Immunogen conjugates comprising N-substituted-amino-3-desoxydigoxigenin derivatives coupled to conventional immunogenic carrier materials, and antibodies raised against such conjugates. Also provided are labeled digoxigenin conjugates for use with the digoxigenin antibodies in preferred immunoassay techniques for determining digoxin in biological fluids.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
This invention relates to immunogen conjugates comprising digoxigenin 
derivatives, particularly carboxy- and amino-functionalized derivatives 
coupled to conventional immunogenic carrier materials, and anti-digoxin 
antibodies prepared against such immunogen conjugates. Such antibodies are 
useful in immunoassays for determining digoxin in biological fluids. The 
invention also relates to labeled digoxigenin conjugates useful in 
particularly preferred immunoassay techniques. 
2. Background of the Invention 
It is of established medical value to monitor the concentration of digoxin 
in the bloodstream of patients under treatment with the drug. Immunoassays 
are the currently most common methods used to determine digoxin in blood 
samples, e.g., serum or plasma, and are based on the specific binding of 
anti-digoxin antibodies to the drug in the test sample. 
Digoxin is a cardiac glycoside consisting of an aglycone, digoxigenin, and 
three glycosidic digitoxose residues linked to the aglycone at the C-3 
position. The glycoside is a hapten, that is, it is incapable of 
stimulating antibody production unless it is injected into the host animal 
in the form of a conjugate with an immunogenic carrier material, e.g., a 
protein such as albumin from an animal of a different species. The 
conventional immunogen conjugate for preparing anti-digoxin antibodies 
comprises the glycoside chemically linked through a modification of the 
terminal glycosidic residue to a carrier. The terminal residue is oxidized 
with periodate to open the ring and form a dialdehyde derivative which is 
readily couplable to free amino groups in the carrier [Butler and Chen, 
Proc. Natl. Acad. Sci. USA 57:71(1967), and Smith et al, Biochem 
9:331(1970)]. The result is a digoxin-carrier conjugate. 
Antibodies against the related cardiac glycoside digitoxin have been 
prepared from a conjugate of the aglycone coupled at the C-3 position to 
the carrier. The aglycone 3-o-succinoyl-digitoxigenin has been coupled by 
conventional techniques to protein carriers [Oliver et al, J. Clin. 
Invest. 47:1035(1968)]. 
3-Substituted digoxigenin and digitoxigenin derivatives have been proposed 
and used for the purpose of preparing labeled conjugates to be used in 
conjunction with anti-digoxin and anti-digitoxin antibodies in performing 
immunoassays. Representative of this art are U.S. Pat. Nos. 3,981,982 and 
4,064,227 and U.S. Pat. Nos. 4,039,385; 4,213,893; and 4,273,866 relating 
to various nonradioisotopically labeled conjugates. 
U.S. Pat. Nos. 4,217,280 and 4,219,549 describe the synthesis of various 
3-amino-3-desoxydigoxigenin derivatives and their use in cardiotonic 
therapy. The preparation of 3-digoxigenone is known from Tamm and Gubler, 
Helv. Chim. Acta 42:239(1959) and Shimizu and Mituhashi, Tetrahedron 
24:4207(1968). Boutique and Koenig, Bull. Chem. Soc. Fr. 1973(2), part 2, 
750 describe the reductive amination of steroidal ketones. The use of 
sodium cyanoborohydride in the reductive amination of aldehydes and 
ketones is described by Borch et al, J. Amer. Chem. Soc. 93:2987(1971). 
3-Aminocardenolides were prepared by Hanser et al, Helv. Chim. Acta 
56(8):2782(1973). 
SUMMARY OF THE INVENTION 
The present invention provides digoxigenin immunogen conjugates comprising 
3-[N-(carboxyalkyl)]-amino-3-desoxydigoxigenin or 
3-[N-(aminoalkyl)]-amino-3-desoxydigoxigenin derivatives coupled to 
conventional immunogenic carrier materials. The immunogens provided are of 
the general formula: 
##STR1## 
wherein Carrier is the carrier material, R is a bond or a linking group, 
one of l and m is one and the other is zero, n is an integer from 2 
through 10, and p is on the average from 1 to about 50. When linking group 
R is a bond, the digoxigenin derivative is coupled directly to the carrier 
material, for example by amide linkages between the amino or carboxyl 
group of the derivative and carboxyl or amino groups, respectively, on the 
carrier, which in such case is usually a protein or a polypeptide. When 
bridge group R is other than a simple bond, it may comprise a wide variety 
of conventional structures, for example, the residue of a bifunctional 
coupling agent linking the amino or carboxyl terminal group of the 
digoxigenin moiety to appropriate groups on the carrier, usually amino or 
carboxyl groups. 
Also provided are anti-digoxin antibodies prepared against such conjugates 
according to conventional antiserum or monoclonal techniques. Such 
antibodies are useful in immunoassay methods and reagent means, such as 
test kits and test devices, for determining digoxin. 
Labeled conjugates useful in particularly preferred homogeneous, 
nonradioisotopic immunoassay techniques are also provided, as well as 
novel digoxigenin derivatives used in the synthesis of such labeled 
conjugates and the immunogen conjugates. 
DESCRIPTION OF THE PREFERRED EMBODIMENTS 
The N-functionalized-amino-3-desoxydigoxigenin derivatives are the 
carboxyalkyl and aminoalkyl derivatives of the formula: 
##STR2## 
wherein Z is amino or carboxyl and n is an integer from 2 through 10. Such 
derivatives are prepared by reductive amination of 3-digoxigenone with the 
appropriate, .alpha.,.omega.-alkanediamine or .omega.-aminoalkanoic acid 
in the presence of sodium cyanoborohydride. 
In a preferred embodiment, the functionalized digoxigenin derivatives are 
coupled directly to corresponding amino or carboxyl groups in the carrier 
material by formation of an amide or peptide couple. The resulting 
preferred immunogen conjugates have the formula: 
##STR3## 
wherein Carrier and n are as defined above, Y is an amide group linking 
carboxyl or amino groups on the carrier material, which usually is an 
immunogenic protein or polypeptide, to the digoxigenin moiety, and p is on 
the average from 1 to the number of available amide coupling sites (e.g., 
available carboxyl or amino groups, as the case may be, under the coupling 
conditions) on the carrier, usually less than about 50. 
The peptide condensation reactions available for performing the direct 
coupling of the digoxigenin derivative to a carboxyl group-containing 
carrier are well known and include, without limitation, the carbodiimide 
reaction [Aherne et al, Brit. J. Clin. Pharm. 3:56(1976) and Science 
144:1344(1974)], the mixed anhydride reaction [Erlanger et al, Methods in 
Immunology and Immunochemistry, ed. Williams and Chase, Academic Press 
(New York 1967) p. 149], and the acid azide and active ester reactions 
[Kopple, Peptides and Amino Acids, W. A. Benjamin, Inc. (New York 1966)]. 
See also Clin. Chem. 22:726(1976). 
Alternatively, the digoxigenin derivatives can be coupled through the use 
of a conventional linking reagent that forms a bond at one end with the 
amino or carboxyl group in the derivative and a bond at the other end with 
an appropriate functional group present on the carrier. For example, 
bifunctional coupling reagents are well known for coupling amine 
derivatives to amine macromolecules, including bis-imidates, 
bis-isocyanates, and glutaraldehyde [Immuno-chem. 6:53(1959)]. Other 
useful coupling reactions are thoroughly discussed in the literature, for 
instance in the above-mentioned Kopple monograph; Lowe and Dean, Affinity 
Chromatography, John Wiley and Sons (New York 1974); Means and Feeney, 
Chemical Modification of Proteins, Holden-Day (San Francisco 1971); and 
Glazer et al, Chemical Modification of Proteins, Elsevier (New York 1975). 
The quantity p in the above formulas represents the number of digoxigenin 
moieties that are conjugated to the carrier, i.e., the epitopic density of 
the immunogen, and in the unsual situation will be on the average from 1 
to about 50, more normally from 1 to about 20. Optimal epitopic densities, 
considering the ease and reproducibility of synthesis of the immunogen and 
antibody response, fall between about 2 and about 15, more usually between 
4 and 10. 
The immunogenic carrier material can be selected from any of those 
conventionally known having available functional groups for coupling to 
the digoxigenin derivatives. In most cases, the carrier will be a protein 
or polypeptide, although other materials such as carbohydrates, 
polysaccharides, lipopolysaccharides, nucleic acids and the like of 
sufficient size and immunogenicity can likewise be used. For the most 
part, immunogenic proteins and polypeptides will have molecular weights 
between 4,000 and 10,000,000, preferably greater than 15,000, and more 
usually greater than 50,000. Generally, proteins taken from one animal 
species will be immunogenic when introduced into the blood stream of 
another species. Particularly useful proteins are albumins, globulins, 
enzymes, hemocyanins, glutelins, proteins having significant 
nonproteinaceous constituents, e.g., a glycoproteins, and the like. The 
albumins and globulins of molecular weight between 30,000 and 200,000 are 
particularly preferred. Further reference for the state-of-the-art 
concerning conventional immunogenic carrier materials and techniques for 
coupling haptens thereto may be had to the following: Parker, 
Radioimmunoassay of Biologically Active Compounds, Prentice-Hall 
(Englewood Cliffs, N.J. USA, 1976); Butler, J. Immunol. Meth. 
7:1-24(1974); Weinryb and Schroff, Drug Metab Rev. 10:271-283(1975); 
Broughton and Strong, Clin. Chem. 22:726-732(1976); and Playfair et al, 
Br. Med. Bull. 30:24-31(1974). 
Preparation of specific antibodies using the present immunogen conjugates 
may follow any conventional technique. Numerous texts are available 
describing the fundamental aspects of inducing antibody formation; for 
example reference may be made to Parker, Radioimmunoassay of Biologically 
Active Compounds, Prentice-Hall (Englewood Cliffs, N.J. USA, 1976). In the 
usual case, a host animal such as a rabbit, goat, mouse, guinea pig, or 
horse is injected at one or more of a variety of sites with the immunogen 
conjugate, normally in mixture with an adjuvant. Further injections are 
made at the same site or different sites at regular or irregular intervals 
thereafter with bleedings being taken to assess antibody titer until it is 
determined that an acceptable titer has been reached. The host animal is 
bled to yield a suitable volume of specific antiserum. Where desirable, 
purification steps may be taken to remove undesired material such as 
nonspecific antibodies before the antiserum is considered suitable for use 
in performing actual assays. 
The antibodies can also be obtained by somatic cell hybridization 
techniques, such antibodies being commonly referred to as monoclonal 
antibodies. Reviews of such monoclonal antibody techniques are found in 
Lymphocyte Hybridomas, ed. Melchers et al, Springer-Verlag (New York 
1978), Nature 266:495(1977), and Science 208:692 (1980). 
The antibodies prepared from the immunogens of the present invention can be 
used in any immunoassay method, and the corresponding reagent means, for 
determining digoxin, including agglutination techniques, 
radioimmunoassays, heterogeneous enzyme immunoassays (see U.S. Pat. No. 
3,654,090), heterogeneous fluorescent immunoassays (see U.S. Pat. Nos. 
4,201,763; 4,133,639 and 3,992,631), and homogeneous (separation-free) 
immunoassays. The latter-most are particularly preferred and include such 
techniques as fluorescence quenching or enhancement (see U.S. Pat. No. 
4,160,016), fluorescence polarization (see J. Exp. Med. 122:1029(1965), 
enzyme substrate-labeled immunoassay (see U.S. Pat. No. 4,279,992 and U.K. 
Pat. No. 1,552,607), prosthetic group-labeled immunoassay (see U.S. Pat. 
No. 4,238,565), enzyme modulator-labeled immunoassay, e.g., using 
inhibitor labels (see U.S. Pat. Nos. 4,134,972 and 4,273,866), 
enzyme-labeled immunoassay (see U.S. Pat. No. 3,817,837), energy transfer 
immunoassay (see U.S. Pat. No. 3,996,345), chemically-excited fluorescence 
immunoassay (see U.S. Pat. No. 4,238,195) and double antibody steric 
hindrance immunoassay (see U.S. Pat. Nos. 3,935,074 and 3,998,943). 
Moreover, the derivatives of the present invention can be used to prepare 
the labeled conjugates needed to perform the various immunoassays 
described above. Appropriate derivatives can be radio-labeled or labeled 
with fluorescent moieties in accordance with standard methods. Likewise 
the appropriate labeling moiety for the preferred homogeneous techniques, 
e.g., an enzyme substrate, a prosthetic group, an enzyme modulator, or an 
enzyme (which is a protein and can be coupled similarly to the immunogenic 
carrier as described above) can be coupled to the derivatives to yield 
labeled conjugates. 
One type of preferred labeled conjugate is that labeled with 
.beta.-galactosyl-umbelliferone (.beta.GU), having the general formula: 
##STR4## 
and n is as defined above. Preferably, such conjugates are prepared by 
conventional peptide condensations of .beta.GU-carboxylic acid (U.S. Pat. 
No. 4,226,978) with the appropriate 
3-[N-(aminoalkyl)]-amino-3-desoxydigoxigenin derivative. The 
.beta.BGU-labeled conjugates are useful as labeled reagents in 
substrate-labeled fluorescent immunoassays (SLFIA-see U.S. Pat. No. 
4,279,992). 
Another type of labeled conjugate is that labeled with flavin adenine 
dinucleotide (FAD), having the general formula: 
##STR5## 
wherein Ribose --(Phos).sub.2 Riboflavin represents the 
riboflavin-pyrophosphate-ribose residue in FAD, r is an integer from 2 
through 10, and n is as defined above. Such conjugates are prepared by 
peptide condensation of an appropriate N.sup.6 -.omega.-aminoalkyl-FAD 
derivative (U.S. Pat. No. 4,255,566) with the appropriate 
N-(carboxyalkyl)-amino-3-desoxydigoxigenin derivative. The FAD-labeled 
conjugates are useful as labeled reagents in apoenzyme reactivation 
immunoassay systems (ARIS-see U.S. Pat. No. 4,238,565). 
The reagent means of the present invention comprises all of the essential 
chemical elements required to conduct a desired digoxin immunoassay method 
encompassed by the present invention. The reagent means or system is 
presented in a commercially packaged form, as a composition or admixture 
where the compatibility of the reagents will allow, in a test device 
configuration, or as a test kit, i.e., a packaged combination of one or 
more containers holding the necessary reagents. Included in the reagent 
means are the reagents appropriate for the binding reaction system 
desired, e.g., an antibody and labeled conjugate of the present invention. 
Of course, the reagent means can include other materials as are known in 
the art and which may be desirable from a commercial and user standpoint, 
such as buffers, diluents, standards, and so forth. Particularly preferred 
is a test kit for the homogeneous competitive binding immunoassay of the 
present invention comprising (a) an anti-digoxin antibody of the present 
invention and (b) a labeled digoxigenin conjugate which has a detectable 
property which is altered when bound with the antibody. Also preferred is 
a test device comprising the reagent composition and a solid carrier 
member incorporated therewith. The various forms of such test device are 
described in U.S. patent application Ser. No. 202,378, filed Oct. 30, 
1980, which is incorporated herein by reference and which has published as 
European patent application No. 51,213. The specific label used in the 
preferred test kit and test device will depend on the technique followed, 
as described hereinabove. 
The present invention will now be illustrated, but is not intended to be 
limited, by the following examples.

EXAMPLE 1 
Preparation of digoxigenin derivatives 
3-N-(6-Aminohexyl)amino-3-desoxydigoxigenin 
A mixture of 1 gram (g) 3-digoxigenone [Tamm and Gubler, Helv. Chim. Acta, 
42:239(1959) and Shimizu and Mituhashi, Tetrahedron 24:4207(1968)] (2.58 
mmol), 3.21 g 1,6-hexanediamine (27.6 mmol), and 50 milliliters (ml) 
methanol (CH.sub.3 OH) was adjusted to pH 6 with glacial acetic acid and 
treated with 125 milligrams (mg) sodium cyanoborohydride (1.99 mmol). 
After stirring overnight, the mixture was preadsorbed onto 5 g silica gel 
(SiO.sub.2) and chromatographed over 200 g SiO.sub.2 -60 (E. Merck, West 
Germany) eluted first with 3 liters (L) of the lower phase of a 2:1:1 
chloroform (CHCl.sub.3)--CH.sub.3 OH-14% ammonium hydroxide (NH.sub.4 OH) 
mixture followed by 3 L of the lowr phase of a 2:1:1 CHCl.sub.3 --CH.sub.3 
OH-28% NH.sub.4 OH mixture. A partial separation of one of the two 
3.alpha. and .beta.-isomers of the product was achieved. The fractions 
containing this product were pooled, concentrated, and precipitated as an 
amorphous white powder from isopropanol-1:1 (ether-hexane). Yield 336 mg 
(27%). 
Analysis: Calculated for C.sub.29 H.sub.28 N.sub.2 O.sub.4.1/2H.sub.2 O: C, 
69,98; H, 10.06; N, 5.63. Found: C, 69.68; H, 9.61; N, 5.71. 
.sup.1 H NMR (90 mHz, DMSO-d.sub.6): .delta.0.66 (s,3H); 0.85 (s,3H); 1.29 
(m,28H); 2.35 (m,3H); 7.11 (m,7H); 4.09 (m,1H); 4.87 (s,2H); 5.81 (s,1H). 
IR (KCl): 2940, 2870, 1740, 1625, 1395, 1025 cm.sup.-1. 
3-N-(5-Carboxypentyl)amino-3-desoxydigoxigenin 
To a suspension of 300 mg 3-digoxigenone (0.77 mmol) and 506 mg 
6-aminocarproic acid (3.86 mmol) in 30 mL CH.sub.3 OH was added 253 mg 
sodium cyanoborohydride. The mixture was allowed to stir overnight at room 
temperature. The reaction mixture was quenched with 0.5 Ml H.sub.2 O and 
evaporated under reduced pressure. The residue was taken up in 10 ml 
CH.sub.3 OH, preadsorbed on 1 g SiO.sub.2, and chromatographed over a 
3.5.times.18 cm column of SiO.sub.2 eluted with the lower phase of a 
1:1:1: CHCl.sub.3 CH.sub.3 OH-conc.NH.sub.4 OH mixture. The fractions 
containing the desired product were pooled and evaporated to give 596 mg 
of a colorless glass. The sample was dissolved in 2 ml H.sub.2 O and 
chromatographed over a 2.5.times.90 cm column of Sephadex.RTM. LH-20 
(Pharmacia, Piscataway, NJ, USA) eluted with H.sub.2 O. The fractions 
containing the product as a mixture of C-3 isomers were pooled and 
evaporated. The product was obtained as a white powder from CH.sub.3 
OH/ether-petroleum ether. Yield 279 mg (72%). mp 172.degree.-180.degree. 
C. 
Analysis: Calculated for C.sub.29 H.sub.45 NO.sub.6.1/2H.sub.2 O: C, 68.01; 
H, 9.04; N, 2.73. Found: C, 68.14; H, 9.26; N, 2.47. 
.sup.1 H NMR (90 mHz, DMSO-d.sub.6): .delta.0.65 (s,3H; 0.85 (s,3H); 1.38 
(m,31H); 2.5 (m,2H); 2.97 (m,1H); 3.23 (m,2H); 4.02 (H.sub.2 O); 4.87 
(s,2H); 5.82 (s,1H). 
(IR (KCl): 2940, 2860, 1780, 1745, 1625, 1570, 1455, 1400 cm.sup.-1. 
EXAMPLE 2 
Preparation of anti-digoxin antibodies 
Thirteen milligrams of 3-N-(5-carboxypentyl) amino-3-desoxydigoxigenin was 
dissolved in 0.3 ml of dry dimethylformamide containing 7 .mu.l of 
tri-n-butylamine, and this mixture was cooled in an ice bath. Ten 
microliters (.mu.l) of ethyl chloroformate was added and allowed to react 
for 15 minutes. This reaction mixture was added dropwise to a stirred 
solution of 125 mg bovine serum albumin in 5 ml water containing 125 .mu.l 
1.0N sodium hydroxide (NaOH). Twenty-four hours later the reaction was 
chromatographed on a 2.5.times.55 cm column of Sephadex.RTM. G-25 (coarse) 
(Pharmacia) equilibrated with 0.1M sodium phosphate buffer, pH 7.0, and 
10.5 ml fractions were collected. The absorbance at 280 nanometers (nm) of 
fraction 13 was recorded. Also, 200 .mu.l of this fraction was mixed with 
0.8 ml of concentrated sulfuric acid and the optical absorption spectrum 
was recorded from 340 to 450 nm. The absorbance at 380 nm taken from this 
spectrum was used to calculate the amount of digoxigenin derivative 
coupled to bovine serum albumin assuming that the millimolar extinction 
coefficient at 380 nm for the digoxigenin derivative is 3.2. The 
hapten:protein molar ratio was 8.6. 
Fractions 12 to 15 from the chromatography were pooled and used for 
immunization. This pool was diluted with 0.1M sodium phosphate buffer, pH 
7.0, to give a protein concentration of 0.4 mg/ml. One milliliter of this 
immunogen was combined with 1 ml of complete Freund's adjuvant and 
injected subcutaneously into a rabbit. A booster immunization was 
administered at four week intervals and for these the immunogen was 
combined with incomplete Freund's adjuvant. Test bleedings were taken one 
week after the boosters. Five months after the initial immunization, 
antiserum with suitable titer was obtained. 
EXAMPLE 3 
Preparation of labeled digoxigenin conjugates 
3-N-6-(7-.beta.-galactosylcoumarin-3-carboxamidohexyl)amino-3-desoxydigoxig 
enin. 
To 243 mg 7-.beta.galactoxylcoumarin-3-carboxylic acid (U.S. Pat. No. 
4,226,978) (0.66 mmol) and 84 mg N-hydroxysuccinimide (0.73 mmol) in 5 ml 
dimethylformamide under argon gas at 0.degree. C. was added 143 mg 
dicyclohexylcarbodiimide (0.69 mmol). The mixture was allowed to warm from 
0.degree. C. to room temperature over 4 hours. To this mixture was added 
322 mg 3-N-(6-aminohexyl)amino-3-desoxydigoxigenin (0.66 mmol), and the 
resulting solution allowed to stir overnight. The crude reaction mixture 
was adsorbed onto 1 g SilicAR.RTM. CC-7 (Mallinckrodt, St. Louis, Mo. USA) 
and applied to a 2.5.times.60 cm column of SilicAR.RTM. CC-7 packed with 
ethanol. The column was eluted with a linear gradient of 2 L ethanol to 2 
L of a 4:1 ethanol-1M triethylammonium bicarbonate (pH 7.5) mixture. 
Fractions containing the product were pooled, evaporated, and the product 
was precipitated from methanol-1:1 ether/petroleum ether. Yield 124 mg. 
The sample was applied to a 2.5.times.60 cm column of Sephadex.RTM. LH-20 
(Pharmacia) packed and eluted with methanol. The product was obtained by 
evaporation of solvent and precipitation from methanol-1:1 ether/hexane. 
Yield 72 mg (13%). 
MP 152.degree. C. (decomposed). 
Analysis: Calculated for C.sub.45 H.sub.62 N.sub.2 O.sub.13.4H.sub.2 O: C, 
59.5; H, 7.8; N, 3.1. Found: C, 59.7; H, 7.0; N, 3.8. 
.sup.1 NMR (90 mHz, DMSO-d.sub.6): .delta.0.65 (s,3H); 0.86 (s,3H); 1.35 
(m,26H); 2.3 (m,2H); 3,55 (m,13H); 4.06 (m,2H); 4,69 (m,3H); 4,87 (s,2H); 
5.04 (d,J=7 Hz,1H); 5.82 (s,1H); 6.41 (m,1H); 7.15 (s,1H); 7.21 (d,J=8 
Hz,1H); 7.93 (d,J=8 Hz,1H); 8.63 (bt,J=6 Hz,1H); 8.82 (s,1H). 
IR(KCl): 1735, 1710, 1645, 1615, 1545, 1220, 1075 cm.sup.-1. 
Digoxigenin-FAD conjugate 
To 25.2 mg 3-N-(5-carboxypentyl)amino-3-desoxdigoxigenin (50 .mu.mol) and 
6.3 mg N-hydroxysuccinimide (56 .mu.mmol) in 0.5 ml dry dimethylformamide 
under argon gas at 0.degree. C. was added a solution of 10.8 mg 
dicyclohexylcarbodiimide (52 .mu.mol) in 0.6 ml dry dimethylsulfoxide. The 
reaction was stirred 30 minutes at 0.degree. C. and 30 minutes at room 
temperature. A solution of 10 .mu.mol N.sup.6 -(6-aminohexyl)-flavin 
adenine dinucleotide (U.S. Pat. No. 4,255,566) in 1 ml H.sub.1 O was added 
to the above solution, and the mixture was allowed to stir overnight at 
room temperature. The reaction mixture was then diluted to 450 ml with 
H.sub.2 O and applied to a 1.5.times.30 centimeter (cm) column of 
DEAE-cellulose (bicarbonate form). The column was eluted with a linear 
gradient of 1.5 L H.sub.2 O to 1.5 L 0.1M triethylammonium bicarbonate (pH 
7.5) at a flow rate of 2.8 ml/min. Fractions of 17 ml/min were taken. 
Fractions 16-32 contained the product and were pooled, concentrated, and 
adjusted to pH 7.0 and 25 ml volume. From the absorbance measurement at 
450 nm (0.860) the yield was calculated as 1.903 .mu.mol (19% yield) based 
on the millimolar extinction coefficient and FAD as 11.3. 
EXAMPLE 4 
Characterization of Digoxigenin-FAD 
A sample (10 .mu.l) of reaction mixture of the NOS ester of the digoxigenin 
carboxylic acid derivative and aminohexyl-FAD was chromatographed by thin 
layer chromatography using isobutyric acid-H.sub.2 O-triethylamine 
(70:29:1). The spot with R.sub.f =0.2 was scraped from the plate and 
suspended in 1.0 ml 0.1M phosphate buffer, pH 7.0. A 0.35 ml aliquot of 
the supernatant was mixed with 100 .mu.l of glucose oxidase assay reagent 
comprised of: 
0.1M phosphate buffer, pH 7.0 
2.1 mM sodium 3,5-dichloro-2-hydroxybenzene sulfonate 
1.1% (w/v) bovine serum albumin 
21 .mu.g/ml peroxidase 
0.105M glucose 
Apoglucose oxidase reagent was prepared consisting of 2 micromoler (.mu.M) 
FAD binding sites; 10% (w/v) glycerol; 4 micromolar (mM) 
4-aminoantipyrine, 0.1 molar (M) Phosphate, pH 7.0. Antiserum to digoxin 
(Atlantic Antibodies, Scarborough, Maine USA) was used diluted 10-fold 
with 0.1M phosphate, pH 7.0. A solution of digoxin (130 .mu.M) in 0.1M 
phosphate, pH 7.0, was diluted 10-fold with the same buffer. 
Assays were performed by placing 100 microliters (.mu.l) of apoenzyme 
reagent and diluted digoxin antiserum in one corner of disposable 
cuvettes. Buffer or diluted digoxin solution (10 .mu.l) was placed in the 
opposite corner. 1.90 ml of glucose oxidase assay reagent was added to 
start the reaction. The assays were incubated at 25.degree. C. for 10 
minutes and the absorbance at 520 nm recorded. The final concentration of 
digoxin was 65 nM. 
______________________________________ 
mA.sub.520nm at 10 min.* 
Without With 
Antiserum to Digoxin (.mu.l) 
Digoxin Digoxin 
______________________________________ 
-- 797 740 
5 463 716 
10 391 505 
20 354 378 
30 344 339 
______________________________________ 
*Average of duplicates corrected for blank activity in absence of label 
(mA.sub.520nm = 105). 
EXAMPLE 5 
Titration of Antiserum to 3-N-(5-carboxypentyl)amino-3-desoxydigoxigenin 
The titration was conducted with a substrate-labeled fluorescence 
immunoassay SLFIA; see U.S. Pat. No. 4,279,992) method using 
.beta.-GU-digoxigenin conjugate, supra, as the labeled conjugate. Three 
milliliters of 50 nM Bicine buffer, pH 8.3, was measured into six cuvettes 
and the volumes of antiserum listed below were added to appropriate 
cuvettes. Then 100 .mu.l of .beta.-GU-digoxigenin with an absorbance at 
340 nm of 0.01 was added. Finally, 100 .mu.l of .beta.-galactosidase from 
E. coli was added and the reactions were allowed to stand at ambient 
temperature for 20 minutes. (One unit of enzyme activity hydrolyzes 1 
.mu.mol of o-nitrophenyl-.beta.-galactosidase per minute at 25.degree. C. 
in 50 nM Bicine buffer, pH 8.3). At the end of this incubation period, the 
fluorescence was recorded using 400 nm light for excitation and 450 nm for 
emission. The following results were obtained: 
______________________________________ 
Antiserum Fluorescence 
(ml/assay) (arbitrary units) 
______________________________________ 
0 94 
1 80 
2 66 
5 32 
10 35 
20 32 
______________________________________ 
The fluorescence decreased as the antiserum level increased; indicating 
that antibody was binding to the .beta.-GU-digoxigenin conjugate. 
EXAMPLE 6 
Digoxin immunoassay 
Various amounts of 2 .mu.M digoxin were added to cuvettes containing 3.0 ml 
of 50 mM Bicine buffer, pH 8.3, to give the final digoxin concentrations 
listed below. One hundred microliters of .beta.-galactosidase (0.6 
unit/ml) in the Bicine buffer was added to each cuvette and the reactions 
were allowed to stand at ambient temperature for 20 minutes. At the end of 
this period, the fluorescence was recorded. The following results were 
obtained: 
______________________________________ 
Digoxin Fluorescence 
(mM) (arbitrary units) 
______________________________________ 
0 39 
7.6 44 
15 52 
38 65 
152 85 
______________________________________ 
As the digoxin level increased, the fluorescence increased, indicating that 
the digoxin and the .beta.-GU-digoxigenin conjugate were competing for 
antibody binding sites.