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A CHEMIFLUORESCENT IMMUNOASSAYFOR THE DETERMINATION OFMARINOBUFAGENIN IN BODY FLUIDSDaad Abi-Ghanem a , Xinzhong Lai b , Luc R. Berghman a c , DarijanaHorvat d , Jing Li b e , Daniel Romo b e , Mohammad N. Uddin d ,Yoshiaki Kamano f , Toshihiko Nogawa f , Jun-Ping Xu f , George R.Pettit f & Jules B. Puschett d ga Department of Poultry Science , Texas A&M University , CollegeStation, Texas, USAb Department of Chemistry , Texas A&M University , College Station,Texas, USAc Department of Veterinary Pathobiology , Texas A&M University ,College Station, Texas, USAd Department of Medicine , Texas A&M University Health ScienceCenter, Texas A&M University , College Station, Texas, USAe The Natural Products LINCHPIN Laboratory , Texas A&M University ,College Station, Texas, USAf Department of Chemistry and Biochemistry , Arizona StateUniversity , Temple, Arizona, USAg The Scott & White Memorial Hospital and Clinic , Temple, Texas,USAPublished online: 19 Jan 2011.

To cite this article: Daad Abi-Ghanem , Xinzhong Lai , Luc R. Berghman , Darijana Horvat , Jing Li ,Daniel Romo , Mohammad N. Uddin , Yoshiaki Kamano , Toshihiko Nogawa , Jun-Ping Xu , George R.Pettit & Jules B. Puschett (2011) A CHEMIFLUORESCENT IMMUNOASSAY FOR THE DETERMINATION OFMARINOBUFAGENIN IN BODY FLUIDS, Journal of Immunoassay and Immunochemistry, 32:1, 31-46, DOI:10.1080/15321819.2010.538107

To link to this article: http://dx.doi.org/10.1080/15321819.2010.538107

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A CHEMIFLUORESCENT IMMUNOASSAY FOR THEDETERMINATION OF MARINOBUFAGENIN IN BODY FLUIDS

Daad Abi-Ghanem,1 Xinzhong Lai,2 Luc R. Berghman,1,3 Darijana Horvat,4

Jing Li,2,5 Daniel Romo,2,5 Mohammad N. Uddin,4 Yoshiaki Kamano,6

Toshihiko Nogawa,6 Jun-Ping Xu,6 George R. Pettit,6 andJules B. Puschett4,7

1Department of Poultry Science, Texas A&M University, College Station, Texas, USA2Department of Chemistry, Texas A&M University, College Station, Texas, USA3Department of Veterinary Pathobiology, Texas A&M University, College Station,Texas, USA4Department of Medicine, Texas A&M University Health Science Center, Texas A&MUniversity, College Station, Texas, USA5The Natural Products LINCHPIN Laboratory, Texas A&M University,College Station, Texas, USA6Department of Chemistry and Biochemistry, Arizona State University, Temple,Arizona, USA7The Scott & White Memorial Hospital and Clinic, Temple, Texas, USA

& We describe here the development of a chemifluorescent competitive enzyme-linked immunosorbentassay (ELISA) that quantifies marinobufagenin (MBG) levels in biological fluids. Based on a poly-clonal antibody raised against a novel MBG–bovine serum albumin conjugate, this assay achievedan MBG detection limit of less than 9 pg=mL. MBG levels in various rat urine and serum sampleswere effectively determined using this methodology. Interassay variability averaged 9.8%, whileintra-assay variability averaged 1.9 and 2.5% in representative serum and urine samples, respect-ively. Recovery of exogenously added MBG averaged 106%, and parallelism data further establishedthe accuracy of the assay. Employment of this assay to detect MBG abnormalities represents a powerfultool for the possible diagnosis, prevention and management of human hypertensive states, parti-cularly preeclampsia.

Keywords bufodienolides, cardiac glycosides, diagnostic, hypertension, immunoassay,marinobufagenin

Address correspondence to Jules B. Puschett, Texas A&M Health Science Center College ofMedicine=Scott & White, 2401 S. 31st St. Medical Education Center, Room 407 L, Temple, TX 76508,USA. E-mail: jbpuschett@aol.com

Journal of Immunoassay and Immunochemistry, 32:31–46, 2011Copyright # Taylor & Francis Group, LLCISSN: 1532-1819 print/1532-4230 onlineDOI: 10.1080/15321819.2010.538107

Journal of Immunoassay and Immunochemistry, 32:31–46, 2011Copyright # Taylor & Francis Group, LLCISSN: 1532-1819 print/1532-4230 onlineDOI: 10.1080/15321819.2010.538107

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INTRODUCTION

The bufodienolides are a group of cardiotonic steroids that circulate inmammalian blood, are excreted in the urine, and are well-known potentconstituents of toad venoms and some plants.[1,2] They have been increas-ingly recognized as playing important roles in cardiovascular and kidneydisease, including hypertensive states and the cardiomyopathy of kidneyfailure.[3] The most widely studied of these compounds, marinobufagenin(MBG), plays an especially prominent role in the causation of thepregnancy-specific syndrome preeclampsia. The latter consists in the denovo development of hypertension and proteinuria after 20 weeks ofgestation.[4] Indeed, evidence has accrued that MBG is etiologically impor-tant in the elevation in blood pressure associated with hypertensive statescharacterized by excessive volume expansion.[5–10] Importantly, MBGblood levels are elevated in human volume expansion-mediated hyperten-sion,[8–10] and in experimental hypertensive states in animal models.[5,11]

MBG is one of the cardiac glycosides identified in the ancient Chinesemedicine Ch’an Su, which has been used for centuries in the treatmentof heart failure.[12,13] Furthermore, the extraction of toad skin and venomresults in mixtures of cardenolides (e.g., ouabain) and bufodienolides,which have been described in the medical literature as sources of poisoningand death.[13–15] Therefore, we have developed a method for the determi-nation of MBG in blood and urine. The methodology employed in thisassay is detailed in this communication.

While an assay for MBG has been previously reported,[16] the majoradvantage of our approach lies in the use of a novel point of attachmentof MBG to the carrier protein. The current strategy differs significantlyfrom previous work, given that the point of attachment to carrier proteinswas in close proximity to the only structural difference between MBG andresibufogenin (RBG; i.e., the C5 b-hydroxyl group). This potentiallyobscures the essential difference between RBG and MBG, and the resultingantibodies would not be expected to differentiate these closely analogousstructures.

MATERIALS AND METHODS

Marinobufogenin

One of the traditional Chinese medicinal preparations known as Ch’anSu prepared from the dried secretion of Chinese toad species (especiallyBufo gargarizans Cantor and B. melanostrictus Schneider) was purchased asthin plate in the Hong Kong folk-medical market and extracted withCH2Cl2

[17] or 1:1 ethanol-water.[18] Very careful separation of the extracts

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was performed by a series of column chromatographic proceduresemploying gel permeation with Sephadex LH-20 and where necessary usingHP-Cellulofine[17] followed by HPLC.[19] Penultimate separation stepsutilized silica gel column chromatography and TLC on the same sub-strate.[20] When pure, the marinobufagenin was found to be identical withboth natural and synthetic[21] specimens by a series of spectral methods:nuclear magnetic resonance (NMR)[22] and high-resolution (HR) massspectrometry.[23]

MBG–Protein Conjugates

A C16 N-hydroxysuccinimide (NHS) ester of cinobufotalin (CINO,Specs, Delft, HT, the Netherlands) was used to attach the hapten to acarrier protein, bovine serum albumin (BSA). CINO is very closely relatedto MBG in structure, only differing by an additional hydroxyl group at C16protected as an acetate group. To prepare the BSA–MBG antigen, thecinobufotalin-NHS ester was reacted with the surface lysines of BSA (Sigma,St. Louis, MO) by first dissolving the activated ester (1mg, 0.0016mmol) in100 mL dimethyl sulfoxide (DMSO), followed by the addition of 1mL stocksolution of BSA (10mg=mL in phosphate buffer, 0.1M phosphate, pH 7.2;0.1mM NaC1) at 23�C for 3–4 hr. The mixture was thoroughly stirred at thesame temperature for 10hr. A 20-mL aliquot was analyzed by MALDI massspectrometry to verify successful conjugation. In a similar manner, theMBG linker was conjugated to b-lactoglobulin (BLG, Sigma, St. Louis, MO).

Production of Polyclonal Antisera

Polyclonal anti-MBG–BSA antisera were produced by immunization ofsix New Zealand White rabbits with the BSA–MBG conjugate. Rabbits wereimmunized at 3-week intervals by subcutaneous administration of conju-gate in complete Freund’s adjuvant for the first injection, followed byincomplete Freund’s adjuvant for all subsequent injections. Decreasingimmunogen doses (100 mg for the first 4 injections, 50mg for the sub-sequent ones) were used to promote affinity maturation. One week aftereach immunization, blood samples were collected from the central earvein, and the resulting polyclonal sera were assessed for the presence ofMBG-specific antibodies by ELISA against the MBG–BLG conjugate.BLG has no sequence similarity with BSA, which eliminates potentialinterference of anti-BSA antibodies present in the antisera.

Briefly, Microfluor2 black ELISA plates (Thermo Scientific, Rockford,IL) were coated for 1 hr at 37�C with MBG–BLG (2mg=mL 0.1M NaHCO3

buffer, pH 9.5). Serum samples were serially diluted with StartingBlock

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buffer (Thermo Scientific, Rockford, IL), and a standard curve of MBGranging from 9 to 6561 pg=ml was prepared in StartingBlock buffer. TheMBG–BLG-coated ELISA plates were rinsed with TBST (0.01M Tris-HC1,pH 7.4; 0.9% (w=v) NaCl; 0.1% Triton-X), and blocked with StartingBlockbuffer. The various MBG dilutions were then added (50mL=well), directlyfollowed by the addition of the various dilutions of the respective primaryantibodies (50 mL=well), and the plates were incubated for 3 hr at ambienttemperature. Finally, the plates were rinsed as already described and incu-bated for 1 hr with a peroxidase-conjugated goat anti-rabbit immunoglob-ulin G (IgG) (1:3000 in StartingBlock buffer; Jackson ImmunoResearchLaboratories, West Grove, PA). Detection of bound antibody was completedby addition of QuantaRed enhanced chemifluorescent HRP substrate(Thermo Scientifc, Rockford, IL), and chemifluorescence was measuredin relative fluorescence units (excitation 544nm and emission 600nm)using a Victor2 plate reader (PerkinElmer, Waltham, MA). Negative con-trols included substitution of primary antibodies with preimmune sera orwith TBST. Data were analyzed and a five-parameter logistic curve fittingwas generated using the StatLIA software (Brendan Technologies,Carlsbad, CA). The final bleed of the best-responding rabbit was used forsubsequent assay development.

Biological Samples

Various rat urine and serum samples were randomly obtained fromexperiments performed at Texas A&M Health Science Center=Scott &White, Temple, TX. Animal care and all animal manipulations were conduc-ted in accordance with institutional guidelines. All samples were stored at�20�C until the time of use. Since urine did not show any matrix inter-ference in recovery and parallelism tests (vide infra), rat urine samples werefurther analyzed unprocessed, except for dilution with StartingBlock buffer.In contrast, rat serum samples were purified by solid-phase extraction (SPE)on C18 Empore disk cartridges (Varian, Inc., Palo Alto, CA). The disk wasfirst conditioned by addition of 150 mL acetonitrile, centrifuged at 280� gfor 1min, then washed with Milli-Q water and centrifuged as alreadydescribed. The serum sample (300mL) was then added to the disk with anequal volume of 10% acetonitrile in water, and centrifuged as described.Interfering substances were removed by sequential washes with Milli-Q waterand 10% acetonitrile, and the sample was twice eluted with 150 mL of aceto-nitrile each. Samples were then vacuum-dried and reconstituted in 100mL ofStartingBlock buffer. Diluted urine samples and extracted serum sampleswere run in duplicate, and average MBG concentrations (pg=mL) wereextrapolated from the standard curve using the StatLIA software.

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Sensitivity

The sensitivity of the assay was defined as the concentration of MBG (pg=mL) producing a response that was significantly different from the responseobtained in the absence of MBG. This was calculated by constructing a 95%confidence interval for the response at zero doses, and extrapolating thelower confidence limit to a detection limit value.

Specificity

To determine the specificity of the assay, the following substances weretested for potential cross-reactivity: cinobufotalin, resibufogenin (RBG,ChromaDex, Santa Ana, CA), digoxigenin, proscillaridin A, ouabain, bufa-lin, and digoxin (all from Sigma, St. Louis, MO). Cross-reactivity assays werecarried out as described earlier, except that a standard curve for each of therespective MBG congeners was prepared and incubated with the polyclonalantibody on the MBG–BLG-coated plate. Bound antibody was detected fol-lowing sequential incubation with HRP-goat anti-rabbit IgG and QuantaRedenhanced chemifluorescent HRP substrate, as described earlier. Dose-response curves were constructed for the cross-reactants and were comparedto the MBG dose-response curve. Percentage cross-reactivity was calculatedas 100 divided by the fold increase in the dose of cross-reactant requiredto produce the same signal as a certain dose of MBG.

Analytical Recovery from Serum

Normal rat serum (Jackson ImmunoResearch Laboratories, West Grove,PA) was extracted on SPE columns, as described earlier. This MBG-free ratserum was then spiked with 81, 243, and 729pg=mL of MBG. Followingsolid-phase extraction, serum samples or a standard curve of MBG wasincubated with anti-MBG–BSA on an MBG–BLG-coated plate, and boundantibody was detected as described earlier. MBG concentrations in serumsamples were determined, and percentage MBG recovery was calculated as100�measured MBG concentration=predicted MBG concentration.

Parallelism

To establish dilutional linearity of the assay, random urine samples werediluted 1:3, 1:9, and 1:27 in StartingBlock buffer. MBG-free normal ratserum (prepared as described earlier) was spiked with threefold dilutionsof MBG (ranging from 27 to 6561pg=mL), and each dilution was purifiedby SPE. MBG concentrations in urine=serum samples were determinedusing our polyclonal antibody assay, as described earlier.

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Assay Precision

The overall precision of the assay was estimated using within- andbetween-assay precision profiles. For intra-assay variability, the percent coef-ficient of variability (%CV¼ 100� SD=Mean) between sample duplicateswas computed using the StatLIA software. Ten representative serum and 10representative urine samples were selected to illustrate this measure of theassay’s precision. For interassay variability, six rat urine samples were assayedin duplicates in five consecutive assays. Overall mean MBG concentrationsobtained from the five assays were calculated for each sample. The average%CV (100� SD=Mean) for each sample and the overall%CV were calculated.

RESULTS

Synthesis of Bovine Serum Albumin (BSA)–MBG andb-Lactoglobulin (BLG)–MBG Conjugates

Unlike previous approaches to produce anti-MBG antisera using theC3-hydroxyl group of MBG for conjugation with a carrier protein(Figure 1, 4), our strategy consisted of activating the C16-hydroxyl group(Figure 1, 5) in order to maximally favor the production of antibodiescapable of discriminating between MBG and its antagonist, RBG(Figure 1, 2). Furthermore, we identified the excellent potential utility ofa commercially available bufodienolide, cinobufotalin (CINO) (Figure 1,3) for immunogen synthesis. CINO, differing from MBG by a mere acetylprotected hydroxyl group at C16, maintains all structural features ofMBG including the critical C5-hydroxyl group, the only functional group

FIGURE 1 Structures of MBG, RBG, and CINO (structural differences highlighted in red), a previouslydescribed C3-substituted MBG conjugate [25], and a newly designed immunogen starting with cinobu-fotalin.

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that distinguishes it from RBG. Most importantly, the alternative point ofattachment, namely, the C16-OH of CINO versus the C3-OH of MBG(Figure 1), extends the distance between the linker and the criticalC5-hydroxyl group, which was expected to increase the probability of gener-ating antibodies that recognize this subtle difference between MBG and RBG.

We synthesized the required CINO-NHS ester as shown in Figure 2A.CINO was first protected as the C3 t-butyldimethylsilyl ether 6, and thendeacetylation under mild basic conditions revealed the C16-hydroxyl as a

FIGURE 2 (A) Synthesis of a novel BSA-MBG conjugate 5 from cinobufotalin. The estimated molarratio of BSA: MBG was 10:1 as assessed by MALDI. (B) Structure of a novel BLG-MBG antigen 11 fromcinobufotalin. The estimated molar ratio of BLG: MBG as assessed by MALDI was 5–6:1.

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handle for conjugation to carrier proteins. The carboxylic acid-NHS ester 8was coupled to the C16-hydroxyl of 7 under standard conditions. Thisprovided an MBG hapten carrier 9 that bears an activated ester readiedfor coupling to carrier proteins following a final mild deprotection of thet-butyldimethylsilyl ether at C3.[24] The synthesis was readily scaled to pro-vide multiple of milligrams of the NHS ester activated MBG-hapten 9,which was used to conjugate to both BSA and BLG (vide infra).

To prepare the BSA-MBG antigen, the hapten carrier 9 (Figure 2A) wasallowed to react with the surface lysines of BSA by first dissolving theactivated ester in DMSO, followed by addition of the DMSO solution to asolution of BSA (10mg=mL) in PBS buffer. Mass spectrometric (MALDI)comparison of the native carrier protein and the resulting BSA–MBGconjugate 5 (Figure 1) indicated that the MBG hapten 9 (Figure 2A) wasconjugated on average to BSA in �10:1 molar ratio, that is, an average of�10 molecules of MBG to every molecule of protein. In a similar manner,we prepared a second antigen (Figure 2B) based on an alternative carrierprotein, b-lactoglobulin (BLG). Mass spectrometric (MALDI) analysisindicated a loading of �5–6 molecules of the MBG-hapten to everymolecule of BLG.

FIGURE 3 Assay sensitivity and working range. A standard curve of MBG and rabbit anti-BSA-MBG(diluted 1:4� 106) were incubated on an MBG-BLG coated plate. Bound antibody was detected follow-ing sequential incubation with HRP-conjugated goat anti-rabbit IgG and QuantaRed enhanced chemi-fluorescent HRP substrate. The assay can detect less than 9pg of MBG=mL and its dynamic range isclose to three orders of magnitude. Values are means of triplicates� SE.

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Sensitivity

Polyclonal anti-MBG–BSA antibody was produced by immunization of NewZealand White rabbits with the BSA–MBG conjugate. Following nine immuni-zations, an antiserum that could be diluted down to 4� 10�6 was obtained. Atthis concentration and using chemifluorescence as the endpoint, a minimumdetectable dose of less than 9pg=ml was achieved. A typical standard curvefeaturing a wide dynamic range from 9 pg to 7ng=mL is shown in Figure 3.

Specificity

The specificity of the antisera was of particular concern, given the largenumber of physiologically active congeners of MBG. Specificity wasestimated as the percentage of cross-reaction with the following substances:cinobufotalin, resibufogenin (RBG), digoxigenin, proscillaridin A, oua-bain, bufalin, and digoxin. As shown in Table 1, the cross-reactivity profileof the rabbit antiserum was remarkably favorable. Cinobufotalin generatedimmunoreactivity comparable to that of MBG, while all the other com-pounds tested generated minimal cross-reactivity (less than 5% for RBG,less than 0.5% for digoxin and digoxigenin, less than 2% for proscillaridinA, and less than 1% for bufalin and ouabain).

Analytical Recovery in Serum

Recovery is the ability of an assay to measure a known amount of analytefrom the matrix. Since urine samples did not show any matrix interference

TABLE 1 Cross-Reactivity of the Anti-MBG Antiserum with Selected MBG Congeners

Percent cross-reactivity

Cinobufotalin 100%Resibufogenin <5%Proscillaridin A <2%Ouabain <1%Bufalin <1%Digoxin <0.5%Digoxigenin <0.5%

TABLE 2 Recovery of MBG from Exogenously Spiked Rat Serum

Added MBG (pg=mL) Recovered MBG (pg=mL) Recovery (%)

81 84� 7.8 104243 270� 65 111729 756� 47.8 104

Average recovery (%) 106

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FIGURE 4 Parallelism. (A) Random rat urine samples (U1, U2, and U3) were diluted 1:3, 1:9, and 1:27in StartingBlock buffer. (B) MBG-free serum was spiked with threefold dilutions of MBG (ranging from27 to 6561 pg=mL), and each dilution was purified by SPE. Urine=serum samples or a standard curve ofMBG was incubated with anti-MBG–BSA on an MBG–BLG-coated plate. Bound antibody was detectedfollowing sequential incubation with HRP-goat anti-rabbit IgG and QuantaRed enhanced chemifluores-cent HRP substrate.

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effects and were analyzed unprocessed, whereas serum samples neededpretreatment, the analytical recovery of the assay was determined by addingknown amounts of MBG (81, 243, and 729 pg=mL) to MBG-free normal ratserum. MBG concentrations were determined following solid-phase extrac-tion, and the percent recovery was calculated. The recovery profile of theassay ranged from 103 to 107% (Table 2).

Parallelism

Dilution was used as an additional measure of the accuracy of the assay.Random urine samples were diluted 1:3, 1:9, and 1:27 in StartingBlockbuffer. MBG-free serum was spiked with threefold dilutions of MBG(ranging from 27 to 6561pg=mL), and each dilution was purified on SPEcolumns. MBG concentrations in urine and serum samples were determ-ined using our polyclonal antibody assay. Dilution curves parallel to thestandard curve were obtained (Figure 4, A and B), thus establishing thedilutional linearity of the assay and further documenting its accuracy.

TABLE 3 Intra-Assay Variability in Rat Serum (Top) and Urine (Bottom) Samples

Serum sample Mean [MBG] pg=mL %CV

Uk-1 208.5� 0.5 0.3Uk-2 239� 3.0 1.8Uk-3 229.5� 3.5 2.2Uk-4 183.5� 2.5 1.9Uk-5 218.5� 0.5 0.3Uk-6 147� 3.0 2.9Uk-7 219.5� 3.5 2.3Uk-8 216.5� 4.5 2.9Uk-9 234� 3.0 1.8Uk-10 238� 5.0 3.0

Average %CV 1.9

Urine sample Mean [MBG] pg=mL %CV

Uk-1 209.5� 2.5 1.7Uk-2 212� 6.0 4.0Uk-3 191� 2.0 1.5Uk-4 211.5� 4.5 3.0Uk-5 205.5� 2.5 1.7Uk-6 217.5� 2.5 1.6Uk-7 173.5� 0.5 0.4Uk-8 190� 2.0 1.5Uk-9 168.5� 8.5 7.1Uk-10 147� 3.0 2.9

Average %CV 2.5

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Precision

Precision, which describes the repeatability of an assay, was estimated asthe variation within and between assays. The intra-assay precision wasdefined as the precision between duplicates of the same sample run withinthe same assay. The percent coefficient of variability (%CV) between dupli-cates was typically less than 10% for all urine and serum samples, and aver-aged 1.9 and 2.5% in representative serum and urine samples, respectively(Table 3). Interassay precision reflects the ability of an assay to reproducethe same result on the same sample from run to run. Six urine samples wererun in five consecutive assays, and the %CV for each sample was calculated.The overall interassay variability was estimated to be 9.8% (Table 4).

DISCUSSION

An analysis of the literature regarding the bufodienolides indicatestheir potential involvement in a number of processes that lead to the devel-opment of vascular abnormalities and disorders.[3] However, because thesecompounds appear to exert their effects as a result of inhibition of theubiquitous transport enzyme Naþ=Kþ-ATPase,[1] their importance in otherphysiological and pathophysiological processes has been noted. Thus, uti-lizing the enzyme as a signalsome, the effects of the bufodienolides, butespecially of marinobufagenin (MBG), have been extended to cell differen-tiation,[25] cardiomegaly in the setting of kidney failure,[26] fibrosis ofinjured organs,[27] and the epithelial-to-mesenchymal transformation lead-ing to organ failure.[28] Furthermore, MBG has been determined to play arole in ‘‘vascular leak.’’[29] This action, in various vascular beds, appears toresult from its effect on endothelial cells, causing disruption of their ‘‘tightjunctions.’’[30] The latter process has been determined to result in upregu-lated apoptosis involving p38 and the MAPK system.[30] Thus, the need formethodology to determine the levels of MBG in blood and urine is bothtopical and appropriate.

TABLE 4 Interassay Variability

Mean [MBG] pg=mL Interassay variability

357� 11.1 7.0515� 12.7 5.5242� 10.4 9.6125� 7.7 13.8160� 6.2 8.679� 5 14.4Average %CV 9.8

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The polyclonal antibody-based assay described in this communication issensitive, specific, reproducible, and reliable. Antibodies to MBG werepreviously generated using an immunogen prepared directly from MBG(Figure 1, 1).[16] This particular antigen 4 (Figure 1) was prepared byattachment of a five-carbon linker to the C3-hydroxyl group of MBG usingglutaric anhydride. The resulting carboxylic acid derivative was thencoupled to the surface lysine groups of the carrier protein BSA by use ofthe N-hydroxysuccinimidyl ester method as previously employed forsteroid conjugation.[31] While the generated antibodies exhibited lowcross-reactivity with some other bufodienolide natural products and carde-nolides, there were no data reported for resibufogenin (RBG) (Figure 1, 2),an antagonist to MBG and hence a compound of great interest to us. Giventhat the only difference between RBG and MBG is the absence of theC5-hydroxyl group, it is challenging to produce an antibody that will recog-nize MBG with minimum cross-reactivity to RBG, especially when the pointof attachment for an antigen (the C3-hydroxyl) is close to this site of differ-ence. Thus, we have designed and synthesized an MBG immunogen 5(Figure 1) with an alternative point of attachment that maximally displaysthe subtle difference (the C5-hydroxyl group) between MBG and RBG tothe immune system. This approach is quite different from previous workin this area,[16,32] where the C3-hydroxyl was used for activation and conju-gation of MBG, hence obscuring the essential difference between RBG andMBG at C5 from immune recognition in the immunized host.

As a logical consequence of our immunization strategy, MBG and CINOare equipotent in this assay. Nevertheless, the ability of the assay todistinguish between MBG and its antagonist, RBG, makes it superior andvery pertinent from biological and clinical standpoints. In fact, our immu-noassay showed minimal cross-reactivity with MBG congeners, includingRBG, an advantage bestowed by the alternative point of attachment ofMBG to the linker. Furthermore, the use of the MBG–BLG conjugate tocoat the ELISA plates allowed the measurement of MBG immunoreactivitywithout the interference of anti-BSA antibodies. The immunoassay has awide dynamic range (close to three orders of magnitude) and can detectas little as 9 pg=mL MBG. It is easy to perform, affordable, and is amenableto automation, making it an attractive and novel method to determineMBG levels in various biological fluids. Urine samples can be analyzedunprocessed, save for dilution, whereas serum samples need to be purifiedby solid-phase extraction.

The utility of this immunoassay in the detection and monitoring ofdisease processes as diverse as the pregnancy-specific syndrome of pree-clampsia[5] and organ failure due to the acceleration of the fibroticprocess[27,33] demonstrates its need as well as its potential. In thepreeclamptic syndrome, for example, the determination of levels of MBG

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early in pregnancy may possess not only a great predictive value, but alsothe possibility that early intervention with an MBG antagonist may resultin the prevention of disease.[34] Thus, the development of this assay istimely and useful. Its utility is now being tested in animal models and,shortly, in human disease processes.

ACKNOWLEDGMENTS

This work was supported (in part) by grants-in-aid from the Joe W. andDorothy Dorsett Brown Foundation, New Orleans, LA, Dialysis Clinic, Inc.,Nashville, TN, from Scott & White Healthcare, Inc., Temple, TX, the TexasA&M Health Science Center College of Medicine, College Station, TX (toJBP), and by grants from the USDA (grant 2008-35204-04554 to LRB), fromthe National Institutes of Health (RO1 GM086307 to DR), grants R01 CA90441-01-04, 5R01, and CA090441-07-08 from the Division of Cancer Treat-ment and Diagnosis, NCI, DHHS (GRP), and the Welch Foundation(A-1280 to DR). In addition, funding from the College of Science, TexasA&M University (for JL) is gratefully acknowledged. The authors thankLonnie Doyle for the production of this article.

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