terminus with the a-carboxyl group of the plete adjuvant. (Pharmacia, Piscataway, NJ) were then standard troblotted onto Immobilon-P PVDF amino acid attached to the solid support, (mAbs) generated using entire was membranes Rink resin and methods (26). This procedure (Millipore, Marlborough, MA) using 9-fluorenylmethoxy- replicated 3 times. In an attempt to induce using the PhastSystem immunoblot appa- carbonyl (FMOC) protected amino acids. class shift and increase the ratus Membranes were blocked in An additional Cys residue was added at the antibody affinity (25). to acti- of the antibodies, the final set of immu- phosphate-buffered saline (PBS) (0.01 M N-terminus for coupling the peptide nizations was modified the 0.15 M NaCl, pH 7.3) with vated carrier (29). The peptide was by increasing proteins Na.H.PO4, time between boosts to a minimum of 3 3% bovine serum albumin overnight, incu- cleaved from the resin with trifluoroacetic a third boost bated with TCSN diluted in PBS with acid (TFA) (30), purified by reverse-phase weeks and incorporating prior to fusion. 0.5% Tween-20 (wash buffer; range high performance liquid chromatography colonies were screened for for 2 hr, and then probed (rpHPLC) on a Whatman partisil 10 Hybridoma 1:3-1:10, v/v) in VTG cross-reactivity using an antibody with secondary antibody (1:4000 wash ODS-2 column fitted to an HPLC system immunosorbent and avidin-biotin in wash (Waters, Milford, MA) and equilibrated capture enzyme-linked buffer) complex Vector A TFA in buffer Elite Kit, Laboratories, with 20% solvent (0.1% assay (ELISA) (27) against purified VTGs, (ABC from immature or male for min each. and 80% solvent B (0.1% TFA in blood plasma Burlingame, CA) 30 CH3CN) was with a linear bass and trout injected with Antibody binding visualized dH20) followed by 20-min gradient striped 170- to induce diaminobenzidene Vector to solvent A at a flow rate of 1 ml/min. estradiol (E2) vitellogenesis (E2- (DAB Kit, 35% and of plasma) (15), or plasma from normal male Laboratories). Compositional quantitative analysis and trout as the reference Preliminary ELISA and Western blot the peptide was conducted on a Pico Tag bass prepara- collect- screens of the of the mAbs to differ- work station after a 22-hr tions. The mAbs were harvested ability (Waters) hydroly- by the tissue culture bind to vitellogenic plasma and sis and phenyl isothiocynate (PITC) ing supernatant (TCSN), entially in from a of derivitization which was stored at 40C the of nonvitellogenic plasma variety (31). presence for to months until use. other were also done. Additional Aliquots of purified peptide (2 mg) 0.01% NaN3 up 3 species 1:2 with tested ELISA included several to maleimide- TCSN (100 pl) was mixed 10% species by were conjugated (1:2, w/w) normal serum and added to the wells fishes Morone activated carrier proteins, keyhole limpet goat (white perch, americana; 10 Environmental Health Perspectives VITELLOGENINAS A BIOMARKER FOR ESTROGENS Table 1. Reactivity in the antibody capture ELISA of the hemocyanin (KLH; mw 450-13,000 cultures were designated 2C1 1, 2D8, 3E8, Rl monoclonal antibody (OD 490) to plasma samples kDa), or ovalbumin (OVA; mw 45 kDa) 5G7, and 8G7. All five were isotyped as from vitellogenic females and males of several verte- using an Imject Activated Immunogen IgMs. Because 2D8 gave the most consis- brate species. Plasma samples were diluted 1:100. Conjugation kit (Pierce Chemical Co., tent results in initial screens, it was chosen Plasma source Female Male in All of the Rockford, IL). The KLH peptide conju- for use subsequent analyses. gate was used to immunize two female hybridoma cultures that were cloned were Brown trout, 1.613 0.163 Salmo trutta New Zealand white rabbits sc with 200 derived from mice immunized with trout pg protein in 0.5 ml sterile distilled water VTG. None of the hybridoma colonies Pollock, 0.768 0.213 with bass Theragra chalcogramma mixed 1:2 with Freund's complete adju- derived from mice immunized vant. Subsequent immunizations (n = 8), VTG gave significant levels of cross-reac- White croaker, 0.745 0.219 Genyonemus lineatus done 2 weeks later and then at approxi- tivity to both trout and bass VTG, the mately 3- to 4-week intervals, used 100 pg criterion used to select colonies for doning. White perch, 1.123 0.143 The Rl mAb showed an excellent Morone americana protein and Freund's incomplete adjuvant. Rabbits were bled periodically through the dose-response profile of binding to VTG Tuatara, 0.473 0.160 were showed Sphenodon punctatus marginal ear vein and serum samples purified from trout and bass and diluted 1:100 in ELISA buffer, very low levels of reactivity to male plasma collected, 0.492 0.107 Snake, the 1). When the Crotalus molossus and evaluated for antibody titers using from both species (Figure ELISA with the OVA-peptide conjugate as ELISA was used to screen plasma from nat- Chicken, 0.464 0.186 female or male white Gallus domesticus the reference protein. When the titer was urally vitellogenic judged to be sufficient, the rabbits were perch, white croaker, brown trout, pollock, and their serum was iso- the exsanguinated snake, tuatara, and chicken, reactivity lated and frozen at -80°C until use. of RI to female plasma was a minimum of VTGs and serum samples 2 to 3 times higher than to male plasma Purified 0.7- obtained from striped bass males or (Table 1). 2D8 immunoactivity in the VTG E2- females were SDS- ELISA to plasma from vitellogenic and 0.6 -._ Control treated separated by PAGE (32) and Western blots (33) were nonvitellogenic striped bass was similar to 0.5- the of the that of Rl but exhibited substantially used to test immunoreactivity 0.4- antisera to the VTGs and other serum pro- higher background levels (Figure 2). o 0.3e used the antiserum Similar results were obtained for trout teins. Western blots 0.2 \ diluted 1:3000 and were visualized using plasma and a variety of purified control 0.1 to alkaline proteins including OVA, bovine serum goat anti-rabbit IgG conjugated 0.0- X 1 2 o3 4 5 6 phosphatase as the secondary antibody. albumin, and KLH, which gave the same dilution Log, plasma profile as male trout or bass plasma over Results the same range of total protein concentra- tion (data not shown). The higher back- 2. to Figure Binding of the 2D8 monoclonal antibody Monodonal Antibodies accompanied by ground for 2D8 was not serial dilutions of from (VTG) and plasma vitellogenic Three sets of fusions resulted in the genera- any detectable nonspecific binding to in the nonvitellogenic (control) immature striped bass The first set of tion of seven mAb cultures. plasma proteins from nonvitellogenic bass ELISA. Vitellogenesis was induced in antibody capture with that even with high fusions rise to two lines or trout in Western blots. The 2D8 mAb the fish by injection E2. Note gave hybridoma background levels for nonvitellogenic fish there is still culture was ELISA from which one monoclonal was also screened by against plasma in to from a distinct increase binding plasma E2- The mAb by this cul- from vitellogenic and nonvitellogenic black developed. produced treated fish. was as an rat snakes with similar results (Figure 3). ture, designated Rl, isotyped class antibody with kappa light SDS-PAGE of purified trout or bass IgM RI demonstrated cross reac- VTG or from fish injected chains. strong plasma samples to VTG from bass and rain- tivity striped in the ELISA and was used to bow trout 0.6 - -U- RBTVTG the Western blot Initial 0.4- develop procedure. -o- SBVTG VTG 0.5 - --- RBT Male that reacted RI with - Control ELISA screens plasma C- SB Male -+- 0.3- from vitellogenic females or males of sev- 0.4 - from diverse taxa eral vertebrate species 0.3- 0.2- showed to vitellogenic specific reactivity 0.2 - for all tested. Rl was lost plasma species 0.1- due to an malfunction. The 0.1 - equipment resulted in one mAb second set of fusions T2 4 5 6 but lost 0.1 1 designated T2, specific 0.001 0.01 10 100 dilution Log, plasma to immunoactivity vitellogenic plasma VTG, ig/ml after its or VTGs shortly develop- samples it was never or used for ment, and isotyped Figure 1. Binding of the Rl monoclonal antibody (OD Figure 3. Binding of the 2D8 monoclonal antibody to resulted The third set of fusions screening. to bass and rainbow trout serial dilutions of from and 490) purified striped (SB) plasma vitellogenic (VTG) from in one positive hybridoma colony in Dilutions of (RBT) VTG the antibody capture ELISA. nonvitellogenic (control) black rat snakes in the anti- were which five monoclonal cultures male male and SB are serial from ELISA. was induced in the plasma (RBT male) body capture Vitellogenesis not The mAbs these 1:10 to 1:1280 (scale shown). snakes by injection with E2. derived. produced by Volume October 1995 1 1 103, Supplement 7, HEPPELL ETAL. with E2 revealed distinct high molecular Negative controls for the Western blots a c d e weight bands (MA involved using fresh culture 170,000-200,000) not media in place present in plasma from male trout or bass of TCSN from the monoclonal cell cul- (Figure 4A). We identified these bands as tures producing the 2D8 mAb or substitut- 20 the primary VTG subunit and its degrada- ing TCSN from a monoclonal culture tion products (24,25,34). Western producing an IgM class blots of mAb directed the SDS-PAGE gels done using the 2D8 against a recombinant hepatitis B core anti- mAb revealed immunoreactivity to the gen. These substitutions eliminated purified VTGs and the high molecular immunoreactivity on the Western blots. weight bands for VTG in plasma, but no Antisera Polyonal binding to plasma proteins from male trout or bass was detected (Figure 4B). This The results of pat- microsequencing of the fish 69.l. tern in SDS-PAGE and Western blots is VTGs verified the strong degree of conser- identical to that obtained using homolo- vation at the N-terminal region of the pro- in gous antisera raised rabbits against tein (Table 2). The region of highest purified trout or bass VTG A homology the 46-: [(24,25,34); among fishes extends Hara and CV Sullivan, unpublished data] between amino acid residues 7 and 20 of with the exception that the homologous the striped bass VTG molecule, showing antisera reacted only very weakly with the 100% identity in sequence with mummi- heterologous VTG, producing a barely vis- with SD)S-PAGE and corresponding Western blots chog VTG, 87% pinfish VTG, 93% Figure 5. om striped bass done using the ible band in anti-VIG Western blots (35). SDS- with brown bullhead VTG, and 60% with of plasma fr ;erum. Lane a, mw markers; lane b, plasma PAGE and Western blots using the 2D8 white peptide VTG. This of antis sturgeon degree ature striped bass injected with E2; lane c, mAb and from an imm plasma E2-treated and con- homology made the N-terminus a primary from a male striped bass; lane d, Western blot trol bullfrogs, black rat snakes, and chick- candidate for generating an antipeptide of b; /ane e Western blot of c. Numbers with horizontal ens also showed immunoreactivity to high antiserum for detecting VTG in a wide bars indicate the mass of the mw markers. molecular weight, E2-inducible proteins, variety of species (23). presumably VTG, but not to plasma pro- The antiserum generated against the teins in control samples not consensus N-terminal VTG y received (data shown). sequence effluent rich in polynu- historically conjugated to KLH reacted preferentially clear aromnatic hydrocarbons (PAHs) from with the VTG band in an iated (Mr=: 170,000) associ coking facility. VTG is Western blots of plasma from immature in serum samples from the apparent striped bass injected with E2; this band is tumor-be;aring adult males and females not visible in SDS-PAGE or Western blots after SDS;-PAGE, but not in serum from of plasma from control male bass (Figure adult femaales collected from a pristine con- 5). The fainter immunoreactive band trol creek at the same time and outside of 36,000) visible on the Western blot the norm al vitellogenic season for this (Mr in only the lane for male plasma appears to species (Fiigure 6). Corresponding Western have a lower affinity to the antiserum, fad- blots e the anti-VTG don using peptide more as ing rapidly the antiserum is diluted antiserum revealed strong immunoreactiv- out. We are in the process of and to the VTG band in purifying ity 170,000) (Mr- identifying this protein. the lane f4or plasma from a tumor-bearing We on selective female an d also to a lower band that previously reported is Mr induction of vitellogenesis in tumor-bear- presumablly a breakdown product of VTG ing (hepatocellular- and cholangio-carcino- (Figure 7) This was ). immunostaining not mas) male and female brown bullhead evident isn the lane for plasma from a collected from the Black River near control maale. Ohio The Lorraine, (23). collection site We are currently examining the extent receives effluent from a steel plant and of cross-re of the anti-VTG activity peptide Table 2. N-terminal amino acid sequences for several fish vitellogenins. Source Sequence Homology, % bass: Striped HNVNFAPEFAAGKTYVYKYEAL 4. of rainbow trout and (100) Figure (A) SDS-PAGE striped Mummichog: MKAVVLALTLASYA -GONFAPEFAAGKTYVYKYEAL (100) bass VTG and blood Lane plasma proteins. a, purified Pinfish: YQVNLAPEFAAGR_THVYKYEAF (87) bass lane from bass striped VTG; b, plasma striped Bullhead: HQJILVPEFAAGKTFVYKYE (93) injected with E2; lane c, male striped bass plasma; Yellow perch: QVNFAPEFA lane rainbow trout lane from d, purified VTG; e, plasma Medaka: QLVFAPEFA rainbow trout injected with lane male rainbow E2; f, Sturgeon: QQTKYEPSFSGSKTYOYKYEG (60) trout The were stained with Coomassie plasma. gels Brilliant Blue. Western blots of a set of the Underlining indicates regions of similarity with the striped bass. Numbers in are the (B) parallel parentheses percent similarity same shown in A done the mono- with the bass between 7 samples using 2D8 striped sequence positions and 20. The sequence MKAWLALTLASYA for mummichog is clonal antibody. the putative leader sequence (23). Environmental Health Perspectives VITELLOGENIN AS A BIOMARKER FOR ESTROGENS reacts to purified VTG and pos- specifically a b c d 200-i sibly other estrogen-inducible proteins in representatives of at least four vertebrate classes. Because the monoclonal cell line 2D8 was initially selected on the producing 97j_ basis of its ability to recognize VTG from 69-.1 bass, species from lineages both trout and that have evolved independently for 200 million years, we con- approximately 468- sider it likely that 2D8 is targeted at a con- served on the VTG molecule. epitope Whether this epitope is completely unique to VTG remains to be rigorously verified. *30 - that we developed were All of the mAbs found to be IgM class antibodies. Such antibodies, while having strong epitope specificity, have a tendency toward high 21.5- nonspecific binding in immunoas- levels of says because of their large mass. This is a factor in the relatively high likely to be 14.3- background seen in our ELISA (Figure 2). into We plan to cleave the pentameric IgM its monomeric Fab fragments in an to reduce the high background. In attempt 6.5- our final series of immunizations, we modified our protocol to increase the prob- ability of generating high affinity-high titer mw marker or serum Figure 6. SDS-PAGE of proteins Figure 7. SDS-PAGE (lanes a-c) and corresponding mAbs but we were unable to accom- IgG bullhead. Lane a, mw markers; lane b, blots dand of mw marker or from brown Western (lanes e) proteins plish this goal. The last five monoclonal from a control serum from a female collected pristine serum from brown bullhead using the anti-VTG peptide derived from the final fusion, all cultures, serum from a female with mw lane serum from a creek; lane c, hepatocellu- antiserum. Lane a, markers; b, produced IgM class mAbs. collected from the lar- and cholangio-carcinoma pol- female with hepatocellular- and cholangio-carcinoma IgM induction is part of the initial luted Black River; lane d, serum from a male with from the Black lane serum collected polluted River; c, to a from the Black River. immune response following exposure cholangio-carcinoma collected from a male collected from a pristine control creek; Numbers with horizontal bars indicate the mass of the lane Western blot of b; lane e, Western blot of c. novel antigen and, upon subsequent expo- d, mw markers. Numbers with horizontal bars indicate the mass of the is a sure to the same antigen, there typically mw markers. class shift towards synthesis of IgGs (26). that our We have developed the concept from other fish the of vertebrate inability to induce antibody class shift and antiserum with plasma property recognizing of Initial studies indicate that the VTG without regard to species of origin. generate an IgG mAb may be a function species. We both monoclonal antibodies the or nature of antigenicity of con- antiserum may have wide cross-reactivity developed degree the VTG molecule. with VTGs from several teleost fish includ- and a polyclonal antiserum possessing this served epitopes on and Such antibodies have the poten- Because there are extensive gene sequences ing medaka 193,000 130,000) property. (Mr.- VTG between VTG and ' 120,000 and tial to form the basis of a universal that are well conserved and yellow perch (Mr VTGs tested so far all immunoassay able to detect estrogen some mammalian serum proteins (16,17), 104,000). The fish in vertebrate. inducible share a of at their N- exposure any oviparous some of which are also estrogen high degree similarity a Our ELISA results for the mAbs (RI it is possible that there is no antibody termini (Table 2); however, preliminary (37), to lack of test with rainbow trout serum gave nega- and 2D8) show much higher absorbance class shift due strong antigenicity work is in values for VTG or plasma from of the conserved VTG epitopes. They may tive results. Further progress purified than for "self' to an with this and others that fill the E2-treated fish or snakes plasma be too close to generate IgG species between rainbow trout from untreated immature animals or males response. There are natural autoantibodies phylogenetic gap but do not in most individuals; in and bass. (Figures 1-3), they provide present healthy striped conclusive evidence that the mAbs mice, the vast majority of these are IgM Discussion VTG in the class specifically react to plasma. (38). VTG is a need for a bio- the results of Western blotting It should also be noted that There is a demonstrable However, that the 2D8 mAb large and complex phospholipoglycopro- chemical marker that can be used to iden- clearly demonstrate in has to before their binds to VTG in plasma of tein, one that our hands proved tify estrogenic compounds preferentially and bass 4) be There is no release into the environment (36). The rainbow trout striped (Figure highly antigenic (24,25). corroborate as well as to high MW E2-induced pro- reason to presume that dominant epitopes results of this study generally in of bull- on the are conserved. on VTG as a biomarker for teins, presumably VTG, plasma protein Polyclonal prior reports in trout or and frogs, black rat snakes, and chickens (35). antisera raised rabbits against to estrogens (6-9), they exposure ELISA these bass VTG show levels of demonstrate the feasibility of two Combined with the results, very high to their to antibodies with observations indicate that the 2D8 mAb immunoreactivity homologous approaches developing October 1995 Volume 103, Supplement 7, HEPPELL ETAL. lead to production of numerous hybrid- the reason(s) for its cross-reactivity VTG but are also weakly reactive to het- evaluate erologous VTG from the other species oma colonies secreting antibodies specific to the anti-VTG peptide antiserum. to bass but not trout VTG. Salmonids are Although the cross-reactivity may preclude (35), lending credence to the concept that conserved epitopes contribute little to the considered primitive to the temperate utilization of this antiserum in ELISA the conserved struc- screens for VTG in some species, the anti- overall immune response to VTG. During basses, and perhaps development of the mouse immune tural epitope that our mAbs were directed serum is clearly useful for Western blotting may be anti- or less protected in to detect VTG in diverse lineages of fishes. response, many VTG epitopes against is less variable genic enough to induce IgG synthesis, these fish. This may account for the better In summary, we have demonstrated the we select immune response that trout VTG elicited of universal antibod- whereas the conserved epitopes feasibility generating for may not exhibit this degree of anti- in mice as compared to striped bass VTG. ies directed against the VTG molecule- antibodies that can be exploited to detect genicity. We are currently exploring this Our polyclonal antiserum directed for exposure possibility using denatured and delipidated against the short, synthetic, consensus N- VTG as a general biomarker VTG as the for terminal sequence of VTG (Table 2) of oviparous animals to endogenous and or deglycosylated antigen generating universal mAbs. Alternatively, appears to specifically recognize the high environmental estrogens and estrogen will to suppress the immune molecular weight, E2-inducible protein that mimics. Both mAbs directed against con- we attempt response of mice to species-specific we identified as VTG in plasma from served epitopes on the protein and poly- VTG epitopes by female or fish (Figures 5,6) and clonal antiserum directed against its immunodominant E2-treated treatment with cyclophosphamide (39). serum from fish with cancer (hepatocellu- conserved N-terminal amino acid sequence is interesting to note that although lar- and cholangio-carcinoma) collected were developed. Further research is needed It The both trout and bass VTGs were used as from a contaminated site (Figure 7). to produce higher affinity mAbs and more we were only able to obtain mAbs faint protein band (-36 kDa) visible in specific antisera to more easily develop antigens, bass plasma may for VTG as a biochemical marker with the desired specific cross-reactivity Western blots of striped bioassays from mice immunized with trout VTG. be a VTG-related protein. Once we have for estrogen exposure. Our results clearly and identified this that this is an achievable goal. The bass VTG was however able to gener- purified, sequenced, pro- indicate ate an immune response in mice, which tein, we will be able to more conclusively REFERENCES 1. Colburn Clement C, eds. Chemically-induced Alterations in gous radioimmunoassay and immunological cross-reactivity T, Sexual and Functional Development: The Wildlife/Human with vitellogenin from other teleosts. Comp Biochem Physiol Connection. Princeton, NJ:Princeton Scientific Publishing, 1992. 81B:63-71 (1985). 2. Schmidt WA. Hormone Copycats. Ann Arbor, MI:National 13. Benfey TJ, Donaldson EM, Owen TG. An homologous Wildlife Federation, 1994. radioimmunoassay for coho salmon (Oncorynchus kisutch) vitel- 3. Wallace RA. Vitellogenesis and oocyte growth in non-mam- logenin, with general applicability to other Pacific salmonids. malian vertebrates. In: Developmental Biology. Vol 1 (Browder Gen Comp Endocrinol 75:78-82 (1989). LW, ed). New York:Plenum Press, 1985;127-177. 14. Lee KBH, Lim EH, Lam TJ, Ding JL. Vitellogenin diversity in 4. Wahli W. Evolution and expression of vitellogenin genes. the perciformes. J Exp Zool 264:100-106 (1992). Trends Genet 4:227-232 (1988). 15. Wahli W, David IB, Ryffel GU, Weber R. Vitellogenesis and 5. Lazier CB, MacKay ME. Vitellogenin gene expression in teleost the vitellogenin gene family. Science 212:298-304 (1981). fish. In: Biochemistry and Molecular Biology of Fishes; 16. Baker ME. Is vitellogenin an ancestor of apolipoprotein B-100 Molecular Biology Frontiers. Vol 2 (Hochachka PW, of human low-density lipoprotein and human lipoprotein Mommsen TP, eds). Amsterdam:Elsevier, 1991;391-405. lipase? Biochem J 255:1057-1060 (1988). 6. Pelissero C, Le Menn Kaushick F. Estrogenic effect of 17. Baker ME. Invertebrate vitellogenin is homologous to human F, dietary soya bean meal on vitellogenesis in cultured siberian von Willebrand factor. Biochem J 256:1059-1060 (1988). sturgeon Acipenser baeri. Gen Comp Endocrinol 83:447-457 18. Byrne BM, Gruber M, Ab G. The evolution of egg yolk pro- (1991). teins. Prog Biophys Mol Biol 53:33-69 (1989). 7. Jobling Sum ter JP. Detergent components in sewage 19. Wallace RA, Deufel RA, Misluvin Z. Protein incorporation by S, of autologous and effluent are weay oestrogenic to fish: an in vitro study using isolated amphibian oocytes-VI. Comparison rainbow trout (Oncorynchus mykiss) hepatocytes. Aquat Toxicol xenogenic vitellogenins. Comp Biochem Physiol 65B:151-155 27:361-372 (1993). (1980). 8. Pelissero C, Flouriot G, Foucher JL, Bennetau B, Dunogues J, 20. Covens M, Covens L, Ollevier F, De Loof A. comparative yolk Legac F, Sumpter JP. Vitellogenin synthesis in cultured hepato- study of some properties of vitellogenin (Vg) and proteins cytes: an in vitro test for the estrogenic potency of chemicals. J in a number of freshwater and marine teleost fishes. Comp Steroid Biochem Mol Biol 44:263-272 (1993). Biochem Phsyiol 88B:75-80(1987). Regulation 9. Purdom CE, Hardiman PA, Bye VJ, Eno NC, Tyler CR, 21. Stifani S, LeMenn F, Rodriguez JN, Schneider WJ. Biochim Sumpter JP. Estrogenic effects of effluents from sewage treat- of oogenesis: the piscine receptor for vitellogenin. ment works. Chem Ecol 8:275-285 (1994). Biophys Acta 1045:271-279 (1990). in the P. Comparative studies of fish, amphib- 10. Sumpter J. Estrogenic surfactant-derived chemicals 22. Carnevali 0, Belvedere vitellogenins. J Exp Zool 259:18-25 (1991). aquatic environment. Environ Health Perspect 103 (Suppl 7): ian, and reptilian Wallace LaFleur Gross 173-178 (1995). 23. Folmar LC, Denslow ND, RA, G, T, of an estradiol- Bonomelli Sullivan CV. A conserved N-terminal 11. Campbell CM, Idler DR. Characterization S, highly serum as value to the bio- induced protein from rainbow trout vitellogenin by sequence for teleost vitellogenin with potential to the composition and radioimmunological cross reactivity chemistry, molecular biology and pathology of vitellogenesis. J ovarian yolk fractions. Biol Reprod 22:605-617 (1980). Fish Biol 46:255-263 (1995). in 24. Hara A, Sullivan CV, DickhoffWW. Isolation and some char- 12. So YP, Idler DR, Hwang Plasma vitellogenin landlocked Sj. homolo- its Atlantic salmon (Salmo saklr Ouananiche): isolation, acterization of vitellogenin and related egg yolk proteins 14 Environmental Health Perspectives VITELLOGENINAS A BIOMARKER FOR ESTROGENS from coho salmon (Oncorynchus kisutch). Zool Sci 10:245-256 33. Johnson DA, Gautsch JW, Sportsman JR, Elder JH. Improved technique utilizing non-fat dry milk for analysis of proteins and (1993). 25. Tao Y, Hara A, Hodson RG, Woods LC III, Sullivan CV. nucleic acids transferred to nitrocellulose. Gene Anal Tech Purification, characterization and immunoassay of striped bass 1:3-8 (1984). (Morone saxatilis) vitellogenin. Fish Physiol Biochem 12:31-46 34. Hara A. Studies on female-specific serum proteins (vitel- logenin) and egg yolk proteins in teleosts: immunochemical, (1993). 26. Harlow E, Lane D. Antibodies: A Laboratory Manual. Cold physiochemical and structural studies. Mem Fac Fish Spring Harbor, NY:Cold Spring Harbor Laboratory Press, 1988. Hokkaido Univ 34: 1-59 (1987). Kah F. 27. Nunez-Rodriguez J, 0, Geffard M, Le Menn Enzyme- 35. Heppell SA. Development of universal vertebrate vitellogenin linked immunosorbent assay (ELISA) for sole (Solea vulgaris) antibodies. Masters Thesis, North Carolina State University, vitellogenin. Comp Biochem Physiol 92B:741-746 (1989). Raleigh, NC, 1994. A in 28. Devereux J, Haeberlie P, Smithies 0. comprehensive set of 36. Colburn T, Clement C, eds. Chemically-induced Alterations for the VAX. Nucleic Acids Res sequence analysis programs Sexual and Functional Development: The Wildlife/Human 12:387-395 (1984). Connection. Princeton, NJ:Princeton Scientific Publishing, Hashida Yoshitake 29. Ishikawa E, Imagawa M, S, S, Hamaguchi 1992;1-8. Ueno T. Enzyme-labeling of antibodies and their fragments 37. Walsh BW, Schiff Y, I, Rosner B, Greenberg L, Ravnikar V, Sacks for and immunohistochemical FM. Effects of the enzyme immunoassay staining. J postmenopausal estrogen replacement on Immunoassay 4:209-327 (1983). concentrations and metabolism of plasma lipoproteins. Engl Fields Fields GB. A mini- 30. King D, CG, cleavage method which J Med 325:1196-1204 (1991). FMOC solid phase synthesis. Lim mizes side reactions following Int 38. Kaushik A, A, Poncet P, Ge X-R, Dighiero G. Protein Res of J Pept 36:255-266 (1990). Comparative analysis natural antibody specificities among 31. Bidlingmeyer BA, Cohen SA, Tarvin TL. Pre-column derivati- hybridomas originating from spleen an peritoneal cavity of zation for amino acids adult NZB and BALB/c mice. Scand analysis. J Chromatogr 336:93-104 J Immunol 27:461-471 (1984). (1988). Tricine-sodium sulfate- 39. Neil GA, Urnovitz HB. Recent improvements in the produc- 32. Schagger H, von Jagow G. dodecyl for the of tion of cells. polyacrylamide gel electrophoresis separation proteins antibody-secreting hybridoma Trends Biotechnol in the range from 1 to 100 kDa. Anal Biochem 166:368-379 6:209-213 (1988). (1987). Volume 103, Supplement 7, October 1995 15 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Environmental Health Perspectives Unpaywall

Universal assay of vitellogenin as a biomarker for environmental estrogens.

Heppell, S A; Denslow, N D; Folmar, L C; Sullivan, C V
Environmental Health Perspectives – Oct 1, 1995
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Abstract

Universal Assay of Vitellogenin as a Biomarker for Environmental Estrogens A. Heppell,1 Nancy D. Denslow,2 Leroy C. Folmar,3 and Scott V. Sullivan' Craig 'Department of Zoology, North Carolina State University, Raleigh, North Carolina; 2Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida; 3U.S. Environmental Protection Agency, Gulf Breeze, Florida Vitellogenin (VTG), the serum phospholipoglycoprotein precursor to egg yolk, is potentially an ideal biomarker for environmental estrogens. This study was undertaken to develop antibodies against conserved regions on the VTG molecule that could form the basis for establishing bioassays to detect estrogen exposure in any oviparous vertebrate. We developed monoclonal antibodies (mAbs) generated against purified rainbow trout VTG purified from two phylogenetically distant vertebrates, mykiss) VTG and selected for the property of specifically recognizing (Oncorhynchus trout and striped bass (Morone saxatilis). Results of enzyme-linked immunosorbent assay and Western blotting indicated that these mAbs specifically recognize purified VTG and VTG or other estrogen-inducible proteins in plasma or serum from representative species of four vertebrate classes (fish, amphibians, reptiles, and birds). All of the mAbs generated were IgM class. A polyclonal antiserum was raised against a synthetic consensus peptide representing the conserved N-terminal amino acid sequence of VTG. The results of Western blotting indicate that this antiserum teleost fish of diverse families. It was used to detect VrG in Western blots of serum from specifically recognizes VTG in plasma or serum from with cancer and cholangio-carcinoma) collected from a contaminated industrial site outside of brown bullhead (Ameiurus nebulosus) (hepatocellular their normal vitellogenic season. Our results indicate that it is feasible to generate antibodies capable of recognizing VTG without regard to species and that development of a universal VTG assay is an achievable goal. - Environ Health Perspect 1 03(Suppl 7):9-15 (1995) Key words: vitellogenin, universal assay, environmental estrogens, xenobiotics, monoclonal, immunoassay, synthetic peptide, biomarker, polyclonal, hormone mimics effects on the reproduc- their use or release into the environment. and their potential Introduction their notoriously weak struc- tive biology of vertebrates. However, that the Vitellogenin (VTG) is potentially an Environmental estrogens, chemicals with ture-activity relationship requires of similar to estrogen, identity and potency of environmental ideal biomarker for the estrogenicity bioactivity endogenous The chemicals. It is a large serum phospholipo- are known to affect and sexual estrogens be defined by bioactivity. development pre- of vertebrates and are impli- current lack of effective bioassays is consid- glycoprotein that serves as the major maturation be a cause of the lack of cursor to the egg-yolk proteins of cated as cancer promoters (1). They ered to proximal of detergents, regulatory action regarding these com- oviparous vertebrates (3). VTG is synthe- indude breakdown products An effective needs to sized and secreted by the liver in response and a variety of chlo- pounds (2). bioassay pesticides, plasticizers, such chemicals be developed to assess the estrogenicity of to circulating estrogens in maturing rinated compounds. Ideally, before or their derivatives females and is normally undetectable would be evaluated for estrogenicity synthetic compounds (4,5) and in the plasma of immature animals males. The presence of this estrogen- in the of an ani- inducible protein serum on in the Environment, III: Global Health Implications This paper was presented at the Symposium Estrogens in DC. received: March 15, 1995; manuscript accepted: mal can be taken as evidence of exposure to held 9-11 January 1994 Washington, Manuscript April 4, 1995. or or endogenous exogenous estrogens assistance in either samples, fish, or both. The following peo- We thank several people for obtaining plasma estrogen mimics. This potential ofVTG as and animals: M. tuatara; P. Licht, snake; E. donated plasma from vitellogenic nonvitellogenic Brown, ple has been white T. F. and A. J. Hinshaw donated the a biomarker already explored and croaker; Siopes, Edens, Youngblood, chicken; Casillas, pollock A. Hara for the of antiserum to trout VTG and to L. Greene rainbow trout. We are grateful to gift homologous several fish for which both in using species, mAbs. We thank B. Parten for Protein sequencing was for the antihepatitis B-core protein microsequencing. have been devel- vivo and in vitro assays Core in the Center for Biotechnology Research at carried out in the Protein Chemistry Facility Interdisciplinary However, immunological Florida. C. Bidwell the for white vitellogenin cDNA. This work oped (6-10). the University of provided sequence sturgeon of an Electric Power Research Institute Graduate Research Fellowship was supported by the University Florida, and structural of VTG can properties vary North Carolina Center 13ARIG 0803 to and a cooperative agreement SAH), a Biotechnology grant (#91 CVS) (to greatly even among closely related species Environmental Protection and the of Florida to NDD). between the U.S. Agency University (#CR821437 which limits broad scale applica- (11-14), to Dr. V. of Zoology, Campus Box 7617, North Carolina Address correspondence Craig Sullivan, Department NC 27695. 515-7186. Fax: (919) 515-2698. E-mail: vertebrate taxa. State University, Raleigh, Telephone: (919) tion of these assays across [email protected] it has become apparent that Recently used: avidin-biotin complex; DAB, diaminobenzidene; ELISA, enzyme-linked immunosor- Abbreviations ABC, VTG is an ancient protein belonging to a FMOC, fluorenylmethoxycarbonyl; IgG, bent assay; E2, 17>-estradiol; Fab, antibody binding fragment; limpet hemocyanin; mAb, monoclonal antibody; Mr, multigene family that includes a variety of immunoglobulin G; IgM, immunoglobulin M; KLH, keyhole aromatic saline; relative mobility; OVA, ovalbumin; PAH, polynuclear hydrocarbons; PBS, phosphate-buffered vertebrate lipoproteins, including various fluoride immunoblot membranes; rpHPLC, reverse-phase PITC, phenylisothiocyanate; PVDF, polyvinilydene and invertebrate VTGs as well as the sodium dodecyl sulfate-polyacrylamide gel electrophore- high performance liquid chromatography; SDS-PAGE, trifluoroacetic human serum proteins, apolipoprotein TCSN, tissue culture supernatant; TFA, acid; VTG, vitellogenin. sis; Environmental Health Perspectives HEPPELL ETAL. B-100 and von Willebrand factor (15-18). of a 96-well plate that had been previously white croaker, Genyonemus lineatus; brown Coupled with prior evidence for conserva- coated with antigen. The second antibody trout, Salmo trutta; and pollock, Theragra tion of some functional and immunologi- was a goat antimouse IgM, p-chain specific as well as representative rep- chalcogramma) cal properties of the molecule (19-22), antibody (Sigma Chemical Co, St. Louis, tiles (tuatara, Sphenodon punctatus; and rat- these findings suggest that VTG may pos- MO) conjugated to horseradish peroxidase, tlesnake, Crotalus molossus) and a bird sess conserved epitopes that could be and antibody binding was visualized with (chicken, Gallus domesticus). Additional to antibodies and O-phenylenediamine/H202 in 0.1 M cit- screened by Western blot included exploited develop species immunoassays of a more universal nature. rate-phosphate buffer, pH 5.0. the bullfrog (Rana catesbeiana), the black This study was undertaken to develop Hybridoma colonies whose TCSN rat snake (Elaphe obsoleta), and the chicken. antibodies against conserved regions on the demonstrated immunoreactivity in the To initiate generation of polyclonal VTG molecule that could form the basis ELISA to VTG and E2-plasma from both antisera known conserved regions against for establishing bioassays to detect estrogen bass and trout but did not react to normal on the VTG molecule, we microsequenced exposure in any oviparous vertebrate. We male plasma were selected for cloning. amino acids in the N-terminal regions of a cho- VTGs purified from several phy- attempted to develop both monoclonal Rainbow trout and striped bass were number of antibodies generated against purified VTG sen as the for initial screenings logenetically diverse teleost fish (23). We species and polyclonal antisera raised against the because they represent distinct vertebrate obtained sequences for several teleost fish conserved N-terminal amino acid sequence lines that have evolved independently over induding the striped bass, brown bullhead pinfish (Lagodon of VTG (23). Using these two approaches a considerable period of time (-200x 106 (Ameiurus nebulosus), we demonstrated that it is feasible to gener- methods for their respec- rhomboides), yellow perch (Perca years), purifying flavescens), as as ate antibodies capable of recognizing VTG tive VTGs were well established (24,25), and medaka (Oryzias latipes), well without regard to species of origin and that and they were available because they were sequences deduced from the cDNA of heteroclitus) and development of a universal VTG assay is subjects of other endocrine investigations mummichog (Fundulus an achievable goal. in our laboratory. Positive colonies were white sturgeon (Acipenser transmontanus). to bass was used as the template subjected to limiting dilution produce The striped Methods monoclonal cell lines secreting mAbs with for comparison because it is the most highly immunized ip with characteristics. The mAbs from the in To determine an Balb/C mice were desired evolved fish our study. either 100 of rainbow trout monoclonal cultures were isotyped using optimal peptide segment for antibody tar- pg as we a consensus N-terminal (Oncorynchus mykiss) VTG purified commercial kits (Life Technologies, geting, analyzed Hara et al. or 100 of MD, or Sigma). with a protein structure program described by (24) Gaithersberg, sequence pg for a second which an antigenicity index striped bass (Morone saxatilis) VTG Western blots were used (28), provides as described by Tao et al. (25) series of mAb screens. Samples of purified at each position of the peptide. Using this purified or male from information we a consensus mixed 1:2 with Freund's complete adju- VTG, E2-plasma, plasma designed vant. Mice were boosted twice at 2- to 3- trout and bass were normalized for protein peptide fifteen amino acids long. with of mixed dode- The consensus peptide was assembled week intervals 50 pg antigen concentration, separated by sodium 1:2 with Freund's incomplete adjuvant. cyl sulfate 4 to 15% polyacrylamide gradi- on a Model 431A automated peptide syn- were on a thesizer Foster Four days before fusion, they again ent gel electrophoresis (SDS-PAGE) (Applied Biosystems, City, N- boosted with 100 pg of antigen in incom- PhastSystem electrophoresis apparatus CA) from the C-terminus towards the Monoclonal antibodies and then elec- terminus with the a-carboxyl group of the plete adjuvant. (Pharmacia, Piscataway, NJ) were then standard troblotted onto Immobilon-P PVDF amino acid attached to the solid support, (mAbs) generated using entire was membranes Rink resin and methods (26). This procedure (Millipore, Marlborough, MA) using 9-fluorenylmethoxy- replicated 3 times. In an attempt to induce using the PhastSystem immunoblot appa- carbonyl (FMOC) protected amino acids. class shift and increase the ratus Membranes were blocked in An additional Cys residue was added at the antibody affinity (25). to acti- of the antibodies, the final set of immu- phosphate-buffered saline (PBS) (0.01 M N-terminus for coupling the peptide nizations was modified the 0.15 M NaCl, pH 7.3) with vated carrier (29). The peptide was by increasing proteins Na.H.PO4, time between boosts to a minimum of 3 3% bovine serum albumin overnight, incu- cleaved from the resin with trifluoroacetic a third boost bated with TCSN diluted in PBS with acid (TFA) (30), purified by reverse-phase weeks and incorporating prior to fusion. 0.5% Tween-20 (wash buffer; range high performance liquid chromatography colonies were screened for for 2 hr, and then probed (rpHPLC) on a Whatman partisil 10 Hybridoma 1:3-1:10, v/v) in VTG cross-reactivity using an antibody with secondary antibody (1:4000 wash ODS-2 column fitted to an HPLC system immunosorbent and avidin-biotin in wash (Waters, Milford, MA) and equilibrated capture enzyme-linked buffer) complex Vector A TFA in buffer Elite Kit, Laboratories, with 20% solvent (0.1% assay (ELISA) (27) against purified VTGs, (ABC from immature or male for min each. and 80% solvent B (0.1% TFA in blood plasma Burlingame, CA) 30 CH3CN) was with a linear bass and trout injected with Antibody binding visualized dH20) followed by 20-min gradient striped 170- to induce diaminobenzidene Vector to solvent A at a flow rate of 1 ml/min. estradiol (E2) vitellogenesis (E2- (DAB Kit, 35% and of plasma) (15), or plasma from normal male Laboratories). Compositional quantitative analysis and trout as the reference Preliminary ELISA and Western blot the peptide was conducted on a Pico Tag bass prepara- collect- screens of the of the mAbs to differ- work station after a 22-hr tions. The mAbs were harvested ability (Waters) hydroly- by the tissue culture bind to vitellogenic plasma and sis and phenyl isothiocynate (PITC) ing supernatant (TCSN), entially in from a of derivitization which was stored at 40C the of nonvitellogenic plasma variety (31). presence for to months until use. other were also done. Additional Aliquots of purified peptide (2 mg) 0.01% NaN3 up 3 species 1:2 with tested ELISA included several to maleimide- TCSN (100 pl) was mixed 10% species by were conjugated (1:2, w/w) normal serum and added to the wells fishes Morone activated carrier proteins, keyhole limpet goat (white perch, americana; 10 Environmental Health Perspectives VITELLOGENINAS A BIOMARKER FOR ESTROGENS Table 1. Reactivity in the antibody capture ELISA of the hemocyanin (KLH; mw 450-13,000 cultures were designated 2C1 1, 2D8, 3E8, Rl monoclonal antibody (OD 490) to plasma samples kDa), or ovalbumin (OVA; mw 45 kDa) 5G7, and 8G7. All five were isotyped as from vitellogenic females and males of several verte- using an Imject Activated Immunogen IgMs. Because 2D8 gave the most consis- brate species. Plasma samples were diluted 1:100. Conjugation kit (Pierce Chemical Co., tent results in initial screens, it was chosen Plasma source Female Male in All of the Rockford, IL). The KLH peptide conju- for use subsequent analyses. gate was used to immunize two female hybridoma cultures that were cloned were Brown trout, 1.613 0.163 Salmo trutta New Zealand white rabbits sc with 200 derived from mice immunized with trout pg protein in 0.5 ml sterile distilled water VTG. None of the hybridoma colonies Pollock, 0.768 0.213 with bass Theragra chalcogramma mixed 1:2 with Freund's complete adju- derived from mice immunized vant. Subsequent immunizations (n = 8), VTG gave significant levels of cross-reac- White croaker, 0.745 0.219 Genyonemus lineatus done 2 weeks later and then at approxi- tivity to both trout and bass VTG, the mately 3- to 4-week intervals, used 100 pg criterion used to select colonies for doning. White perch, 1.123 0.143 The Rl mAb showed an excellent Morone americana protein and Freund's incomplete adjuvant. Rabbits were bled periodically through the dose-response profile of binding to VTG Tuatara, 0.473 0.160 were showed Sphenodon punctatus marginal ear vein and serum samples purified from trout and bass and diluted 1:100 in ELISA buffer, very low levels of reactivity to male plasma collected, 0.492 0.107 Snake, the 1). When the Crotalus molossus and evaluated for antibody titers using from both species (Figure ELISA with the OVA-peptide conjugate as ELISA was used to screen plasma from nat- Chicken, 0.464 0.186 female or male white Gallus domesticus the reference protein. When the titer was urally vitellogenic judged to be sufficient, the rabbits were perch, white croaker, brown trout, pollock, and their serum was iso- the exsanguinated snake, tuatara, and chicken, reactivity lated and frozen at -80°C until use. of RI to female plasma was a minimum of VTGs and serum samples 2 to 3 times higher than to male plasma Purified 0.7- obtained from striped bass males or (Table 1). 2D8 immunoactivity in the VTG E2- females were SDS- ELISA to plasma from vitellogenic and 0.6 -._ Control treated separated by PAGE (32) and Western blots (33) were nonvitellogenic striped bass was similar to 0.5- the of the that of Rl but exhibited substantially used to test immunoreactivity 0.4- antisera to the VTGs and other serum pro- higher background levels (Figure 2). o 0.3e used the antiserum Similar results were obtained for trout teins. Western blots 0.2 \ diluted 1:3000 and were visualized using plasma and a variety of purified control 0.1 to alkaline proteins including OVA, bovine serum goat anti-rabbit IgG conjugated 0.0- X 1 2 o3 4 5 6 phosphatase as the secondary antibody. albumin, and KLH, which gave the same dilution Log, plasma profile as male trout or bass plasma over Results the same range of total protein concentra- tion (data not shown). The higher back- 2. to Figure Binding of the 2D8 monoclonal antibody Monodonal Antibodies accompanied by ground for 2D8 was not serial dilutions of from (VTG) and plasma vitellogenic Three sets of fusions resulted in the genera- any detectable nonspecific binding to in the nonvitellogenic (control) immature striped bass The first set of tion of seven mAb cultures. plasma proteins from nonvitellogenic bass ELISA. Vitellogenesis was induced in antibody capture with that even with high fusions rise to two lines or trout in Western blots. The 2D8 mAb the fish by injection E2. Note gave hybridoma background levels for nonvitellogenic fish there is still culture was ELISA from which one monoclonal was also screened by against plasma in to from a distinct increase binding plasma E2- The mAb by this cul- from vitellogenic and nonvitellogenic black developed. produced treated fish. was as an rat snakes with similar results (Figure 3). ture, designated Rl, isotyped class antibody with kappa light SDS-PAGE of purified trout or bass IgM RI demonstrated cross reac- VTG or from fish injected chains. strong plasma samples to VTG from bass and rain- tivity striped in the ELISA and was used to bow trout 0.6 - -U- RBTVTG the Western blot Initial 0.4- develop procedure. -o- SBVTG VTG 0.5 - --- RBT Male that reacted RI with - Control ELISA screens plasma C- SB Male -+- 0.3- from vitellogenic females or males of sev- 0.4 - from diverse taxa eral vertebrate species 0.3- 0.2- showed to vitellogenic specific reactivity 0.2 - for all tested. Rl was lost plasma species 0.1- due to an malfunction. The 0.1 - equipment resulted in one mAb second set of fusions T2 4 5 6 but lost 0.1 1 designated T2, specific 0.001 0.01 10 100 dilution Log, plasma to immunoactivity vitellogenic plasma VTG, ig/ml after its or VTGs shortly develop- samples it was never or used for ment, and isotyped Figure 1. Binding of the Rl monoclonal antibody (OD Figure 3. Binding of the 2D8 monoclonal antibody to resulted The third set of fusions screening. to bass and rainbow trout serial dilutions of from and 490) purified striped (SB) plasma vitellogenic (VTG) from in one positive hybridoma colony in Dilutions of (RBT) VTG the antibody capture ELISA. nonvitellogenic (control) black rat snakes in the anti- were which five monoclonal cultures male male and SB are serial from ELISA. was induced in the plasma (RBT male) body capture Vitellogenesis not The mAbs these 1:10 to 1:1280 (scale shown). snakes by injection with E2. derived. produced by Volume October 1995 1 1 103, Supplement 7, HEPPELL ETAL. with E2 revealed distinct high molecular Negative controls for the Western blots a c d e weight bands (MA involved using fresh culture 170,000-200,000) not media in place present in plasma from male trout or bass of TCSN from the monoclonal cell cul- (Figure 4A). We identified these bands as tures producing the 2D8 mAb or substitut- 20 the primary VTG subunit and its degrada- ing TCSN from a monoclonal culture tion products (24,25,34). Western producing an IgM class blots of mAb directed the SDS-PAGE gels done using the 2D8 against a recombinant hepatitis B core anti- mAb revealed immunoreactivity to the gen. These substitutions eliminated purified VTGs and the high molecular immunoreactivity on the Western blots. weight bands for VTG in plasma, but no Antisera Polyonal binding to plasma proteins from male trout or bass was detected (Figure 4B). This The results of pat- microsequencing of the fish 69.l. tern in SDS-PAGE and Western blots is VTGs verified the strong degree of conser- identical to that obtained using homolo- vation at the N-terminal region of the pro- in gous antisera raised rabbits against tein (Table 2). The region of highest purified trout or bass VTG A homology the 46-: [(24,25,34); among fishes extends Hara and CV Sullivan, unpublished data] between amino acid residues 7 and 20 of with the exception that the homologous the striped bass VTG molecule, showing antisera reacted only very weakly with the 100% identity in sequence with mummi- heterologous VTG, producing a barely vis- with SD)S-PAGE and corresponding Western blots chog VTG, 87% pinfish VTG, 93% Figure 5. om striped bass done using the ible band in anti-VIG Western blots (35). SDS- with brown bullhead VTG, and 60% with of plasma fr ;erum. Lane a, mw markers; lane b, plasma PAGE and Western blots using the 2D8 white peptide VTG. This of antis sturgeon degree ature striped bass injected with E2; lane c, mAb and from an imm plasma E2-treated and con- homology made the N-terminus a primary from a male striped bass; lane d, Western blot trol bullfrogs, black rat snakes, and chick- candidate for generating an antipeptide of b; /ane e Western blot of c. Numbers with horizontal ens also showed immunoreactivity to high antiserum for detecting VTG in a wide bars indicate the mass of the mw markers. molecular weight, E2-inducible proteins, variety of species (23). presumably VTG, but not to plasma pro- The antiserum generated against the teins in control samples not consensus N-terminal VTG y received (data shown). sequence effluent rich in polynu- historically conjugated to KLH reacted preferentially clear aromnatic hydrocarbons (PAHs) from with the VTG band in an iated (Mr=: 170,000) associ coking facility. VTG is Western blots of plasma from immature in serum samples from the apparent striped bass injected with E2; this band is tumor-be;aring adult males and females not visible in SDS-PAGE or Western blots after SDS;-PAGE, but not in serum from of plasma from control male bass (Figure adult femaales collected from a pristine con- 5). The fainter immunoreactive band trol creek at the same time and outside of 36,000) visible on the Western blot the norm al vitellogenic season for this (Mr in only the lane for male plasma appears to species (Fiigure 6). Corresponding Western have a lower affinity to the antiserum, fad- blots e the anti-VTG don using peptide more as ing rapidly the antiserum is diluted antiserum revealed strong immunoreactiv- out. We are in the process of and to the VTG band in purifying ity 170,000) (Mr- identifying this protein. the lane f4or plasma from a tumor-bearing We on selective female an d also to a lower band that previously reported is Mr induction of vitellogenesis in tumor-bear- presumablly a breakdown product of VTG ing (hepatocellular- and cholangio-carcino- (Figure 7) This was ). immunostaining not mas) male and female brown bullhead evident isn the lane for plasma from a collected from the Black River near control maale. Ohio The Lorraine, (23). collection site We are currently examining the extent receives effluent from a steel plant and of cross-re of the anti-VTG activity peptide Table 2. N-terminal amino acid sequences for several fish vitellogenins. Source Sequence Homology, % bass: Striped HNVNFAPEFAAGKTYVYKYEAL 4. of rainbow trout and (100) Figure (A) SDS-PAGE striped Mummichog: MKAVVLALTLASYA -GONFAPEFAAGKTYVYKYEAL (100) bass VTG and blood Lane plasma proteins. a, purified Pinfish: YQVNLAPEFAAGR_THVYKYEAF (87) bass lane from bass striped VTG; b, plasma striped Bullhead: HQJILVPEFAAGKTFVYKYE (93) injected with E2; lane c, male striped bass plasma; Yellow perch: QVNFAPEFA lane rainbow trout lane from d, purified VTG; e, plasma Medaka: QLVFAPEFA rainbow trout injected with lane male rainbow E2; f, Sturgeon: QQTKYEPSFSGSKTYOYKYEG (60) trout The were stained with Coomassie plasma. gels Brilliant Blue. Western blots of a set of the Underlining indicates regions of similarity with the striped bass. Numbers in are the (B) parallel parentheses percent similarity same shown in A done the mono- with the bass between 7 samples using 2D8 striped sequence positions and 20. The sequence MKAWLALTLASYA for mummichog is clonal antibody. the putative leader sequence (23). Environmental Health Perspectives VITELLOGENIN AS A BIOMARKER FOR ESTROGENS reacts to purified VTG and pos- specifically a b c d 200-i sibly other estrogen-inducible proteins in representatives of at least four vertebrate classes. Because the monoclonal cell line 2D8 was initially selected on the producing 97j_ basis of its ability to recognize VTG from 69-.1 bass, species from lineages both trout and that have evolved independently for 200 million years, we con- approximately 468- sider it likely that 2D8 is targeted at a con- served on the VTG molecule. epitope Whether this epitope is completely unique to VTG remains to be rigorously verified. *30 - that we developed were All of the mAbs found to be IgM class antibodies. Such antibodies, while having strong epitope specificity, have a tendency toward high 21.5- nonspecific binding in immunoas- levels of says because of their large mass. This is a factor in the relatively high likely to be 14.3- background seen in our ELISA (Figure 2). into We plan to cleave the pentameric IgM its monomeric Fab fragments in an to reduce the high background. In attempt 6.5- our final series of immunizations, we modified our protocol to increase the prob- ability of generating high affinity-high titer mw marker or serum Figure 6. SDS-PAGE of proteins Figure 7. SDS-PAGE (lanes a-c) and corresponding mAbs but we were unable to accom- IgG bullhead. Lane a, mw markers; lane b, blots dand of mw marker or from brown Western (lanes e) proteins plish this goal. The last five monoclonal from a control serum from a female collected pristine serum from brown bullhead using the anti-VTG peptide derived from the final fusion, all cultures, serum from a female with mw lane serum from a creek; lane c, hepatocellu- antiserum. Lane a, markers; b, produced IgM class mAbs. collected from the lar- and cholangio-carcinoma pol- female with hepatocellular- and cholangio-carcinoma IgM induction is part of the initial luted Black River; lane d, serum from a male with from the Black lane serum collected polluted River; c, to a from the Black River. immune response following exposure cholangio-carcinoma collected from a male collected from a pristine control creek; Numbers with horizontal bars indicate the mass of the lane Western blot of b; lane e, Western blot of c. novel antigen and, upon subsequent expo- d, mw markers. Numbers with horizontal bars indicate the mass of the is a sure to the same antigen, there typically mw markers. class shift towards synthesis of IgGs (26). that our We have developed the concept from other fish the of vertebrate inability to induce antibody class shift and antiserum with plasma property recognizing of Initial studies indicate that the VTG without regard to species of origin. generate an IgG mAb may be a function species. We both monoclonal antibodies the or nature of antigenicity of con- antiserum may have wide cross-reactivity developed degree the VTG molecule. with VTGs from several teleost fish includ- and a polyclonal antiserum possessing this served epitopes on and Such antibodies have the poten- Because there are extensive gene sequences ing medaka 193,000 130,000) property. (Mr.- VTG between VTG and ' 120,000 and tial to form the basis of a universal that are well conserved and yellow perch (Mr VTGs tested so far all immunoassay able to detect estrogen some mammalian serum proteins (16,17), 104,000). The fish in vertebrate. inducible share a of at their N- exposure any oviparous some of which are also estrogen high degree similarity a Our ELISA results for the mAbs (RI it is possible that there is no antibody termini (Table 2); however, preliminary (37), to lack of test with rainbow trout serum gave nega- and 2D8) show much higher absorbance class shift due strong antigenicity work is in values for VTG or plasma from of the conserved VTG epitopes. They may tive results. Further progress purified than for "self' to an with this and others that fill the E2-treated fish or snakes plasma be too close to generate IgG species between rainbow trout from untreated immature animals or males response. There are natural autoantibodies phylogenetic gap but do not in most individuals; in and bass. (Figures 1-3), they provide present healthy striped conclusive evidence that the mAbs mice, the vast majority of these are IgM Discussion VTG in the class specifically react to plasma. (38). VTG is a need for a bio- the results of Western blotting It should also be noted that There is a demonstrable However, that the 2D8 mAb large and complex phospholipoglycopro- chemical marker that can be used to iden- clearly demonstrate in has to before their binds to VTG in plasma of tein, one that our hands proved tify estrogenic compounds preferentially and bass 4) be There is no release into the environment (36). The rainbow trout striped (Figure highly antigenic (24,25). corroborate as well as to high MW E2-induced pro- reason to presume that dominant epitopes results of this study generally in of bull- on the are conserved. on VTG as a biomarker for teins, presumably VTG, plasma protein Polyclonal prior reports in trout or and frogs, black rat snakes, and chickens (35). antisera raised rabbits against to estrogens (6-9), they exposure ELISA these bass VTG show levels of demonstrate the feasibility of two Combined with the results, very high to their to antibodies with observations indicate that the 2D8 mAb immunoreactivity homologous approaches developing October 1995 Volume 103, Supplement 7, HEPPELL ETAL. lead to production of numerous hybrid- the reason(s) for its cross-reactivity VTG but are also weakly reactive to het- evaluate erologous VTG from the other species oma colonies secreting antibodies specific to the anti-VTG peptide antiserum. to bass but not trout VTG. Salmonids are Although the cross-reactivity may preclude (35), lending credence to the concept that conserved epitopes contribute little to the considered primitive to the temperate utilization of this antiserum in ELISA the conserved struc- screens for VTG in some species, the anti- overall immune response to VTG. During basses, and perhaps development of the mouse immune tural epitope that our mAbs were directed serum is clearly useful for Western blotting may be anti- or less protected in to detect VTG in diverse lineages of fishes. response, many VTG epitopes against is less variable genic enough to induce IgG synthesis, these fish. This may account for the better In summary, we have demonstrated the we select immune response that trout VTG elicited of universal antibod- whereas the conserved epitopes feasibility generating for may not exhibit this degree of anti- in mice as compared to striped bass VTG. ies directed against the VTG molecule- antibodies that can be exploited to detect genicity. We are currently exploring this Our polyclonal antiserum directed for exposure possibility using denatured and delipidated against the short, synthetic, consensus N- VTG as a general biomarker VTG as the for terminal sequence of VTG (Table 2) of oviparous animals to endogenous and or deglycosylated antigen generating universal mAbs. Alternatively, appears to specifically recognize the high environmental estrogens and estrogen will to suppress the immune molecular weight, E2-inducible protein that mimics. Both mAbs directed against con- we attempt response of mice to species-specific we identified as VTG in plasma from served epitopes on the protein and poly- VTG epitopes by female or fish (Figures 5,6) and clonal antiserum directed against its immunodominant E2-treated treatment with cyclophosphamide (39). serum from fish with cancer (hepatocellu- conserved N-terminal amino acid sequence is interesting to note that although lar- and cholangio-carcinoma) collected were developed. Further research is needed It The both trout and bass VTGs were used as from a contaminated site (Figure 7). to produce higher affinity mAbs and more we were only able to obtain mAbs faint protein band (-36 kDa) visible in specific antisera to more easily develop antigens, bass plasma may for VTG as a biochemical marker with the desired specific cross-reactivity Western blots of striped bioassays from mice immunized with trout VTG. be a VTG-related protein. Once we have for estrogen exposure. Our results clearly and identified this that this is an achievable goal. The bass VTG was however able to gener- purified, sequenced, pro- indicate ate an immune response in mice, which tein, we will be able to more conclusively REFERENCES 1. Colburn Clement C, eds. Chemically-induced Alterations in gous radioimmunoassay and immunological cross-reactivity T, Sexual and Functional Development: The Wildlife/Human with vitellogenin from other teleosts. Comp Biochem Physiol Connection. Princeton, NJ:Princeton Scientific Publishing, 1992. 81B:63-71 (1985). 2. Schmidt WA. Hormone Copycats. Ann Arbor, MI:National 13. Benfey TJ, Donaldson EM, Owen TG. An homologous Wildlife Federation, 1994. radioimmunoassay for coho salmon (Oncorynchus kisutch) vitel- 3. Wallace RA. Vitellogenesis and oocyte growth in non-mam- logenin, with general applicability to other Pacific salmonids. malian vertebrates. In: Developmental Biology. Vol 1 (Browder Gen Comp Endocrinol 75:78-82 (1989). LW, ed). 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