The exogenous digitalis glycosides, ouabain and digoxin, have been widely used in humans to treat congestive heart failure and cardiac arrhythmias. Several reports have also pointed to the existence of endogenous ouabain- and digoxin-like compounds, but their precise roles in mammalian physiology and various disorders of the circulation are not clear. In an attempt to produce specific Abs for the purification and identification of endogenous ouabain-like compounds, somatic cell fusion was used to produce mAbs specific for ouabain. Our attempts to produce ouabain-specific mAbs were unsuccessful when ouabain was coupled to exogenous proteins such as bovine γ-globulins, BSA, and human serum albumin. However, when ouabain was coupled to an Ab of A/J mice origin and the same strain of mouse was used for immunization with ouabain-Ab conjugate, three Abs (1-10, 5A12, and 7-1) specific for ouabain were obtained. In assays of fluorescence quenching and saturation equilibrium with tritiated ouabain, Ab 1-10 exhibited 200 nM affinity for ouabain. These three mAbs are distinguished from existing Abs to ouabain and digoxin by their specificity for ouabain and lack of cross-reactivity with digoxin. Specificity studies showed that the loss of cross-reactivity was correlated with the presence of a hydroxyl group at either position 12β (digoxin) or 16β (gitoxin) of the steroid ring. These Abs can be used to develop assays for detection and characterization of ouabain-like molecules in vivo.

Digitalis glycosides have been used widely in the clinical treatment of congestive heart failure and certain cardiac arrhythmias for hundreds of years. The therapeutic index of these drugs is narrow, and clinical toxicity is frequent (1). The advent of somatic cell fusion techniques (2) permitted the production of mAbs with high sensitivity and specificity to digoxin (Dig)4 (3) for use in the study of Ab structure-function relationships, development of clinical assays, and emergency treatment for life-threatening digitalis overdoses (4, 5).

In recent years, a body of evidence has accumulated to suggest the existence in mammals, including man, of compounds with biological properties similar to or identical with those of the plant-derived cardiotonic steroids. Structural analysis of tissue and plasma extracts has indicated that one of these compounds is identical in structure to the plant-derived cardenolide, ouabain (Oua) (6, 7) (Fig. 1). This Oua-like compound (OLC) has been implicated in the control of renal sodium excretion, blood pressure regulation, cardiac muscle performance, and the pathogenesis of hypertension through endogenous regulation (or dysregulation) of Na+,K+-ATPase (sodium pump) in pertinent target tissues (8).

FIGURE 1.

Schematic representation of Oua and Dig structures, showing the numbering system of the cardenolide steroid rings.

FIGURE 1.

Schematic representation of Oua and Dig structures, showing the numbering system of the cardenolide steroid rings.

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Whether this endogenous Oua is truly endogenous, vs accumulated from the environment, or exists in the circulation as a regulatory hormone is currently debated. In an effort to answer these crucial questions, several groups have developed polyclonal anti-Oua Abs for use in RIA and ELISA-type biological assays. Using different Abs, findings have continued to be contradictory. Some laboratories have obtained immunoassay data supporting not only the existence of OLC in mammalian plasma and tissues (9, 10), but that OLC can be secreted by adrenal cells in culture in response to receptor stimulation (11) and feeding of steroid hormone precursors (12). To our knowledge, only one group has provided physicochemical structural analysis of immunoreactive isolates. Liquid chromatography mass spectrometry using selected ion recording and positive electrospray mass spectrometry indicated a compound in tissue isolates and adrenal cell culture supernatants, respectively, with a molecular mass identical with that of authentic plant-derived Oua (12).

Other investigators, using their own polyclonal Abs to Oua, have questioned the existence of authentic Oua in plasma and adrenal cell culture supernatants and have provided data that structural identity of OLC from these sources with plant Oua is unlikely. The primary basis for these conclusions rests on the demonstration that HPLC retention times for immunoreactive OLC and authentic plant Oua spiked into the chromatographic samples are different (13, 14, 15)

One obvious explanation for these discrepant results is that the assays employed do not recognize the same compounds; that is, that the polyclonal Abs are not specific for plant Oua or a putative Oua isomer of mammalian origin. One approach to enhance the specificity of and/or provide a standardization for immunoassay detection of OLC would be the development of anti-Oua mAbs with high specificity for Oua. To our knowledge, there is only one report in the literature of an mAb to Oua, but this Ab showed a high degree of cross-reactivity with Dig, the cardiac glycoside in prevalent clinical use (16) (Fig. 1).

We set out to raise such Abs by techniques previously used in our laboratory for the production of anti-Dig mAbs (3). Initial attempts were unsuccessful; all the mAbs recognized the Oua-protein conjugate, but not the hapten Oua itself. By using a novel Ag presentation technique, we were able to overcome this problem of specificity for the Oua-protein complex. We report here the identification of mAbs with high specificity for Oua that do not recognize the clinically used cardiac glycoside, Dig. We propose that further development of these Abs could make possible standardization in bioassays and allow clarification of ambiguities in the literature regarding the presence, source, pathogenetic role, and mammalian biosynthetic possibilities of OLC.

The generation, selection, and characterization of cell lines producing the 26-10 (IgG2a, κ) and 36-71 (IgG1, κ) mAbs were previously reported (3, 17). Ab 26-10, which was obtained from the spleen cells of A/J mice immunized with Dig-coupled BSA (Dig-BSA), exhibits an affinity of 9.1 × 10−9 M for Dig and cross-reacts with Oua (Ka = 6.0 ± 0.4 × 10−8 M) (18). Ab 36-71 was also derived from spleen cells of A/J mice and is specific for the hapten p-azophenylarsonate, with a binding constant Ka = 1–4 × 10−7 M (19, 20).

Oua, Dig, other cardiac glycosides (Table II), and steroid hormones (cortisone, corticosterone, and progesterone) were purchased from Sigma (St. Louis, MO). Oua was covalently coupled through its terminal rhamnose moiety to a number of proteins as previously described (21). Ags included Oua-BGG (bovine γ-globulin; United States Biochemical, Cleveland, OH), Oua-HSA (Miles Laboratories, Elkhart, IN), and Oua-BSA. Oua was also coupled to the affinity-purified mAb 26-10. Oua-BGG contained an average of 2.5 Oua residues/molecule of BGG; Oua-BSA, Oua-HSA, and Oua-26-10 Ab conjugates contained 0.5, 1.0, and 1.5 Oua residues/molecule of protein, respectively, as determined by their absorption spectrum in concentrated H2SO4 (22).

Table II.

Structural characteristics of Oua and Dig analoguesa

AnalogueSubstitutions at Steroid Positions
11α12β16β19
Oua —OH l-rhamnose —OH —OH   —OH 
Ouabagenin —OH —OH —OH —OH   —OH 
Strophanthidin  —OH —OH    ⋕O 
Acetylstrophanthidin  —OCOCH3 —OH    ⋕O 
Acovenoside A —OH 6-deoxy-3-O-methyl-l-talose     CH3 
Convallatoxin  l-rhamnose —OH    ⋕O 
Helveticoside  Digitoxose —OH    ⋕O 
Digitoxin  Tridigitoxose      
Digitoxigenin  —OH      
Dig  Tridigitoxose   —OH   
Digoxigenin-3,12-diacetate —OH —OCOCH3   —OCOCH3   
Gitoxin  Tridigitoxose    —OH  
Gitoxigenin-3,16-diacetate  —OCOCH3    —OCOCH3  
16-Acetylgitoxin  Tridigitoxose    —OCOCH3  
Oleandrin  Oleandrose    —OCOCH3  
Oleandrigenin  —OH    —OCOCH3  
AnalogueSubstitutions at Steroid Positions
11α12β16β19
Oua —OH l-rhamnose —OH —OH   —OH 
Ouabagenin —OH —OH —OH —OH   —OH 
Strophanthidin  —OH —OH    ⋕O 
Acetylstrophanthidin  —OCOCH3 —OH    ⋕O 
Acovenoside A —OH 6-deoxy-3-O-methyl-l-talose     CH3 
Convallatoxin  l-rhamnose —OH    ⋕O 
Helveticoside  Digitoxose —OH    ⋕O 
Digitoxin  Tridigitoxose      
Digitoxigenin  —OH      
Dig  Tridigitoxose   —OH   
Digoxigenin-3,12-diacetate —OH —OCOCH3   —OCOCH3   
Gitoxin  Tridigitoxose    —OH  
Gitoxigenin-3,16-diacetate  —OCOCH3    —OCOCH3  
16-Acetylgitoxin  Tridigitoxose    —OCOCH3  
Oleandrin  Oleandrose    —OCOCH3  
Oleandrigenin  —OH    —OCOCH3  
a

Cardenolide numbering scheme is shown in Fig. 1.

All immunizations were given i.p. For production of Oua-specific mAbs, two strains of mice and different Oua-protein conjugates were used. In the first attempt, BALB/c mice (The Jackson Laboratory, Bar Harbor, ME) were immunized i.p. with 100 μg of Oua-BSA emulsified in IFA. They were again immunized 3 wk later with 50 μg of Oua-BSA in CFA. Ten days later mice received 10 μg of soluble Oua-BSA. Two weeks later (3 days before fusion) mice were boosted with 10 μg of soluble Oua-BSA. In subsequent fusion experiments, a similar immunization protocol was used, but a different strain of mice (A/J, The Jackson Laboratory) and different immunizing Ags (Oua-BGG, Oua-HSA, or Oua coupled to 26-10 Ab) were used. Mice that were immunized with Oua-26-10 Ab conjugate received six additional booster injections of 10 μg of Ag in soluble form every 15 days (hyperimmunized). Before fusion, mouse sera were tested for Ab titers. Fifty percent binding to Oua-protein conjugates was achieved at 30,000- to 45,000-fold serum dilutions.

Fusions were conducted using Sp2/0-Ag14(Sp2/0) cell lines (23). After fusion, cells were distributed into 96-well microtiter plates.

Clones producing Oua-specific mAbs were selected by testing the ability of culture supernatants from wells showing cell growth to bind to immobilized Oua-protein conjugates in ELISA assays. Fifty microliters of a solution of Oua-protein conjugates (5 μg/ml in PBSA (0.15 M NaCl, 0.1 M sodium phosphate, and 0.02% sodium azide, pH 7.2)) were immobilized in the wells of microtiter plates. The binding of mAbs in the culture supernatants was detected using HRP-goat anti-mouse Ab (Sigma) (24). The end point of the reaction was determined after addition of 25 μl of 2 M phosphoric acid in an ELISA reader at 450 nm. Clones were selected for further study if the OD450 was ≥1.0 for Oua-protein conjugates and ≤0.2 for uncoupled protein. Clones from the wells that tested positive in direct binding assays were transferred to 48-well microtiter plates.

Inhibition ELISA was used to determine whether the binding of Abs in the culture supernatants to immobilized Oua-protein conjugates was inhibited by free Oua. Thus, the binding of 25 μl of culture supernatants to immobilized Oua-coupled protein was tested in the presence of either 25 μl of a solution of 100 μM Oua or 25 μl of 1% BSA, both in PBS. Clones that exhibited ≥40% inhibition were subcloned and studied further.

The isotypes of mAbs were determined using an isotyping ELISA kit (Zymed, San Francisco, CA).

Oua-specific Abs were purified from 1 l of culture supernatant by affinity chromatography on Oua-BGG-Sepharose. Abs were concentrated using Centriprep (30,000 m.w. cut-off; Amicon, Beverly, MA) and were subjected to gel filtration on Ultrogel ACA34 columns (LKB, Bromma, Sweden) to separate the monomer mAbs from aggregated ones.

Competition ELISA was used first to determine the relative affinity of each mAb for Oua and Dig. The 96-well PVC plates were coated with 50 μl of 5 μg/ml Oua-BGG in PBSA. First, we determined the Ab concentration that was not in excess of immobilized Ag. Using the direct binding assay described above, the concentration of Ab at which 50% binding was achieved was ascertained. Inhibition of binding of Abs to Oua-BGG was determined by adding 25 μl of Ab (concentrations as determined above) and 25 μl of free Oua (0.001–200 μM, 2-fold dilutions). The percent inhibition is the ratio (OD450 in the presence of 1% BSA − OD450 in the presence of Oua)/(OD450 in the presence of 1% BSA) × 100. The relative affinity (IC50) is the Oua concentration that inhibits 50% of the binding of Ab to Oua-BGG.

The equilibrium binding constant (Ka) of Oua-specific mAb 1-10 was also determined by fluorescence quenching using a Hitachi F-4500 fluorescence spectrophotometer (Hitachi, San Jose, CA). The excitation and emission wavelengths were 270 and 340 nm, respectively. Eight incremental additions of 20 μl of 10−6 M Oua in 2 ml of Ab solution in PBSA (12–20 μg) followed by four incremental additions of 20 μl of 10−5 M Oua were made. The initial fluorescence reading was diminished by 70–75%. Control titrations were conducted by adding Oua to 2 ml of an mAb solution with unrelated specificity (36-71 mAb). Fluorescence quenching was repeated with 1-10 and 36-71 mAbs using 10−6 and 10−5 M Dig in PBSA. The Ka was calculated using a curve-fitting program (19).

The affinity for the 1-10 mAb was confirmed using an equilibrium saturation method with [3H]Oua or [3H]Dig (DuPont-NEN, Boston, MA) as described previously (18, 25). Briefly, 22 μg of mAbs (1-10 or 36-71) were added to different concentrations of either tritiated Oua or Dig (0.08–20 nM, 4.5 × 102 to 4.5 × 105 cpm, 2-fold dilutions). Following incubation at room temperature for 1 h, samples were filtered through glass fiber to separate bound from free hapten, and the filters were washed with 10 ml of cold PBSA. 3H-labeled ligand in the filters was measured by liquid scintillation counting. Affinity data were analyzed using the LIGAND program (26).

Competition ELISA was used to determine the cross-reactivity of the mAbs with different digitalis glycosides (Oua, Dig, gitoxin, and digitoxin and their derivatives listed in Tables I and II) and with endogenous steroid hormones (cortisone, corticosterone, and progesterone). In these assays binding of mAbs to Oua-BGG was determined in the presence or the absence of various concentrations (0.00035–200 μM) of free digitalis glycosides and steroid hormones as described above.

In an attempt to produce Oua-specific mAbs, Oua was coupled to different protein carriers. From the fusion of spleen cells of A/J and BALB/c mice that were immunized with Oua-BSA, Oua-HSA, or Oua-BGG, >1000 clones exhibited significant specific binding to Oua-protein conjugates, but the binding of very few clones could be inhibited by free Oua. These clones had low relative affinity for Oua (IC50 = 10−4 M). It appeared that Oua was being recognized by the Ab-producing cells in vivo mainly in the context of the epitopes on the protein carrier. To overcome the problems associated with protein carrier immunogenicity, we coupled Oua to the anti-Dig 26-10 mAb that was derived from A/J mice (3) and then hyperimmunized A/J mice with the Oua-26-10 Ab conjugate. From the fused splenocytes of two immunized mice in two different fusion experiments a total of 600 clones were screened for their binding to Oua-BGG and BGG. Sixty clones were found to produce Abs that bound to Oua-BGG but not to BGG (data not shown).

Inhibition assays were performed to identify clones that produce mAbs, the binding of which to Oua-BGG could be inhibited with free Oua at micromolar concentrations. Four clones (5A12 and 2H8 from the first fusion, and 7-1 and 1-10 from the second fusion) were selected for further studies. The mAb 2H8 was IgG2a κ, and the other mAbs were IgG1 κ. Fig. 2 shows the inhibition pattern of each mAb with Oua. The high affinity Dig-specific mAb 26-10, which cross-reacts with Oua (Ka for Oua = 6 × 10−8 M) (18), was used as control. The binding of all four mAbs to Oua-BGG could be inhibited with free Oua in a concentration-dependent manner. Approximately 7–25 μM Oua was required to achieve 50% inhibition for Oua-specific mAbs. For 26-10 mAb, 0.37 μM Oua was required for 50% inhibition (Fig. 2 and Table I).

FIGURE 2.

Inhibition of binding of mAbs to Oua-BGG by free Oua. Abs were titrated in direct binding assays to determine the concentration equivalent to 35–50% binding to Oua-BGG. Binding of Abs to Oua-BGG was determined in the presence or the absence of 0.00035–200 μM free Oua. Ab binding was detected using HRP-goat anti-mouse Ab. The control mAb 26-10 was raised against Dig-BSA and cross-reacts with Oua. The percent inhibition was calculated as described in Materials and Methods.

FIGURE 2.

Inhibition of binding of mAbs to Oua-BGG by free Oua. Abs were titrated in direct binding assays to determine the concentration equivalent to 35–50% binding to Oua-BGG. Binding of Abs to Oua-BGG was determined in the presence or the absence of 0.00035–200 μM free Oua. Ab binding was detected using HRP-goat anti-mouse Ab. The control mAb 26-10 was raised against Dig-BSA and cross-reacts with Oua. The percent inhibition was calculated as described in Materials and Methods.

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Table I.

Fine specificity of Oua-specific mAbsa

AnaloguebIC50 (μM)
26-10c1-10c7-1c5A12c
Oua 0.37 25 20 10 
Ouabagenin 0.40 30 30 16 
Strophanthidin 0.20 10 10 
Acetylstrophanthidin 0.20 
Acovenoside 0.20 
Convallatoxin 0.10 
Helveticoside 0.20 150 150 150 
Digitoxin 0.02 
Digitoxigenin 0.04 
Dig 0.006 NId NI NI 
Digoxigenin-3,12-diacetate NI NI NI 
Gitoxin 1.5 NI NI 100 
Gitoxigenin-3,16-diacetate NI NI NI NI 
16-Acetylgitoxin 0.5 NI NI NI 
Oleandrin >100 NI NI NI 
Oleandrigenin >100 NI NI NI 
Cortisone 100 NI NI NI 
Corticosterone NI NI NI NI 
Progesterone NI NI NI 
AnaloguebIC50 (μM)
26-10c1-10c7-1c5A12c
Oua 0.37 25 20 10 
Ouabagenin 0.40 30 30 16 
Strophanthidin 0.20 10 10 
Acetylstrophanthidin 0.20 
Acovenoside 0.20 
Convallatoxin 0.10 
Helveticoside 0.20 150 150 150 
Digitoxin 0.02 
Digitoxigenin 0.04 
Dig 0.006 NId NI NI 
Digoxigenin-3,12-diacetate NI NI NI 
Gitoxin 1.5 NI NI 100 
Gitoxigenin-3,16-diacetate NI NI NI NI 
16-Acetylgitoxin 0.5 NI NI NI 
Oleandrin >100 NI NI NI 
Oleandrigenin >100 NI NI NI 
Cortisone 100 NI NI NI 
Corticosterone NI NI NI NI 
Progesterone NI NI NI 
a

Specificity of mAbs was determined in ELISA competition assays. Binding of mAbs to Oua-BGG was determined in the presence or absence of increasing concentrations (0.00035–200 μM, 2-fold dilutions) of inhibitors using HRP-goat anti-mouse Abs. The μM concentration required for 50% inhibition of the binding of Abs to Oua-BGG was calculated as described in Materials and Methods.

b

Analogues were prepared at 2–5 mM concentrations in 70% ethanol and diluted into PBS.

c

mAbs were diluted in 1% BSA/PBS and titrated using direct binding ELISA to determine the Ab concentration equivalent to 35–50% binding to immobilized Oua-BGG.

d

NI, no inhibition was observed at highest inhibitor concentration (100 μM).

The specificity of these mAbs was tested in inhibition assays using Dig. As shown in Fig. 3 and Table I, three mAbs showed minimal (5A12) or absent (7-1 and 1-10) cross-reactivity with Dig, as their binding to Oua-BGG could not be inhibited with concentrations of free Dig as high as 100 μM. One mAb (2H8) cross-reacted with Dig (IC50 = 3 μM). Approximately 0.006 μM Dig was required for 50% inhibition of 26-10 binding. The relative affinity of 26-10 for Dig in inhibition assays is in agreement with the previously reported Ka values using [3H]Dig (9.1 × 10−9 M) (25). The Ka of 26-10 for Oua was previously reported to be 40-fold less than that for Dig using [3H]Oua (18); in the inhibition assays reported here this difference is 62-fold (Table I).

FIGURE 3.

Inhibition of binding of mAbs to Oua-BGG by free digoxin. Binding of Abs to Oua-BGG was determined in the presence or the absence of 0.0001–100 μM free Dig as described in Fig. 2. The results for Ab 7-1 are identical with those for 1-10 mAb (symbols obscured).

FIGURE 3.

Inhibition of binding of mAbs to Oua-BGG by free digoxin. Binding of Abs to Oua-BGG was determined in the presence or the absence of 0.0001–100 μM free Dig as described in Fig. 2. The results for Ab 7-1 are identical with those for 1-10 mAb (symbols obscured).

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Because the 2H8 mAb cross-reacts with Dig, we excluded this Ab from further study. mAbs 5A12, 7-1, and 1-10 were affinity purified, concentrated, and applied to a gel filtration column (ACA34). Fig. 4 demonstrates the aggregation pattern of these Abs in neutral buffer (PBSA). All mAbs formed aggregates, but mAb 1-10 had the lowest amount of aggregates and the highest amount of monomer Ab. The cell line producing mAb 1-10 secreted high levels of Ab, ∼15 mg of purified Ab from 1 l of culture supernatant. In contrast, hybridoma clones 5A12 and 7-1 were low producers. The level of production of mAbs and their aggregation patterns are important for the practicality of large scale production, purification, and stability. The inhibition assays were repeated using all three affinity-purified Abs. Similar relative affinity values (IC50 = 7–25 μM) were obtained for all affinity-purified Abs (data not shown).

FIGURE 4.

Elution patterns of Oua-specific mAbs from an ACA34 gel filtration column. One liter of Ab-containing culture supernatants was passed through an Oua-BGG-Sepharose column. Abs were eluted with 0.2 M ammonia into tubes containing 1.5 M Tris, pH 4.5, and concentrated using Centriprep. Concentrated Abs were loaded onto ACA34 columns, which were equilibrated with PBSA. One-milliliter fractions were collected.

FIGURE 4.

Elution patterns of Oua-specific mAbs from an ACA34 gel filtration column. One liter of Ab-containing culture supernatants was passed through an Oua-BGG-Sepharose column. Abs were eluted with 0.2 M ammonia into tubes containing 1.5 M Tris, pH 4.5, and concentrated using Centriprep. Concentrated Abs were loaded onto ACA34 columns, which were equilibrated with PBSA. One-milliliter fractions were collected.

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Fluorescence quenching analysis of anti-Oua Abs indicated that the fluorescence emission only of 1-10 mAb (but not that of 5A12, 7-1, or 26-10) can be quenched upon addition of free Oua. The Ka of 1-10 mAb for Oua was 3 ± 1 × 10−7 M using fluorescence quenching (Fig. 5). The fluorescence emission of 1-10 mAb was comparable to that of control Ab upon addition of 10−6 and 10−5 M free Dig, confirming that this mAb does not cross-react with free Dig in solution.

FIGURE 5.

Plots of quenching of fluorescence of 1-10 anti-Oua mAb and control (36-71) mAb vs free hapten. Hapten (10−6 and 10−5 M) was added to 12–20 μg of Ab (in 2 ml of PBSA) as described in Materials and Methods. Quench data were transformed using a computer-assisted curve-fitting program (19 ) to determine the intrinsic affinity (Ka) of 1-10 mAb for Oua and digitoxin.

FIGURE 5.

Plots of quenching of fluorescence of 1-10 anti-Oua mAb and control (36-71) mAb vs free hapten. Hapten (10−6 and 10−5 M) was added to 12–20 μg of Ab (in 2 ml of PBSA) as described in Materials and Methods. Quench data were transformed using a computer-assisted curve-fitting program (19 ) to determine the intrinsic affinity (Ka) of 1-10 mAb for Oua and digitoxin.

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Using a saturation equilibrium assay with tritiated Oua, the Ka of 1-10 mAb was measured (2.4 × 10−7 M) and was similar to that obtained by fluorescence quenching (data not shown). The saturation equilibrium assay was repeated using [3H]Dig. Ab 1-10 did not capture sufficient tritiated ligand for measurement.

The binding specificity of Oua-specific mAbs to closely related analogues of Oua and Dig was determined by competition ELISA. Table I shows the relative affinity (IC50; i.e., micromolar concentration of free inhibitor required for 50% inhibition) of mAbs for each inhibitor, compared with that of 26-10 mAb. All three mAbs exhibited similar, but not identical, fine specificities for Oua analogues. The absence of the rhamnose sugar of Oua at position 3 of the steroid ring (Fig. 1) did not substantially affect binding, as ouabagenin binding was indistinguishable from that of Oua (Tables I and II). However, the relative affinities of Abs for helveticoside (strophanthidin digitoxoside) were reduced 6- to 15-fold compared with their affinities for Oua, indicating that the nature of the attached sugar affects binding for Oua analogues lacking the 1β- and 11α-OH substitutions (Tables I and II). Neither Dig nor gitoxin inhibited the binding of mAbs to Oua-BGG, although Ab 5A12 exhibited cross-reactivity with gitoxin (IC50 = 100 μM; Table I). None of the three mAbs reacted with the endogenous steroid hormones cortisone, corticosterone, and progesterone (Table I). Surprisingly, all three mAbs bound to digitoxin at micromolar concentrations (2–4 μM). The cross-reactivity of 1-10 mAb with digitoxin was confirmed using fluorescence quenching. As shown in Fig. 5, digitoxin, but not Dig or gitoxin, inhibited the fluorescence emission of 1-10 mAb in a pattern similar to that of Oua. A Ka of 4.9 ± 0.8 × 10−7 M was obtained for digitoxin.

Oua is a cardiac glycoside (m.w., 584.7) found in certain plant species, such as the seeds of Strophanthus gratus (27). Oua and OLC have also been found in humans and animals (7, 28, 29, 30, 31). Although its function in plants is not known, in mammals Oua and OLC may play a role in the regulation of sodium balance, arterial pressure, and vascular smooth muscle tone under normal circumstances and have a pathophysiologic role in common clinical disorders, such as essential hypertension, pregnancy-induced hypertension, cardiac failure, and chronic renal failure (8, 32). Two major issues that need to be clarified are 1) the source(s) (including in vivo vs ex vivo) of Oua or OLC molecules found in humans and animals, and 2) the actual function of Oua or OLC in vivo as a regulator of cardiovascular physiology. The availability of specific molecular probes and reliable methods of detecting and measuring endogenous or exogenous Oua is the prerequisite to successfully investigating these issues.

Three anti-Oua mAbs were produced by somatic cell fusion. Each Ab was analyzed for its affinity and fine specificity for Oua and related cardiac glycosides. Using solid phase competition assays, an IC50 range of 7–25 μM for Oua was obtained for these mAbs (Fig. 2 and Table I). The affinity (Ka) of one mAb (1-10) was measured by two other methods (fluorescence quenching and saturation equilibrium) and was found to range from 0.24–0.3 × 10−8 M (240–300 nM; Fig. 5). These affinities are sufficiently high to allow the Ab to be used in different methods of Oua detection. Although two mAbs with high affinities (2.0 × 10−7 and 1.2 × 10−9 M) for Oua were previously reported (16), both cross-reacted with Dig, the form of cardiac glycoside widely prescribed for the treatment of heart failure and certain arrhythmias. Such cross-reactivity would probably be problematic, particularly in human studies. The mAbs here reported are distinguished from the earlier ones in their specificity for Oua and their lack of cross-reactivity with Dig (Fig. 3 and Table I).

In some cases, a combination of methods of Ab engineering using phage display and molecular modeling have been used to change the specificity of Abs that bind to closely related analogues of an Ag. For example, the affinity and specificity of an anti-cortisol mAb that cross-reacted with prednisolone and dexamethasone was improved by 8- and 5-fold, respectively, using random mutagenesis of its V region genes (33). An anti-estradiol Ab fragment that cross-reacted with testosterone was mutagenized to improve its specificity (34). Similar methods have been used for anti-testosterone mAb cross-reactive with dehydroepiandrosterone sulfate (35) and anti-hydroxyprogestrone mAb cross-reactive with cortisol (36) to improve affinity and specificity. Our own efforts (data not shown) in engineering Oua-specific Ab fragments using the existing Dig-binding mAbs 26-10 and 40-50 (37) have not been successful to date. Maintaining high affinity and increasing the Oua specificity relative to that of Dig of 26-10 Ab would require extensive alterations of the binding site. Thus, we used the traditional somatic cell fusion method to produce Oua-specific mAbs.

Fusion of the spleen cells of mice immunized with Oua coupled to BSA, HSA, or BGG with plasmacytomas yielded a very large number of clones secreting mAbs specific for the Oua-protein carrier. In every fusion three kinds of specificities could be detected. The first group (32%) secreted Abs that bound to Oua-protein conjugates; they did not cross-react with either Dig-protein conjugates or protein carriers alone (data not shown). The specificity of the second group of mAbs, which constituted 54% of the clones, was directed against Oua-protein conjugates, which cross-reacted with Dig-proteins but not with protein carrier. The third group of mAbs (14%) bound only the protein carrier. We were surprised by the fact that the binding of mAbs to Oua-protein conjugates could not be inhibited by micromolar concentrations of free Oua. Because we were searching for high affinity Abs, all the inhibition screenings were performed in the presence of 100 μM free Oua. This indicated that either Oua is not immunogenic in vivo or the immunogenicity of the protein carriers is greater than that of Oua, thus shifting the specificity of the Abs toward the protein.

To avoid the problems associated with protein carrier immunogenicity, Oua was coupled to 26-10 Ab. Because the 26-10 Ab was derived from A/J mice, the same mouse strain was used for immunization with the Oua-26-10 conjugate. Among 60 clones that secreted mAbs exhibiting specific binding to Oua-BGG, only the binding of four Abs was inhibited with free Oua. These results can be explained in two different ways. First, because OLC exists in vivo, the immune system may be tolerant to Oua, and thus Oua can be recognized only in the context of exogenous proteins. This explains why the specificity of Abs secreted by clones isolated from mice immunized with Oua coupled to BSA, HSA, or BGG was directed against Oua-protein carriers and not Oua alone. An alternative explanation is that in Oua the steroid ring is attached through a single sugar (rhamnose; Fig. 1), which may allow the attached proteins to sterically hinder the cardenolide moiety of Oua. Anti-Dig mAbs can be elicited more easily, because in Dig the steroid ring is attached via the tridigitoxose (Fig. 1); thus, the sugars may act as a spacer between the steroid ring of Dig and the protein. Also, there are significant structural differences between the steroid ring substitutions of Oua and those of Dig (Fig. 1 and Table II). Oua has four OH groups at steroid positions 1β, 5β, 11α, and 19, while Dig does not share any of these OH groups; Dig has an OH group at steroid position 12β. Such differences could be sufficient for a molecule to be recognized as self or non-self by the cells of the immune system.

Cross-reactivity of anti-Oua mAbs with digitoxin was an unexpected finding. Comparison of digitoxin with Oua, Dig, and gitoxin reveals that Oua and digitoxin both lack OH groups at position 12β or 16β of the steroid ring, while Dig and gitoxin contain OH groups at 12β and 16β, respectively (Table II). Oua and digitoxin differ with respect to their sugars at position 3β (rhamnose vs digitoxose, respectively), and digitoxin also lacks the OH group at positions 5β, 11α, and 19 (Table II).

The reason for heteroclicity of anti-Oua mAbs is not known. We cannot rule out the possibility that the chemical identity of Oua is altered in the Oua-protein conjugate, but assays of fluorescence quenching and saturation equilibrium demonstrated that anti-Oua mAbs can bind free Oua in native form in solution, thus indicating that the ouabain structure has to some extent been preserved. Although only speculation, the observation that immunization of mice with Oua-26-10 complex resulted in Abs with higher affinity for digitoxin, which was not the immunizing Ag, could be explained if Oua were modified to a digitoxin-like compound in vivo after the complex was processed for presentation to T and B cells. However, to our knowledge there is no experimental evidence for targeted modification of self-Ags by the immune system.

mAbs elicited against Dig (3, 25, 37, 38) exhibit varying specificity patterns for related cardiac glycosides. Such mAbs can bind Dig and digitoxin equally well or distinguish these two analogues by up to a 1000-fold difference. The three anti-Oua mAbs reported here are unique in binding with high affinity to digitoxin, but not to Dig. In addition, they do not cross-react with gitoxin as do mAbs elicited against Dig (3, 38). This indicates that in anti-Oua Abs, binding site complementarity around the 12β OH is probably very tight.

The chemical nature and the structure of endogenous digitalis-like factors have remained elusive. Some investigators have identified an OLC in human plasma (7), while others (8) have isolated a compound from human urine that was indistinguishable from Dig based on physico-chemical analysis and immunoreactivity with anti-Dig IgG. These Abs also neutralized the potency of the Dig-like compound. In addition, a Dig-like immunoreactive factor was isolated from mammalian adrenal cortex that exhibited similar chromatographic and spectral properties as Dig (39). Thus, it is possible that both endogenous OLC and Dig-like compounds exist in vivo in mammals; if this is so, only specific probes would distinguish between them. The purpose of having a panel of anti-Oua mAbs that do not cross-react with Dig is to assure that the structural nature of the purified OLC is that of Oua and not Dig. In addition, we focused on the production of anti-Oua mAbs to aid in determining the molecular identity of the OLC we previously isolated from hypothalamus (29).

From the clinical point of view, OLC has been implicated in the pathophysiology of human essential hypertension and congestive heart failure (8, 32). Patients with these disorders, who will be subjects of clinical studies to verify a role for OLC, are often treated with Dig. Thus, the availability of our mAbs may allow study of these patients to verify a role for OLC even if they are treated with Dig.

Recently, we produced an additional anti-Oua mAb (8E4) with an affinity of 1.8 × 10−8 M for Oua (unpublished observations). The pattern of specificity of 8E4 is similar to that of the mAbs reported here; i.e., 8E4 binds to Oua and digitoxin, but not to Dig and gitoxin, in contrast to the 26-10 Ab.

It is surprising that a panel of mAbs obtained from three independent fusions all exhibited the same unique specificity. This may indicate that the in vivo immune response to Oua is restricted, explaining the low frequency of Oua-specific clones in fusion experiments.

Using a novel Ag presentation technique, we have been able to obtain mAbs to the cardiac glycoside, Oua, whereas immunization with more traditional hapten-protein complexes produced mAbs to the complex, but not to Oua itself. The particular advantage of these mAbs is that they do not cross react with either endogenous adrenal steroids or Dig, the primary cardiac glycoside used in clinical practice. Although they are heteroclitic and do react with some of the cardenolides tested, none of these latter has been reported as an isolate of mammalian origin. The mAbs herein described can thus provide more specific molecular probes to assess the putative role of endogenous Oua in mammalian physiology and in the pathophysiology of the prevalent human cardiovascular diseases, hypertension and congestive heart failure. It is important that anti-Oua Abs be validated by demonstrating their utility as reagents to detect OLC.

We thank Lihua Zhang and Rou-Fun Kwong for technical assistance.

1

This work was supported by National Institutes of Health Grant RO1HL52282 (to G.T.H) and in part by National Institutes of Health Grants R29AI33175 (to B.P.S.) and RO1 CA24432 and HL47415 (to M.N.M.).

4

Abbreviations in this paper: Oua, ouabain; Dig, digoxin; BGG, bovine γ-globulin; OLC, ouabain-like compound(s); HSA, human serum albumin.

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