Apparatus for performing fluorescent assays which separates bulk and evanescent fluorescence

This invention uses the evanescent wave detection of particles to distinguish bound from free in an analyte-binding assay. Illumination below the critical angle is employed, and a beveled window is used to eliminate bulk fluorescence from the emitted evanescent wave liquid. The sample is held in a non-rigid film cuvette.

FIELD OF THE INVENTION 
This invention relates to the use of evanescent wave detection of 
fluorescent particles to distinguish bound from free material in an 
analyte-binding assay. 
BACKGROUND OF THE INVENTION 
In order to determine the concentration of analytes in biological fluids, 
specific binding partners for analytes are often used. The analyte 
concentration is determined by generating a signal which is modulated in 
accordance with the amount of analyte bound to the binding partner. Many 
forms of binding assays have been described, most of which depend on a 
physical technique such as centrifugation or filtration to separate bound 
from free material. These techniques can be complex and expensive to 
automate. On the other hand, the optical phenomenon known as "total 
internal reflection" has been used to distinguish bound from free material 
in several immunoassay systems that do not require a mechanical separation 
step. Immunoassay systems employ antibodies as the analyte binding 
partners; the principles used, however, can be applied to other forms of 
binding assays such as those using hormone receptors or DNA probes. 
Total internal reflection is a phenomenon that occurs when light is aimed 
at a glancing angle, above the so-called "critical angle", from a medium 
of high refractive index, such as glass, toward a medium of lower 
refractive index, such as water. The beam of light is reflected at the 
interface between the two media. Total internal reflection is described in 
E. Hecht and A. Zajac, Optics, Addison-Wesley Publishing Co., Reading, 
Mass. (1974), pp. 81-84. 
Under conditions of total internal reflection, it can be demonstrated that 
a portion of the light called the "evanescent wave" penetrates the 
low-refractive-index medium to a depth of a fraction of a wavelength, 
typically 100 nm or so. This light will therefore illuminate materials 
which are bound at the interface between the two media; materials not at 
the interface will not be illuminated. This provides a separation, a means 
of distinguishing bound from free material, without the need for a 
mechanical separation device. 
U.S Pat. No. 3,939,350 (Kronick et al.) describes an immunoassay system 
employing haptens or antibodies attached to a glass prism having a surface 
in contact with an aqueous medium. Immunologically-bound fluorescent 
antibodies are detected by their presence within the region illuminated by 
the evanescent wave. To achieve this, Kronick designed a system such that 
light enters the sample chamber above the critical angle. This requires 
sophisticated light sources such as lasers or arc lamps to produce a small 
diameter, collimated beam for illumination of a small sample. The samples 
are placed on a slide but nevertheless the sample chamber must be cleaned 
after each assay. 
U.S. Pat. No. 4,451,434 (Hart) utilizes fluorescent latex particles as a 
label, giving potentially much greater signal per binding event than that 
obtainable by Kronick et al. Although an improvement over Kronick et al., 
Hart still is faced with the problem of using sophisticated light sources 
with the inherent disadvantages just related. Also, high quality sample 
cuvettes must be used which are formed to incorporate prisms. Even when 
well manufactured, plastic devices will not in general have the high 
optical quality of the glass, quartz or sapphire prisms. 
EP 0 326 375 and EP 0 254 430 (Schutt et al.) describe a similar 
immunoassay system in which light-scattering particles, such as polymer 
latex or colloidal gold, are used in place of the fluorophores described 
by Kronick et al. The examples in these patents indicate that assays 
employing evanescent wave phenomena can achieve sensitivity otherwise 
obtainable only in assays that employ a mechanical separation step. 
Otherwise, Schutt et al. suffer from the same disadvantages as Hart and 
Kronick et al. 
U.S Pat. No. 4,447,546 (Hirschfeld) is an example of the use of an optical 
fiber or rod-like waveguide in an immunoassay. Since light is confined 
inside an optical fiber by a series of internal reflections, an evanescent 
wave field exists along the entire surface of the fiber. Antibodies or 
antigens are attached to the fiber, and the fiber is then immersed in the 
sample to be tested. Fluorescence or other optical changes can be detected 
at an end of the fiber. Because some such devices can be immersed directly 
into a neat biological fluid, they are sometimes referred to as 
"biosensors". Whatever Hirschfeld's advantages, his system still requires 
cleaning or replacement of the biosensor after each use. 
U.S Pat. No. 4,810,658 (Shanks et al.) describe a waveguide which is placed 
in contact with an illuminated sample. Fluorescence from bound material 
produces an evanescent wave in the waveguide which exits the waveguide 
above the critical angle. Fluorescence from unbound material is refracted 
at the interface, and can therefore only exit the waveguide at an angle 
below the critical angle. 
Systems such as those described by Schutt et al. and Shanks et al. rely 
upon optical apertures to limit the acceptance angle of the detector so 
that non-evanescent waves are excluded. The edge of the waveguide in these 
cases is an extended light source, as opposed to a point source. That is, 
light is emitted from regions that do not lie on the optical axis of the 
system. Under these conditions, no aperture can be designed that accepts 
all rays up to a given incidence angle, a, and rejects all others. Thus 
separation of evanescent and non-evanescent radiation will be less than 
ideal. Under most assay conditions, the evanescent signal is much weaker 
than the non-evanescent background, so good separation is essential. 
Furthermore, apertures must be properly aligned with respect to the 
waveguide and the detector in order to function well. 
Another disadvantage of Shanks et al. is that the illuminating beam passes 
through the sample in order to reach the waveguide. This increases the 
interfering effects in the bulk solution of light scattering or absorbing 
substances on the evanescent wave signal. 
SUMMARY OF THE INVENTION 
Many of these disadvantages of the prior art are overcome by the apparatus 
of this invention. According to this invention, an apparatus is provided 
for detecting an analyte of interest in a sample by using a source of 
excitation radiation and a tag capable of causing inelastic scattering of 
the excitation radiation, the apparatus comprising: an optically 
transparent sample holder having an interior volume and an inner wall, an 
optically transparent member adapted to contact the sample holder to 
provide an optical interface with the sample, the transparent member 
having a refractive index greater than the refractive index of the sample, 
means for directing radiation from the source to the transparent member at 
angles below the critical angle relative to the optical interface, thereby 
to illuminate the interior of the sample holder, first and second binding 
members, the tag being attached to the first binding member, the second 
binding member being immobilized on the inner wall of the sample holder at 
the optical interface, such that the presence of analyte in the sample 
modulates the attachment of the tag to the wall, and a first detector for 
detecting radiation produced by such inelastic scattering, the transparent 
member being shaped to direct evanescent wave radiation, from the inner 
wall of the sample holder, lying between the plane of the analyte binding 
inner wall and the total internal reflection critical angle of the optical 
interface, to the detector. 
Using this apparatus, low F-number illumination optics can be employed. The 
low F-number permits the use of inexpensive lamps such as quartz-halogen 
lamps. Furthermore, the illuminating beam need not pass through the sample 
prior to reaching the transparent member. Thus the illuminating beam may 
strike the bioactive surface before passing through the sample. This 
reduces the interfering effects of light scattering or absorbing 
substances on the evanescent wave signal. 
The window geometry employed in this invention uses the internal reflection 
principle to efficiently collect rays below a given angle of incidence a, 
and to reject all rays of greater incidence angle, even for an extended 
light source. No precise alignment is necessary to achieve this 
separation, since the angle a is determined solely by the shape and 
refractive index of the window. 
The apparatus also takes advantage of a unique form of sample container not 
previously utilized for evanescent wave immunoassays. The sample is 
contained in a bag formed from transparent, flexible film. Such a bag, or 
sample pack, can be pressurized between two parallel windows to form a 
cuvette for spectrophotometric analysis. In the present invention, the 
second window may be non-transparent. A refractive-index-matching oil may 
be used to improve the optical coupling between the window and the sample 
pack. The window incorporates a prism that transmits incident illumination 
in one direction, and transmits the evanescent wave fluorescence in 
another direction. The advantage here is in using a window which is 
separate from the biochemically-reactive sample pack wall. 
The pack wall can be made of a cast or blown film, which can easily achieve 
the required optical transparency and flatness at low cost. Freedom from 
optical defects is essential to achieving high-sensitivity evanescent-wave 
assays, since optical defects couple signal from the bulk solution into 
the detector, and hence contribute background noise. This invention 
eliminates the challenge of mass-producing high-optical-quality sample 
cuvettes which incorporate prisms, waveguides or optical fibers, as in the 
prior art devices, respectively. Even well-molded, plastic devices will 
not in general have the high optical quality of the glass or quartz prisms 
that can be incorporated in this invention. 
Use of a pack film separate from the prism also simplifies the problem of 
coating bioactive material onto the surface, since, in general, coating 
film is easier to mechanize than coating discrete devices. Also, the 
chemistry of the film for coating can be controlled without regard to its 
molding or extrusion properties that would affect the formation of a 
prism, waveguide, or fiber. Likewise, since the prism is not in contact 
with the sample, it can be designed without considerations of 
biocompatibility or coating. 
In short, illumination below the critical angle is employed. A shaped 
window is used to eliminate bulk fluorescence from the emitted evanescent 
wave signal. Finally, the sample is held within a non-rigid film cuvette.

DESCRIPTION OF THE INVENTION 
FIG. 1 shows a schematic representation of the present invention in which 
there is shown a light source 10. The invention may employ any light 
source capable of exciting fluorescence or phosphorescence. These sources 
10 include tungsten or quartz-halogen lamps, arc lamps, flashlamps, 
light-emitting diodes, lasers, etc. The light source 10 will generally be 
stable and continuous, but may be pulsed or chopped to permit synchronous 
detection as a means of noise reduction. Light 11 from the source 10 is 
directed toward a sample cuvette 26 which is more clearly shown in FIG. 2. 
In the case shown, a quartz-halogen lamp 10 is used, since this type of 
lamp generates adequate light power in a simple and inexpensive 
configuration. Since the rays from such a lamp are divergent, a lens 12 is 
used to collect rays and direct them toward the sample cuvette 26. The 
light may be filtered by a color filter 14 to remove any incident 
radiation at the emission wavelength which would interfere with detection, 
excess infrared radiation that could overheat the sample, and any 
non-exciting wavelengths that could cause nonspecific fluorescence of the 
sample, window, cuvette, or other components. 
A beamsplitter 16 or other device may be used to sample the incident beam 
as by the split light 17 in order to establish a reference signal for 
ratio measurement or synchronous detection by reference diode detector 18. 
The incident light then passes through an aperture 19. 
Incident light leaving the aperture 19 passes through a first member or 
window 20 to a sample cuvette 26. This window 20 must be transparent at 
the excitation and emission wavelengths of the fluorophore or phosphor, 
must be substantially non-fluorescent and non-phosphorescent, and must be 
of good optical quality, since scattering centers may couple extraneous 
light into the evanescent wave signal. The window must be of higher 
refractive index than the bulk sample 25 contained in the cuvette 26 in 
order for evanescent wave generation to take place. Generally, optical 
glass, quartz or sapphire will be used for the window 20. 
The aperture 19 is used so that portions of the sample cuvette 26 not in 
contact with the windows 20, 22 will not be illuminated. Areas of poor 
contact between the cuvette and the windows can produce small, flexible 
prisms which couple unpredictable amounts of bulk fluorescence into the 
evanescent wave. This can lead to spurious signals. 
After the light leaves the window 20, it enters the sample 25 through the 
cuvette wall 29, which is in contact with the window 20 and preferably is 
made of a flexible film 60. A liquid such as microscope immersion oil may 
be used to ensure good optical coupling, i.e., optical interface, between 
the film 60 and the window 20, since the evanescent wave will not cross 
any air gaps. 
The cuvette film 60 must be transparent, substantially non-fluorescent, and 
of good optical quality. The blowing or casting processes used to form 
films can be designed so that the surface tension of the film 38 generates 
a flat, high-quality optical surface. Such processes are well known and 
described, for example, by Baird, R. J. and Baird, D. T., Industrial 
Plastics, The Goodheart-Willcox Co., Inc., South Holland, Ill. (1982), p. 
106, 177-183. The film 60 must be of higher refractive index than the bulk 
sample 25 so that an evanescent wave will be formed. The film 60 must be 
mechanically strong and flexible if high pressures are used to force good 
contact between the film 60 and the window 20. Surfactant treatment of the 
film 60 may be necessary to eliminate air bubbles in the sample, and to 
reduce nonspecific binding of fluorescent material. The film 60 must be 
capable of binding a bioactive molecule, without substantial shedding or 
loss of activity of that molecule. A blown, ionomeric film such as Surlyn 
(R) can be used successfully. Other films such as polyvinyl chloride or 
polystyrene may also be used. Bioactive materials can be coupled to such 
polymers by absorption or by covalent bonding to moieties such as 
carboxyl, hydroxyl or amino groups present on or added to the polymer 
surface. Bioactive materials may be applied to the film 60 by a number of 
coating processes, including spraying, soaking, or printing. 
Light 11 passes through the first window 20 normal (perpendicular) to the 
window. This maximizes the light reaching the sample cuvette 26. Beyond 
the sample cuvette 26 is a second window 22 made of the same material as 
the first window 20. Light passing directly through the sample 25 in the 
sample cuvette 26 and the second window 22 is directed to a sample diode 
detector 36. This permits sample absorbance to be measured simultaneously 
or alternatively with the evanescent wave measurement which will be 
described below. Thus, more than one analyte may be detected at one time 
or assay quality measurements may be made. 
Inside the cuvette, an assay or biochemical process is used to bind the 
fluorescent tag particles to the inner film wall 29. Examples of such 
processes are shown schematically in FIG. 3. The extent of binding of the 
particles is modulated by the presence of an analyte of interest. FIG. 3a 
shows a "sandwich"-type assay, which is a well-known form of assay 
typically for antigens or nucleic acids. Attached to the film inner wall 
29 is a substance 40 capable of binding to the analyte of interest 46; 
attached to a fluorescent latex particle 42 is a second binding partner 44 
capable of simultaneously binding to the analyte 46. The use of the latex 
particles is preferred. In this case, a "sandwich" is formed in the 
presence of the analyte 46, and the extent of fluorescent particle binding 
increases with increasing analyte concentration. 
FIG. 3b shows a "competitive binding" assay, typically used for 
immunochemical detection and also for assays involving hormones and 
receptors, lectins, sugars, etc. In this type of assay, the surface of the 
film inner wall 29 bears a binding partner 50 for the analyte 52. 
Fluorescent latex particles 54 bear the analyte 52 or an analog thereof. 
In the absence of analyte 52 in the sample, the particles 54 bind 
extensively to the surface 29. The presence of analyte 52 inhibit the 
binding of particles 54 by occupying receptors of the binding partners 50 
on the film inner surface 29. Thus high analyte concentrations result in a 
lower extent and rate of binding. 
FIG. 3c shows a competitive-binding assay with the roles of the fluorescent 
particles 54 and the film inner wall 29 reversed, that is, the film inner 
wall 29 bears the analog 52' of an analyte and the particles 54 bear the 
analyte receptor or binding partner 50. 
In this invention, a fluorescent latex is used to generate signal. The 
fluorescent latex particles 42, 54 give a much greater signal per binding 
event than does a single fluorescent molecule per binding molecule. The 
fluorescent latex may consist of particles from about 0.01 to 1.0 u in 
diameter. Larger particles may settle during the assay, depending on their 
density. Latex particles are well known and described by Bangs, L. B., 
Uniform Latex Particles, Seradyne, Inc., Indianapolis, Ind. (1987) pp. 
3-8. They can be easily made by emulsion polymerization processes, from 
materials such as polystyrene or polymethyl methacrylate. Care must be 
taken to ensure that the particles do not irreversibly aggregate during 
their manufacture, dyeing, labelling, storage, or use. Proper buffer ionic 
strength and surfactant concentration can prevent aggregation. 
Particles can be dyed with fluorophores by a variety of methods. One such 
method is described, for example, by Bangs, L. B., Op. Cit., pp. 40-42. 
The efficiency of evanescent wave detection of fluorescence will vary with 
the particle refractive index in a manner described by in E. H. Lee, et 
al., "Angular distribution of fluorescence from liquids and monodispersed 
spheres by evanescent wave excitation", Applied Optics 18 (6), Mar. 15, 
1979, pp. 862-868. Therefore the refractive index of the particles should 
be chosen to maximize the amount of fluorescent emission which is directed 
toward the detector. The size of the particle will affect assay 
sensitivity in a complex way, because size will affect the diffusion 
coefficient of the particle and the surface-to-volume ratio of the system. 
Therefore size affects the reaction rate, as well as the amount of 
fluorescence per particle. Since the particle diameter may be on the same 
order of magnitude as the wavelength of incident illumination, one may 
take advantage of resonance effects which result in enhanced brightness 
for certain particle sizes. 
The fluorescent dye in the latex should be chosen to have a high extinction 
coefficient for the exciting wavelength, high quantum yield, and a 
sufficient Stokes shift to simplify the excitation and emission color 
filters for minimization of scattered light from passing through pair. It 
may be preferable to use an emission wavelength longer than about 550 nm, 
to reduce fluorescence from biological samples and plastic materials in 
the instrument and cuvette. The dye may have a fluorescence lifetime 
greater than that of biological materials and plastics, so that 
time-resolved detection can distinguish the dye from background sources of 
fluorescence. The dye may be phosphorescent rather than fluorescent, with 
a lifetime on the order of seconds or more. Since the dyes may be embedded 
in the polymeric latex, it is not required that the dye be water-soluble 
or fluorescent in an aqueous phase. 
Alternatively, a chemiluminescent molecule may be used in place of the dye. 
In this case, an external light source need not be used, but rather, the 
chemicals required to activate luminescence must be added to the sample. 
As noted by Hart, an absorbing dye may be added to the sample in order to 
reduce interference from fluorescence in the bulk of the sample. 
A fluorescent latex having a spectrum distinct from that of the particles 
used in the assay, but which binds to the binding moiety on the cuvette 
film, may be added to the sample, and measured at a second set of 
wavelengths, to correct for variations in system gain and in the density 
of binding sites on the film. Care must be taken to ensure that such a 
latex does not interfere with the binding of analyte to its partner. 
A fluorescent dye or non-binding fluorescent latex having a spectrum 
distinct from that of the bioactive particles may be added to the sample, 
and measured at a second set of wavelengths, to correct for fluorescence 
background from the bulk. 
Because light is directed into the sample, both bound and free latex 
particles will emit radiation, i.e., fluoresce, if a fluorescent material 
is used, and will emit their radiation at all possible angles. However as 
described by Hecht et al., fluorescence from the bulk of the sample cannot 
enter the window at an angle above the critical angle of the sample-window 
optical interface. Fluorescent material bound to the surface 29, on the 
other hand, generates an evanescent wave that can be emitted above the 
critical angle. The difficulty in efficiently separating and detecting 
only the evanescent wave emission from such wall surface 29 is obviated by 
this invention. 
In accordance with this invention, the first transparent member or window 
20 is formed to have a beveled edge 28 which is used to separate emission 
above the critical angle from that below. The bevel angle is chosen so 
that all light crossing the sample-window interface less than the critical 
angle will be internally reflected at the beveled edge or face 28, and 
will thus not enter a detector 36. The rays from the exiting evanescent 
wave are directed through a condenser lens 32 and color filter 34 to a 
suitable detector 36. Nearly all rays entering the window above the 
critical angle will pass through the beveled face 28, and will reach the 
detector 36. These rays will be bent (refracted), and this must be kept in 
mind in determining the proper position for the detector. 
Because the beveled window edge 28 excludes bulk fluorescence of the sample 
in the interior of the sample cuvette from the detector, in the absence of 
particle binding to the cuvette wall 29 there would ideally be no 
evanescent wave, and therefore no background fluorescence signal. In 
practice, there are two main sources of background. First, any microscopic 
defects in the cuvette film or window may act as prisms which couple the 
bulk fluorescence into the detector. Second, a certain number of particles 
will be close enough to the film to generate an evanescent wave, even 
though they are unbound, merely as a consequence of the uniform spatial 
distribution of particles within the cuvette. There may also be binding of 
particles to the film which is not mediated by the presence of analyte. 
Such so-called "nonspecific binding" is a nearly universal phenomenon in 
binding assays. The assay buffer is formulated to maximize the assay 
signal while minimizing nonspecific binding. Buffer salts, proteins, and 
surfactants are generally used for this purpose. 
The evanescent emission, whether collected with the lens 32 or an optical 
fiber (not shown), may be passed through an aperture (not shown) to remove 
stray light. The light must be filtered as by filter 34 to remove any 
light at the excitation wavelength, so that scattered light is not 
detected. This will remove the typically large scatter background signal. 
Suitable filters may include interference filters and colored glass 
filters. Light then reaches the detector 36 sensitive to the fluorescent 
emission wavelength. Examples of detectors 36 include photomultipliers 
with either current (analog) or photon-counting electronics, vacuum 
photodiodes, silicon or other photodiodes, or photoconductive materials. 
Low light levels usually require high sensitivity normally provided by the 
PMT. 
Electronic signals from the evanescent wave detector 36, and from the 
reference detector 18 and absorbance detector 24, if any, are typically 
sent to a digital computer for processing. One advantageous form of 
processing is to measure the ratio of the evanescent signal to that of the 
reference detector 18, to cancel out signal variations due to changes in 
lamp intensity. Another advantageous form of signal processing is to 
measure the rate of change of the evanescent fluorescence signal over 
time, as the biochemical reaction takes place. This effectively removes 
any background signal, since the background signal does not generally 
change with time. 
In a preferred embodiment of this invention, as best seen in FIG. 2, the 
first and second windows 20, 22 may be part of a cell or sample cuvette 26 
forming device of the type described in U.S. Pat. Nos. 3,770,382 and 4,066 
362 issued to Carter et al and used in the aca.RTM. Automatic Clinical 
Analyzer. As is described by Carter et al. the windows 20, 22 define 
recesses in respective jaws 70, 93, the jaw 70 which forms the first 
window 20 being fixed, and the jaw 93 which may form the second window 22 
being movable. If desired, the jaw 93 may be formed of an optical 
material, as previously described, with a recessed formed for the window 
22. The sample cuvette in this case is an aca.RTM. pack which has walls 
formed of flexible, transparent plastic film 38 (FIG. 1) as previously 
described. Thus when the jaws 70, 93 are brought together on the 
deformable part of a sample cuvette 26, the jaws 70, 93 squeeze the walls 
of the pack into the shape of the recess in the jaws thus forming the 
sample cell 26. The jaws may be actuated by any suitable mechanism. The 
mechanism used in the Automatic Clinical Analyzer sold by E. I. du Pont de 
Nemours and Company, Wilmington, Del. is suitable for this purpose. A 
simplified version of this mechanism is illustrated in FIG. 2 and includes 
a pivoted mechanical linkage 100 which is moved upwardly by a motor drive 
102 to pivot about the pivot 104. This causes the jar of movable platen 93 
to move toward the fixed platen 70 and form the aca.RTM. pack 26 into an 
optical window as described. 
As described in Carter et al., a fluid applicator is included to coat the 
surface between the pouch of the sample cuvette 26 and the windows 20, 22 
with an optical coupling fluid. This fluid stabilizes any recesses between 
the pouch and the window formed by imperfections in the film from which 
the pouch is made and which would otherwise change dimensions during 
transmission measurements enough to introduce substantial error or form 
interference fringes. 
The advantages of the apparatus of this invention are many. The use of a 
film pack that is separate from the windows 20, 22 simplifies the problem 
of coating bioactive material onto surfaces. It is generally easier to 
mechanize such coating onto film. Also, the cavity for the sample chamber 
26 and prisms need not be cleaned after each use since they never become 
contaminated in the first place. The window geometry employed efficiently 
collects rays below a given angle of incidence. Precise alignment of the 
system is not necessary. Finally by allowing the light to enter the sample 
region below the critical angle, low F-number illumination optics can be 
employed. Further the exciting light is not attenuated before it strikes 
the area of interest. 
EXAMPLE 
Experiments were carried out by modifying the photometer of an aca.RTM. IV 
Clinical Analyzer (E. I. du Pont de Nemours and Company, Wilmington, 
Del.). This photometer contains the means for pressurizing a sample pack 
between two windows, and for applying a refractive-index-matching oil 
between the film and the windows. The lamp was used with an excitation 
filter having a center wavelength of 530 nm, and a bandwidth of 10 nm 
(Corion Corp., Holliston, Mass., #P10-530-F). The standard lamp-side 
window platen was removed, and was modified to hold a quartz circular 
window with a face beveled 70.degree.. from the plane of the window face. 
The beamsplitter holder was modified to hold detection optics at a 
50.degree. angle from the plane of the window face. Evanescent light was 
collected through a double-convex lens (Edmund Scientific Co., Barrington, 
N.J., #32860, 6 mm diameter, 6 mm focal length), an interference filter, 
(Corion Corp. #P70-650-A, 650 nm center wavelength, 70 nm bandwidth), and 
an aperture approximately 1 mm wide and 3 mm high. The lens was positioned 
to image the sample-window interface onto the aperture with a 2:1 
demagnification. 
The detector was a R1547 side-on 1/2"-diameter photomultiplier (Hamamatsu 
Corp., Bridgewater, N.J.) with a spectral response from 185 to 850 nm. The 
dynode resistors were each 200,000 ohms, and the anode was terminated in 
50 ohms. Coaxial cable carried pulses to a photon counting system (Modern 
Instrumentation Technology, Inc., Boulder Colo., F-100T or equivalent). 
The photomultiplier was operated at 1000 volts, with a discriminator 
threshold that gave approximately 120 dark counts per second. Photon count 
rate was measured with a digital frequency meter (Hewlett-Packard Co., 
Palo Alto, Calif., 5300B, 5306, 5312A or equivalent). This was connected 
via HP-IB interface to a desktop computer (Hewlett-Packard Co. HP-85) 
which plotted the photon count rate as a function of time, and calculated 
the slope of the curve at one-minute intervals. 
In order to demonstrate a sandwich immunoassay for Thyroid-Stimulating 
Hormone (TSH), a monoclonal antibody to TSH known as 972 was used. This 
antibody was developed by E. I. du Pont de Nemours and Company. Reagent 
chemicals were from Sigma Chemical Co., St. Louis, Mo. except as noted. 
The antibody was diluted to 1 mg/mL in a pH 7.0 phosphate buffer (5.42 g 
monosodium dihydrogen phosphate, 16.3 g disodium monohydrogen phosphate 
heptahydrate, 990 mL water). A 10% suspension of latex particles of 0.41 u 
diameter with a --COOH surface and a rhodamine dye were obtained from 
Bangs Laboratories, Inc., Indianapolis, Ind. One mL of antibody solution 
was mixed gently with 0.1 mL of 10% latex particle solution. To this was 
added 0.011 mL of a 10 mg/mL solution of EDAC, 
1-ethyl-3(-3-dimethylaminopropyl) carbodiimide-HCl, in water. The 
particles, antibody, and EDAC were incubated overnight at room 
temperature, then centrifuged for 10 min at 10000 RPM. This resulted in 
coating of the particles with the antibody. 
The next step was to overcoat the particles with bovine serum albumin 
(BSA), to reduce nonspecific binding. Ten mg/mL BSA was dissolved in 
phosphate buffer at pH 7.0. The particles were resuspended by vigorous 
vortex mixing and sonication in 1 mL of this solution, incubated for 2 hr 
at room temperature on a rocker table, and centrifuged as above. 
It is necessary to wash the particles thoroughly, since any antibody shed 
during the assay will inhibit signal generation. This was accomplished by 
resuspending the particles in 1 mL of 15 mg/mL glycine and 0.1 mg/mL 
sodium dodecyl sulfate in phosphate buffer, pH 7.0, and then washing (by 
centrifugation as above) the particles three times in this buffer. 
The particles were stored at 4.degree. C. in a solution of 15 mg/mL 
glycine. 
To form the other half of the sandwich assay, aca.RTM. packs were coated 
with a second monoclonal antibody to TSH, known as 4/46. This antibody was 
diluted to 0.1 mg/mL in citrate-phosphate buffer, pH 4.8, 0.15 M NaCl, 0.1 
mg/mL sodium azide (antibody coating buffer). Unsealed, unfilled aca.RTM. 
sample packs were obtained. A rubber dam was placed over the inside of the 
pack, with a 15 mm diameter hole over the area of the pack which is 
illuminated in the photometer. Into that hole was placed 0.75 mL of the 
antibody solution. This was incubated for 1 hr at room temperature, 
aspirated, and then washed three times with the antibody coating buffer 
described above. 
The film was then treated to minimize nonspecific binding with 0.75 mL of 
blocking solution, 50 mg/mL BSA, 1 mg/mL Tween.RTM.-20, 0.05 M NaCl in 
phosphate buffer, pH 7.0. This was incubated for 1 hr at room temperature, 
and aspirated. A second coating was done in a similar blocking solution, 
except the Tween.RTM.-20 was replaced with 50 mg/mL trehalose, and the 
NaCl was omitted. This solution was aspirated, and the packs were allowed 
to dry for 3 hr in a dry room at 22.degree. C. The packs were then sealed 
on a heat-sealing machine used in pack manufacture. 
An assay buffer of Sigma phosphate-buffered saline (12 mM phosphate, 120 mM 
NaCl, pH 7.5) with 1 M added NaCl and 0.05% Tween.RTM.-20 was used. 
Calibrators consisted of horse serum containing added amounts of purified 
TSH. Two mL of assay buffer and 0.5 mL of each calibrator were pre-mixed 
and then added to an aca pack prepared as above. The packs were incubated 
for 15 min at room temperature. This was followed by the addition of 0.5 
mL of the antibody-coated latex prepared above in 2 mL of assay buffer. 
The packs were then placed into the photometer, and the jaws were closed 
to pressurize the cuvette. Reaction rates were measured from 120 sec to 
420 sec after the jaws were closed. Results were as follows: 
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Photon Counts/sec/min 
TSH Concentration 
Rate Signal 
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50 uIU/mL 1097 CPS/min 
0.5 uIU/mL 530 CPS/min 
0 uIU/mL 420 CPS/min 
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These results demonstrate a relationship between the dose of TSH present 
and the rate of particle binding to the cuvette wall.