Note: Descriptions are shown in the official language in which they were submitted.
Background Of The Invention
This disclosure relates generally to fluorescent
compounds which have proved particularly useful in a variety
of immunofluorescence procedures to detect and determine
quantities of small biologically interesting molecules in body
fluids. The low molecular weight substances which are desired
to be detected or monitored include therapeutically adminis-
tered drugs, drugs having a known potential for abuse, and
various other compounds which are of interest in diagnosing
diaease or monitoring therapy.
While the claimed compounds can be utilized in a
variety of immunofluorescence techniques; they are particularly
useful in a procedure called fluorescence polarization immuno-
assay. A fluorescence polarization immunoassay combines the
specificity of an immunoassay with the speed and convenience
of a homogeneous method. When a fluorescent conjugate is bound
to an antibody, linearly polarized light which is used to
excite the fluorescent conjugate remains highly polarized upon
emission because the fluorophore is constrained from rotating
between the time the light is adsorbed and emitted. On the
other hand, when the fluorescent conjugate is not bound by
antibody, its rotation is much faster , molecules are more
randomly oriented, and the emitted light is depolarized.
Fluorescence polarization, then, provides a direct measure of
bound and free fluorescent conjugate in a competitive binding
immunoassay. Fluorescent conjugate competes for antibody
binding sites with the compound of interest in the patient
sample. The greater the concentration of the compound being
assayed, the larger the fraction of fluorescent conjugate un-
bound by antibody, and therefore, a greater degree of de-
polarized light is emitted. The precise relationship between
the concentration of analyte and polarization is established
by generating a standard curve. Calibrators with known amounts
of analyte are read and the polarization recorded. When an
unknown is read, its concentration is estimated by inter-
polation between standards.
-- 2 --
Description Of The Prior Art
-
Fluorescent conjugates of derivatives of low molecu-
lar weight compounds are not unique. U. S. Patent 3,998,943
(1976) specifically describes the preparation of fluorescently
labeled insulin using fluorescein isothiocyanate (FITC) and
fluorescently-labeled morphine using 4-amino-fluorescein
hydrochloride.
The fluorescent derivatives of the present invention
are labeled with dichlorotriazinylaminofluorescein which is
commonly referred to as DTAF. DTAF is the product of the re-
action of aminofluorescein and cyanuric chloride. In an
article by Blakeslee, et al, Jounal of Immunological Methods,
13, 305-320, ~1976), DTAF was described as an effective reagent
for conjugating fluorescein to immunoglobulins (IgG) having
molecular weights of at least 160,000.
Summary Of The Invention
The present invention discloses and claims the use
of DTAF to impart fluorescence to a wide variety of biologically
interesting compounds which, when labeled, can be characterized
by the formula:
Cl
~N=(~
N~
z
wherein X is a biologically interesting moiety having a molecu-
lar weight of less than 2,000 and having at least one reactive
substituent selected from the group consisting of a primary
or secondary amine or hydroxyl group by which it attaches to acarbon
atom of the triazine ring; and Z is selected from the group
consisting of 4-amino fluorescein and 5-aminofluorescein
attached through the 4-amino or 5-amino, res~ectivelv to the
number 2 carbon of the triazine ring.
6~
-- 3 --
Description Of The Preferred Embodiments
DTAF can be readily prepared according to the method
adopted by Blakeslee, et al (supra). DTAF Isomer I is prepared
from 5-amino fluorescein; Isomer II is derived from 4-amino
fluorescein.
The conjugation of either isomer of DTAF to compounds
of interest can generally be accomplished by dissolving equal
molar amounts of DTAF and a compound having a reactive amine
(primary or secondary) or a hydroxyl group in an appropriate
solvent such as water, methanol, dimethylformamide or dimethyl-
sulfoxide. If the reactive amine is a salt, a suita~le base
ls added to the reaction mixture to form the free base of the
reactive amine. After completion of the reaction, the conju-
gates can be purified by using either thin-layer or column
chromatography.
The biologically interesting compounds employed in
the claimed invention must have at least one primary or second-
ary amino group or hydroxyl group by which it can react with
and attach to a carbon atom of the triazine ring. The reactive
group may be inherent in the biologically active compounds as
it is in the quinidine, procainamide, thyroxine and the amino-
glycoside antibiotics, or it may be introduced into the com-
pound by derivatization. Examples of biological compounds that
require the formation of amino derivatives before DTAF con-
jugates can be prepared include theophylline, valproic acid,
phenobarbital, phenytoin, primidone, disopyramide, digoxin,
chloramphenicol, aspirin, acetaminophen, carbamazepine, des-
imprmine, and nortriptyline.
Structures illustrating the most preferred compounds
of the disclosed invention include the following:
8-AMINOMETHYLTHEOPHYLINE - DTAF
CH3 ¦l ~N
N ~ - ~ ~ N \
CH3 \0
4 ~
2-ETHYL-5-AMINOPENTANOIC ACID - DTAF
/OH
C>== ~
Ho-C/0 ~===
CHOEI2CH2cH2NH ( ~
COOH / \\
CH3CH
~0
and
2-PROPYL-5-AMINOPENTANOIC ACID - DTAF
Cl ~ OH
~ -N ~
HO-C-CHCH2CH2CH2N~
¦ COOH / \\
C~2CH2CH3 ~
The ~ollowing examples will demonstrate the
preparation of specific DTAF conjugates within the scope of
the claimed invention. Note that in the following examples,
DTAF may be either isomer unless specifically stated.
-- s --
EXAMPLE I
_NTAMICIN - DTAF
Gentamicin sulfate (200 mg) was dissolved in 1 ml
of distilled water. The pH was adjusted to 9.0 with
approximately 0.8 ml of l.OM sodium hydroxide. DTAF (20 mg)
was dissolved in 1.5 ml of methanol and added dropwise to the
gentamicin solution with stirring and allowed to react for
one hour. 'rhe reaction mixture was added to a DEAE cellulose
medium mesh column and the resulting gentamicin-DTAF eluted
with 0.lM phosphate buffer at pH 8Ø
EXAMPLE II
TOBR~MYCIN - DTAF
Tobramycin (250 mg) was dissolved in 2 ml of carbo-
nate buffer, 0.lM, pH 9Ø DTAF (20 mg) was dissolved in
1 ml of methanol and added to 1 ml of the tobramycin solution.
After about five minutes, the reaction mixture was purified by
application to a 20 ml DEAE cellulose column and equilibrated
with 0.lM phosphate buffer at pH 8Ø The reaction product
was eluted with the same buffer.
EXAMPLE III
AMIKACIN - DTAF
Amikacin (24 mg) was dissolved in 0.2 ml of water,
and 4.5 mg of DTAF were suspended in 0.2 ml of methanol. The
methanoli~ suspension of DTAF was added to the amikacin solu-
tion with stirring. The small particles of DTAF rapidly
dissolved, so the reaction mixture was not stirred. After
thirty minutes the reaction mixture was applied to a 17 ml
column of DEAE cellulose equilibrated with pH 8.1 phosphate
buffer, 0.1M. The reaction product was eluted with the same
buffer.
EXAMPLE IV
DESETHYL-N-ACETYL-PROCAINAMIDE - DT~F
-
Para-acetamidobenzoic acid (1.79 g) and 1.15 g of
N-hydroxysuccinimide were dissolved in 15 ml of pyridine. Then,
2.3 g N,N'-dicyclohexylcarbodiimide was added and dissolved.
6 --
The reaction mixture was chilled in a refrigerator for two
hours and then filtered. Crystals were rinsed with about
2 ml of acetone. N-ethylethylenediamine (0.88 g) was added to
the combined pyridine-acetone filtrate. The mixture was stirred
for two hours and then chilled in a refrigerator for about
twenty-four hours. The resulting crystals were filtered and
rinsed with acetone. The crystals ~2.0 g) were dissolved in
50 ml of distilled water, and the solution adjusted to pH 10
with 6N sodium hydroxide solution. A white precipitate was
filtered off and dried in a dessicator. The DTA~ conjugate
was formed by dissolving equal molar amounts of desethyl-N-
acetylprocainamide and DTAF in methanol. The reaction was
completed in about ten minutes. Purification was performed
by silica gel thin-layer chromatography, using a developing
solvent of chloroform/acetone (1:1).
EXAMPLE V
N-p-ACETAMIDOBENZOYL ETHYLENE DIAMINE - DTAF
The procedure of example IV was repeated using 0.9 g
of ethylenediamine in place of N-ethylethylenediamine. The
reaction mixture was stirred for one hour, and chilled for
one and one-half hours. The precipitate was filtered and rinsed
with acetone. The DTAF conjugate was prepared the same as in
Example IV, but methanol was used as the developing solvent.
EXAMPI,E VI
N-p-ACETAMIDOBENZOYL-N'-ETHYL-N'-AMINO-
ACETYL ETHYLENE DIAMINE - DTAF
Desethyl-N-acetyl procainamide (1.25 g) from
example IV and 0.8 g of chloroacetyl chloride were dissolved in
25 ml of acetone and refluxed for two hours. After refluxing,
the mixture was filtered and the filtrate evaporated to dry-
ness. The yellow residue from the filtrate and 0.75 g of
sodium iodide were dissolved in 20 ml of acetone and refluxed
for one hour. The solution was filtered and the filtrate
evaporated to dryness. The red residue was dissolved in
20 ml of methanol. Concentrated ammonium hydroxide (20 ml)
was added to the solution, which was refluxed for one and one-
half hours. After cooling, the mixture was extracted twice
with 20 ml of chloroform. The combined extracts were dried
- 7
over sodium sulfate, filtered, and evaporated. The DTAF
conjugate was prepared the same as in example IV and purified
by thin-layer chromatography (chloroform/acetone (1:1)).
EXAMPLE VII
AMINO-PRIMIDONE - DTAF
.
Primidone (1.1 g) was dissolved in 10 ml of con-
centrated sulfuric acid. Another solution of 1 ml concentrated
nitric acid and 2 ml of concentrated sulfuric acid was added to
the reaction mixture slowly without coolinq. The mixture was
then shaken at room temperature for forty-five minutes, and
the reaction mixture was poured over 50 ml ice and crystals
of para-nitro-primidone were filtered and rinsed with water.
The crystals (1.17 g), melting point 225 - 228 C, were dis-
solved in 200 ml of hot ethanol. Iron powder (1.5 y) and
100 ml of water were added. The mixture was heated to boiling,
2 ml of concentrated hydrochloric acid were added, and the
mixture was refluxed for two hours. The hot mixture was
filtered and the filtrate evaporated to dryness. The residue
was dissolved in 100~ ethanol and precipitated out by adding
diethyl ether. The precipitate was filtered to give 0.8 g of
brown hydroscopic crystals. The DTAF conjugate was formed by
dissolving 5 mg of DTAF and 5 mg of the brown crystals in
0.5 ml of methanol. The reaction was complete in 10 minutes
and the conjugate was purified by silica gel thin-layer chroma-
tography using chloroform/methanol (3:1) as the developing
solvent. Final purification was performed by thin-layer
chromatography using chloroform/methanol (2:1).
EXAMPLE VIII
2-ETHYL-5-AMINO-PENTANOIC ACID - DTAF
Delta-5-valerolactam ¢7.5 g) was dissolved in 60 ml
of dry tetrahydrofuran, under a dry nitrogen atmosphere and
n-butyllithium (1.6M, 90 ml) in hexane ~ere added dropwise
to the reaction flask and chillPd with 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 (still under
dry nitrogen atmosphere). l-Bromoethane (8.0 g) was slowly
6~
-- 8 --
added to the reaction flask while the flask was chilled in an
ice bath. The mixture was then stirred for sixteen hours at room
temperature and then 100 ml of w`ater was added slowly. This mix-
ture was stirred at room temperature for thirty minutes and the
organic layer separated. The aqueous layer was extracted with
50 ml of diethyl ether and the organic layers combined and dried
over sodium sulfate. The solvent was evaporated to give a
dark oil,which crystallized on standing,and was recrystallized
from petroleum ether to give 3.8 g of product. This product
~2.8 g) was refluxed in 25 ml of 6N hydrochloric acid for six
hours. The water was evaporated to give a dark, thick oil.
The DTAF conjugate was formed by dissolving equimolar amounts
of 2-ethyl-5-amino-pentanoic acid and DTAF in methanol. The
reaction was completed in about ten minutes. The product was
purified by silica gel thin-layer chormatography with chloroform/
methanol (3:1) as the developing solvent.
EXAMPLE X
D-THYROXINE - DTAF
DTAF (5 mg) was dissolved in 0.5 ml of methanol. 5 mg
of D-thyroxine was added and then dimethylsulfoxide was added
dropwise until a clear solution was formed. Two drops of tri-
ethylamine were added and a conjugate formed after about
sixteen hours. The product was purified by silica gel thin-
layer chromatography using chloroform/methanol (3:1) as the
developing solvent.
EXAMPLE XI
L-THYROXINE - DTAF
This conjugate was prepared following the procedure
of Example X, but substituting L-thyroxine.
EXL~PLE XII
3,3',5-TRIIODO-L-THYRONINE - DTAF
This conjugate was prepared following the procedure
of Example X, but substituting 3,31,5-triiodo-L-thyronine.
- 9 -
EXAMPLE XIII
5-AMINO-DIBENZOCYCLOHEPTANE - DTAF
The following were dissolved in 50 ml of methanol:
8.00 g of ammonium acetate, 630 mg of sodium cyanoborohydride,
and 2.10 g of 10,11-dihydro-5H-dibenzo [a,d] cycloheptent-5-
one. The solution was refluxed for twenty-four hours and then
evaporated to dryness. A tan residue remained, which was dis-
solved in 25 ml of 2N hydrochloric acid and extracted twice
with 25 ml dichloromethane. 6N sodium hydroxide was added to
the aqueous phase unitl the pH reached 14. A brown oil began to
form and the solution was chilled in a freezer for 16 hours.
All water was evaporated and the residue was taken up in metha-
nol and filtered. The filtrate was evaporated to give a white
residue. The DTAF conjugate was formed by dissolving equimolar
amounts of DTAF and the white residue in methanol. The product
was purified by silica gel thin-layer chromatography using
chloroform/acetone (1:1) as developing solvent.
EXAMPLE XIV
DIBENZOSUBERONE HYDRAZONE - DTAF
10,11-Dihydro-5H-dibenzo [a,d] cyclohepten-5-one
~10 g) and 18 g of dimethylhydrazine were refluxed for twenty-
four hours in 100% ethanol. 100 ml of distilled water were
added and the yellow solution was extracted with diethyl ether
until extracts were colorless. The combined ether extracts
were washed with 25 ml of 2N hydrochloric acid. The organic
phase was then dried over sodium sulfate and evaporated.
The residue was a thick orange oil, dibenzosuberone dimethyl-
hydrazone. This product ~2.0 g) was refluxed for twelve hours
with 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
give a yellow oil, dibenzosuberone hydrazone~ The DTAF con-
jugate was prepared the same as in Example XIII, but using
chloroform/methanol (3:1) as the developing solvent.
- ~6~
-- 10 --
EXAMPLE XV
5-(y-AMINOPROPYLIDENE)-5H-DIBENZO[a,d]-
10,11-DIHYDRO-CYCLOHEPTENE - DTAF
5-(y-Bromopropylidene)-5H-dibenzo [a,d]-10,11
dihydro-cycloheptene and its precursor 5-cyclopropyl-5-
hydroxy-5H-dibenzo ~a,d]-10,11-dihydrocycloheptene were pre-
pared by the procedure described in Jounal 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-(y-bromopropylidene~-5H-dibenzo [a,d]-10,11-dihydrocyclo-
heptene, was followed substituting the bromopropylidene com-
pound for the chloropropylidene compound. The DTAF conjugate
was formed by dissolving equimolar amounts of DTAF and the
amine in methanol. An excess amount of triethylamine was
added and the reaction was completed in thirty minutes. The
reaction product was purified by silica gel thin-layer chroma-
tography using chloroform/methanol (2:1) as the developing
solvent.
EXAMPLE XVI
N-AMINOACETYLIMINOSTILBENE - DTAF
Iminodibenzyl (6.0 g) was dissolved in 30 ml of
chloroform. Chloroacetyl chloride (6 ml) was added and the
mixture was refluxed for forty-five minutes. Water (60 ml)
was added and the mixture was stirred for thirty minutes at
room temperature. The chloroform layer was separated and
dried over sodium sulfate and evaporated to dryness. The
residue was dissolved in 25 ml of acetone and a solution of
4.5 g sodium iodide dissolved in 25 ml of acetone was added. This
mixture was refluxed for thirty minutes, 100 ml of water was
added, and the reaction product was extracted twice with 50 ml
of chloroform and evaporated to dryness. The residue was dis-
solved in 40 ml of methanol. Concentrated ammonium hydroxide
(60 ml) was added and the mixture was refluxed for one hour.
The solution was evaporated to dryness and the residue was
taken up in 100 ml of chloroform and washed twice with 30 ml
of 2N hydrochloric acid. The organic phase was dried over
sodium sulfate and evaporated to dryness. The DTAF conjugate
was prepared by dissolving 5 mg of DTAF and 5 mg of the amine
in 0.5 ml of methanol. Conjugate formed in ten minutes and
was purified the same as Example XIII.
EXAMPLE XVII
N-AMINOACETYLDESIPRAMINE - DTAF
Desipramine hydrochloride (1~33 g) and 0.80 g of
chloroacetyl chloride were dissolved in 25 ml chloroform.
This was refluxed for two hours, then the chloroform was
evaporated. The residue was dissolved in 25 ml of acetone.
Sodium iodide (0.75 g) was added, and the solution was refluxed
for thirty minutes. The solution was filtered and the
precipitated salt was rinsed with acetone. The acetone filtrate
was evaporated and the residue was taken up in 20 ml of
methanol. Concentrated ammonium hydroxide (20 ml) was added
and the 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. The conjugate was prepared by dissolving 5 mg
each of DTAF and the amine in 0.5 ml of methanol. About five
drops of dimethylsulfoxide were added to dissolve the precipi-
tate. The reaction was completed in ten minutes and purified
by using the procedure of Example XIII using chloroform/
methanol (3:1) as the developing solvent.
EXAMPLE XVIII
8-AMINOMETHYL-THEOPHYLLINE - DTAF
DTAF ~1~ mg) and 8-aminomethyl-theophylline (5 mg)
were dissolved in 0.5 ml of dimethylsulfoxide. After five
minutes the reaction was complete and the conjugate was puri-
fied by silica gel thin-layer chromatography, using chloroform/
acetone ~1:1) as the developing solvent.
EXAMPLE IXX
8-AMINOETHYL-THEOPHYLLINE - DTAF
Procedure same as Example XVIII substituting
8-aminoethyl-theophylline for the methyl derivative.
;2~
- 12 -
EXAMPLE XX
QUINIDIN~ - DTAF
DTAF (5 mg) and anhydrous quinidine (5 mg) were
dissolved in 0.5 ml of dimethylformamide. After sixteen hours
the reaction was complete and the conjugate was purified by
silica gel thin-layer chromatography, using chloroform/
methanol (3:1) as the developing solvent.
As mentioned above, the fluorescently labeled
tracers prepared according to this invention can be used in a
variety of immunoassay procedures. The following assays are
offered to demonstrate the suitability of the representative
sampling of these tracers in fluorescence polarization assays.
All examples followed the same basic protocol:
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 containing surfactant is then added to each tube;
3~ Finally, a volume of diluted antiserum is
added; and
4~ The reaction mixture is incubated at room
temperature.
EXAMPLE XXI
VALPROIC ACID ASSAY
2-ETHYL-5-AMINO-PENTANOIC ACID DTAF CONJUGATE
Materials Required:
1) Buffer: 0.1M phosphate, pH 7.5, containing
0.01% (w/v~ sodium azide and 0.01% ~w/v) bovine gamma globulin
(BGGl-
2) Tracer: 2-ethyl-5-amino-pentanoic acid DTAF
conjugate 50 x 10 9M in 0.lM tris hydrochloride buffer, pH
7.8, containing 0.1% (w/v~ sodium dodecyl sùlfate, 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.
~ ) Standards or unknowns: Human serum (or other
biological fluid~ containing valproic acid in the concentration
range 0 to 15~ ~g/ml.
~ ~ 6 ~
- 13 -
5) Fluorescence polarimeter: Instrument capable
of reading the polarization of fluorescense of a 1 x 10 9M
fluorescein solution to + 0.001 polarization unit.
Protocol:
1) 0.75 ul of standard or unknown sample placed
in a 12 x 75 nm disposable culture tube (cuvette). This
is accomplished by pipetting 20 ul of standard or unknown into
a predilution container followed by 500 ~1 of buffer. Next,
20 ~1 of diluted sample is pipetted into the 12 x 75 culture
tube followed by 400 ~1 of buffer~
2~ 40 ~1 of tracer and 800 ~1 of buffer are added
to the cuvette~
3) 40 ~1 of antiserum and 800 ~1 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. (-~q/ml)Polarization
0 0.217
12.5 0.186
0.165
0.132
100 ~ 099
15a 0.081
The polarization changes in a regular manner as the
concentration o~ valproic acid is varied allowing the con-
struction 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.
~0~
-- 14 --
EXAMPLE XXI I
_ENTAMICI~ ASSAY
Materials:
1) suffer: (See valproic acid assay).
2) Tracer: Gentamicin-DTAF at 100 nM in a tris-
hydrochloride buffer pH 1.5 containing 0.125% sodium dodecyl
sulfate, 0.01~ sodium azide, and 0.01~ bovine gamma globulin.
3) Antibody: Rabbit or sheep antisera to genta-
micin diluted appropriately in buffer.
4) Standards or unknowns: Human serum (or other
biological fluid) containing gentamicin.
5) Fluorescence polarimeter: (See valproic acid
assay).
P~otocol:
1) 1.8 ~l of standard or unknown sample is placed
in a 12 x 75 nM disposable culture tube (cuvette). This is
done by pipetting 20 ul of sample followed by 200 ~1 of buffer.
Next 20 ~1 of diluted sample is pipetted into the cuvette
followed by 200 ~1 of buffer.
2) 40 ~l of tracer and 1000 ~1 of buffer are added
to the cuvette.
3) 40 ~1 of antibody and 1000 ul 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 (~ /ml) Polarization
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 allow-
ing construction of a standard curve. Unknown samples are
_ 15 _
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 concentra-
tion of gentamicin in biological samples is thereby illustrated.
EXAMPLE XXIII
N-ACETYL PROCArNAMIDE ASSAY
Materials required:
1) Buffer: (See valproic acid assay).
2) Traeer: Desethyl-N-acetyl proeainamide - DTAF
conjugate at a coneentration of 50 X 10 9M in a 5.75~ (w/v)
solution of sodium toluene sulfonate.
3) Antiserum: Rabbit antiserum to N-aeetyl-
proeainamide 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 ul of standard or unknown is placed in a
euvette by pipetting 10 ~1 of sample into a predilution eon-
tainer and mixing with ~00 ~1 of buffer. Ten ~1 of diluted
sample is next pipetted into the euvette followed by 200 ~1
of buffer.
2) 40 ~1 of tracer and 1000 ~1 of buffer are added
to the cuvette.
3~ 40 ~1 of antiserum and 1000 ul of buffer are
next added to the euvette. The contents of the cuvette are
mixed and incubated at room temperature for approximately 15
minutes at room temperature.
3) The fluorescence polarization is read following
the 15-minute incubation period. Typical results for N-acetyl-
procainamide are presented in Table III.
2~
- 16
TABLE III
N-Acetyl Procainamide (ug/ml) Polarization
0 0.239
1 0.218
2 0.209
4 0.190
8 0.173
16 0.158
A standard curve can be constructed ~rom the data
in Table III. Unknown samples treated identically to the
standards can be quantitated by reference to the standard
curve, thereby illustrating the usefulness of the des-ethyl-
N-acetyl procainamide-DTAF conjugate for the determination of
N-acetyl procainamide in biological fluids.
