Theophylline antigens and antibodies

Theophylline derivatives are provided for preparing reagents for use in competitive protein binding assays and for use as reagents in competitive protein binding assays. Theophylline is substituted at the 3 position and conjugated to antigens for production of antibodies which specifically recognize theophylline as distinct from structurally similar analogs such as caffeine. Enzyme conjugates are provided which find use for measuring the amount of theophylline in a sample suspected of containing theophylline. A method is provided employing the reagents for the determination of theophylline.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
Theophylline (1,3-dimethylxanthine) is a drug commonly used in the 
treatment of asthma, hypertension and nephrotic edema. At elevated plasma 
levels, theophylline will sometimes produce nausea and serious toxic 
effects may occur at high plasma concentrations ranging from about 
25-70.mu.g/ml. Serum theophylline shows considerable individual variation 
in patients, due to wide differences in the extent of metabolism and 
secretion as well as fluctuations during dosing intervals in the rate of 
absorption and distribution. With infants, the problems are further 
exacerbated, due to the infants low body fluid level. 
In view of the serious side effects as a result of elevated serum 
theophylline levels, it is important that sensitive techniques be provided 
for monitoring theophylline levels. The technique should be rapid, 
accurate, and readily distinguish theophylline from its normal metabolites 
and widely prevalent analogs, such as xanthine and caffeine. 
2. Description of the Prior Art 
U.S. Pat. Nos. 3,690,834, 3,817,837, 3,850,752, and 3,766,162, and the 
references cited therein, describe a series of different immunoassays. The 
disclosure of U.S. Pat. No. 3,817,837 describing a homogeneous enzyme 
immunoassay is incorporated herein by reference. Synthesis of xanthine 
derivatives may be found in Advances in Heterocyclic Chemists 1966 Vol. 
VI, Lister et al, Rev. Pure & Applied Chem. (Australia). See also U.S. 
Pat. Nos. 2,517,410 and 2,673,848, as well as Holmes and Leonard, J. Org. 
Chem. 41 568 (1976) and Cavalieri et al, J. Am. Chem. Soc. 76, 1119 (1954) 
for the preparation of xanthene derivatives. Rasmussen and Leonard, J. Am. 
Chem. Soc. 89 5439 (1967) discloses the use of pivaloyloxymethyl as a 
protecting group. 
SUMMARY OF THE INVENTION 
Theophylline derivatives are conjugated to antigens and enzymes. The 
antigenic theophylline conjugates are employed for the production of 
antibodies for specific recognition of theophylline. The enzymes 
conjugates are employed in competitive protein binding assays for the 
determination of theophylline, particularly in serum. 
DESCRIPTION OF THE SPECIFIC EMBODIMENTS 
The subject invention concerns 3-substituted-1-methyl-xanthines, which 
includes precursors, antigens for the preparation of antibodies to 
theophylline and enzyme conjugates which find use in competitive protein 
binding assays. The 3-substituent involves a short chain, usually, but not 
necessarily, involving a non-oxocarbonyl group, including the nitroger and 
sulfur analogs thereof, joined to a poly(amino acid), which encompasses 
both natural and synthetic polypeptides, proteins and their combinations 
with prosthetic groups. The poly(amino acids) of particular interest are 
antigens and enzymes. The linking group may be a hydrocarbon group, that 
will normally have from 1 to 4 heteroatoms, which are oxygen, nitrogen, or 
sulfur, involved in the linking chain or bonded to carbon atoms in the 
chain. Usually, the chain will be of from about 1 to 10 atoms other than 
hydrogen, more usually from about 2 to 6 atoms other than hydrogen. 
For the most part, the poly(amino acid) derivatives employed in this 
invention will have the following formula; 
##STR1## 
wherein: 
PAA refers to a poly(amino acid), particularly antigens and enzymes, 
wherein the antigens will normally be from about 5,000 to 10,000,000 
molecular weight, more usually from about 10,000 to 500,000 molecular and 
preferably from about 25,000 to 300,000; while enzymes will normally be 
from about 10,000 to 600,000 molecular weight, more usually from about 
10,000 to 300,000 molecular weight, and preferably from about 10,000 to 
200,000 molecular weight; 
n is the number of xanthine groups bonded to PAA and will be on the average 
at least 1 and up to the molecular weight of PAA divided by 1,500, more 
usually divided by 2,000; for antigens n will be generally on the average 
from 4 to 250, more usually from about 6 to 100; while for enzymes, n will 
generally be from about 1 to 30, more usually from about 2 to 20, and 
preferably from about 2 to 12; and 
R is a linking group which may be a bond, but is normally of from 1 to 12, 
more usually from 2 to 10 atoms other than hydrogen which are carbon, 
oxygen, nitrogen and sulfur, generally having from 0 to 1 sites of 
aliphatic unsaturation, usually ethylenic, and wherein oxygen will be 
present normally solely bonded to carbon i.e. ether or non-oxocarbonyl, 
nitrogen will be present as amido or tertiary-amino, and sulfur will be 
present as thioether or thiono; usually only oxygen and nitrogen will be 
present; R may be aliphatic, alicyclic, aromatic or combinations thereof, 
but will normally be aliphatic having not more than one site of aliphatic 
unsaturation e.g. ethylenic, and preferably saturated aliphatic. 
The compounds prepared in accordance with this invention will be free or 
substantially free of xanthine substituted at the 7 and 8 positions. For 
the purpose of this invention, it is necessary that the products be solely 
3-xanthine substituted. 
For the most part, R groups will have the following formula: 
##STR2## 
wherein: 
X and X.sup.1 may be the same or different and are oxygen, imino(NH) or 
sulfur, particularly oxygen or imino; 
T and T.sup.1 may be the same or different and may be a bond, a hydrocarbon 
radical of from 1 to 10 carbon atoms, more usually of from 1 to 4 carbon 
atoms, having a total of from 1 to 8 carbon atoms, more usually of from 1 
to 6 carbon atoms, generally aliphatic, and preferably saturated 
aliphatic, particularly methylene or polymethylene, or T.sup.1 may be 
hydrocarbylamino where the nitrogen is bonded to (CX.sup.1) wherein said 
hydrocarbyl group has the same limitation as for said hydrocarbon radical; 
Y is a bond, amido or oxy; 
m and p are integers of from 0 to 1, preferably p being 1 and m being 0, 
wherein CX is bonded to the xanthine and CX.sup.1 is bonded to the 
poly(amino acid). 
(By hydrocarbyl is intended an organic radical composed solely of hydrogen 
and carbon, which may be aliphatic, alicyclic, aromatic or combinations, 
saturated or unsaturated. For this invention, the hydrocarbyl groups will 
have not more than one site of unsaturation e.g. ethylenic) 
Illustrative linking groups have the following formulae: 
--CH.sub.2 CO-- 
--ch.sub.2 c(nh)-- 
--coch.sub.2 ch.sub.2 co-- 
--cochchco-- 
--ch.sub.2 conhch.sub.2 co-- 
--coch.sub.2 n(ch.sub.3)ch.sub.2 co-- 
--ch.sub.2 ch.sub.2 c(nh)-- 
--coch.sub.2 ch.sub.2 och.sub.2 ch.sub.2 co-- 
--coch.sub.2 och.sub.2 co-- 
--ch.sub.2 chchco-- 
the compounds which find use for conjugation to the poly(amino acid) will 
for the most part have the following formula: 
##STR3## 
wherein: 
R.sup.1 is a bond or linking group of from 1 to 12 atoms other than 
hydrogen, more usually of from 1 to 6 atoms other than hydrogen and 
preferably of from about 1 to 5 atoms other than hydrogen, which are 
carbon, oxygen, nitrogen and sulfur, wherein the characteristics set forth 
for carbon, oxygen, nitrogen and sulfur are as set forth for R; 
Z is oxocarbonyl (CHO), or non-oxo-carbonyl (includes the nitrogen-imido- 
and sulfur-thiono-analogs) carboxyl, carboxyester, wherein the ester group 
is nitrophenyl or N-succinimidyloxy, alkoxyimido, wherein the alkoxy group 
is of from 1 to 3 carbon atoms, isothiocyanate, or isocyanate. 
The oxo group may be linked to available amino groups by reductive 
amination. The carboxylic acid groups and their nitrogen and sulfur 
analogs may be linked directly to available amino groups either by 
employing active esters, activating with carbodiimide or preparing a mixed 
anhydride with a chlorocarbonate ester e.g. alkoxycarbonyl of from 2 to 7 
carbon atoms. 
As indicated previously, of particular interest are compounds where the 
oxo-carbonyl group (other than keto) and the non-oxo-carbonyl group are 
bonded to an amino group, which is part of an antigenic polypeptide or 
protein. By bonding the carbonyl derivative of xanthine to the polypeptide 
or protein, antibodies can be formed to theophylline, which distinguish 
from caffeine in a competitive protein binding assay. A narrower class of 
proteins, which also can be used as antigens, but will be normally be used 
as such, are enzymes which are employed as the detector in an immunoassay 
system. As antigens, inactive enzymes can be used. 
Polypeptides (referred to generally in the invention as poly(amino acid)) 
usually encompass from about 2 to 100 amino acid units (usually less than 
about 12,000 molecular weight). Larger polypeptides are arbitrarily called 
proteins. Proteins are usually composed of from 1 to 20 polypeptide chains 
called subunits, which are associated by covalent or noncovalent bonds. 
Subunits are normally of from about 100 to 300 amino acid groups (or 
10,000 to 35,000 molecular weight). For the purposes of this invention, 
poly(amino acid) is intended to include individual polypeptide units and 
polypeptides which are subunits or polypeptide units in combination with 
other functional groups, such as porphyrins, as in haemoglobin or 
cytochrome oxidase. 
The number of xanthine groups will vary depending upon whether the 
poly(amino acid) is an enzyme or antigen. The maximum number of groups 
will be limited by the effect of substitution on solubility, activity, and 
the like. For the formation of antibodies, a sufficient number of xanthine 
groups should be present, so as to provide a satisfactory harvest of 
antibodies to the theophylline (anti(theophylline)). Otherwise, the 
proportion of antibodies to theophylline as compared to antibodies to 
other compounds may be undesirably low. 
The first group of protein materials or polypeptides which will be 
considered are the antigenic polypeptides. These may be joined to the 
carbonyl group of the theophylline analog through an amino group. The 
product can be used for the formation of antibodies to theophylline. The 
protein materials which may be used will vary widely, and will normally be 
from 5,000 to 10 million molecular weight, more usually 25,000 to 500,000 
molecular weight. 
Enzymes will normally be of molecular weights in the range of about 10,000 
to 600,000, usually in the range of about 10,000 to 150,000, and more 
usually in the range of 12,000 to 80,000. Some enzymes will have a 
plurality of enzyme subunits. It is intended when speaking of enzyme 
molecular weights to refer to the entire enzyme. There will be on the 
average at least about one xanthine per enzyme, usually at least about two 
xanthines per enzyme, when the labeling is not limited to a specific amino 
group, and rarely more than 40 xanthines per enzyme, usually not more than 
30 xanthines per enzyme. For example with lysozyme the average number of 
xanthine groups will be in the range of about 2 to 5. For 
glucose-6-phosphate dehydrogenase and malate dehydrogenase the average 
number will be in the range of 2 to 20, usually 2 to 12. 
While the theophylline analog may be bonded through the non-oxo-carbonyl 
group to hydroxyl or mercapto groups, which are present in the proteins, 
for the most part the bonding will be to amino. Therefore, the compounds 
are described as amides (including nitrogen and thioanalogs e.g. amidine 
and thioamide. Also included within the non-oxo-carboxyl derivatives are 
urea, guanidine and thiourea.), although esters and thioesters may also be 
present. The aldehyde derivative will be bonded solely to amino to form 
alkylamine groups through reductive amination. 
Amino acids present in proteins which have free amino groups for bonding to 
the carboxy modified xanthine includes lysine, N-terminal amino acids, 
etc. The hydroxyl and mercaptan containing amino acids include serine, 
cysteine, tyrosine and threonine. 
Various protein and polypeptide types may be employed as the antigenic 
material. These types include albumins, enzymes, serum proteins, e.g., 
globulins, ocular lens proteins, lipoproteins, etc. Illustrative proteins 
include bovine serum albumin, keyhole limpet hemocyanin, egg albumin, 
bovine gamma-globulin, etc. Small neutral polypeptides which are 
immunogenic such as gramicidins may also be employed. Various synthetic 
polypeptides may be employed, such as polymers of lysine, glutamic acid, 
phenylalanine, tyrosine, etc., either by themselves or in combination. Of 
particular interest is polylysine or a combination of lysine and glutamic 
acid. Any synthetic polypeptide must contain a sufficient number of free 
amino groups as, for example, provided by lysine. 
The second group of protein molecules are the detectors. These are the 
enzymes to which the carbonyl modified xanthine may be conjugated. As 
indicated, the xanthine modified enzyme is useful for immunoassays. A 
description of the immunoassay technique will follow. 
Various enzymes may be used such as peptidases, esterases, amidases, 
phosphorylases, carbohydrases, oxidases, e.g. dehydrogenase, reductases, 
and the like. Of particular interest are enzymes such as lysozyme, 
peroxidase, .alpha.-amylase, dehydrogenases, particularly malate 
dehydrogenase and glucose-6-phosphate dehydrogenase, alkaline phosphatase, 
.beta.-glucuronidase, cellulase and phospholipase. In accordance with the 
I.U.B. Classification, the enzymes of interest are: 1. Oxidoreductases, 
particularly Group 1.1, and more particularly 1.1.1, and 1.11, more 
particularly, 1.11.1; and 3. Hydrolases, particularly 3.2, and more 
particularly 3.2.1. 
The enzyme-bound-theophylline will for the most part have the following 
formula: 
##STR4## 
wherein: 
ENZ is an enzyme, preferably an oxidoreductase or hydrolase, particularly 
oxidoreductases employing DPN or DPNP e.g. dehydrogenases, oxiodases and 
peroxidases or hydrolases, including esterases, e.g. phosphatases, 
lysozyme, and the like; the enzyme has at least 2% of its original 
activity, generally at least 10%, more usually at least 20% and preferably 
at least 30% of its original activity; 
n.sup.1 is an integer which on the average is in the range of 1 to the 
molecular weight of the enzyme divided by about 2,000, more usually in the 
range of about 1 to 30, preferably in the range of 1 to 20, and more 
preferably in the range of about 2 to 16; 
R.sup.2 is a linking group which may be a bond or a divalent organic 
radical of from 1 to 10 atoms other than hydrogen, which are carbon, 
oxygen, nitrogen and sulfur, particularly carbon, oxygen and nitrogen, and 
more particularly carbon and oxygen, the oxygen being present as oxy or 
non-oxocarbonyl, that is, bonded solely to carbon, and the nitrogen is 
amido or bonded solely to carbon as tertiary-amino, while sulfur is 
present as thiono; usually, R.sup.2 is aliphatic having from 0 to 1 site 
of ethylenic unsaturation, preferably saturated, and will be of from 1 to 
6 carbon atoms, more usually of from 1 to 4 carbon atoms, preferably 
methylene or polymethylene; and 
X, X.sup.1, m have been defined previously. 
The enzymes which are employed are preferably inhibited when the xanthine 
groups conjugated to the enzyme are bound to antibodies for the xanthine 
groups ie. anti(theophylline). The inhibition at saturation with 
anti(theophylline) should be at least 20% of the activity of the 
conjugated enzyme, usually at least 30% and preferably at least 40%, 
generally not more than 90%. 
In forming the various amide product which find use in the subject 
invention, the carboxylic acid will normally be activated. This can be 
achieved in a number of ways. Two ways of particular interest are the 
reaction with a carbodiimide, usually a water soluble dialiphatic or 
dicycloaliphatic carbodiimide in an inert polar solvent, e.g. 
dimethylformamide, acetonitrile, THF, DMSO, and hexamethylphosphoramide. 
The reaction is carried out by bringing the various reagents together 
under mild conditions and allowing sufficient time for the reaction to 
occur. 
A second method is to form a mixed anhydride employing an alkyl 
chloroformate, e.g. isobutyl chloroformate. The mixed anhydride is formed 
by combining the carboxy substituted xanthine, the alkyl chloroformate and 
tertiary amine. The temperature is normally below ambient temperature and 
a small amount of carbitol may be used. 
At least a stoichiometric amount of the chloroformate is employed based on 
the xanthine derivative, and usually an excess, which ususally does not 
exceed three times stoichiometric. The tertiary amine is present in at 
least equimolar amount to the chloroformate. 
The mixture is then combined with the amino compound to be conjugated and 
the reaction allowed to proceed under mild conditions. 
Also, esters of the carboxy modified xanthine can be employed which are 
operative in water for acylating amine functions. Illustrative hydroxylic 
groups are p-nitrophenyl and N-hydroxy succinimide which can be used to 
prepare the p-nitrophenyl and N-succinimidyloxy esters respectively. For 
the aldehyde conjugation, a reductive amination is carried out in a polar, 
usually aqueous medium, employing sodium cyanoborohydride as the reducing 
agent. 
A novel and simple procedure is provided for the production of 
1-methyl-3-substituted-xanthines free of other isomers. The starting 
material is 1-methylxanthine which is condensed with an approximately 
stoichiometric amount halomethyl pivalate (halo of atomic number 17 to 35, 
particularly chloro) under basic conditions in a polar anhydrous 
non-hydroxylic organic medium e.g. DMF, THF, DMSO, HMP, etc. Mild 
temperatures are employed 0.degree. t 50.degree. C., ambient temperatures 
being convenient. The base may be the nitrogen salt, sodium carbonate, 
tert-amine, etc. The reaction is allowed to proceed to completion and the 
mono-substituted-pivaloyloxymethyl derivative separated from any minor 
amounts of disubstituted material. 
The 7-substituted product is isolated and combined with an alkyl ester of 
halosubstituted aliphatic carboxylic acid. The halo is of atomic number 17 
to 53 preferably iodo, and is preferably co-substituted. The alkyl group 
of the alcohol portion is of from 1 to 6 carbon atoms, preferably 1 to 2 
carbon atoms and the acid group is of from 2 to 7 carbon atoms. A basic 
anhydrous non-hydroxylic polar medium is employed, as described above, 
employing analogous reaction conditions. 
After isolating the product, the ester groups are hydrolyzed under 
conventional conditions. Aqueous base may be employed at a temperature of 
from about 75.degree. to 100.degree. C. Upon acidification of the solution 
the desired 1-methyl-3-carboxylalkylxanthine may be isolated. 
The antibodies which are prepared in response to the conjugated antigens of 
this invention have strong specific binding to the parent drug, the 
conjugated antigen, the compound or derivative thereof used to conjugate 
to the antigen, and the enzyme conjugate. 
The assay will be able to detect theophylline in the concentration range 0 
to 100 .mu.g/ml and should be able to distinguish between no theophylline 
and 5 .mu.g/ml, preferably 2.5 .mu.g/ml. Normally, the sample of interest 
will be serum, although other sample sources may be employed. 
The assay will generally be carried out by combining in an aqueous buffered 
medium, generally at a pH in the range of 5 to 10, more usually 6 to 9, 
the sample to be assayed, enzyme-bound-theophylline and 
anti(theophylline). Besides water up to 20 volume percent of polar organic 
solvents may be included in the assay medium, e.g. alkanols, ethers, 
amides, etc. 
The amounts of the reagents will vary depending upon the enzyme activity of 
the enzyme-bound-theophylline, the degree of inhibition resulting from 
binding of anti(theophylline) to the enzyme-bound-theophylline, the manner 
of measurement, the sensitivity of the reagent combination to variations 
in the concentration of theophylline, the binding constant of the 
anti(theophylline) and the like. The primary concern is that a reasonable 
spread of measured values can be obtained over the theophylline range of 
interest. When measuring changes in optical density due to changes in the 
light absorption of the assay medium as a result of an enzymatic 
transformation, over a theophylline concentration range of about 0 to 50 
.mu.g/ml, the measured change in absorption should be at least about 
0.25.DELTA.OD, preferably at least 0.5.DELTA.OD. 
Usually, the mole ratio of anti(theophylline) based on binding sites to 
theophylline in enzyme-bound-theophylline will be about 0.01-100:1. The 
concentration of enzyme-bound-theophylline will generally be in the range 
of about 10.sup.-5 to 10.sup.-10, 
Included in the assay medium will be the enzyme substrates. Usually, one of 
the substrates will be transformed to a product which has a distinctive 
absorption in the ultraviolet or visible region. Conveniently enzymes can 
be employed which transform NAD or NADP to NADH or NADPH and the formation 
of NADH or NADPH followed spectrophotometrically. By taking two readings 
at a particular wavelength over a predeterminted time period, a rate value 
can be obtained which relates to the enzymatic activity. By employing the 
same protocol with an unknown sample as employed with samples spiked with 
known concentrations of theophylline, the result obtained can be 
translated into a theophylline concentration. 
Temperatures for the assay will generally be in the range of about 
10.degree.-50.degree. C., usually in the range 25.degree.-40.degree. C.

EXPERIMENTAL 
The following examples are offered by way of illustration and not by way of 
limitation. (All percents not otherwise indicated are by weight, except 
for mixtures of liquids which are by volume. All temperatures not 
otherwise indicated are centigrade. Abbreviations include DMF for 
N,N-dimethylformamide; TLC, thin layer chromotography; ECDI, 
1-ethyl-3-(dimethylaminopropyl) carbodiimide hydrochloride; THF, 
tetrahydrofuran. 
EXAMPLE 1 
Preparation of 1-methyl-7-pivaloyloxymethyl xanthine 
To a mixture of 1-methylxanthine (844 mg, 5.08 mmoles) and sodium carbonate 
(538 mg, 5.08 mmoles) in 20 ml of dry DMF under nitrogen was slowly added 
over a period of 1 hour a solution of chloromethylpivalate (828 .mu.l, 
5.59 mmole) in 6 ml DMF at room temperature and the reaction mixture 
stirred overnight. The mixture was filtered, the filtrate evaporated to 
dryness and the residue triturated with 50 ml of 10 vol. %. 
EtOH/CHCl.sub.3. The solids obtained were starting material and the 
organic phase was chromatographed (10 vol. % EtOH/CHCl.sub.3) on silica 
gel plates. Two products were observed by eluting with 125 ml of 10 vol. % 
EtOH/CHCl.sub.3 and the band R.sub.f, 0.48 obtained as 365 mg of the 
desired product. The other band proved to be the 3,7-disubstituted 
material. 
EXAMPLE 2 
Preparation of 1-methyl-3-(carboethoxypropyl)-7-(pivaloyloxymethyl)xanthine 
A mixture of 1-methyl-7-(pivaloyloxymethyl)xanthine (350 mg, 1.25 mmes) 
sodium carbonate (265 mg, 2.50 mmoles) and ethyl 4-iodobutyrate (384 
.mu.l, 2.50 mmole) in 5.8 ml dry DMF was stirred under nitrogen at room 
temperature for 18 hours. Water was added and the reaction mixture 
extracted with chloroform. After drying, the extracts were stripped to 
leave a yellow oil which goes to chromotographed on four thick silica gel 
plates with 10% EtOH/CHCl.sub.3. The band was scraped off and eluted with 
100 ml of 25% EtOH/CHCl.sub.3 to yield 609 mg of the desired product as an 
oil. 
EXAMPLE 3 
Preparation of 1-methyl-3-(3'-carboxypropyl)xanthine 
An oily suspension of 
1-methyl-3(carboethoxypropyl)-7-(pivaloyloxymethyl)xanthine (508 mg) in 2 
N NaOH(36.2 ml) was heated under nitrogen at 95.degree.-100.degree. for 2 
hrs. The oil dissolved and the reaction was shown to be complete by TLC. 
After cooling the reaction mixure, it was acidified to pH 2-3 with 15 ml 
12% hydrochloric acid and the solution extracted with 3.times.5 ml 
chloroform to remove the pivalic acid. After washing the chloroform 
extracts with 5 ml 0.5 N HCl, the aqueous solutions were combined and 
stripped to dryness and the residue dried in vacuo at room temperature. 
The crude product was dissolved in about 22 ml hot water, decolorized and 
concentrated to 15 ml, at which time 159 mg of a crystalline product was 
obtained upon cooling. m.p. 220.degree.-221.degree.. 
EXAMPLE 4 
Conjugation of 1-methyl-3-(3'-carboxypropyl)xanthine with bovine gamma 
globulin 
To a clear solution of 1-methyl-3-(3-carboxypropyl)xanthine (45 mg, 0.178 
mmole) in 1.5 ml DMF was added N-hydroxysuccinimide (20.5 mg, 0.178 mmole) 
and EDCI (39.1 mg, 0.20 mmole) at 0.degree. under nitrogen. After stirring 
the solution at 5.degree. for 18 hours, the reaction mixture was added to 
a solution of bovine gamma globulin (550 mg) in a mixture of 27 ml 
carbonate buffer (ph9, 0.05 M) and DMF at 0.degree. and the mixture 
maintained at this temperature for a period of 1.5 hours, while 
maintaining the pH at 8.5-9.0 using 1 N NaOH. After stirring the mixture 
overnight at 5.degree., the product was dialyzed against 10.times.4 l. 
water and 3.times.4 l. ammonium hydroxide. Lyophilization of the conjugate 
yielded 470 mg of protein. Employing a spectrophotometric technique, the 
hapten number was determined to be 15. 
EXAMPLE 5 
Preparation of 3-(2'-cyanoethyl)-1-methylxanthine 
Into a reaction flask was introduced 7-pivaloyloxymethyl-1-methylxanthine 
(100 mg) dissolved in 1.42 ml DMF, 44.17 mg sodium carbonate and 47 .mu.l 
acrylonitrile. After heating at 100.degree. for 16 hours, the mixture was 
poured into water and the aqueous mixture extracted with chloroform. The 
chloroform extracts were dried and evaporated and the oily residue 
chromatographed twice on silica gel plates, and the band eluted with 75 ml 
of 25% ethanol/chloroform (v/v) to give 109 mg of an oily product which 
solidified on standing. mp 118-120. 
The above nitrile (109 mg) was suspended in 0.35 ml 1 N sodium hydroxide 
and 0.35 ml of THF added. The mixture was stirred at room temperature for 
3.5 hours, followed by the addition of 0.2 ml of 1 N sodium hydroxide and 
stirring continued for an additional 0.5 hour. The mixture was then poured 
into about 2 ml water, the aqueous solution acidified and extracted 
exhaustively with chloroform. The chloroform extracts were washed with 8% 
sodium bicarbonate, followed by drying and evaporation to dryness. The 
residue was chromotographed on two silica gel plates (10% EtOH/HCCl.sub.3) 
and eluted with 125 ml 25% EtOH/HCCl.sub.3. Evaporation to dryness yield 
50 mg of the desired product. 
The subject nitrile can be readily modified to the imidoester and used for 
conjugation to amino groups present in poly(amino acids) to provide 
amidine linkages. 
EXAMPLE 6 
Conjugation of 3-carboxypropyl-1-methylxanthine to glucose-6-phosphate 
dehydrogenase 
A lyophilized powder of G6PDH was dissolved in 0.055 M tris-HCl, pH 8.1, to 
a protein concentration of about 2-3 mg/ml. The mixture was allowed to 
stand overnight at 4.degree.. 
Into a reaction flask was introduced 3-carboxypropyl-1-methylxanthine (36 
mg, 0.14 mmoles), 16.4 mg of N-hydroxy succinimide, 31.4 mg of ECDI and 
400 .mu.l of DMF and the mixture stirred overnight at 4.degree.. 
To 5 ml of the above G6PDH solution is added 50 mg glucose-6-phosphate 
disodium salt, 100 mg of NADH and 1.5 ml of carbitol and the pH adjusted 
to about 8.5-9 with 2 N sodium hydroxide. Almost the entire 400 .mu.l of 
the ester prepared above is added to the stirring enzyme solution in 10 
.mu.l increments over a 2 hour period while maintaining the solution at 
4.degree.. During the reaction the pH drops to 7.5-8. The resulting 
conjugate is then dialyzed at 4.degree. against 0.055 M tris HCl, pH 8.1, 
containing as preservatives 0.5% sodium azide and 0.005% thimerosal. 
In order to demonstrate the effectiveness of the subject compositions in an 
assay for theophylline, a number of assays were carried out. 
In carrying out the assay, a Gilford 300 N Microsample Spectrophotometer is 
employed, with a Thermo-cuvette with a flow cell. All readings are made at 
340 nm. The following solutions are prepared as reagents for use in the 
assay: 
TABLE I 
______________________________________ 
Buffer: 0.055M tris-HCl, pH 8.1 (RT) 
0.05% sodium azide 
0.005% Thimerosal 
Enzyme conjugate: 
Buffer 
0.9% NaCl 
1.0% RSA,* pH 8.1 (RT) 
sufficient enzyme conjugate (Ex. 1) 
to give a rate of .DELTA.OD in the 
range of 350-500 
in the assay medium. 
Assay Buffer: 
Buffer 
0.5% NaCl 
0.01% (v/v) Triton X-100, pH 8.1 (RT) 
Antibody Reagent: 
Buffer 
1.0% RSA 
1-methylxanthine 1.67.mu.g/ml 
G6P (Na) 0.066M 
NAD 0.04M 
pH 5 (RT) 
antitheophylline optimized for assay 
All % unless otherwise indicated 
are w/v (g/ml) 
______________________________________ 
*RSA-rabbit serum albumin 
The protocol employed for carrying out an assay is as follows. 50 .mu.l of 
the sample is drawn up into a diluter and dispensed with 250 .mu.l of the 
assay buffer into a 1 ml Croan cup. A 50 .mu.l aliquot of the diluted 
sample is drawn up and dispensed with a 250 .mu.l portion of assay buffer 
into a second Croan cup. Into the second Croan cup is introduced 50 .mu.l 
of the antibody reagent with 250 .mu.l of the assay buffer, followed by 
the addition of 50 .mu.l of the enzyme reagent and 250 .mu.l of the assay 
buffer. Immediately after the enzyme addition, the entire sample is 
aspirated into the flow cell. After 15 sec. a first reading is taken, 
followed by a second reading after a thirty second interval. The results 
are reported as the difference in absorbance.times.2.667. 
The following table indicates the results obtained with a number of samples 
having known amounts of theophylline. 
TABLE II 
______________________________________ 
Theophylline 
Conc. in Sample .DELTA.ODx 
.mu.g/ml 2.667 
______________________________________ 
0 163 
2.5 201 
5 221 
10 244 
20 267 
40 292 
______________________________________ 
By graphing the above results on semilog paper, one can then determine the 
concentration of theophylline in a sample suspected of containing 
theophylline by comparing the .DELTA.OD obtained with the unknown sample 
with the concentration-.DELTA.OD plot obtained with the above results. 
It is found that the cross-reactivity due to 1-methylxanthine can be 
effectively damped by the addition of a small amount of 1-methylxanthine 
to the antibody reagent, generally a sufficient amount to provide from 
about 1 to 20, preferably 2 to 15 .mu.g/ml in the assay medium. In this 
manner, false positives can be avoided, where 1-methylxanthine is present 
to any extent in the serum sample to be assayed. 
A cross-reactivity study was made with a wide variety of similarily 
structured compounds. The subject assay must be able to distinguish 
between ubiquitous compounds of analogous structures to theophylline, such 
as caffeine and its metabolites, in order to insure an accurate result. 
Compounds studied for cross-reactivity were caffeine, 1,3-dimethyluric 
acid, theobromine, 1-methylxanthine and 3-methylxanthine. When samples 
were spiked with 100 .mu.g/ml of these compounds, with the one exception 
of 1-methylxanthine, the observed value was below the value obtained with 
2.5 .mu.g/ml of theophylline. The one exception was 1-methylxanthine where 
100 .mu.g/ml of 1-methylxanthine is equivalent to 5-10 .mu.g/ml 
concentration of theophylline. However, as indicated above, when a small 
amount of 1-methylxanthine is employed in the assay medium, substantially 
no effect is seen by the addition of further 1-methylxanthine. 
It is evident from the above results, that the subject invention provides a 
sensitive assay for measuring theophylline in physiological fluids, 
particularly serum. The assay protocol provides for distinguishing 
theophylline at extremely low concentrations from compounds having 
extremely similar structures. Furthermore, the protocol is simple, fast, 
and is readily carried out on a conventional spectrophotometer. 
Although the foregoing invention has been described in some detail by way 
of illustration and example for purposes of clarity of understanding, it 
will be obvious that certain changes and modifications may be practiced 
within the scope of the appended claims.