Fluorescence polarization immunoassays for multiple analytes using sequential addition of tracers

A methodology is presented which relates in general to fluorescence polarization immunoassays (FPIA) and modifications of current processes wherein detection and quantification of several commonly abused or therapeutic drugs in a single biological fluid sample are determined utilizing manual or current software or related equipment to allow the sequential and simultaneous performance of more than one FPIA assay. The methodology involves combining the reagents either separately or pre-mixed, for multiple assays in a single reagent package, these reagents being used to assay quantitative amounts of each of the assay analytes in a sequential step manner. The assay being performed by mixing the sample with a combination reagent and then initiating a specific reaction for each of the separate analytes by sequentially adding a specific reagent, i.e. tracer for each and reading the results during each separate stage to determine the specific reaction taken place and utilization of manual or software modification monitoring to subtract out the contribution to total signal of each specific reagent added previously to the stage of interest.

BACKGROUND OF THE INVENTION 
This invention relates in general to fluorescence polarization immunoassays 
(FPIA) and modifications of current processes for detection of several 
commonly abused drugs in biological fluid wherein sequential or 
simultaneous performance of more than one FPIA assay is achieved utilizing 
a single cuvette. Combination of FPIA reagents and manual or software 
technologies allows for performance of at least two FPIA assays per 
cuvette. In another aspect, the invention relates to combination FPIA on 
analytes other than abused drugs. 
Description of the Prior Art 
Nonisotopic immunoassays widely used in clinical and research arenas for 
the determination of both the presence and the quantity of analytes such 
as proteins, nucleotide sequences, drugs, steroids and the like can be 
divided into two types: heterogeneous assays and homogeneous assays. 
The heterogeneous assays utilize for example, a solid support such as beads 
in order to bind labeled reagents to the support while the remainder of 
the reagents remains unbound. A procedure is required to separate bound 
and free labeled reagents. In homogeneous assays, no separation is 
required, thus eliminating the need for an additional step. There are at 
least five types of homogeneous immunoassays routinely used. One of these, 
fluorescence polarization immunoassays (FPIA), can be used to measure 
small quantities of substances for example, in a nanogram-per-milliliter 
range. Use is made of the fact that molecules can exist in a ground or low 
energy state and, after exposure to incident radiation, an excited or high 
energy state. Absorption of energy from this source results in promotion 
of one or more electrons in a molecule to higher energy levels. As this 
jump occurs, the electron may lose a small percentage of the absorbed 
energy, for example, collisions with other molecules and the like. As its 
electrons return from higher energy levels to ground state, the excited 
molecule can radiate energy. The energy generated in this way is, however, 
less than that ordinarily involved in exciting the molecule. As a result, 
the wavelength of the light emitted, fluorescence light, is longer than 
that of the light used to excite the molecule. Emitted light energy can be 
detected using standard equipment, such as a detector positioned at a 
right angle to the incident light beam. 
Understanding of FPIA also requires understanding of polarized light. 
Ordinary light can be thought of as a number of electromagnetic waves, 
each in a single plane; each wave passes through the central axis or path 
of the light beam. Polarized light, however, is light in which only one 
wave plane occurs (the others having been eliminated or screened out). 
When a fluorescence molecule is orientated such that its dipoles lie in 
the same plane as the light waves, it absorbs the polarized light. As it 
returns to its ground state, the molecule emits light in the same plane. 
Two additional factors of importance in FPIA are time related. First, the 
fluorescence lifetime of the molecule being used must be considered. The 
lifetime is the interval between excitation of the molecule by a polarized 
light burst and emission by the molecule of a similar burst. Second, the 
rotational relaxation time of the molecule, the time necessary for an 
excited molecule to move out of alignment so that the emitted polarized 
light is emitted in a direction different from its excitation must be 
taken into account. Small molecules rotate rapidly in solution; their 
rotational relaxation times are shorter than molecular fluorescence 
lifetime. As a result, after having absorbed polarized light, such small 
molecules become randomly orientated by the time a burst of polarized 
emitted light is obtained. Larger molecules, for example immunoglobulins, 
rotate relatively slowly and have rotational times longer than the typical 
fluorescence lifetime. Fluorescence polarization measurements rely on the 
fact that the polarized excitation radiation gives rise to polarized 
emission radiation if no molecular rotation of the fluorophore occurs. A 
fluorophore is a fluorescence molecule or a compound which has the 
property of absorbing light at one wave length and emitting it at a longer 
wave length. As described above, the fluorophore bound to a small molecule 
experiences molecular rotation at a rate that is rapid compared to the 
lifetime of the excited state prior to emission. Thus, the light is 
depolarized when bound to a small molecule. When antibody binds the 
fluorophore-antigen, rotation decreases dramatically because of the large 
size of the antibody, causing the emitted light to remain polarized. 
This phenomenon is utilized in immunoassays, for example, with antibody and 
fluorophore-labeled hapten or fluorophore-labeled antigen present, binding 
occurs between antibody and fluorophore hapten or between antibody and 
fluorophore-antigen and total fluorescence polarization occurs. As antigen 
to be analyzed is added, the antigen binds to antibody competitively, 
fluorophore-antigen is not bound and depolarization is observed. The 
depolarization is a function of antigen concentration and constitutes a 
quantitative assay. 
Methods exist which are useful for detection of specific antigens, i.e. 
substances whose introduction into an animal stimulates the production of 
antibodies capable of reacting specifically with the antigens. Competitive 
binding immunoassays are used for measuring ligands in a test sample. For 
purposes of this disclosure, a "ligand" is a substance of biological 
interest to be determined quantitatively by a competitive binding 
immunoassay technique. The ligands compete with a labeled reagent or 
ligand analog, or tracer, for a limited number of receptor binding sites 
on antibodies specific to the ligand and ligand analog. In recent years, 
such methods have become of utmost importance in both research and 
clinical environments involving the detection of specific antigens in 
biological fluids such as blood, sputum, urine and the like. The detection 
of antigens or antibodies capable of specifically combining can often be 
related to various disease states and consequently is extremely useful in 
diagnosis, in gaining basic understanding concerning the genus of the 
disease, and in monitoring the effectiveness of therapies. Many schemes 
for detecting ligands or anti-ligands have evolved over recent years based 
on the selective, immunological reactivity which characterizes these 
substances. Generally, these schemes are collectively termed immunoassays. 
The class of immunoassays discussed measure changes in fluorescence 
polarization and depolarization for the detection of ligands, in 
particular, the fluorescence depolarization methods have become most 
useful in connection with therapeutic drug and abused drug monitoring. 
In such fluorescence depolarization immunoassay, the observation of a 
decrease in depolarization indicates increased binding of anti-ligand to 
the fluorescencely labeled ligand since such a binding results in a large 
molecule which rotates slowly and is thus, a less efficient depolarizing 
agent. If, on the other hand, the sample contains ligands which compete 
with the fluorescencely labeled ligands for binding sites on the 
anti-ligand, then fewer anti-ligand molecules are available to bind the 
fluorescencely labeled ligands and an increasing level of depolarization 
is observed. As may be readily appreciated, quantitation of such an assay 
may be conveniently accomplished using standard preparations for 
comparisons of samples containing known levels of the ligand. In fact, 
this technique is currently being used by Abbott Laboratories in its 
commercially available TDx.RTM. instrument such as described in U.S. Pat. 
Nos. 4,269,511 and 4,420,568. 
FPIA procedures have traditionally been limited to or directed to single 
analyte studies, however, more recently for example, multiple drug urine 
studies have been approached with a single reagent and fluorescent 
polarization process. The single reagent used, prepared by mixing four 
different antisera and three fluorescein-labeled derivatives, produced a 
polarized signal that is the average of the individual signals of the 
derivatives. The urine sample is added to the premixed reagent, incubated 
at room temperature for a few minutes, then fluorescence polarization is 
measured. The presence of any of several abused drugs at concentrations of 
one mg/L or more noticeably decreases the signal. Although other 
combinations are possible, the assay detects the presence of cocaine 
metabolite, amphetamine, and(or) several barbiturates. 
Urines are screened for abused drugs by quantitative techniques followed if 
necessary, by a confirmatory assay. Among the screening techniques 
developed are radioimmunoassays involving separation, and non-separation 
enzymoimmunoassays. Rapid, easily automated assays are desirable and, for 
simplicity, several investigators have attempted to develop combination 
assays capable of detecting any one of several abused drugs. The use of 
fluorescence polarization immunoassays (FPIA) for individual drugs of 
abuse is well established. More recently, the use of a combination reagent 
capable of detecting benzoylecgonine (a cocaine metabolite), amphetamine, 
and several commonly abused barbiturates in urine has been published. The 
use of a single assay tube to detect the presence of one or more of 
several drugs simplifies the initial screening procedure and reduces cost. 
Such a mixture approach i.e., four different antiserum and three different 
fluorescein-labeled derivatives, provides a reaction soup which may detect 
the presence of more than one of several drugs. However, it does not 
detect or quantify the amount of each drug once the drug presence is 
known. 
Other single analyte fluorescence polarization immunoassay methodology is 
utilized in, for example, the analyte digoxin in a biological fluid. 
Digoxin is a potent cardiac glycoside widely prescribed for the treatment 
of patients suffering from congestive heart failure as well as from some 
types of cardiac arrhythmias. Digoxin toxicity is a common and serious 
problem in a clinical setting. 
As previously mentioned, the methodology of FPIA for measuring the 
concentration of drugs or other analytes in biological fluids such as 
human serum or plasma is well known. Fully-automated assays can be used in 
conjunction with an analytical instrument such as the TDx.RTM. analyzer 
available from Abbott Laboratories, Abbott Park, Ill. The TDx.RTM. is 
suitable as a batch analyzer utilizing one reagent set per run while the 
ADx.RTM. has the ability to utilize multiple reagent sets per run. Both 
the TDx.RTM. and the ADx.RTM. provide suitable analyzer technology for use 
according to the invention. Following, is a full discussion of such an 
assay for the analyte digoxin; however, the TDx.RTM. procedures are 
suitable for use in the combination fluorescence polarization immunoassays 
method of the present invention. 
ANALYTE CALIBRATION 
An art taught FPIA system utilizing the TDx.RTM. is presented in the 
following discussion to more clearly focus on FPIA and microprocessor 
controlled testing using the FPIA system for the assay of the analyte 
digoxin. Controls and a series of human serum samples containing unknown 
digoxin levels were prepared. A centrifuge tube was designated for each 
sample to be tested, and placed in a suitable rack. A pipettor was filled 
with the precipitation reagent prepared as previously described in this 
example, and 200 microliters of the reagent were dispensed into each 
centrifuge tube by touching the tip of the pipettor to the wall of the 
centrifuge tube and depressing the dispensing button on the pipettor. 
Then, 200 microliters of each serum sample were pipetted into its 
corresponding centrifuge tube containing the precipitation reagent. After 
pipetting of the samples, each centrifuge tube was capped and mixed on a 
vortex mixer for 3-5 seconds, to ensure thorough mixing. The tubes were 
then placed into a centrifuge head, and centrifuged for about ninety (90) 
seconds at 9.500.times.g, until a clear supernatant and a hard compact 
pellet of denatured protein was obtained. After centrifugation was 
complete, each tube was uncapped and 250 microliters of the supernatant 
decanted into the corresponding sample well of a TDx.RTM. Sample Cartridge 
(commercially available from Abbott Laboratories;, in preparation for 
performing the digoxin assay. The remainder of the digoxin assay procedure 
was performed substantially as a routine assay or calibration run on the 
TDx.RTM. Analyzer. In this regard, reference is made to the "Procedures 
for Operation" section of the "TDx.RTM. System Operation" manual, 
previously described, for further details of the protocol used. The 
following is a description of the major aspects of the performance of the 
assay. 
ASSAY OPERATION 
All assay steps are controlled by the microprocessor and protocols 
programmed into the software of a TDx.RTM. Analyzer. A specific pattern on 
a barcode label is scanned by a barcode reader and the corresponding 
protocol is retrieved from the computer memory of the Analyzer. Each 
protocol contains detailed instructions for movement of pipetting syringes 
of the Analyzer which determines the volume of sample and reagents used in 
the pipetting steps, instruction for movement of a boom arm containing an 
aspirating probe, and for movement of a rotating carousel containing 
reaction cuvettes, as well as calibrator concentrations used for a 
calibration curve. The carousel has a unique barcode and set of 
instructions. Stepper motors, directed by an internal computer, move the 
carousel, syringes and boom arm. A light beam, focused on the carousel and 
controlled by the microprocessor, is used to monitor the number and 
placement of reaction cuvettes as the carousel rotates past. Two 
electrodes attached near the end of the probe serve as a liquid sensor 
which determines the presence of a liquid by electrical conductivity, 
thereby minimizing penetration of the probe into the samples and reagents. 
The TDx.RTM. Analyzer automatically pipettes reagents and test samples 
while simultaneously pipetting dilution buffer, then dispenses both into a 
reaction cuvette positioned in a rotating carousel. Two pipetting syringes 
are driven down and the sample or reagent is aspirated at the same time 
the buffer is being drawn. When both syringes have been filled with the 
correct volumes, a boom arm moves the probe to the reaction cuvette, and 
the syringes are driven up, expelling their volumes. The liquids are 
dispensed at a high velocity creating sufficient turbulence for complete 
mixing. 
Pipetting operations are performed with a dual syringe pump in conjunction 
with a boom assembly. A 250 microliter syringe is used to aspirate the 
sample and reagent, and a 2500 microliter syringe dispenses dilution 
buffer. The syringes are driven by two stepper motors which are 
computer-controlled for precise pipetting as determined by the assay 
parameters. The boom assembly consists of an arm which moves vertically 
and horizontally, teflon tubing which is integral with the probe and 
liquid level sensor, and a separate barcode reader which moves 
horizontally with the boom arm. For aspiration of samples or reagents, the 
boom arm moves in a horizontal arc until the probe is positioned over the 
proper sample cartridge well. The probe is then moved vertically until the 
tip comes in contact with the liquid and sample is aspirated. Upon 
completion of the aspiration, the boom moves up and horizontally, to 
position the probe over the dilution well or the cuvette. Coordinated 
rotation of the carousel occurs to bring a sample cartridge or cuvette 
into the correct position for receipt of the dispensed liquid. 
The dilution buffer rests on a platform controlled by a microswitch which 
signals when the buffer is empty. Teflon tubing connects the buffer bottle 
to a valve block, liquid heater and boom arm. An integral valve directs 
the flow of liquids in and out of the syringe and tubing. 
Some biological fluids, such as patient blood sera, evidence substantial 
background fluorescence which must be taken into consideration in order to 
obtain an accurate measurement of the analyte level in the sample. In the 
TDx.RTM. Analyzer, a sample blank for each sample, calibrator or control 
is automatically made and read before a fluorescence tracer is added to 
the reaction mixture in each cuvette. To obtain an indication of the true 
background fluorescence present in the final sample, the mixture on the 
first reading must be at the final diluted concentration. This is 
accomplished by adding half the sample volume to a reaction mixture 
representing half the final reaction mixture. The blank mixtures are 
measured by the fluorescence detector and the intensities are stored in 
the computer memory of the instrument. 
After the remaining reagents and the rest of the sample are added and 
incubated, the final reading is made. The blank intensities are subtracted 
from the final reaction mixture intensities before polarization values are 
calculated by the TDx.RTM. Analyzer. The polarization equation becomes as 
follows: 
##EQU1## 
The net and blank intensity for each cuvette is also calculated and 
printed, using the equation 
EQU I.sub.Net or I.sub.Blk) =2I.sub.hv +I.sub.vv 
MEASURING FLUORESCENCE POLARIZATION 
The light source (excitation beam) used for the tungsten fluorescence 
polarization reading on the TDx.RTM. Analyzer is a halogen lamp. The light 
passes through a filter which selects the correct excitation wavelength 
(usually 485 nanometers), and a reference detector signal is used to 
monitor the intensity of the lamp. The computer of the instrument can 
adjust the lamp intensity to provide a constant and accurate measure of 
the background intensity of samples with naturally fluorescing substances. 
A liquid crystal-polarizer combination in the light path rapidly polarizes 
the excitation beam horizontally and then vertically many times in 
sequence for each reaction cuvette measured. The polarized excitation beam 
is focused with a lens into the center of the sample, in the reaction 
cuvette of the carousel. Baffles bordering the cuvette serve as light 
traps preventing the excitation beam from entering the emission optics 
(polarization detection means of the instrument. The light path for the 
emission optics is at a 75 degree angle to the excitation light path. 
Another lens collimates emitted light and passes it through an emission 
filter which selects light of a wavelength corresponding, e.g., to the 
emission peak of fluorescein (525-550 nanometers). Emitted light is then 
passed through a vertical polarizer, and a photomultiplier tube converts 
the fluorescence into an electrical current which is recorded as numbers 
to be entered into the polarization equation by the computerized 
electronics of the instrument, providing a polarization value for each 
reaction cuvette measured. 
CALCULATION OF ANALYTE CONCENTRATION 
The calibration curve for each assay is stored in permanent computer memory 
of the TDx.RTM. Analyzer. The stored curve equation is generated by 
assaying samples with increasing concentrations of the analyte and 
measuring the polarization value for each concentration. The appropriate 
data reduction for that assay calculates a best-fit curve equation using 
six calibrator concentrations, one of which is zero. Curve parameters of 
slope, span of polarization value between high and low calibrators, and 
polarization value of the zero calibrator are used to determine the best 
fit. Concentrations of the analyte in unknown samples are calculated from 
this curve equation using the polarization values generated for each 
sample in the assay. 
Although the principle of FPIA has been known since the 1970's, and 
feasible instrumentation for the assay with automatic testing combined 
with microprocessing is available, multiple analyte and quantitative 
assays are only available by running separate reagents in separate 
cuvettes. The prior art disadvantage is based on through-put and 
disposable usage. None of the prior art deals with sequential FPIA with 
the obvious advantage of increasing efficiency of reagent and disposable 
use for clinics and laboratories along with cost savings and reduced 
packaging requirements. These advantages are achieved through a process 
for obtaining determinations of the presence or absence of multiple 
analytes as well as quantitative amounts in a single sample cuvette. 
SUMMARY OF THE INVENTION 
In accordance with the principles of the present invention, there is 
provided an accurate, relatively low cost and reliable methodology 
utilizing manual or current TDx.RTM./ADx.RTM. FPIA reagent and software or 
related equipment to allow the sequential or simultaneous performance of 
more than one FPIA assay in a single cuvette. The process involves 
combining the reagents, either separately or premixed, for multiple assays 
in a single reagent package. These reagents are then used to assay 
quantitative amounts of each of the assay analytes in a sequential 
process. The assay is performed by mixing the sample with the combination 
reagent initially (i.e. mixture of antisera) and then initiating a 
specific reaction for each of the separate analytes by sequentially adding 
a specific reagent (i.e. tracer) for each and reading the results during 
each separate stage to determine the specific reaction taking place. 
Software modifications monitor and subtract out the contribution to total 
signal of each specific reagent added previously to the stage of interest.

DETAILED DESCRIPTION OF THE INVENTION 
The combination FPIA will be presented in discussions of running two or 
more analytes in sequential process as well as the presentation of 
configurations of three analytes, although, theoretically the limit on the 
number of analytes achievable will be due only to constraints of reaction 
cuvette volume and mixing constraint=associated therewith. 
Original combination FPIA according to the invention involved the mixing of 
equal parts of antiserum (S pot) reagents from two assays, cocaine and 
cannabinoids, to make the combo S reagent. A 4 pot R-pack was then 
constructed consisting of the combination S reagent, cocaine tracer 
(CocMT), cannabinoids tracer (CannbT), and the cannabinoids pretreatment 
(P pot). The sequence of the test is shown in the following Example I: 
EXAMPLE I 
The sample was mixed with the pretreatment in the predilute well and 
incubated. A portion of this mixture was transferred to the cuvette and a 
blank reading taken. The combo S reagent was added to the cuvette along 
with the CocMT reagent and the mixture was incubated. After the 
incubation, a reading was taken after which the cocaine metabolite 
concentration was calculated. The intensities from this reading were 
stored for use later. Another portion of the combo S reagent, the CannbT 
reagent and an additional portion of CocMT reagent were added to the 
cuvette and the mixture again incubated. After incubation a reading was 
taken and after correction for the value stored from the preceding 
reading, the cannabinoids concentration was calculated. The basic 
methodology of the FPIA and the estimation of concentrations from a stored 
calibration curve were identical to the current AD.sub.x .TM. 
technologies. 
Following, is a description of the actual sequence of events utilized to 
perform a combination assay. 
1) 25 microliters of sample and 25 microliters of a pretreatment solution 
were mixed together with 450 microliters of buffer in a predilution 
vessel. 
2) 100 microliters of the diluted sample were transferred to the cuvette 
with 900 microliters of buffer. 
3) Incubation for 30 seconds. 
4) A reading was taken and stored. 
5) 37.5 microliters of the combined antiserum reagent and 18.8 microliters 
of the first analyte (cocaine) tracer, along with 50 microliters of the 
diluted sample were added to the cuvette with 843.7 microliters of buffer. 
6) Incubation for 420 seconds. 
7) A second reading was taken and after correcting by the first reading, an 
mP value was calculated. The second reading was also stored. 
8) 12.5 microliters of the combined antiserum reagent, 25 microliters of 
the second analyte (cannabinoids) tracer, 6.2 microliters of the first 
analyte tracer, and 50 microliters of the diluted sample were added to the 
cuvette along with 406.3 microliters of buffer. 
9) Incubation for 300 seconds. 
10) A third reading was taken and after correcting by the second reading, 
an mP value was calculated. 
The data below is comprised of the mP values produced during two runs of 
the assay as previously described. The column labelled COC contains the mP 
calculated from the second readings for their respective cuvettes, and 
that labelled CANNB contains the mP calculated from the third readings. 
The readings were produced by the ADx.RTM. instrument having already been 
corrected by the first, or blank, reading. In run #1. the samples were 6 
calibrators containing increasing concentrations of cocaine metabolite 
(benzoylecgonine). One can see the progressive lowering of the mP values 
for the 6 samples in the second or "COC" stage of the assay with little or 
no reaction in the third "cannab" stage. Conversely, for run #2 the 
samples are cannabinoid calibrators. One can see the lack of reaction in 
the second stage, while a progressive lowering of the mp values is seen in 
the third stage. This data demonstrates the selectivity of the assay 
technology. 
______________________________________ 
Sample CONCENTRATIONS COC CANNB 
______________________________________ 
Run #1 
COC A 0 ng/ml 181.6 170.6 
COC B 300 ng/ml 134.0 169.3 
COC C 1000 ng/ml 101.6 164.0 
COC D 2000 ng/ml 84.8 165.6 
COC E 3000 ng/ml 74.5 160.8 
COC F 5000 ng/ml 64.8 164.1 
Run #2 
CANNB A 0 ng/ml 180.3 166.5 
CANNB B 25 ng/ml 179.9 157.7 
CANNB C 40 ng/ml 179.2 143.6 
CANNB D 60 ng/ml 178.0 134.6 
CANNB E 80 ng/ml 178.6 122.6 
CANNB F 150 ng/ml 179.7 91.4 
______________________________________ 
Representative of ligands determinable by the methods of the present 
invention include steroids such as estrone, estradiol, cortisol, 
testosterone, progesterone, digoxin, digitoxin, thyroxine, 
triiodothyronine, antiasthmatic 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; antidepresant drugs including 
tricyclics such as nortriptyline, 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, hydromorphone, 
oxymorphone, codeine, hydrocodone, dextromethorphan, cannabinoids, various 
barbiturates, phencyclidine and and their metabolites and the like. 
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. In 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. 
In addition to the concentration range of the 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 and 
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. 
For purposes of presenting the combination FPIA invention for three 
analytes the following abbreviations will be utilized in the text: 
______________________________________ 
A.sub.1 = Analyte 1 Various unknown 
A.sub.2 = Analyte 2 quantities and/or 
A.sub.3 = Analyte 3 combinations of 
these may be in a 
given sample. 
S.sub.1 = Antiserum to analyte 1 
S.sub.2 = Antiserum to analyte 2 
S.sub.3 = Antiserum to analyte 3 
S.sub.1 S.sub.2 S.sub.3 = Combined antiserum 
reagent 
T.sub.1 = Tracer for analyte 1 
T.sub.2 = Tracer for analyte 2 
T.sub.3 = Tracer for analyte 3 
.multidot. = Denotes a bound pair 
(i.e., S.sub.1 .multidot. T.sub.1) 
mP = milli-Polarization, the unit 
of measure in FPIA 
[ ] = As the equilibrium formulas 
become more involved, brackets 
will be used to delineate the 
analyte specific parts of the total 
reaction. 
______________________________________ 
The following examples provide a description of the methodology according 
to the invention in stoichiometric terms: 
EXAMPLE II: A NEGATIVE SAMPLE 
Sample and combined antiserum are mixed and incubated. The resulting 
equilibrium is: 
EQU S.sub.1 S.sub.2 S.sub.3 &lt;-&gt;S.sub.1 +S.sub.2 +S.sub.3 
After incubation, a reading is taken and stored as Blank Reading "B1" 
T.sub.1 is added and the mixture incubated. The resulting equilibrium is: 
EQU S.sub.1 +S.sub.2 +S.sub.3 +T.sub.1 &lt;-&gt;[S.sub.1 +S.sub.1 .multidot.T.sub.1 
+T.sub.1 ]+S.sub.2 +S.sub.3 
After incubation, a reading is taken; "R1". This reading is corrected for 
"B1" and an mP value is calculated. In this scenario, the concentration of 
S.sub.1 .multidot.T.sub.1 is high, therefore the mP value is high denoting 
no concentration of A.sub.1 in the sample. The reading "R1" is then stored 
as "B2" T.sub.2 is added and the mixture incubated. The resulting 
equilibrium is: 
EQU [S.sub.1 +S.sub.1 .multidot.T.sub.1 +T.sub.1 ]+S.sub.2 +S.sub.3 +T.sub.2 &lt;- 
&gt; 
EQU [S.sub.1 +S.sub.1 .multidot.T.sub.1 +T.sub.1 ]+[S.sub.2 +S.sub.2 
.multidot.T.sub.2 +T.sub.2 ]+S.sub.3 
After incubation, a reading is taken; "R2". This reading is corrected for 
"B2", thus removing the fluorescence contribution of T.sub.1 from the 
total, and an mP value is calculated. In this scenario, the concentration 
of S.sub.2 .multidot.T.sub.2 is high, therefore the mP value is high 
denoting no concentration of A.sub.2 in the sample. The reading "R2" is 
then stored as "B3" T.sub.3 is added and the mixture incubated. The 
resulting equilibrium is 
EQU [S.sub.1 +S.sub.1 .multidot.T.sub.1 +T.sub.1 ]+[S.sub.2 +S.sub.2 
.multidot.T.sub.2 +T.sub.2 ]+ 
EQU S.sub.3 +T.sub.3 &lt;-&gt;[S.sub.1 +S.sub.1 .multidot.T.sub.1 +T.sub.1 ]+ 
EQU [S.sub.2 +S.sub.2 .multidot.T.sub.2 +T.sub.2 ]+[S.sub.3 +S.sub.3 
.multidot.T.sub.3 +T.sub.3 ] 
After incubation, a reading is taken; "R3". This reading is corrected for 
"B3", thus removing the fluorescence contribution of T.sub.1 an T.sub.2 
from the total, and an mP value is calculated. In this scenario, the 
concentration of S.sub.3 .multidot.T.sub.3 is high, therefore the mP value 
is high denoting no concentration of A3 in the sample. 
EXAMPLE III: A SAMPLE CONTAINING ANALYTE 2 ONLY 
Sample and combined antiserum are mixed and incubated. The resulting 
equilibrium is: 
EQU S.sub.1 S.sub.2 S.sub.3 +A.sub.2 =S.sub.1 +[S.sub.2 +S.sub.2 
.multidot.A.sub.2 +A.sub.2 ]+S.sub.3 
After incubation, a reading is taken and stored as Blank Reading "B1" 
T.sub.1 is added and the mixture incubated. The resulting equilibrium is: 
EQU S.sub.1 +[S.sub.2 +S.sub.2 .multidot.A.sub.2 +A.sub.2 ]+S.sub.3 +T.sub.1 
&lt;-&gt; [S.sub.1 +S.sub.1 .multidot.T.sub.1 +T.sub.1 ]+[S.sub.2 +S.sub.2 
.multidot.A.sub.2 +A.sub.2 ]+S.sub.3 
After incubation, a reading is taken; "R1". This reading is corrected for 
"B1" and an mP value is calculated. In this example, the concentration of 
S.sub.1 .multidot.T.sub.1 is high, therefore the mP value is high denoting 
no concentration of A.sub.1 in the sample. The reading "R1" is then stored 
as "B2" T.sub.2 is added and the mixture incubated. The resulting 
equilibrium is: 
EQU [S.sub.1 +S.sub.1 .multidot.T.sub.1 +T.sub.1 ]+[S.sub.2 +S.sub.2 
.multidot.A.sub.2 +A.sub.2 ]+ 
EQU S.sub.3 +T.sub.2 &lt;-&gt;[S.sub.1 +S.sub.1 .multidot.T.sub.1 +T.sub.1 ]+ 
EQU [S.sub.2 +S.sub.2 .multidot.A.sub.2 +S.sub.2 .multidot.T.sub.2 +A.sub.2 
+T.sub.2 ]+S.sub.2 
After incubation, a reading is taken; "R2". This reading is corrected for 
"B2", thus removing the fluorescence contribution of T.sub.1 from the 
total, and an mP value is calculated. In this example, the concentration 
of S.sub.2 .multidot.T.sub.2 has been lowered due to some of S.sub.2 being 
bound to A.sub.2, therefore the mP value is lowered denoting a 
concentration of A.sub.2 present in the sample. The reading "R2" is then 
stored as "B3" T.sub.3 is added and the mixture incubated. The resulting 
equilibrium is: 
##EQU2## 
After incubation, a reading is taken; "R3". This reading is corrected for 
"B3", thus removing the fluorescence contribution of T.sub.1 and T.sub.2 
from the total, and an mP value is calculated. In this example, the 
concentration of S.sub.3 .multidot.T.sub.3 is high, therefore the mP value 
is high denoting no concentration of A.sub.3 in the sample. 
EXAMPLE IV: A SAMPLE CONTAINING ANALYTE 1 AND ANALYTE 3 
Sample and combined antiserum are mixed and incubated. The resulting 
equilibrium is: 
EQU S.sub.1 S.sub.2 S.sub.3 +A.sub.1 +A.sub.3 &lt;-&gt;[S.sub.1 +S.sub.1 
.multidot.A.sub.1 +A.sub.1 ]+ 
EQU S.sub.2 +[S.sub.3 +S.sub.3 .multidot.A.sub.3 +A.sub.3 ] 
After incubation, a reading is taken and stored as Blank Reading "B1" 
T.sub.1 is added and the mixture incubated. The resulting equilibrium is: 
EQU [S.sub.1 +S.sub.1 .multidot.A.sub.1 +A.sub.1 ]+S.sub.2 + 
EQU [S.sub.3 +S.sub.3 .multidot.A.sub.3 +A.sub.3 ]+T.sub.1 &lt;-&gt; 
EQU [S.sub.1 +S.sub.1 .multidot.A.sub.1 +S.sub.1 .multidot.T.sub.1 +A.sub.1 
+T.sub.1 + 
EQU +S.sub.2 +[S.sub.3 S.sub.3 .multidot.A.sub.3 +A.sub.3 ] 
After incubation, a reading is taken; "R1". This reading is corrected for 
"B1" and an mP value is calculated. In this scenario, the concentration of 
S.sub.1 .multidot.T.sub.1 has been lowered due to some of S.sub.1 being 
bound to A.sub.1, therefore the mP value is lowered denoting a 
concentration of A.sub.1 present in the sample. 
The reading "R1" is then stored as "B2" 
T.sub.2 is added and the mixture incubated. The resulting equilibrium is: 
##EQU3## 
After incubation, a reading is taken; "R2". This reading is corrected for 
"B2", thus removing the fluorescence contribution of T.sub.1 from the 
total, and an mP value is calculated. In this example, the concentration 
value is high denoting no concentration of A.sub.2 present in the sample. 
The reading "R2" is then stored as "B3" 
T.sub.3 is added and the mixture incubated. The resulting equilibrium is: 
##EQU4## 
After incubation, a reading is taken; "R3". This reading is corrected for 
"B3", us removing the fluorescence contribution of T.sub.1 and T.sub.2 
from the total, and an mP value is calculated. In this example, the 
concentration of S.sub.3 .multidot.T.sub.3 has been lowered due to some of 
S.sub.3 being bound to A.sub.3, therefore the mP value is lowered denoting 
a concentration of A.sub.3 present in the sample. 
Following, are actual constituents of reagents for use in Example II, III 
and IV for two separate reagent systems. 
______________________________________ 
Constituents: 
______________________________________ 
AMPHETAMINE/METHAMPHETAMINE 
COCAINE METABOLITE 
CANNABINOIDS 
(ACC) 
S Antibody Pot 
Citrate diluent 974.12 mLs/Ltr 
AM/MT II Antibody Stock 
11.28 mLs/Ltr 
Cocaine Metabolite Antibody 
13.68 mLs/Ltr 
Stock 
Cannabinoids Antibody 
0.92 mLs/Ltr 
Stock 
Riboflavin Binding Protein 
10.0 gms/Ltr 
Citrate Diluent 
Citric Acid Anhydrous Powder 
19.2 gm/Ltr 
Sodium Hydroxide Pellets 
11.6 gm/Ltr 
Sodium Azide 1.0 gm/Ltr 
distilled water 995.0 gm/Ltr 
T1 AM/MT II Tracer Pot 
distilled water 1.0 Ltr/Ltr 
Sodium phosphate (monobasic) 
8.53 gm/Ltr 
Sodium phosphate (tribasic) 
14.53 gm/Ltr 
Bovine gamma globulin 
0.10 gm/Ltr 
Sodium Azide 1.05 gm/Ltr 
Amphetamine/Methampheta 
mine II Fluorescein 
T2 Cocaine Metabolite Tracer Pot 
distilled water 1.0 Ltr/Ltr 
Tris Ultra Pure 12.11 gm/Ltr 
6N HCl 13.0 ml/Ltr 
Bovine Gamma Globulin 
0.10 gm/Ltr 
Sodium Azide 1.00 gm/Ltr 
Cholic Acid (Sodium Salt) 
100.0 gm/Ltr 
T3 Cannabinoids - GS Tracer Pot 
distilled water 1.0 Ltr/Ltr 
Sodium phosphate (monobasic) 
8.53 gm/Ltr 
Sodium phosphate (tribasic) 
14.53 gm/Ltr 
Bovine Gamma Globulin 
0.10 gm/Ltr 
Sodium Azide 1.05 gm/Ltr 
Cholic Acid (Sodium Salt) 
50.0 gm/Ltr 
Cannabinoids - GS Fluorescein 
P AM/MT II Pretreatment Pot 
distilled water 0.992 Ltr/Ltr 
Sodium Periodate 42.78 gm/Ltr 
W Cannabinoids - GS Wash Pot 
distilled water 515.35 gm/Ltr 
Dimethyl Sulfoxide 550.05 gm/Ltr 
Sodium Chloride 4.50 gm/Ltr 
BARBITURATES 
PCP 
OPIATES 
(BPO) 
S1 Antibody Pot 
0.5M Hepes Buffer 979.92 mLs/Ltr 
Barbs II Antibody Stock 
15.80 mLs/Ltr 
PCP II Antibody Stock 
4.28 mLs/Ltr 
Riboflavin Binding Protein 
10.0 gms/Ltr 
0.5M Hepes Buffer Diluent 
distilled water 1000. 
HEPES, Powder 11.92 gm 
Ovalbumin 10.0 gm 
Bovine Serum Albumin 
8.0 gm 
Sodium Azide 1.0 gm 
Glycerol 119.0 gm 
6N Sodium Hydroxide 
as required 
S2 Antibody Pot 
OPIATES Antibody Diluent 
965.80 mLs/Ltr 
OPIATES Antibody STOCK 
4.20 mLs/Ltr 
Normal Sheep Serum 30.0 mLs/Ltr 
containing 0.15 mg/ml 
Dextromethorphan 
OPIATES Antibody Diluent 
distilled water 1.0 LTR/LTR 
Ethylene Glycol 20.0 mL/LTR 
(density = 1.114) 
Sodium Phosphate (tribasic) 
14.53 gm/LTR 
Bovine Gamma Globulin, 
0.10 gm/LTR 
Cohn FRACTION 2 
Sodium Azide 1.05 gm/LTR 
Sodium Phosphate (monobasic) 
8.53 gm/LTR 
T1 Barbiturates Tracer Pot 
distilled water 0.892 LTR/LTR 
Sodium Phosphate (dibasic) 
26.80 gm/LTR 
Heptahydrate 
6N Sodium Hydroxide 
55.0 mL/LTR (approx) 
Bovine Gamma Globulin 
0.1 gm/LTR 
Sodium Azide 1.00 gm/LTR 
5-Sulfo-Salicylate 50.0 gm/LTR 
Barbiturates Fluorescein 
T2 PCP Tracer Pot 
Tris Ultra Pure 12.11 gm/LTR 
6N HCl 13.0 ml/LTR (approx) 
Bovine Gamma Globulin 
0.1 gm/LTR 
Sodium Azide 1.00 gm/LTR 
distilled water 1.00 LTR/LTR 
Cholic Acid, (Sodium Salt) 
100.0 gm/LTR 
6N Hydrochloric Acid 
As required 
6N Sodium Hydroxide 
As required 
Phencyclidine Fluorescein 
T3 OPIATES Tracer Pot 
distilled water 0.965 LTR/LTR 
Citric Acid Anhydrous Powder 
9.6 gm/LTR 
Sodium Hydroxide Pellets 
21.8 gm/LTR 
Bovine Gamma Globulin 
0.1 gm/LTR 
Sodium Azide 1.00 gm/LTR 
5-Sulfo-Salicylate 50.0 gm/LTR 
6N Sodium Hydroxide 
As required 
Opiates Fluorescein 
W PCP II WASH POT 
distilled water 410.0 gm/LTR 
1-Butanol 81.0 gm/LTR 
Dimethylformamide 472.0 gm/LTR 
Sodium Chloride 4.5 gm/LTR 
______________________________________ 
The preceding provides a description of the inventive process in simple 
terms. In addition to the specific tracer added at each of the stages, 
quantities of the combined antiserum and the tracers from the previous 
stages must also be added to balance and maintain the various equilibria 
that are being established. Without the balancing additions of these 
previous reagents, the reagent concentrations would become progressively 
more dilute and the equilibria would shift from one stage to the next. 
This would cause a corresponding change to the mP of the previous 
tracer/antiserum component. An mP shift would be manifest in a change in 
the ratio of the horizontal and vertical readings contribution from that 
particular tracer and it could not then be back subtracted based on the 
previous reading's values. This is of major importance to the assay 
technology since, without the back subtraction, one cannot differentiate 
one tracer's contribution from another's as the second and third stage 
readings are taken. 
The balancing additions of antiserum and tracer are determinable 
mathematically from the volumes of the reaction mixture and the previously 
added volumes for the respective reagents. In simple terms, maintain the 
concentration of each of the reagents in the cuvette throughout the assay 
time. This is true however, only when each stage of the reaction is 
allowed to continue to complete equilibrium. In reality, each stage of the 
reaction is read slightly before equilibrium is reached and the reaction 
continues after the reading is taken. This is not a problem when the 
concentration of the analytes in the sample tested are negative or at 
moderate levels. The reactions approach their equilibrium levels rapidly 
and are almost complete when the reading is taken. Therefore, there is 
very little change in the mP of the tracer/antiserum combination and thus, 
no problem with the back subtraction which is important to the next stage. 
However, if one of the analytes is present in the sample at a very high 
level, the reaction approaches its final equilibrium more slowly and there 
is more of a continuation of the mP change after the reading is taken. 
This causes the back subtraction of this reading from the reading at the 
next stage to be in error and affects the answer obtained. In some cases 
this results in sufficient error in the subsequent mP calculated to give a 
false answer. 
It is this fact that requires a special optimization of the balancing 
additions of the reagents. Either of the antiserum or tracer reagents can 
be adjusted to effect the equilibrium, or position relative to the 
reaction. It has been chosen to keep the concentration of the combined 
antiserum reagent at the mathematically determined level and utilize the 
separate tracer reagents to modulate the reactions relative to 
equilibrium. It has been determined experimentally that adding less than 
the calculated volume of the balancing addition of the tracers at 
subsequent stages reduces to acceptable levels, or eliminates, the 
interferences caused by very high analyte concentrations. Based on the 
data it has been found that by adding differing volumes of the balancing 
tracer additions it is possible to change either or both of the previous 
and subsequent reactions in such a way that the mP obtained in the second 
or third stage of the assay is not affected by, or is affected to only a 
slight degree, by the concentration of the analytes reacting in the 
previous stages. 
EXAMPLE V 
The following is a presentation of actual data of the optimization of the 
balancing sips of tracers for a typical Combination FPIA assay in 
accordance with Examples II, III, or IV. The assay in this example is 
designed to test sequentially for Barbiturates, Phencyclidine, and 
Opiates. The optimization is performed in three segments. 
1) Optimize the second sip of tracer 1 to prevent a significant effect of a 
high concentration of analyte 1 on the detection of analyte 2. 
2) Optimize the second sip of tracer 2 to prevent a significant effect of a 
high concentration of analyte 2 on the detection of analyte 3. 
3) Optimize the third sip of tracer 1 to prevent a significant effect of a 
high concentration of analyte 1 on the detection of analyte 3. 
The optimization is done by varying the amount of the sip in question and 
monitoring the difference in the mP readings of the subsequent stage for a 
sample containing none of the analyte for the tracer in optimization and 
for a sample containing a very high concentration of that analyte. The 
tables below are comprised of the data from the actual runs in the three 
segments of the optimization of an assay. 
______________________________________ 
Optimize T.sub.1 2nd Sip 
Phencyclidine mP 
mP 
2nd Sip vol 
NHU* Barb Stock** diff*** 
______________________________________ 
1.0 163.17 174.92 11.75 
1.5 166.37 176.28 9.91 
1.6 163.14 174.34 11.20 
1.7 167.11 174.33 7.22 
1.8 169.76 173.32 3.56 
1.9 162.34 172.50 10.16 
2.0 161.25 165.48 4.24 
2.1 164.91 161.28 -3.63 .rarw. 
4.0 170.66 148.60 -22.06 
______________________________________ 
*Normal Human Urine 
**Secobarbital @ 1000 mg/ml 
***mPNHU minus mP Barb Stock 
.rarw. = optimum sip volume 
Optimize T.sub.2 2nd Sip 
Opiates mP 
mP 
Sip vol NHU Barb stock* 
diff 
______________________________________ 
1.0 292.98 317.95 24.97 
1.9 281.24 281.79 0.55 .rarw. 
2.0 278.37 275.83 -2.54 
2.1 279.42 271.30 -8.12 
______________________________________ 
*Phencyclidine @ 300 mg/ml 
.rarw. = optimum sip volume 
Optimize T.sub.1 3rd Sip 
Opiates mP 
mP 
Sip vol NHU Barb stock 
diff 
______________________________________ 
4.9 275.96 309.49 33.53 
6.0 275.81 277.39 1.59 .rarw. 
6.1 280.44 277.21 -3.23 
7.4 264.68 240.52 -24.16 
______________________________________ 
Based on the results of this testing the volumes of the tracer sips chosen 
for this particular set of reagents were as follows: 
______________________________________ 
Volumes Chosen (microliters) 
T.sub.1 T.sub.2 
T.sub.3 
______________________________________ 
1st 25 25 25 
2nd 2.1 1.9 -- 
3rd 6.0 -- -- 
______________________________________ 
Using these volumes, runs were performed with various samples and the 
following results were obtained, demonstrating, in actual runs, the lack 
of effect of the concentration of one analyte on the detection of another. 
______________________________________ 
mP 
Sample Barb PCP Opts 
______________________________________ 
NHU 229.95 161.99 286.11 
High Barb 59.88 159.56 287.38 
High PCP 232.08 40.51 287.84 
______________________________________ 
EXAMPLE VI 
EXAMPLE OF THE COMBINATION TECHNOLOGY ON ANALYTES OTHER THAN ABUSED DRUGS 
Following are date tables containing runs in the Combination format 
utilizing reagents from two assays currently running on the TDx.RTM. 
instrument. The assays are for the cardiac antiarrhythmic drug 
procainamide and its major metabolite N-Acetylprocainamide (NAPA). The 
reagents used are as designated below. The S-pot (antiserum rgt) was 
composed of a 50/50 mix of the S reagents from the two kits. The tracers 
were used in full strength as T.sub.1 and T.sub.2. The P-pot reagent from 
the procainamide kit was used in this experiment. 
The runs were accomplished by using the pipetting sequence currently 
utilized for the BPO assay, substituting the reagents as described above. 
The T.sub.3 reagent pot was filled with TDx.RTM. Dilution Buffer since 
only two active reagent systems were involved in these runs. The first 
stage is the procainamide reading and the second stage is the NAPA 
reading. 
Run #1 was a run with the procainamide calibrators and controls, and run #2 
was with the NAPA calibrators and controls. In Run #1 the downward trend 
as expected occurred for the calibrators in the mP 1 column. The slight 
upward trend in the mP 2 column (NAPA mP) is due to the nonoptimal 
settings of the T.sub.1 second sip, as noted before in explanation of the 
BPO assay optimization. In Run #2, the downward trend is seen in the NAPA 
mp 2 column. The slight downward trend in the mP 1 column is due to a 
slight cross-reactivity of the procainamide antiserum with the NAPA 
molecule. This points out a need for no, or very minimal, 
cross-reactivities of the antibodies used in Combination Assays. 
Also included in the data tables, is the output from a computer 
curvefitting program showing good curve-fits for both assays as evidenced 
by the controls reading close to their respective targets when read back 
from the fit curve. This demonstrates the selectivity of the Combination 
technology in this application. 
______________________________________ 
Concentrations 
Sample mP 1 mP 2 Target 
Actual* 
______________________________________ 
RUN #1 
Procainamide A Cal 
251.64 227.45 
Procainamide B Cal 
157.08 255.94 
Procainamide C Cal 
119.45 263.23 
Procainamide D Cal 
99.54 265.29 
Procainamide E Cal 
79.20 268.49 
Procainamide F Cal 
67.41 266.76 
Low Control 130.64 257.98 2.0 1.93 
Med Control 90.09 266.03 6.0 6.56 
High Control 75.51 265.11 15.0 12.39 
RUN #2 
NAPA A Cal 251.58 225.27 
NAPA B Cal 249.08 129.92 
NAPA C Cal 240.66 78.64 
NAPA D Cal 228.93 60.31 
NAPA E Cal 221.60 52.47 
NAPA F Cal 206.46 47.23 
Low Control 242.01 95.94 4.0 3.93 
Med Control 237.26 64.42 9.0 9.74 
High Control 211.12 48.80 25.0 25.27 
______________________________________ 
*As calculated from the calibrator mP by a computer curve 
fitting/interpolation program 
The Procainamide reagents consist of the following: 
P Pretreatment Solution. Surfactant in buffer with protein stabilizer. 
Preservative: 0.1% Sodium Azide. 
S Antisera. Procainamide Antiserum (Rabbit) in buffer with protein 
stabilizer. 
Preservative: 0.1% Sodium Azide. 
T Tracer. Procainamide--fluorescein tracer in buffer with surfactant and 
protein stabilizer. 
Preservative: 0.1% Sodium Azide. 
The N-Acetylprocainamide reagents consist of the following: 
P Pretreatment Solution. Surfactant in buffer containing protein 
stabilizer. 
Preservative: 0.1% Sodium Azide. 
S Antisera. N-acetylprocainamide Antiserum (Sheep) in buffer with protein 
stabilizer. 
Preservative: 0.1% Sodium Azide. 
T Tracer. N-acetylprocainamide--fluorescein tracer in buffer containing 
surfactant and protein stabilizer. 
Preservative: 0.1% Sodium Azide. 
Those skilled in the art will recognize or be able to ascertain, using no 
more than routine experimentation, many equivalents to the specific 
embodiments of the invention described herein. Such equivalents are 
intended to be encompassed within the scope of this invention.