An immunochemical method and reagent compounds for determining ligands in a sample. The reagent compounds are derivatives of a triazinylaminofluorescein and are represented by the structural formula ##STR1## wherein Y is halo or lower alkyl; and PA1 R is a ligand-analog wherein said ligand-analog has at least one common epitope with said ligand so as to be specifically recognizable by a common antibody. The reagent compound and an antibody specific to the ligand are added to the sample in a fluorescence polarization immunoassay.

BACKGROUND OF THE INVENTION 
The present invention relates to a method and reagents for determining 
ligands in biological fluids such as serum, plasma, spinal fluid, amnionic 
fluid and urine. In particular, the present invention relates to a 
fluorescent polarization immunoassay procedure and to tracers employed as 
reagents in such procedures. The fluorescent polarization immunoassay 
procedure of the present invention combines the specificity of an 
immunoassay with the speed and convenience of fluorescent polarization 
techniques to provide a means for determining the amount of a specific 
ligand present in a sample. 
Competitive binding immunoassays for measuring ligands are based on the 
competition between a ligand in a test sample and a labeled reagent, 
referred to as a tracer, for a limited number of receptor binding sites on 
antibodies specific to the ligand and tracer. The concentration of ligand 
in the sample determines the amount of tracer that will specifically bind 
to an antibody. The amount of tracer-antibody conjugate produced may be 
quantitatively measured and is inversely proportional to the quantity of 
ligand in the test sample. 
In general, fluorescent polarization techniques are based on the principle 
that a fluorescent labeled compound when excited by linearly polarized 
light will emit fluorescence having a degree of polarization inversely 
related to its rate of rotation. Therefore, when a molecule, such as a 
tracer-antibody conjugate having a fluorescent label is excited with 
linearly polarized light, the emitted light remains highly polarized 
because the fluorophore is constrained from rotating between the time 
light is absorbed and emitted. When a "free" tracer compound (i.e., 
unbound to an antibody) is excited by linearly polarized light, its 
rotation is much faster than the corresponding tracer-antibody conjugate 
and the molecules are more randomly oriented, therefore, the emitted light 
is depolarized. Thus, fluorescent polarization provides a quantitive means 
for measuring the amount of tracer-antibody conjugate produced in a 
competitive binding immunoassay. 
Various fluorescent labeled compounds are known in the art. U.S. Pat. No. 
3,998,943 describes the preparation of a fluorescently labeled insulin 
derivative using fluorescein isothiocyanate (FITC) as the fluorescent 
label and a fluorescently labeled morphine derivative using 
4-aminofluorescein hydrochloride as the fluorescent label. Blakeslee, et 
al, in The Journal of Immunological Methods, 13, 305-320 (1976) described 
the 5-[(4,6-dichlorotriazin-2-yl)-amino]fluorescein derivatives of 
immunoglobulins (IgG) having molecular weights of at least 160,000. 
Blakeslee fails to teach or suggest the use of the various fluorescein 
derivatives in conjunction with fluorescent polarization immunoassay 
techniques. 
SUMMARY OF THE INVENTION 
The present invention encompasses a method for determining ligands in a 
sample comprising intermixing with said sample a biologically acceptable 
salt of a tracer of the formula: 
##STR2## 
wherein Y is halo or lower alkyl; and 
R is a ligand-analog wherein said ligand-analog has at least one common 
epitope with said ligand so as to be specifically reconizable by a common 
antibody; 
and an antibody capable of specifically recognizing said ligand and said 
tracer; and then determining the amount of tracer-antibody conjugate by 
fluorescence polarization techniques as a measure of the concentration of 
said ligand in the sample. 
The invention further relates to a novel class of tracers of formula (I) 
and biologically acceptable salts thereof, which are useful in reagents in 
the above-described method. The methods and tracers of the present 
invention are particularly useful in quantitatively monitoring therapeutic 
drug concentrations in serum and plasma. 
DETAILED DESCRIPTION OF THE INVENTION 
The term "ligand" as used herein refers to a molecule in particular a low 
molecular weight hapten, to which a receptor, normally an antibody, can be 
obtained or formed. Haptens are protein-free bodies, generally of low 
molecular weight that do not induce antibody formation when injected into 
an animal, but are reactive to antibodies. Antibodies to hapten are 
generally raised by first conjugating the haptens to a protein and 
injecting the conjugate product into an animal. The resulting antibodies 
are isolated by conventional antibody isolation techniques. 
The ligands determinable by the method of the present invention vary over a 
wide molecular weight range. Although high molecular weight ligands may be 
determined, for best results, it is generally preferable to employ the 
methods of the present invention to determine ligands of low molecular 
weight, generally in a range of 50 to 4000. It is more preferred to 
determine ligands having a molecular weight in a range of 100 to 2000. 
The novel tracer of the present invention includes compounds of formula (I) 
wherein the ligand-analogs represented by R include radicals having a 
molecular weight within a range of 50 to 4000. The preferred novel tracers 
include compounds of formula (I) wherein the ligand-analogs represented by 
R include radicals having a molecular weight within a range of 100 to 
2000. 
Representative of ligands determinable by the methods of the present 
invention include steroids such as estrone, estradiol, cortisol, 
testosterone, progesterone, chenodeoxycholic acid, digoxin, cholic acid, 
digitoxin, deoxycholic acid, lithocholic acids and the ester and amide 
derivatives thereof; vitamins such as B-12, folic acid, thyroxine, 
triiodothyronine, histamine, serotorin, prostaglandins such as PGE, PGF, 
PGA; antiasthamatic drugs such as theophylline, antineoplastic drugs such 
as doxorubicin and methotrexate; antiarrhythmic drugs such as 
disopyramide, lidocaine, procainamide, propranolol, quinidine, 
N-acetylprocainamide; anticonvulsant drugs such as phenobarbital, 
phenytoin, primidone, valproic acid, carbamazepine and ethosuximide; 
antibiotics such as penicillins, cephalosporins, erythromycin, vancomycin, 
gentamicin, amikacin, chloramphenicol, streptomycin and tobramycin; 
antiarthritic drugs such as salicylate; antidepressant drugs including 
tricyclics such as nortriphyline, amitriptyline, imipramine and 
desipramine; and the like as well as the metabolites thereof. 
Additional ligands that may be determined by the methods of the present 
invention include drugs of abuse such as morphine, heroin, hydromophone, 
oxymorphone, metapon, codeine, hydrocodone, dihydrocodiene, dihydrohydroxy 
codeinone, pholcodine, dextromethorphan, phenazocine and deonin and their 
metabolites. 
The tracers of the present invention generally exist in an equilibrium 
between their acid and ionized states, and in the ionized state are 
effective in the method of the present invention. Therefore, the present 
invention comprises the tracers in either the acid or ionized state and 
for convenience, the tracers of the present invention are structurally 
represented herein in their acid form. When the tracers of the present 
invention are present in their ionized state, the tracers exist in the 
form of biologically acceptable salts. As used herein, the term 
"biologically acceptable salts" refers to salts such as sodium, potassium, 
ammonium and the like which will enable the tracers of the present 
invention to exist in their ionized state when employed in the method of 
the present invention. Generally, the tracers of the present invention 
exist in solution as salts, the specific salt results from the buffer 
employed, i.e., in the presence of a sodium phosphate buffer, the tracers 
of the present invention will generally exist in their ionized state as a 
sodium salt. 
The tracers of the present invention comprise a ligand-analog represented 
by R linked to a triazinylaminofluorescein moiety of the formula: 
##STR3## 
wherein Y is above defined. Representation of the "lower alkyl" groups 
represented by Y include alkyl radicals having from 1 to 4 carbon atoms, 
such as, for example, methyl, ethyl, propyl, isopropyl, n-butyl, 
sec-butyl, t-butyl, and the like. Illustrative of the "halo" groups 
represented by Y include fluoro, chloro, iodo and bromo. It is preferred 
that Y is chloro or bromo and most preferred that Y is chloro. 
The term ligand-analog as used herein refers to a mono- or polyvalent 
radical a substantial proportion of which has the same spatial and polar 
organization as the ligand to define one or more determinant or epitopic 
sites capable of competing with the ligand for the binding sites of a 
receptor. A characteristic of such ligand-analog is that it possesses 
sufficient structural similarity to the ligand of interest so as to be 
recognized by the antibody for the ligand. For the most part, the ligand 
analog will have the same or substantially the same structure and charge 
distribution (spatial and polar organization) as the ligand of interest 
for a significant portion of the molecular surface. Since frequently, the 
linking site for a hapten will be same in preparing the antigen for 
production of antibodies as used for linking to the ligand, the same 
portion of the ligand analog which provides the template for the antibody 
will be exposed by the ligand analog in the tracer. 
In general, the class of ligand analogs represented by R are derived from 
the corresponding ligand by removal of a reactive hydrogen atom, i.e., a 
hydrogen atom bonded to a hydroxy oxygen or a reactive amine (primary or 
secondary) or by the formation of an amino derivative of the ligand 
wherein an imino group 
##STR4## 
replaces one or more atoms originally present in the ligand, at the site 
of binding to the triazinylaminofluorescein moiety. Illustrative of 
ligands which upon the removal of a reactive hydrogen may form a 
ligand-analogs represented by R include for example, procainamide, 
thyroxine, quinidine and the aminoglycoside antibiotics. Illustrative of 
ligands whose amino derivatives are useful as ligand-analog include 
theophylline, valproic acid, phenobarbital, phenytoin, primidone, 
disopyramide, digoxin, chloramphenicol, salicylate, acetaminophen, 
carbamazepine, desimpramine and nortriptyline. In addition, a ligand may 
be structurally modified by the addition or deletion of one or more 
functional groups to form a ligand-analog, while retaining the necessary 
epitope sites for binding to an antibody. However, it is preferred that 
such modified ligand-analogs be bonded to the triazinylaminofluorescein 
moiety through an imino or oxy group. 
The tracers of the present invention are generally prepared in accordance 
with the following procedure: 
EQU R-X (III) 
wherein R is above-defined and X is a reactive hydrogen; is reacted with a 
compound of the formula: 
##STR5## 
wherein Z is halo and Y is above-defined, and wherein the amino group is 
bonded to the 4 or 5 position of the benzoic acid ring; under basic 
conditions in the presence of an intert solvent to yield a compound of 
formula (I). 
It should be noted that if a compound of formula (III) has more than one 
reactive hydrogen, a mixture of products of formula (I) may result upon 
reaction with a triazinylaminofluorescein moiety. For example, when the 
ligand of interest is an antibiotic such as, for example, an 
aminoglycoside, the corresponding ligand-analog is generally derived from 
a ligand having multiple reactive amine hydrogens. Replacing any of such 
reactive amine hydrogens with a triazinylaminofluorescein moiety will 
produce a tracer of formula (I). Therefore, the reaction product resulting 
from the reaction of an antiobiotic such as an aminoglycoside, with 
triazinylaminofluorescein will generally be a mixture of products 
represented by formula (I). All of these reaction products, individually 
or in combination are effective as tracers in a fluorescent polarization 
immunoassay technique. 
The temperature at which the reaction for preparing the tracers of this 
invention proceeds is not critical. The temperature should be one which is 
sufficient so as to initiate and maintain the reaction. Generally, for 
convenience and economy, room temperature is sufficient. In preparing the 
tracers of the present invention, the ratio of reactants is not narrowly 
cirtical. For each mole of a compound of formula (II), one should employ 
one more of a compound of formula (III) to obtain a reasonably yield. It 
is preferred to employ an excess of compound of formula (III) for ease of 
reaction and recovery of the reaction products. 
The compounds of formula (IV) employed as starting materials in the 
production of the tracers of this invention are prepared in accordance 
with the method described by Blakeslee, et al. (supra). It should be noted 
that two isomers of compound (IV) generally exist. Isomer I is prepared 
from 5-aminofluorescein and Isomer II is prepared from 4-aminofluorescein. 
It is preferred to employ Isomer I or Isomer II and mixtures thereof as 
starting materials in the preparation of the compounds of the present 
invention. 
For ease of handling and recovery of product, the process for preparing the 
tracers of the present invention is conducted in the presence of an inert 
solvent. Suitable inert solvents include those solvents which do not react 
with the starting materials and are sufficient to dissolve the starting 
materials, and include for example water, methanol, dimethylformamide, 
dimethylsulfoxide and the like. In order to provide maximum product 
yields, the reaction preferably proceeds under neutral or basic 
conditions. If the compound of formula (III) is a reactive amine salt, a 
suitable base is added to the reaction mixture to form the free base of 
the reactive amine. Suitable bases include for example triethylamine. The 
reaction products of formula (I) are generally purified using either 
thin-layer or column chromatography prior to application in the methods of 
the present invention. 
In accordance with the method of the present invention, a sample containing 
the ligand to be determined is intermixed with a biologically acceptable 
salt of a tracer of formula (I) and an antibody specific for the ligand 
and tracer. The ligand present in the sample and the tracer compete for 
limiting antibody sites resulting in the formation of ligand-antibody and 
tracer-antibody complexes. By maintaining constant the concentration of 
tracer and antibody, the ratio of ligand-antibody complex to 
tracer-antibody complex that is formed is directly proportional to the 
amount of ligand present in the sample. Therefore, upon exciting the 
mixture with fluorescent light and measuring the polarization of the 
fluorescence emitted by a tracer and a tracer-antibody complex, one is 
able to quantitatively determine the amount of ligand in the sample. 
In theory, the fluorescence polarization of a tracer not complexed to an 
antibody is low, approaching zero. Upon complexing with a specific 
antibody, the tracer-antibody complex thus formed assumes the rotation of 
the antibody molecule which is slower than that of the relatively small 
tracer molecule, thereby increasing the polarization observed. Therefore, 
when a ligand competes with the tracer for antibody sites, the observed 
polarization of fluorescence of the tracer-antibody complex becomes a 
value somewhere between that of the tracer and tracer-antibody complex. If 
a sample contains a high concentration of a ligand, the observed 
polarization value is closer to that of the free ligand, i.e., low. If the 
test sample contains a low concentration of the ligand, the polarization 
value is closer to that of the bound ligand, i.e., high. By sequentially 
exciting the reaction mixture of an immunoassay with vertically and then 
horizontally polarized light and analyzing only the vertical component of 
the emitted light, the polarization of fluorescence in the reaction mix 
may be accurately determined. The precise relationship between 
polarization and concentration of the ligand to be determined is 
established by measuring the polarization values of calibrators with known 
concentrations. The concentration of the ligand can be extrapolated from a 
standard curve prepared in this manner. 
The pH at which the method of the present invention is practiced must be 
sufficient to allow the tracers of formula (I) to exist in their ionized 
state. The pH may range from about 3 to 12, more usually in the range of 
from about 5 to 10, most preferably from about 6 to 9. Various buffers may 
be used to achieve and maintain the pH during the assay procedure. 
Representative buffers include borate, phosphate, carbonate, tris, 
barbital and the like. The particular buffer employed is not critical to 
the present invention, but in an individual assay, a specific buffer may 
be preferred in view of the antibody employed and ligand to be determined. 
The cation portion of the buffer will generally determine the cation 
portion of the tracer salt in solution. 
The methods of the present invention are practical at moderate temperatures 
and preferably at a constant temperature. The temperature will normally 
range from about 0.degree. to 50.degree. C., more usually from about 
15.degree. to 40.degree. C. 
The concentration of ligand which may be assayed will generally vary from 
about 10.sup.-2 to 10.sup.-13 M, more usually from about 10.sup.-4 to 
10.sup.-10 M. Higher concentrations of ligand may be assayed upon dilution 
of the original sample. 
In addition to the concentration range of ligand of interest, 
considerations such as whether the assay is qualitative, semiquantitative 
or quantitative, the equipment employed, and the characteristics of the 
tracer and antibody will normally determine the concentration of the 
tracer and antibody to be employed. While the concentration of ligand in 
the sample will determine the range of concentration of the other 
reagents, i.e., tracer and antibody, normally to optimize the sensitivity 
of the assay, individual reagent concentrations will be determined 
empirically. Concentrations of the tracer and antibody are readily 
ascertained by one of ordinary skill in the art. 
As previously mentioned the preferred tracers of the present invention are 
prepared from 5-[(4,6-dichlorotriazin-2-yl)-amino]fluorescein or 
4-[(4,6-dichlorotriazin-2-yl)-amino]fluorescein and exist preferably as 
isomers of the formula: 
##STR6## 
The following illustrative, nonlimiting examples will serve to further 
demonstrate to those skilled in the art the manner in which specific 
tracers within the scope of this invention may be prepared. The symbol 
[DTAF] appearing in the structural formulas illustrating the compounds 
prepared in the following examples, represents a moiety of the formula: 
##STR7## 
wherein the imino nitrogen is attached to the 4 or 5 position in the above 
formula depending on the specific triazinylfluorescein isomer employed as 
the starting material.

EXAMPLE I 
Gentamicin sulfate (200 mg) was dissolved in 1 ml of distilled water and 
the resulting solution was adjusted to pH 9.0 using approximately 0.8 ml 
of 1.0 M sodium hydroxide. 5-[(4,6-dichlorotriazin-2-yl)-amino]fluorescein 
(20 mg) was dissolved in 1.5 ml of methanol and the resulting methanol 
solution was added dropwise to the gentamicin solution with stirring. The 
reaction mixture was allowed to react for one hour. The resultant mixture 
was chormatographed on a DEAE cellulose medium mesh column using 0.1 M 
phosphate buffer at pH 8.0 as the eluent to yield a gentamicin-DTAF 
conjugate. 
EXAMPLE II 
Tobramycin (250 mg) was dissolved in 2 ml of 0.1 M carbonate buffer (pH 
9.0). 5-[(4,6-dichlorotriazin-2-yl)-amino]fluorescein (20 mg) was 
dissolved in 1 ml of methanol and the resulting methanol solution was 
added to 1 ml of the tobramycin solution. After approximately five 
minutes, the reaction mixture was purified by chormatography on a DEAE 
cellulose column using 0.1 M phosphate buffer at pH 8.0 as the eluent to 
yield a tobramycin-DTAF conjugate. 
EXAMPLE III 
Amikacin (9.24 mg) was dissolved in 0.2 ml of water. A suspension 
containing 4.5 mg of dichlorotriazinylaminofluorescein in 0.2 ml of 
methanol was added to the amikacin solution with stirring. The small 
particles of 5-[(4,6-dichlorotriazin-2-yl)-amino]fluorescein rapidly 
dissolved, the reaction mixture was chromatographed on a 17 ml column of 
DEAE cellulose using pH 8.1 phosphate buffer, 0.1 M as the eluent to yield 
an amikacin-DTAF conjugate. 
EXAMPLE IV 
Streptomycin sulfate (200 mg) was dissolved in 15 ml of water and the 
resulting solution was adjusted to pH 10.5 using 1 N sodium hydroxide. To 
the streptomycin solution was dropwise added with stirring, 20 mg of 
5-[(4,6-dichlorotriazin-2-yl)-amino]fluorescein dissolved in 1.5 ml of 
dimethylsulfoxide. 4 ml of the reaction mixture was chromatographed on a 
DEAE cellulose column using a 0.1 M phosphate buffer (pH 8.0) as the 
eluent to yield a streptomycin-DTAF conjugate. 
EXAMPLE V 
Neomycin sulfate (200 mg) was dissolved in 3 ml of water and the resulting 
solution was adjusted to pH 9.0 using 6 N sodium hydroxide. To the 
neomycin solution was dropwise added with stirring 20 mg of 
5-[(4,6-dichlorotriazin-2-yl)-amino]fluorescein dissolved in 1.5 ml of 
dimethylsulfoxide. 2 ml of the reaction mixture was chromatographed on a 
DEAE cellulose column using 0.1 M phosphate buffer (pH 8.0) as the eluent 
to yield a neomycin-DTAF conjugate. 
EXAMPLE VI 
Vancomycin hydrochloride (100 mg) was dissolved in 100 ml of water and the 
resulting solution was adjusted to pH 9.1 using 1 N sodium hydroxide. The 
vancomycin solution was then adjusted to pH 7 using 1 N HCl after which 
time 20 mg of 5-[(4,6-dichlorotriazin-2-yl)-amino]fluorescein in 2 ml of 
dimethylsulfoxide was added with stirring. A crude product formed which 
was purified using silica gel thin-layer chromatography techniques 
employing a developing solvent comprising a chloroform-methanol-water 
(4:4:1) mixture to yield a vancomycin-DTAF conjugate. 
EXAMPLE VII 
To 1.79 g of para-acetamidobenzoic acid and 1.15 g of N-hydroxysuccinimide 
dissolved in 15 ml of pyridine was added 2.3 g of 
N,N'-dicyclohexylcarbodiimide. The reaction mixture was cooled at 4 
C..degree. for two hours and then filtered to remove crystals which had 
formed. The crystals were washed with approximately 2 ml of acetone and 
the pyridine filtrate and acetone washings were then combined. To the 
combined mixture was added 0.88 g of N-ethylethylenediamine. The resulting 
mixture was stirred for two hours and then cooled at 4 C..degree. for 
about twenty-four hours to yield a second crop of crystals. The crystals 
were removed from the mixture by filtration and then rinsed with acetone. 
The two crops of crystals (approximately 2.0 g) were combined and then 
dissolved in 50 ml of distilled water. The pH of the resulting mixture was 
adjusted to pH 10 using a 6 N sodium hydroxide solution. A white 
precipitate, desethyl-N-acetylprocainamide formed and was removed by 
filtration and dried in a dessicator. To 10 mg of the 
desethyl-N-acetyl-procainamide was added 10 mg of 
4-[(4,6-dichlorotriazin-2-yl)-amino]fluorescein dissolved in 1 ml of 
methanol. The mixture was allowed to react for ten minutes, and the crude 
product which had formed was purified using silica gel thin-layer 
chromatographic techniques employing a developing solvent comprising a 1:1 
mixture of chloroform:acetone to yield a 
desethyl-N-acetyl-procainamide-DTAF conjugate of the formula: 
##STR8## 
EXAMPLE VIII 
The procedure of Example IV was employed using 0.9 g of ethylenediamine in 
lieu of N-ethylethylenediamine. The reaction mixture was stirred for one 
hour, and cooled for 1.5 hours. Methanol was used in lieu of a 1:1 mixture 
of chloroform:acetone as the developing solvent in the purification of the 
crude product to yield an N-p-acetamidobenzoyl ethylene diamine-DTAF 
conjugate of the formula: 
##STR9## 
EXAMPLE IX 
A mixture containing desethyl-N-acetyl procainamide (1.25 g) (prepared as 
in Example VII and 0.8 g of chloroacetyl chloride dissolved in 25 ml of 
acetone was refluxed for two hours. The reaction mixture was filtered and 
the filtrate evaporated to yield a yellow residue. The yellow residue and 
0.75 g of sodium iodide were dissolved in 20 ml of acetone and refluxed 
for one hour. The resulting mixture was filtered and the filtrate 
evaporated to dryness to yield a red residue which was then dissolved in 
20 ml of methanol. To the methanol solution was added 20 ml of 
concentrated ammonium hydroxide and the resulting mixture was refluxed for 
1.5 hours. The mixture was cooled and then extracted twice with 20 ml of 
chloroform. The combined extracts were dried over sodium sulfate, filtered 
and evaporated to yield 
N-p-acetamidobenzoyl-N'-ethyl-N'-aminoacetylethylene diamine. To 10 mg of 
N-p-acetamidobenzoyl-N'-ethyl-N'-aminoacetylethylene diamine was added 10 
mg of 5-[(4,6-dichlorotriazin-2-yl)-amino]fluorescein dissolved in 1 ml of 
methanol and the reaction was allowed to proceed for ten minutes until a 
crude product had formed. The crude product was purified using silica gel 
thin-layer chromatographic techniques employing a developing solvent 
comprising a 1:1 mixture of chloroform:acetone to yield an 
N-P-acetamidobenzoyl-N'-ethyl-N'-aminoacetylethylene diamine-DTAF 
conjugate of the formula: 
##STR10## 
EXAMPLE X 
To 1.1 g of primidone dissolved in 10 ml of concentrated sulfuric acid was 
slowly added a solution containing 1 ml of concentrated nitric acid and 2 
ml of concentrated sulfuric acid. The reaction mixture was shaken at room 
temperature for forty-five minutes. The reaction mixture was then poured 
over 50 ml ice and crystals of para-nitroprimidone that had formed were 
filtered and then rinsed with water. The crystals (1.17 g, having a 
melting point 225.degree.-228.degree. C.) were dissolved in 200 ml of hot 
ethanol. To the ethanol solution was added 1.5 g of iron powder and 100 ml 
of water. The resultant mixture was heated to boiling, and then 2 ml of 
concentrated hydrochloric acid was added. The resultant mixture was 
refluxed for two hours and the hot mixture was filtered and the filtrate 
to yield 0.8 g of brown hydroscopic crystals. 5 mg of 
5-[(4,6-dichlorotriazin-2-yl)amino]fluorescein and 5 mg of the brown 
crystals were dissolved in 0.5 ml of methanol. The reaction was complete 
in 10 minutes to yield a crude product which was purified by silica gel 
thin-layer chromatography using 3:1 mixture of chloroform:methanol as the 
developing solvent. The product was further purified using thin-layer 
chromatography employing a 2:1 mixture of chloroform:methanol to yield an 
amino-primidone-DTAF conjugate of the formula: 
##STR11## 
EXAMPLE XI 
To delta-valerolactam (10 g) dissolved in 80 ml of dry tetrahydrofuran, 
under a dry nitrogen atmosphere was deopwise added n-butyllithium (1.6 M, 
125 ml) in hexane while the reaction mixture was chilled in a dry 
ice-acetone bath. After all the n-butyllithium was added, the reaction 
mixture was stirred at room temperature for one hour, refluxed for thirty 
minutes, and cooled to room temperature. 1-Bromopropane (12.3 g) was 
slowly added to the reaction mixture while the mixture was chilled in an 
ice bath. The reaction mixture was then stirred for sixteen hours at room 
temperature. 100 ml of water was added slowly to the reaction mixture and 
the resulting mixture was stirred at room temperature for thirty minutes. 
The layers separated and the aqueous layers were combined, dried over 
sodium sulfate, and then evaporated to yield 12.4 g (88% yield) of a dark, 
heavy oil, which crystallized on standing. The crystals were 
recrystallized from petroleum ether to yield 5.4 g of 
.alpha.-propylvalerolactam (m.p. 75.degree.-76.degree. C.). A portion of 
the .alpha.-propylvalerolactam (2.8 g) was refluxed in 25 ml of 6 N 
hydrochloric acid under a nitrogen atmosphere for six hours. The water was 
evaporated to yield 2-propyl-5-amino-pentanoic acid (2.0 g; 51% yield) and 
recrystalized from ethanol-petroleum ether to yield a solid (m.p. 
71.degree.-73.degree. C.). Equimolar amounts of 2-propyl-5-aminopentanoic 
acid and 5-[(4,6-dichlorotriazin-2-yl)-amino]fluorescein were dissolved in 
methanol. The reaction was completed in about ten minutes to yield a crude 
product which was purified by silica gel thin-layer chromatography with 
chloroform/methanol (3:1) as the developing solvent to yield a 
2-propyl-5-amino-pentanoic acid-DTAF conjugate of the formula: 
##STR12## 
EXAMPLE XII 
The procedure of Example VIII was employed utilizing 
2-ethyl-5-aminopentanoic acid in lieu of 2-propyl-5-amino-pentanoic acid 
to yield a 2-ethyl-5-amino-pentanoic acid-DTAF conjugate of the formula: 
##STR13## 
EXAMPLE XIII 
To a mixture containing 5 mg of 
5-[(4,6-dichlorotriazin-2-yl)-amino]fluorescein dissolved in methanol and 
5 mg of D-thyroxine was dropwise added dimethylsulfoxide until a clear 
solution was formed. Two drops of triethylamine was added to the reaction 
mixture and the reaction was allowed to proceed for 16 hours. A crude 
product has formed which was then purified by silica gel thin-layer 
chromatography using a 3:1 mixture of chloroform:methanol as the 
developing solvent to yield a 3,3',5,5'-tetraiodo-D-thryonine-DTAF 
conjugate of the formula: 
##STR14## 
EXAMPLE XIV 
The procedure of Example XIII was employed utilizing L-thyroxine in lieu of 
D-thyroxine to yield 3,3',5,5'-tetraiodo-L-thyronine-DTAF conjugate which 
is an optical isomer of the conjugate formed in Example XIII. 
EXAMPLE XV 
The procedure of Example XIII was employed utilizing 
3,3',5-triiodo-L-thyronine in lieu of D-thyroxine to yield a 
3,3',5-triiodo-L-thyronine-DTAF conjugate of the formula: 
##STR15## 
EXAMPLE XVI 
A solution containing ammonium acetate (8.0 g), sodium cyanoborohydride 
(630 mg) and 10,11-dihydro-5H-dibenzo [a,d] cyclohepten-5-one (2.1 g) 
dissolved in 50 ml of methanol was refluxed for twenty four hours and then 
evaporated to dryness to yield a tan residue. The residue was dissolved in 
25 ml of 2 N hydrochloric acid and extracted twice with 25 ml 
dichloromethane. 6 N sodium hydroxide was added to the aqueous phase until 
the pH of the solution was 14. A brown oil then began to form and the 
solution was chilled in a freezer for 16 hours. All water in the mixture 
was evaporated and the residue was taken up in methanol and filtered. The 
filtrate was evaporated to yield a white residue. 10 mg of 
5-[(4,6-dichlorotriazin-2-yl)-amino]fluorescein and 10 mg of the white 
residue were dissolved in 1 ml of methanol to yield a crude product which 
was purified by silica gel thin-layer chromatography using a 1:1 mixture 
of chloroform:acetone as a developing solvent to yield a 
5-amino-10,11-dihydro-5H-dibenzo [a,d]-cycloheptene-DTAF conjugate of the 
formula: 
##STR16## 
EXAMPLE XVII 
A mixture containing 10,11-dihydro-5H-dibenzo [a,d]cyclohepten-5-one (10 g) 
and dimethylhydrazine (18%) were refluxed for twenty-four hours in 100% 
ethanol. To the mixture was added 100 ml of distilled water and a yellow 
solution was extracted with diethyl ether until the extracts were 
colorless. The combined ether extracts were washed with 25 ml of 2 N 
hydrochloric acid. The organic phase was then dried over sodium sulfate 
and evaporated to yield dibenzosuberone dimethyl hydrazone as a thick 
orange oil. This oil (2.0 g) was refluxed for twelve hours in a solution 
containing 3 g of hydrazine in 10 ml of 100% ethanol. The reaction mixture 
was poured over 10 ml of ice water then extracted twice with 25 ml of 
diethyl ether. The combined ether extracts were dried over sodium sulfate 
and evaporated to dryness to yield dibenzosuberone hydrazone as a yellow 
oil. 10 mg of 5-[(4,6-dichlorotriazin-2-yl)-amino]fluorescein and 10 mg of 
dibenzosuberone hydrazone in 1 ml of methanol were allowed to react for 
10 minutes to yield a crude product which was purified using silica gel 
thin-layer chromatography using a 3:1 mixture of chloroform:methanol as a 
developing solvent to yield a dibenzosuberone hydrazone-DTAF conjugate of 
the formula: 
##STR17## 
EXAMPLE XVIII 
5-(.gamma.-Bromopropylidene)-5H-dibenzo [a,d]-10,11-dihydro cyclohepten and 
its precursor 5-cyclopropyl-5-hydroxy-5H-dibenzo 
[a,d]-10,11-dihydrocycloheptene were prepared by the procedure described 
in The Journal of Organic Chemistry, Vol. 27, pages 4134-4137 (1962) by R. 
D. Hoffsomer, D. Taub, and N. L. Wendler. Procedure (b) for preparation of 
end product, 5-(.gamma.-aminopropylidene)-5H-dibenzo 
[a,d]-10,11-dihydrocycloheptene, was employed substituting the 
bromopropylidene compound for the chloropropylidene compound. 10 mg of 
5-[(4,6-dichlorotriazin-2-yl)-amino]fluorescein and 10 mg of the end 
product were dissolved in 1 ml of methanol. An excess amount of 
triethylamine was added and the reaction was completed in thirty minutes 
to yield a crude product which was purified by silica gel thin-layer 
chromatography using a 2:1 mixture of chloroform:methanol as a developing 
solvent to yield a 
5-(.gamma.-aminopropylidene)-5-H-dibenzo[a,d]-10,11-dihydrocycloheptene-DT 
AF conjugate of the formula: 
##STR18## 
EXAMPLE XIX 
To a mixture containing 6.0 g of iminodibenzyl in 30 ml of chloroform was 
added 6 ml of chloroacetly chloride and the resultant mixture was refluxed 
for forty-five minutes. To the reaction mixture was added 60 ml of water 
and the resultant mixture was stirred for thirty minutes at room 
temperature. The chloroform layer was separated and dried over sodium 
sulfate and evaporated to dryness to yield a residue. The residue was 
dissolved in 25 ml of acetone. A solution containing 4.5 g sodium iodide 
dissolved in 25 ml of acetone was added to the acetone solution and the 
resultant mixture was refluxed for thirty minutes. To the reaction mixture 
was added 100 ml of water and the reaction mixture was extracted twice 
with 50 ml of chloroform and evaporated to dryness to yield a residue 
which was then dissolved in 40 ml of methanol. To the methanol solution 
was added 60 ml of concentrated ammonium hydroxide and the resultant 
mixture was refluxed for one hour. The reaction mixture was evaporated to 
dryness and the residue was taken up in 100 ml of chloroform and washed 
twice with 30 ml of water. The chloroform layer was dried over sodium 
sulfate and evaporated to dryness to yield 4.5 g of an amine product. A 
mixture containing 5 mg of 5-[(4,6-dichlorotriazin-2-yl)-amino]fluorescein 
and 5 mg of the amine dissolved in 0.5 ml of methanol were allowed to 
react for 10 minutes to yield a crude product which was purified as in 
Example XVI to yield a N-aminoacetyliminostibene-DTAF conjugate of the 
formula: 
##STR19## 
EXAMPLE XX 
A mixture containing desipramine hydrochloride (1.33 g) and chloroacetyl 
chloride (0.8 g) dissolved in 25 ml chloroform was refluxed for two hours. 
The chloroform was evaporated to yield a residue which was dissolved in 25 
ml of acetone. Sodium iodide (0.75 g) was added to the acetone solution 
and the resultant solution was refluxed for thirty minutes and then 
filtered. The precipitated salt was rinsed with acetone and the acetone 
filtrate was evaporated and the residue was taken up in 20 ml of methanol. 
To the methanol solution was added 20 ml of concentrated ammonium 
hydroxide and the resultant solution was refluxed for one hour. The 
reaction mixture was extracted three times with 25 ml of chloroform and 
combined extracts were dried over sodium sulfate, filtered and evaporated. 
5 mg of 5-[(4,6-dichlorotriazin-2-yl)-amino]fluorescein and 5 mg of the 
amine were dissolved in 0.5 ml of methanol. About five drops of 
dimethylsulfoxide were added to the reaction mixture to dissolve the 
precipitate. The reaction was completed in ten minutes and yielded a crude 
product which was purified in accordance with the procedure of Example XVI 
using a 3:1 mixture of chloroform:methanol as the developing solvent to 
yield a N-aminoacetyldesipramine-DTAF conjugate of the formula: 
##STR20## 
EXAMPLE XXI 
5-[(4,6-dichlorotriazin-2-yl)-amino]fluorescein (10 mg) and 
8-aminomethyl-theophylline (5 mg) were dissolved in 0.5 ml of 
dimethylsulfoxide. After five minutes the reaction was complete and 
yielded a crude product which was purified by silica gel thin-layer 
chromatography using a 1:1 mixture of chloroform:acetone as the developing 
solvent to yield an 8-aminomethyl-theophylline-DTAF conjugate of the 
formula: 
##STR21## 
EXAMPLE XXII 
The procedure of Example XXI was employed utilizing 
8-aminoethyl-theophylline in lieu of 8-aminomethyl-theophylline to yield 
an 8-aminoethyl-theophylline-DTAF conjugate of the formula: 
##STR22## 
EXAMPLE XXIII 
5-[(4,6-dichlorotriazin-2-yl)-amino]fluorescein (5 mg) and anhydrous 
quinidine (5 mg) were dissolved in 0.5 ml of dimethylformamide. After 
sixteen hours the reaction was complete and yielded a crude product which 
was purified by silica gel thin-layer chromatography, using a 3:1 mixture 
of chloroform:methanol as the developing solvent to yield a quinidine-DTAF 
conjugate of the formula: 
##STR23## 
The following tracers were also prepared in accordance with the above 
procedures: 
EXAMPLE XXIV 
5-(p-aminobenzamido)-2-propylpentanoic acid-DTAF conjugate 
##STR24## 
EXAMPLE XXV 
1-amino-2-phenyl-2-(2'-pyridyl)-4-diisopropylaminobutane-DTAF conjugate 
##STR25## 
EXAMPLE XXVI 
2-phenyl-2-(2'-pyridyl)-4-(diisopropylamino)butyrylhydrazine-DTAF conjugate 
##STR26## 
EXAMPLE XXVII 
4-aminosalicyclic acid-DTAF conjugate 
##STR27## 
EXAMPLE XXVIII 
procainamide-DTAF conjugate 
##STR28## 
EXAMPLE XXIX 
1-hydroxymethyl-2-hydroxy-2-(4'-nitrophenyl)ethylamine-DTAF conjugate 
##STR29## 
EXAMPLE XXX 
2-.beta.-aminoethylphenytoin-DTAF conjugate 
##STR30## 
EXAMPLE XXXI 
5-aminosalicylic acid-DTAF conjugate 
##STR31## 
EXAMPLE XXXII 
propanolol-DTAF conjugate 
##STR32## 
EXAMPLE XXXIII 
aminophenobarbital-DTAF conjugate 
##STR33## 
EXAMPLE XXXIV 
.alpha.-aminoacetyliminostilbene-DTAF conjugate 
##STR34## 
EXAMPLE XXXV 
1-N-isopropylamino-2-.alpha.-aminoacetyl-3-(1'-naphthoxy)-propane-[DTAF] 
conjugate 
##STR35## 
EXAMPLE XXXVI 
3-dehydroxy-3-aminodigoxigenin-DTAF conjugate 
##STR36## 
As mentioned above, the fluorescently labeled tracers prepared according to 
this invention can be used in a variety of immunoassay procedures in 
particular in a fluorescence polarization immunoassay. The following 
examples demonstrate the suitability of tracers of the present invention 
in assays employing fluorescence polarization techniques. 
All examples followed the same general procedure: 
(1) A small volume of standard or test serum is delivered into a test tube 
and diluted with buffer; 
(2) A small volume of concentrated fluorescent tracer optionally containing 
a surfactant is then added to each tube; 
(3) Finally, a volume of diluted antisera is added; and 
(4) The reaction mixture is incubated at room temperature. 
VALPROIC ACID ASSAY 2-ETHYL-5-AMINO-PENTANOIC ACID DTAF CONJUGATE 
Materials Required 
(1) Buffer: 0.1 M phosphate, pH 7.5, containing 0.01% (w/v) sodium azide 
and 0.01% (w/v) bovine gamma globulin (BGG). 
(2) Tracer: 2-ethyl-5-amino-pentanoic acid-DTAF conjugate 
50.times.10.sup.-9 M in 0.1 M tris hydrochloride buffer, pH 7.8, 
containing 0.1% (w/v) sodium dodecyl sulfate, 0.01% (w/v) bovine gamma 
globulin, and 0.01% (w/v) sodium azide. 
(3) Antibody: Sheep antiserum to valproic acid diluted to 1 to 3.75 in 
buffer. 
(4) Standards or unknowns: human serum (or other biological fluid) 
containing valproic acid in the concentration range 0 to 150 .mu.g/ml. 
(5) Fluorescence polarimeter: Instrument capable of reading the 
polarization of fluorescence of a 1.times.10.sup.-9 M fluorescein solution 
to .+-.0.001 polarization unit. Protocol: 
(1) 0.75 .mu.l of standard or unknown sample placed in a 12.times.75 mm 
disposable culture tube (cuvette). This is accomplished by pipetting 20 
.mu.l of standard or unknown into a predilution container followed by 500 
.mu.l of buffer. Next 20 .mu.l of diluted sample is pipetted into the 
12.times.75 culture tube followed by 400 .mu.l of buffer. 
(2) 40 .mu.l of tracer and 800 .mu.l of buffer are added to the cuvette. 
(3) 40 .mu.l of antiserum and 800 .mu.l of buffer are added to the cuvette. 
The contents of the cuvette are mixed and incubated for approximately 15 
minutes at room temperature. 
(4) The fluorescence polarization is read. Typical results are presented in 
Table I. 
TABLE I 
______________________________________ 
Valproic Acid Conc. (.mu.g/ml) 
Polarization 
______________________________________ 
0 0.217 
12.5 0.186 
25 0.165 
50 0.132 
100 0.099 
150 0.081 
______________________________________ 
The polarization changes in a regular manner as the concentration of 
valproic acid is varied allowing the construction of a standard curve. 
Unknown samples are treated in an identical manner; from the polarization 
of fluorescence of the unknown sample, the concentration of valproic acid 
in the unknown sample may be determined by reference to the standard 
curve. 
GENTAMICIN ASSAY 
Materials Required 
(1) Buffer: (See valproic acid assay). 
(2) Tracer: Gentamicin-DTAF at 100 nM in a trishydrochloride buffer pH 7.5 
containing 0.125% sodium dodecyl sulfate, 0.01% sodium azide, and 0.01% 
bovine gamma globulin. 
(3) Antibody: Rabbit or sheep antisera to gentamicin diluted appropriately 
in buffer. 
(4) Standards or unknowns: human serum (or other biological fluid) 
containing gentamicin. 
(5) Fluorescence polarimeter: (See valproic acid assay). 
Protocol 
(1) 1.8 .mu.l of standard or unknown sample is placed in a 12.times.75 mm 
disposable culture tube (cuvette). This is done by pipetting 20 .mu.l of 
sample followed by 200 .mu.l of buffer. Next 20 .mu.l of diluted sample is 
pipetted into the cuvette followed by 200 .mu.l of buffer. 
(2) 40 .mu.l of tracer and 1000 .mu.l of buffer are added to the cuvette. 
(3) 40 .mu.l of antibody and 1000 .mu.l of buffer are added, the contents 
of the cuvette are mixed and incubated for approximately fifteen minutes 
at room temperature. 
(4) The fluorescence polarization is read following the incubation. Typical 
results are presented in Table II. 
TABLE II 
______________________________________ 
Gentamicin Concentration (.mu.g/ml) 
Polarization 
______________________________________ 
0 0.178 
0.5 0.158 
1.0 0.140 
2.0 0.115 
4.0 0.090 
8.0 0.074 
______________________________________ 
The polarization changes in a regular manner allowing construction of a 
standard curve. Unknown samples are tested in an identical manner, and the 
gentamicin content is determined by reference to the standard curve. The 
utility of the gentamicin-DTAF tracer for determining the concentration of 
gentamicin in biological samples is thereby illustrated. 
N-ACETYL PROCAINAMIDE ASSAY 
Materials required 
(1) Buffer: (See valproic acid assay) 
(2) Tracer: Desethyl-N-acetyl procainamide-DTAF conjugate at a 
concentration of 50.times.10.sup.-9 in a 5.75% (w/v) solution of sodium 
toluene sulfonate. 
(3) Antiserum: Rabbit antiserum to N-acetylprocainamide diluted one to six 
in buffer. 
(4) Standards or unknowns: human serum (or other biological fluid). 
(5) Fluorescence polarimeter: (See Valproic acid assay). 
Protocol 
(1) 0.48 .mu.l of standard or unknown is placed in a cuvette by pipetting 
10 .mu.l of sample into a predilution container and mixing with 200 .mu.l 
of buffer. Ten .mu.l of diluted sample is next pipetted into the cuvette 
followed by 200 .mu.l of buffer. 
(2) 40 .mu.l of tracer and 1000 .mu.l of buffer are added to the cuvette. 
(3) 40 .mu.l of antiserum and 1000 .mu.l of buffer are next added to the 
cuvette. The contents of the cuvette are mixed and incubated at room 
temperature for approximately fifteen minutes at room temperature. 
(4) The fluorescence polarization is read following the fifteen minute 
incubation period. Typical results for the N-acetyl-procainamide are 
presented in Table III. 
TABLE III 
______________________________________ 
N--Acetyl Procainamide (.mu.g/ml) 
Polarization 
______________________________________ 
0 0.239 
1 0.218 
2 0.209 
4 0.190 
8 0.173 
16 0.158 
______________________________________ 
The standard cruve can be constructed from the data in Table III. Unknown 
samples treated identically to the standards can be quantitated by 
references to the standard curve, thereby illustrating the usefulness of 
the standard N-acetyl procainamide-DTAF conjugate for the determination of 
N-acetyl-procainamide in biological fluids. 
The following table summarizes the various fluorescence polarization assays 
that have been carried out in accordance with the above-described 
procedures employing tracers prepared in the preceding examples. The 
tracers employed are identified by Example number and the specific 
ligand(s) determined are indicated. 
______________________________________ 
Tracer Prepared 
In Example Number 
Ligand(s) Assayed 
______________________________________ 
I Gentamicin 
II Tobramycin 
III Amikacin 
IV Streptomycin 
V Neomycin 
VI Vancomycin 
VII N--acetylprocainamide 
VIII N--acetylprocainamide 
IX N--acetylprocainamide 
X Primidone 
XI Valproic acid 
XII Valproic acid 
XIII Thyroxine (T.sub.4) 
XIV Thyroxine (T.sub.4) 
XV Thyroxine (T.sub.3) 
XVI Nortrptyline; Amitriptyline 
XVII Nortrptyline; Amitriptyline 
XVIII Nortrptyline; Amitriptyline 
XIX Imipramine; Desipramine 
XX Imipramine; Desipramine 
XXI Theophylline 
XXII Theophylline 
XXIII Quinidine 
XXIV Valproic acid 
XXV Disopyramide 
XXVI Disopyramide 
XXVII Salicylate 
XXVIII Procainamide 
XXIX Chloroamphenicol 
XXX Phenytoin 
XXXI Salicylate 
XXXII Propranolol 
XXXIII Phenobarbital 
XXXIV Carbamazepine 
XXXV Propranolol 
XXXVI Digoxin 
______________________________________ 
As evident from the above results, the tracers of the present invention are 
effective reagents in fluorescence polarization immunoassays. In addition 
to the properties mentioned above, the tracers of the present invention 
posses a high degree of thermal stability, a high degree of bound 
polarization, high quantum yields and are relatively easy to produce and 
purify. 
Although this invention has been described with respect to specific 
modifications, the details thereof are not to be construed as limitations, 
for it will be apparent that various equivalents, changes and 
modifications may be restorted to without departing from the spirit and 
scope thereof and it is understood that such equivalent embodiments are 
intended to be included therein.