Method and apparatus for heterogeneous chemiluminescence assay

Apparatus and method for performing a chemiluminescence assay involving the immobilization of a chemiluminescent reaction complex to a solid, porous element. The solid, porous element is preferably treated to provide an immobilizing interaction with the chemiluminescent reaction complex wherein the chemiluminescent reaction complex is thereby immobilized to the solid, porous element. The activating and reading of the chemiluminescent reaction are separately performed by evenly distributing a concentrated chemiluminescent activating solution to form a puddle on the surface of the porous element to which the chemiluminescent reaction complex is immobilized.

BACKGROUND OF THE INVENTION 
The present invention relates to methods and apparatus for measuring a 
chemiluminescent signal from a solid surface. In particular, the present 
invention relates to methods and apparatus for use in heterogeneous 
immunoassays wherein a chemiluminescent signal provided by the immobilized 
product of an immunochemical reaction from a solid, porous matrix is 
measured. 
Several automated chemiluminescence instruments use photographic means and 
a densitometer for recording signal. Vogelhut, U.S. Pat. No. 4,231,754; 
Whitehead, et al., U.S. Pat. No. 4,593,728; Thorpe, et al., Clin. Chem., 
30, 806, (1984); and Bunce, et al., Analyst, 110, 65 (1985). 
Clear coated microtitration plates as a solid phase with trigger solution 
port and detector at opposite sides of the plate well may also be employed 
in such instruments. Holley, European Patent Application 025,350; and 
Schroeder, et al., Clin. Chem., 27, 1378-1384 (1981). This technique is 
severely limited due to the slow reaction rates resulting from the limited 
diffusion of analyte molecules to the capturing solid phase. Use of this 
approach has been of limited sensitivity and is generally employed in 
reactions involving the use of ATP and luminol-type tracers. 
Magnetizable microparticles and magnetic separation in a test tube may be 
followed by reading the signal of the suspended particles in a tube 
luminometer as is found in the commercially available Magic.TM. Lite 
system distributed by Ciba-Corning Diagnostics. Because the brown colored 
microparticles optically interfere with the chemiluminescent signal, a 
very low mass of these particles is used. This leads to very slow 
reactions. For example, an assay for thyroid stimulating hormone (TSH) is 
reported to have a three-hour incubation time. In addition, many 
manipulation steps are involved, making this assay configuration difficult 
to automate. Other luminometers in which the signal is generated in a test 
tube are sold by Berthold, Hamilton, Turner Design and Analytical 
Luminescence. 
Enhanced chemiluminescent reactions in a white microtitration plate 
followed by reading the generated signal in a luminometer having a moving 
mask and photomultiplier tube are described in Lisenbee, et al., European 
Patent Application 194,102 and are incorporated in the Amerlite.TM. system 
sold by Amersham Inc. This latter technique suffers from the same 
limitations of an ELISA assay in a coated plate, namely the slow diffusion 
rate of the reactants to the capture phase. 
SUMMARY OF THE INVENTION 
The present invention provides a method for directly exciting and measuring 
a chemiluminescent signal emanating off an immune complex immobilized on 
or in a solid, porous element that is used as a separation means in a 
heterogeneous immunoassay and an apparatus for performing this 
measurement. More specifically, it provides an automated means for 
performing heterogeneous chemiluminescent immunoassays of high 
sensitivity. 
The present invention provides a method and apparatus for measuring a 
chemiluminescent signal produced by an immobilizable immune complex 
comprising analyte from a test sample which is capable of being 
immobilized by a solid, porous element. In particular, analyte is captured 
in a liquid phase using microparticles or polyionic capture agents having 
a binding affinity for the analyte wherein the captured analyte is 
subsequently immobilized by the porous element and a chemiluminescent 
signal is chemically excited and detected. Accordingly, the method of the 
present invention advantageously employs fast diffusion rates in solution 
to provide highly sensitive assays for a wide range of analytes. 
The apparatus for performing a chemiluminescent assay according to the 
present invention includes a container having an aperture and a solid, 
porous element, preferably in the form of a fibrous matrix, capable of 
immobilizing a chemiluminescent generating reaction product complex while, 
at the same time, permitting the passage of other reaction components 
which are not immobilized by the porous matrix. The reaction product is 
immobilized by the porous element through particulate reactants or as the 
result of an interactive property between the porous element and the 
reaction product, such as hydrophilic-hydropholic binding interactions, 
ionic binding interactions, and the like. A detection device is situated 
adjacent to the container which moves to create a light-tight seal with 
the container to allow low light level chemiluminescence measurements. The 
detection device includes means for evenly distributing a chemiluminescent 
activating solution to the porous element. The aperture may be 
funnel-shaped and the means for applying the activating solution may 
include ports disposed toward an interior surface of the funnel. 
The various methods known in the art for forming heterogeneous binding 
reaction systems can be followed in applying the method and apparatus of 
the present invention where a chemiluminescent label is employed as the 
labeled reagent. Typically, the assay reagents for performing such assays 
may take many different forms, but, in general, comprise (1) the analyte 
to be detected, (2) a specific binding partner for the analyte, and (3) a 
labeled reagent, which can be the same or different as the binding partner 
for the analyte. The assay reagents are generally combined simultaneously, 
or sequentially, wherein the labeled reagent becomes bound to its 
corresponding binding partner such that the extent of binding is a 
function of the amount of analyte present. Typically, the bound species 
and the free species are physically separated from each other and the 
amount of label present in either fraction thereof is determined by 
measuring the activity of the particular label being used. Such methods 
include those known as the competitive immunoassay binding technique, the 
sandwich immunoassay technique, and the immunometric technique. In all of 
these heterogeneous immunoassay systems, separation of the free and bound 
species of the labeled reagent is normally accomplished by immobilizing 
one of such species. 
A method for performing a chemiluminescent assay according to the present 
invention includes the steps of binding an analyte to a chemiluminescent 
complex, binding the analyte-chemiluminescent complex to a particulate 
support having binding sites for the analyte-chemiluminescent complex to 
form an immobilizable reaction complex, contacting the immobilizable 
reaction complex with the porous element, and evenly distributing a 
chemiluminescent activating solution on the porous element to provide a 
chemiluminescent signal which is measured and correlated to the amount of 
analyte in a test sample. 
Preferably, a method for performing a chemiluminescent assay according to 
the present invention includes the steps of binding an analyte to a 
chemiluminescent complex, building the analyte-chemiluminescent complex to 
an ionic moiety to form an immobilizable charged reaction complex, and 
contacting the charged reaction complex, with a solid, porous element 
having an ionic charge opposite to that of the immobilizable charged 
reaction complex whereby the charged reaction complex is immobilized by 
the solid, porous element as a result of the ionic binding interaction 
therebetween. A chemiluminescent activating solution is then evenly 
distributed on the porous element to provide a chemiluminescent signal 
which is measured and correlated to the amount of analyte in a test sample 
.

DETAILED DESCRIPTION 
According to the present invention, heterogeneous chemiluminescent 
immunoassays may be easily automated, and hence their precision and 
accuracy improved, by generating and directly detecting a chemiluminescent 
signal from the separated reaction complex immobilized on a solid, porous 
element without the need to transfer the separated complexes to a tube or 
use a tube luminometer. The primary capture reaction is performed in the 
liquid phase to make use of the improved diffusion conditions in the 
liquids, and the immobilizable reaction complex is immobilized by the 
porous element as a result of an interaction between the immobilized 
reaction complex and the porous element, such as a hydrophobic 
interaction, anionic interaction, and the like. The chemiluminescence 
signal is generated from the porous element and is detected by a detector 
facing one side of the porous element and in close proximity to it, while 
the reaction products which are not immobilized, i.e. chemiluminescent 
binding complex not bound to the analyte, are disposed of by an absorbant 
matrix pad in intimate contact with the other side of the porous element. 
The porous element is an integral part of a disposable device that 
comprises reaction chambers and separation and detection chambers. The 
detector device has a shroud which surrounds the separation and detection 
chamber and creates a local light-tight compartment where a 
chemiluminescence signal is generated and detected. Either shroud or the 
whole assembly are movable in the Z-direction to affect a light-tight 
seal. 
The solid, porous element of the present invention is preferably in the 
form of a fibrous matrix and is used to immobilize the immobilizable 
reaction complex as a result of the interaction therebetween, from which 
an assay signal can be generated. The porous element can be selected from 
woven fibrous materials such as glass, cellulose, nylon or other natural 
or synthetic material known to those skilled in the art. It can be also 
chosen from porous glass or ceramic fritted disks or polymer fritted 
disks. Material choice, dimensions and pore size of these porous elements 
can be easily selected by those skilled in the art, to provide an 
effective pore size and adequate void areas to permit proper flow of 
unreacted reagents and sample through the porous element. 
Where the porous element is employed in an ion capture procedure, it is 
treated with a water soluble polycationic polymer. Choice of the water 
soluble polycation, amount of polymer, method and application can be 
determined by those skilled in the art. Derivatization of the surface of 
the porous element to generate positively charged groups and application 
of surface treatment techniques such as plasma treatment can also be 
contemplated and used by those skilled in the art. An important criterion 
is that the porous element is made from translucent or white material thus 
it will not absorb or attenuate the emitted chemiluminescent signal. 
A preferred fibrous materials is H&V product No. HC 4111 glass fiber filter 
paper, which has a nominal thickness of 0.055 inches and is commercially 
available from Hollongsworth and Vose Co., East Walpole, Mass. The 
effective pore size of the fibrous matrix or the spatial separation of the 
fibers is chosen to be larger than the diameter of the microparticles 
employed in the assays. Spatial separation of larger than 10 microns is 
preferred. This is to assure that even after the immobilizable reaction 
complex is immobilized on the fibers, adequate void areas still exist for 
proper flow of reagents and sample through the fibrous matrix, which in 
turn prevents fluid retention on the surface of the matrix which, for 
example, protects the detection device against possible trigger solution 
splashing. 
Ion capture procedures for immobilizing an immobilizable reaction complex 
with a negatively charged polymer tail, as described in copending U.S. 
patent application Ser. No. 150,278, entitled "Ion Capture Assays and 
Devices," filed Jan. 29, 1988, both of which are incorporated by reference 
herein, can be employed according to the present invention to affect a 
fast solution-phase immunochemical reaction. An immobilizable immune 
complex is separated from the rest of the reaction mixture by ionic 
interactions between the negatively charged poly-anion/immune complex and 
the previously treated, positively charged porous element and detected by 
the method described in the present invention. 
Acridinium sulfonamides labeling chemistry, as described in copending U.S. 
patent application Ser. No. 371,763 entitled "Chemiluminescent Acridinium 
Salts", filed June 23, 1989, incorporated herein by reference, may be 
employed according to the present invention for making a stable 
luminescent tracer of high quantum yield. 
Alkaline phosphatase labeling techniques known in the art and use of 
dioxetane catalyzed chemiluminescence may be also used according to the 
present invention to generate a long-lived signal that can be integrated 
to yield high sensitivity assays. 
Chemiluminescent moieties can be used as probes or labels in a specific 
binding assay. For example, such chemiluminescent label can directly react 
with an activating reagent to generate a light signal, such as acridinium 
sulfonamides. Alternatively, such chemiluminescent label serves as a 
catalyst to accelerate the generation of light from a substrate, such as 
alkaline phosphatase, peroxidase and beta-galactosidase. 
The combination of these techniques are particularly useful to permit a 
simple, rapid, highly sensitive immunoassay method for the determination 
of viral particles, macromolecular antigens and haptens. One such assay 
for Hepatitis B Surface antigen has sensitivities which exceed those of 
other methods. For example, one such type of chemiluminescence immunoassay 
for Hepatitis B surface antigen (Clin. Chem, 27, 1378-1384, 1981) involves 
two incubation periods, 1.5 hours each, and has a lowest limit of 
detection of 2 ng/mL. A lowest limit of detection of 1 ng/mL is achieved 
by increasing the incubation time to 16 hours. Using the method and 
detection device described according to the present invention, 
sub-nanogram quantities of Hepatitis B surface antigen may be detected 
within a total assay time of less than one hour. 
According to a preferred embodiment of the present invention, a sandwich 
immunoassay is performed employing a polyanionic acid such as polyglutamic 
acid which is attached to an antibody to the analyte under determination, 
and added to a reaction vessel, either simultaneously or sequentially, 
with the analyte from a test sample and a chemiluminescent-labeled 
antibody. The reaction mixture is incubated for a period of time and under 
conditions which maximize the specific binding reaction. The reaction 
mixture is transferred to a separation and detection chamber. Transfer of 
the reaction mixture into the separation and detection chamber can be 
achieved by mechanical means such as a manual or automated pipettor or by 
a non-contact hydraulic or fluidic means such as described by co-pending 
U.S. patent application Ser. No. 184,726, entitled "Device and Methods for 
Performing a Solid-Phase Immunoassay", filed Apr. 22, 1988, incorporated 
herein by reference. 
The chemiluminescence signal from acridinium labeled assays is triggered 
and simultaneously detection on the porous element in the light-tight 
compartment formed from the disposable device and the detector head. The 
signal is integrated over a period of time longer than the sum of the rise 
and decay time of the chemiluminescence signal, and longer than the 
residence time of the triggered reaction mixture in the porous element. 
Alternatively, a sandwich immunoassay can be performed employing a 
polyanionic acid such as polyglutamic acid which is attached to the 
antibody and added to a reaction vessel, either simultaneously or 
sequentially, with an enzyme-labeled antibody or antigen. Alkaline 
phophatase or .beta.-galactosidase-labeled antigen or antibody may be 
used. The reaction mixture is incubated for a period of time and under 
conditions which maximize the specific binding reaction. The reaction 
mixture is similarly transferred to a separation and detection chamber as 
described above and a chemiluminescent indicator added thereto to generate 
a chemiluminescent signal from the porous element in the separation and 
detection chamber before it is mated with the detector head to form the 
light-tight compartment where the chemiluminescence signal is detected. 
The signal is integrated over a period of time that is generally shorter 
than the residence time of the luminescent reaction mixture in the porous 
element. 
According to the present invention, incubation and separation steps take 
place in two independent compartments. This limits the time during which 
the sample and conjugate are in contact with the porous element and the 
walls of the detection compartment. Thus the amount of sample and labeled 
reagent that binds non-specifically to the porous element and the wall of 
the separation and read compartment is substantially reduced as compared 
to incubation, wash and detection in the same well, as described in U.S. 
Pat. No. 4,652,533, J. Immuno. Methods, 67, 21-35 (1984), or Clin. Chem. 
32, 1682-1686 (1986), and, accordingly, improves assay sensitivity. 
A preferred device for performing the incubation and transferring the 
reaction mixture into the read well is that described in a co-pending U.S. 
patent application entitled "Automated Method and Device for Performing a 
Solid Phase Chemiluminescent Assay" Ser. No. 07/425,651, filed on even 
date herewith, incorporated by reference herein. Such device comprises a 
funnel-like structure, a porous element, and an absorbant material, which 
are assembled to provide intimate contact between the porous element and 
the absorbent material, as well as adequate venting of air displaced by 
fluids absorbed in said absorbent material. The capacity of the absorbent 
material is chosen to be larger than the total volume of sample, reagents 
and wash solution used in the assay steps. This ensures adequate washing 
of the retained reaction product, prevents excessive fluid retention on 
the porous element, and helps to protect the detection device against 
possible trigger solution splashing. The funnel-like structure and the 
porous element are parts of the light-tight compartment formed when the 
device is mated with said detector head of the present invention to detect 
a chemiluminescence signal generated on the porous element. 
It is to be understood that one skilled in the art can design other 
reaction vessels that incorporate a porous element for retaining the 
reaction complexes of an immunochemical reaction as described herein, 
other means for transferring the reaction mixture and other means for 
disposing of the excess reagents and wash solution such as vacuum and 
pressure means, that can be used with the detector head of the present 
invention. 
An example of the chemiluminescence detection device is illustrated in FIG. 
1. The detector device consists of a frame (4), a detector head (5), a 
flexible diaphragm (6), and a lifter mechanism (7). The frame allows 
mounting the detector assembly on a thermostated tunnel where a timing 
belt moves a plurality of disposable reaction devices at a given step 
rate. The detection head (5) consists of a shroud (10), and ports for 
fluid lines (11,12). The lifter mechanism (7) moves shroud (10) up and 
down on a Z-axis. The down movement affects a light seal with the 
separation and detection chambers of a disposable tray where a 
chemiluminescence signal is generated and detected, and the upward 
movement is to clear the disposable to allow its free movement to the 
other positions at the end of measurement. 
FIG. 2 shows an isometric view of a preferred configuration of the 
detection apparatus that is designed to accommodate the preferred 
disposable reaction tray of co-pending U.S. patent application entitled 
"Automated Method and Device for Performing A Solid-Phase Chemiluminescent 
Assay" Ser. No. 07/425,651, filed on even date herewith and incorporated 
by reference herein, which contains two rows of separation and read wells 
at a 36 mm center to center distance. It is to be understood, of course, 
that one skilled in the art can redesign the envelope of the detection 
head to mate with other shapes of disposable devices. 
The shroud (10) is made from machined aluminum or cast polyurethane, and 
comprises four ports for trigger solution injection outlets (11-14), two 
optical detection modules (15, 16), and two boxes (17, 18) containing 
photon counting amplifiers and electric leads for antifog heaters (19). 
Attached to shroud (10) is a flexible black rubber diaphgram (6) of FIG. 1 
that allows the free vertical movement of the shroud (10). 
A cross-sectional view of the signal detection module is shown in FIG. 3 
and comprises a light guide (20), light-pipe protecting sleeve (21), 
injectors (22, 23) that are connected to two of the black Teflon.RTM. 
fluid lines (11-14) of FIG. 2, photomultiplier tube (PMT) (24), a 
photomultiplier tube socket (25) and a collar for holding the PMT socket 
(26). The PMT is springloaded by a 302 stainless steel spring wire (27) to 
ensure proper spacing from the light pipe and is protected from moisture 
by two O-ring seals (28 and 29). O-rings (30 and 31) fix the light pipe 
(20) in space and protect the face of the PMT from moisture. O-ring (30) 
is preferably made of a material with high dielectric constant to prevent 
Ohmic leakage at the photocathode. A mumetal shield (32) surrounds the 
PMT, with a nylon spacer (33) inside (32) which protects the PMT during 
assembly. The mumetal shield (32) is kept electrically isolated by use of 
two O-rings (34,35) and a Teflon sleeve (37) to separate it from the outer 
housing. An electrically conducting outer housing (38) protects the 
detection module. The bottom of shroud (10) has a groove (39) that locates 
the surface feature on the disposable device and a light sealing gasket 
(40) of FIG. 4. The light sealing gasket is made of black inert 
compressable polymer. A preferred material is a nylon nap on a rayon 
backing COE7-1673 (Schlegal Corporation, Rochester, N.Y.). Two low wattage 
anti-fog heaters (41, 42), are used to create a temperature gradient in 
the vicinity of the light pipe to prevent any condensation on the light 
pipe during the measurement. 
FIG. 4 is a cross-sectional view taken along the side of the detector 
device of the present invention and shows the two detector assemblies, 
shroud (19), groove (39) and light sealing gasket (40), the anti-fog 
heating elements (41, 42) and the end of an injector tip (22). FIG. 5 is 
an exploded view of the bottom of the shroud (10), showing the light seal 
groove (39) and the light sealing gasket (40). 
The lifter mechanism assembly (7) of FIG. 1 is shown in FIG. 6 and 
comprises an AC permanent magnet synchronous motor (50), a stainless steel 
shaft (51) and a cam (52) to control the vertical displacement of lifter. 
A stainless steel cam follower roller (53) mounted in the lifter arm (54) 
is in contact with the cam. A stainless steel guide bar (55) is part of 
the base and is in combination with stainless steel rollers for guide bar 
(56, 57). The stainless steel port or shaft (58), guide bar (55) and guide 
bar rollers (56, 57) ensure precise movement along the Z-axis without X or 
Y movements or rotation around the Z-axis. A slotted aluminum disk (60) 
and two diametrically opposed opto-sensors (61, 62) to control motor stops 
for full-up and full-down shroud positions. Shroud down flag (63) and 
shroud down sensor (64) ensure the engagement of the detector head with 
the disposable device. Holes (65, 66) are attachment points to the shroud 
(10) of FIG. 2. 
As shown in FIG. 3, the terminuses of the injection ports (22, 23) are 
directed towards the walls of the detection chamber. The distance these 
ports protrude beyond the lower surface of the light pipe (20) is 0.213 
inches. The lower end of the light pipe (20) is 0.435 inches above the 
surface of the porous matrix. Light pipe (20) is 8 mm in diameter and may 
be made of a quartz, glass or polished plastic rod having a length of 
approximately 3 inches long. 
As indicated in FIG. 7, a trigger solution injection device (70) is used 
such as, for example, a piston pump such as FMI RH pump (Fluid Metering 
Inc., Oyster Bay, N.Y.). A Teflon.TM. tube (71) having an inner diameter 
of 0.03 inches and an outer diameter of 0.062 inches (Cole Palmer, 
Chicago, Ill.) carried trigger solution from pump to a selonoid operated 
three way valve (72), [Angar Scientific, Florham Park, N.J.]. A trigger 
solution was diverted employing Teflon.TM. tubes (73, 74) to two 2-outlet 
manifolds (75, 76). A Minstac multiport manifold (Lee Company, Westbrook, 
Conn.) was used. Two lines per manifold (77, 78) and (79, 80) made of 
Teflon.TM. tubes having an inner diameter of 0.5 mm and an outer diameter 
of 1.59 mm (Aspect Inc., Ann Arbor, Mich.), carried the trigger solution 
from manifold to injection ports. For acridinium labeled chemiluminescence 
reactions, alkaline peroxide solution 80-100 .mu.L was injected through 
the manifold onto separation and detection chamber at a rate of less than 
500 .mu.L per second. 
The trigger solution was dispensed from two injector ports (22, 23), shown 
in FIG. 1, at 180 degrees to each other. Choosing an injection speed less 
than 500 .mu.L per second through a 0.05 mm inner diameter injection 
prevented back-splash of the trigger solution towards the signal pick-up 
optics. This was verified by examination of high speed video recordings of 
the injection process. 
A R647-04 head-on photomultiplier tube (24) (FIG. 3) and an E849-35 socket 
(25) and magnetic shield E989-09 (32) (Hamamatsu Inc., Middlesex, N.J.). 
The PMTs were powered to approximately 1040 V using a high voltage power 
supply (82) (FIG. 7) (Model PMT-20A/N Option 3, Bertan Associates, 
Hicksville, NJ). A .+-.12 V DC power supply (81) (FIG. 7) (Part#12EB50, 
Acopian Inc., Easton, Pa.) was used to power the high voltage power 
supply. The amplifier boards were powered by two isolated .+-.5 volts 
power supplies (83) (Part #5EB 100, Accopian Inc., Easton, Pa.). 
The photon counting amplifier boards (84, 85) comprises a 12 MHz vidio 
amplifier MC 1733 CP (Motorola Inc., Semiconductors Division, Phoenix, 
Ariz.), an AM 686CN high speed comparator (Advanced Micro Devices, 
Sunnyvale, Calif.) is used as a discriminator, and the resultant TTL 
signal is divided by two using a 74F74 flip flop. The output drives a HFBR 
1524 fiber optic transmitter (Hewlett Packard, Palo Alto, Calif.). The 
digital signal was carried on a fiber optic link to an HFBR-2524 fiber 
optic receiver, all are components of a HFBR-0501 kit from Hewlett 
Packard, Palo Alto, Calif. The fiber optic link carries the digital signal 
to the counter/time board, thus avoiding any noise pick-up or ground loop 
effects on this circuitry. The start count signal, integration time, 
trigger pump star/stop and trigger signal counting were controlled by the 
counter/timer board. The counter/timer board was designed and built using 
prior art designs and components. Those skilled in the art can easily 
design equivalent circuits. 
The counter/timer board and the trigger solution pump are controlled by a 
micro computer as, for example, an IBM pc-XT (IBM Corporation, Boca Raton, 
Fla.), or an Intel 310 development system (Intel Corporation, Sunnyvale, 
Calif.) through a standard prior art interface. 
Polyion-bound immune complexes are attracted to the oppositely charge 
porous element and then the porous element is washed with water or aqueous 
solution containing detergents and/or salt compositions to maximize 
efficiency of washing unreacted sample and label away from the porous 
element. Wash solution volume is chosen to be several times the volume of 
the reaction mixture, preferably about three times that volume. Wash 
solution composition is chosen to decrease non-specific binding and to 
increase assay sensitivity. Thus water, saline solution, or hypertonic 
buffered salt solutions can be used. Number of wash cycles are also 
important to assay sensitivity, thus applying multiple aliquots of wash 
solution is preferred. Prewetting the porous matrix with wash solution or 
a protein-containing solution may also help in decreasing non-specific 
binding and increasing specific binding. 
The apparatus of the present invention can be used to detect shortlived 
alkaline peroxide triggered chemiluminescences such as acridinium label 
chemiluminescence or long-lived dioxetane chemiluminescence, such as 
described by WO 881 00694 and EP 0-254-051A2. In the case of short-lived 
chemiluminescence, activating the CL signal is affected within the 
light-tight compartment created by mating the detector device with the 
disposable device. Thus, trigger solution injector ports, fluid lines, 
manifolds and pumps are an integral part of the detection device. 
Long-lived dioxetane-type chemiluminescence is generated by adding an 
enzyme specific chemiluminescent substrate to the enzyme-labeled immune 
complex on the porous element. An example is the commercially available 
alkaline phosphatase substrate 
3-(2'-spiroadamantane)-4-methoxy-4-(3'-phosphoroxy)phenyl-1,2-dioxetane 
disodium salt (AMPPD) from Tropix Inc., Bedford, Mass. The substrate can 
be incubated before the disposable device engages the detector head and 
start of signal integration. The total signal intensity is integrated over 
a portion of the intensity-time curve. 
The acridinium sulfonamide labeled chmiluminescent reaction is triggered on 
the porous element by alkaline peroxide solution using the two injectors 
in the detection device. Trigger solution injection ports may be made from 
chemically inert material that is not affected by high alkaline peroxide 
concentrations. Preferred materials for fabricating these injectors are 
Teflon.TM., Kel-F.TM., nylon, ultra high molecular weight polyethylene or 
Teflon.TM.-coated stainless steel. The assembly of injectors, light guide 
and the porous element is light tight and the aperture of the light guide 
is in close proximity to the surface of the porous element. A preferred 
geometry is to have two trigger solution injectors at 180.degree. to each 
other on the periphery of the light guide. The light guide may be made of 
optical quality plastic, glass, quartz or a highly polished hollow 
metallic tube. The light guide directs emitted chemiluminescence signal to 
the photo detector. 
The trigger solution is directed towards the walls of the separation and 
detection chamber in the disposable and is injected at such a slow rate to 
minimize splashing of the fluid towards the light pick-up optics. 
Injection rates of less than 500 .mu.L per second are preferred. The 
trigger solution flows down the walls of the detection chamber to the 
porous element from more than one direction to form a puddle of fluid that 
uniformly diffuses through the porous element. 
The volume of trigger solution is chosen to be slightly larger than the 
fluid capacity of the porous element. Thus volumes in the range of 50-100 
.mu.L are preferred. Signal integration times longer than the residence 
time of the triggered reaction mixture in the porous element are used. The 
chemiluminescent reaction takes place on the surface of the porous element 
as well as from in the interstice of porous element. 
Hydrogen peroxide concentration in the alkaline peroxide solution used to 
trigger the chemiluminescent reaction on the porous element is kept at 
0.1-1.0% by volume in 0.25N sodium hydroxide solution. A preferred 
concentration is 0.3% hydrogen peroxide solution in 0.25N sodium hydroxide 
solution. Typically chemiluminescent reactions in solution are triggered 
with alkaline hydrogen peroxide containing approximately 0.03% by volume 
hydrogen peroxide in 0.1-0.25N sodium hydroxide solution. Higher peroxide 
concentrations of the trigger solution generate higher signal in a short 
period of time before triggered reaction mixture diffuses through the 
porous element. 
Alkaline phosphtase/dioxetane chemiluminescence can be triggered outside 
the detector head and the signal is collected after the enzyme substrate 
reaction reaches a steady state. In this case trigger solution pump, lines 
and injectors are not used. Signal is collected after the detection head 
mates with the disposable as is indicated by a signal from the shroud-down 
sensor. Alternatively the signal is collected in a rate mode. The trigger 
solution pump and lines can be used to deliver the substrate solution and 
the change in CL intensity as a function of time is calculated by 
performing a series of short duration readings over a period of time. 
Preferred enzyme labels and substrates are alkaline phosphatase and 
3-(2'-spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy)phenyl-1,2-dioxetane 
(AMPPD) substrate. .beta.-galactosidase can also be used as a label and 
3-(2'-spiradamantane)-4-methoxy-4-(3"-.beta.-D'-galactopyrano-yloxy)phenyl 
-1,2-dioxetane (AMPGD) can be used as a substrate. 
The light pipe directs emitted chemiluminescent signal to a photomultiplier 
tube and detection electronics. A preferred signal collection method is 
the use of single photon counting techniques. Detecting low light level 
signals is known to those skilled in the art. Cooled photodiodes, 
avalanche photodiodes, intensified vidicon tubes or microchannel plates 
can be substituted for the photomultiplier tub without deviation from the 
spirit of the invention. A preferred light guide configuration is a highly 
polished quartz rod 8 mm in diameter and 0.5- to 3 inches long. The length 
of the light pipe is not important as it is chosen for packaging 
convenience. Other light guide configurations such shaping the end of the 
rod to encompass a lens-like structure, use of a prism shaped terminus, 
use of fiber optic bundle or gradient index lens can be contemplated and 
applied by those skilled in the art and does not deviate from the spirit 
of this invention. 
The present invention will now be illustrated, but is not intended to be 
limited, by the following examples: 
EXAMPLE I 
Precision Activating And Measuring Short-Lived Acridinium Chemiluminescence 
In A Glass Fiber Matrix 
The precision of measuring a short-lived chemiluminescence signal in a 
porous element was determined as follows: 
50 .mu.L of acridinium labeled anti-HBC antibody conjugate solution 
(Hepatitis B Core antigen Clinical Lots) were manually dispensed on a 
glass fiber matrix in each of 16 separation and detection wells of a 
disposable reaction tray such as described in a co-pending U.S. patent 
application entitled "Automated Method and Device for Performing A 
Solid-Phase Chemiluminescent Assay 07/425,651, filed on even date herewith 
and incorporated by reference herein. Trigger solution was prepared by 
dissolving tablets containing urea peroxide in 0.25M sodium hydroxide to 
yield effective peroxide concentration of 0.3%. The tray was moved under 
the detector head device of the present invention. The detector head was 
lowered to mate with the first two wells of the disposable tray and create 
a light-tight compartment. The high voltage to the PMTs are gated on and 
allowed to equilibrate for 3 seconds. Dark counts were then collected on 
one of the PMTs for 6 seconds. The trigger pump was activated and the 
valve directed the trigger solution to the separation and detection well 
under this PMT. 85 .mu.L of 0.3% alkaline peroxide solution were injected 
through the two injectors and the triggered signal integration started 
simultaneously and continued for 6 seconds integrating. The dark counts of 
the second PMT was then started for 6 seconds followed by activating and 
simultaneously counting the chemiluminescence signal for 6 seconds. The 
detector head was then lifted upward to clear the tray and the tray 
advanced so that two new wells were located under the detector head. The 
detector head was lowered and the process of dark count and triggered 
counts was repeated on the rest of the 8 rows of wells on the disposable 
reaction tray. The data is shown in Table 1. 
TABLE 1 
______________________________________ 
Precision Activating And Measuring Short-Lived 
Chemiluminescence From Acridinium Labeled Anti-HBC 
In A Glass Fiber Matrix 
Side A Side B 
Dark Trigger Dark Trigger 
counts 
counts counts counts 
______________________________________ 
39 38275 37 35548 
29 40177 38 34476 
57 40244 34 34348 
31 39742 31 34650 
39 40141 31 34952 
35 39615 38 33780 
35 40306 32 34990 
28 39839 41 34523 
Mean 37 39792 35 34658 
SD 9 663 6 186 
% CV 1.7 1.5 
______________________________________ 
The low dark counts indicate that a light tight seal has been achieved 
between the disposable device and the detector head. The low % CV indicate 
the reproducibility of activating and detecting a short-lived 
chemiluminescence signal within a porous element. 
EXAMPLE II 
Reproducibility Activating And Measuring A Chemiluminescent Signal From 
Luminescent Microparticles Immobilized On a Porous Element 
Acridinium sulfonamide labeled antibody to Heptatitis B core antigen 
(pooled, 5 .mu.g/mL), was diluted in conjugate diluent, containing 50% 
fetal calf serum (Abbott Laboratories, North Chicago, Ill.) 2% human 
plasma, 0.1% Tween.RTM.-20, 0.1% ethylenediamine tetra acetic acid and 
0.1% sodium azide in phospate buffered saline, pH 6.8. The final conjugate 
concentration was 150 ng/mL. Carboxylated polystyrene microparticles 
coupled to antibody to Hepatitis B core antigen as an undercoat and then 
with recombinant Hepatitis B core antigen were pooled from lots prepared 
for clinical trials and contained 0.3% solids by weight. Microparticles 
were suspended in phosphate buffered saline (Abbott Laboratories, North 
Chicago, Ill.), pH 7.2, containing 16% sucrose. A 0.1% solution of 
Tween.RTM.-20 in phosphate buffered saline, pH 7.2, was used as a transfer 
solution. Luminescent microparticles were prepared by mixing 50 mL of 
conjugate solution and 50 mL of microparticles suspension. The reaction 
mixture was incubated in a water bath at 40.degree. C. for two hours. It 
was then let stand at room temperature for 24 hours to ensure complete 
binding of acridinium sulfonamide labeled antibodies to the antigen 
labeled microparticles. 
100 .mu.L of luminescent microparticles were dispensed on the porous 
fibrous glass matrix in each of the 16 read wells of a disposable reaction 
tray as described in Example 1 were allowed to drain through. The 
microparticles were washed with 100 .mu.L of fetal calf serum, 100 .mu.L 
of de-ionized water and two aliquots 300 .mu.L each of a 0.1% 
Tween.RTM.-20 solution. The disposable tray was linearly moved to a 
subsequent position where the chemiluminescence detection head of the 
present invention was lowered to create a light tight seal with the 
disposable tray. The immobilized and washed microparticles on the glass 
fiber matrix were triggered using 0.3% alkaline peroxide solution and the 
resulting chemiluminescence signal was integrated for a period of six 
seconds. The mean and standard deviation for each eight wells on each side 
of the disposable were calculated. 
TABLE 2 
______________________________________ 
Reproducibility Activating And Measuring A Chemiluminescent 
Signal From Luminescent Microparticles Immobilized On A 
Porous Element. 
Side A Side B 
Dark Trigger Dark Trigger 
counts 
counts counts counts 
______________________________________ 
37 53023 42 49956 
40 54573 47 49473 
38 54214 52 49473 
45 54664 34 48586 
43 55471 37 47624 
44 55446 43 47624 
42 55084 54 47898 
62 54450 44 48053 
Mean 44 54616 44 48588 
SD 7 234 7 220 
% CV 1.4 1.8 
______________________________________ 
The low dark counts indicate that as light tight seal has been achieved 
between the disposable device and the detector head. The low % CV indicate 
the reproducibility of entrapping luminescent microparticles and of 
activating and detecting a short-lived chemiluminescence signal within a 
porous element. 
EXAMPLE III 
Microparticle-Based Sandwich CLIA for Hepatitis B Surface Antigen 
An Ay and an Ad sensitivity panels and positive and negative controls for 
Hepatitis B Surface antigen (Abbott Laboratories, North Chicago, Ill.) 
chemiluminescence conjugate acridinium labeled goat polyclonal antibody 
(0.17 .mu.g/mL) was employed. 
A conjugate diluent was prepared comprising 0.1M monosodium phosphate, 0.1M 
disodium phosphate, 0.1% sodium azide and 53% calf serum (Abbott 
Laboratories North Chicago, Ill.), 10% normal human serum and was filtered 
through 0.45 .mu.m Nalgene disposable sterile filter (Nalge Company, 
Division of Sybron Corporation, Rochester, N.Y.). It was adjusted to a 
final pH of 6.3 and finally filtered through a 0.2 .mu.m Nalgene Filter. 
Carboxylated polystyrene microparticles (0.21 .mu.m) were coupled to IgM 
anti-HBsAg antibodies using EDAC coupling procedure, and having a total 
solids content of 0.24%. 
The washing solution contained 0.1M borate, 0.02% lithium dodecyl sulfate, 
0.9% sodium chloride and 0.1% sodium azide. 
200 .mu.L of control or sample were pipetted into the shallow reaction 
wells of a disposable tray as described in Example 1 using an automatic 
pipettor. 30 .mu.L of latex particles coated with monoclonal IgM 
mouse-antiheptatitis B surface antigen were dispensed into each incubation 
well. The reaction mixture was incubated for 20 minutes in a heated tunnel 
at 40.degree. C. with the disposable device moving into the tunnel by a 
timing belt at increments of 0.8 inches per assay step. It remains in 
position for 72 seconds for performing an assay step, then it increments 
again at 0.8 inches for the next assay step. 
The reaction mixture was transferred and washed from shallow incubation 
well onto the glass fiber matrix of read well, by injecting two pulses 300 
.mu.L each of the wash solutions using the method of transfer as described 
herein. After the transfer and wash solution has drained down the 
absorbant pad, 30 .mu.L acridinium labeled polyclonal goat anti human HBS 
antibodies were dispensed on each fibrous pad. The disposable was moved on 
the timing belt to allow subsequent well pairs to pass under the transfer 
device and to affect transfer of the reaction mixture. The disposable tray 
was incubated for 30 more minutes in the tunnel using the same moving 
timing belt as it is moved to a washing position. The transferred 
microparticles that are retained on the glass fiber matrix and the added 
acridinium labeled antibodies were subsequently washed with three aliquot, 
100 .mu.L each of wash solution containing 0.1% sodium dodecyl sulfate, 
from a wash nozzle. The disposable tray was moved at the same rate to a 
read position where the chemiluminescence detection head of the present 
invention was lowered to mate with the surface feature of the first two 
wells on the disposable to create a light-tight seal. The transferred and 
washed microparticles were triggered using 0.3% alkaline peroxide 
solution. The measured signal for each well was considered to correspond 
to the amount of acridiniium labeled conjugate attached to the 
microparticles and hence directly related to the concentration of the 
Hepatitis B surface antigen in the sample. 
TABLE 3 
______________________________________ 
Microparticle Capture Chemiluminescence Immunoassay for 
Hepatites B Surface Antigen: 
Sensitivity Panel Data. 
Concentration 
Member ng/mL Counts/6 secs 
% CV 
______________________________________ 
ADA 1.90 19270 5.8 
ADB 1.48 15659 6.2 
ADC 0.92 10756 7.1 
ADD 0.74 8945 4.9 
ADE 0.51 6717 5.2 
ADF 0.41 5751 5.3 
ADG 0.31 4547 2.1 
ADH 0.10 2840 4.2 
AYA 2.05 23751 4.5 
AYB 1.11 14242 3.7 
AYC 0.83 11216 4.9 
AYD 0.67 8455 2.2 
AYE 0.53 7132 4.7 
AYF 0.44 6295 4.8 
AYG 0.30 4926 4.9 
AYH 0.14 2911 4.0 
Negative Control 1247 8.3 
Positive Control 115219 6.1 
______________________________________ 
The standard deviation for twelve replicates of the negative control was 
104. The cut-off value of the assay calculated by adding 10 standard 
deviation to the mean of the negative control was 2287 counts. Thus, 
concentrations of Hepatitis B Surface Antigen as low as 0.10 ng/mL of the 
AD subtype and 0.14 ng/mL of the AY subtype can be quantified using a 
microparticle capture chemiluminescence immunoassay and the method and 
device of the present invention. Using the same criterion to the data 
published in Table 4 of Clin. Chem. 27, 1378-1384 (1981), yields a lowest 
limit of quantitation of 5 mg/mL. Thus, a limit of quantitation of 
Hepatitis B surface antigen one order of magnitude lower than reported 
using prior art techniques can be achieved using the device and method of 
this invention. 
EXAMPLE IV 
Ion-Capture Alkaline Phosphatase Labeled Chemiluminescence Competitive 
Binding Immunoassay For a Hapten 
This example shows the use of the detection device and method of this 
invention in a competitive binding assay for the abused drug 
phenylcyclidine (PCP). This assay is performed on urine specimens and uses 
the ion capture immunoassay procedure described herein. The formation of 
the immune complex involves the use of an anionic polymer as a capture 
agent. The reaction mixture is transferred to the detection well of said 
device and the product of the immunochemical reaction is immobilized by 
ionic forces on a porous plug that has been previously treated with a 
solution of a cationic polymer to render it positively charged. 
Anti-phenylcyclidine antibodies were labeled with alkaline phosphatase 
using art procedures known in the art. The labeled antibody solution was 
diluted in a solution containing 1% fish gelatin, 25 mM Tris, 100 mM 
sodium chloride, 1 mM magnesium chloride, 0.1 mM zinc chloride, and 0.1% 
sodium azide. The pH of the solution was 7.2 Prewet and transfer solutions 
were IMx buffer (Abbott Laboratories, North Chicago, Ill.) containing 25 
mM Tris, 0.3M sodium chloride, 0.1% sodium azide, pH 7.2. The cationic 
polymer was a 0.5% aqueous solution of Celquat.TM. L-200 (National Starch 
and Chemical Company; Bridgewater, N.J.) in 10 mM sodium chloride. 
The capture agent, phenylcyclidine-polyglutamic acid, was prepared as 
follows: 
1 gm of polyglutamic acid sodium salt (Sigma Chemical Company, St. Louis, 
Mo.) was added to 7 gms of AG50W-X8 ion exchange resin (Bio-Rad, Richmond, 
Calif.) in 20 mL water and stirred overnight. Liquor was removed and 
lyophilized to give free acid polyglutamic acid (PGAFA). 
Phenylcyclidine-4-chloroformate was prepared by reacting 1.1 mg 
4-hydroxyphenylcyclidine (4.24.times.10.sup.-6 moles) in 0.5 mL 
tetrahydrofuran with 0.5 mL of 10% solution of phosgene in benzene (130 
mole excess). The reaction was allowed to proceed for 2.5 hours at room 
temperature. Solvent was evaporated under a stream of nitrogen to yield a 
residue of phenylcyclidine-4-chloroformate. The residue was dissolved in 
0.5 mL tetrahydrofuran and 1.7 mg of free acid polyglutamic acid 
(molecular weight 40,000) in 0.5 mL 1-methyl-2-pyrrolidinone was added to 
it. The reaction was carried out overnight at room temperature then the 
reaction mixture was evaporated to dryness. The dried mixture was 
dissolved in 1.5 mL 0.1M phosphate buffer, pH 7.0 and dialyzed against a 
volume of the same buffer in a 3,500 molecular weight cut-off dialysis 
bag. The precipitate was filtered. The cloudy aqueous filtrate was 
extracted with methylene chloride until it was clear. The aqueous layer 
was diluted in a buffer containing 1% fish gelatin, 25 mM Tris, 100 mM 
sodium chloride, 1 m magnesium chloride, 0.1 mM zinc chloride and 0.1% 
sodium azide at pH 7.2 to yield 5 5.0 .mu.gmPGA/mL phenylcyclidine-PGA 
capture reagent. 
Sample were phenylcyclicidine calibrators from a TDx.TM. fluorescence 
polarization immunoassay kit (Abbott Laboratories, North Chicago, Ill.). 
Containing 250, 120, 60, 25, and 0 ng/mL phenylcyclidine in human urine as 
confirmed by independent analytical methods. The glass fiber matrix of a 
disposable reaction tray as described herein was treated with 50 .mu.L of 
a 0.5% Celquat.TM. L-200 (National Starch and Chemical Company, 
Bridgewater, N.J. 08807) in a 10 mM solution of sodium chloride. The 
Celquat.TM. L-200 solution was manually applied to each glass fiber 
matrix. 150 .mu.L sample (PCP calibrators), 45 .mu.L IMx buffer, and 420 
.mu.L alkaline phosphatase labeled antiphenylcyclildine antibody solution 
were incubated in test tubes in a water bath at 37.degree. C. for 10 
minutes. 300 .mu.L of the phenylcyclidine polyglutamic acid capture 
reagent and 45 .mu.L IMx buffer were added to the reaction mixture and 
incubated for 10 more minutes at 37.degree.C. 200 .mu.L of the reaction 
mixture was manually transferred onto the treated glass fiber matrix of 
the reaction tray using a manual pipettor. The excess reagents were washed 
by manually dispensing two 75 .mu.L aliquots of IMx buffer on the porous 
fibrous glass plug. 100 .mu.L of a chemiluminescent substrate, 
3-(2'-spiroadamantane)-4-methoxy-4-(3"-phosphoroxoy)phenoxy)phenyl-1,2-dio 
xetane disodium salt (AMPPD) in a 50 mM sodium bicarbonate solution 
containing 1 mM magnesium chloride at a pH of 9.5, was dispensed onto the 
glass fiber matrix using an FMI-RH pump (Fluid Metering Inc., Oyster Bay, 
N.Y.) and was incubated in the glass fiber matrix for 10 minutes. The 
pump, track and detection head functions were controlled by an IBM PC/XT. 
Track stepper motor was controlled with a stepper motor controller board 
(Scientific Solutions, Solon, Ohio). The FMI pump was controlled through a 
Triac interface consisting of MOC 3031 Opto Triac Driver and MAC 3030-8 
triac (Motorola, Inc., Semiconductor Division, Phoenix, Ariz.). The tray 
was then moved under the detector device of the present invention. 
Substrate dispensing and chemiluminescence signal detection were 
controlled in such a way that the substrate incubation time in all glass 
fiber matrices were the same. The detector head was lowered to mate with 
the disposable device and create a light-tight compartment for 
chemiluminescence measurement. The chemiluminescence was integrated for 6 
seconds per well, as shown in Table 4. 
TABLE 4 
______________________________________ 
Ion-Capture Alkaline Phosphatase Labeled Chemiluminescence 
Competitive Binding Immunoassay for Phenylcyclidine in Urine. 
Phenylcyclidine 
Chemiluminescence signal 
Net Polarization 
[ng/mL] counts/6 seconds .DELTA.mP 
______________________________________ 
0 3526 198 
25 2162 173 
60 1440 144 
250 1016 82 
______________________________________ 
The last column shows the change in degree of polarization of a PCP analog 
labeled with a fluorescein as it binds to anti-PCP antibody in a 
competitive binding assay in solution. The trend in the chemiluminescence 
signal of the entrapped product on the glass fiber matrix parallels that 
of fluorescence polarization techniques known in the art in solution using 
a commercially available fluorescence polarization analyzer (TDx Analyzer, 
Abbott Laboratories, North Chicago, Ill.) and commercially available 
fluorescence polarization kits (Abbott Laboratories, North Chicago, Ill.). 
EXAMPLE V 
Ion-Capture Acridinium Labeled Chemiluminescence Competitive Binding 
Immunnoassay for a Hapten 
The present example shows the use of the device and method of this 
invention in a competitive binding assay for the abused drug 
phenylcyclidine (PCP). This assay is performed on urine specimens and uses 
the ion capture immunoassay procedure as described herein. The immune 
complex was formed in the shallow reaction well of the disposable tray as 
described herein and involves the use of an anionic polymer as a capture 
agent. The reaction mixture was transferred to the read well of the device 
and the immunochemical reaction product was immobilized by ionic forces on 
the glass fiber matrix of the device which had been previously treated 
with a solution of a cationic polymer. 
Monoclonal anti-phenylcyclidine antibody was labeled with acridinium 
sulfonamide using EDAC coupling procedures known in the art. It was kept 
in the same buffer used for the anti-core conjugate of Example 3. The 
prewet and transfer solutions were IMx buffer (Abbott Laboratories, North 
Chicago, Ill.) containing 25 mM Tris, 0.3M sodium chloride, 0.1% sodium 
azide, pH 7.2. 
The cationic polymer was a 0.5% aqueous solution of Celquat.TM. L-200 
(National Starch and Chemical Company; Bridgewater, N.J.) in 10 mM sodium 
chloride, and the anionic capture agent phenylcyclidinepolyglutamic acid 
was prepared according to the procedure of Example IV. 
Samples were phenylcyclidine calibrators from a TDx.TM. fluorescence 
polarization immunoassay kit (Abbott Laboratories, North Chicago, Ill.). 
They contained 500, 250, 120, 60, 25, and 0 ng/mL phenylcyclidine in human 
urine. 80 .mu.L of IMx Tris buffer solution followed by 80 .mu.L 
Celquat.TM. L-200 solutions were dispensed on the glass fiber matrices of 
the disposable reaction tray. Solutions were dispensed using two FMI-RH 
pumps and controlled via a triac board by an Intel 310 Development System 
(Intel Inc., Sunnyvale, Calif.). The tray was moved on a linear track 
using a timing belt and a stepper motor. The stepper motor was controlled 
by a board employing components known in the art. After 4.8 minutes, 50 
.mu.L of calibrator (sample) was pipetted into the shallow reaction wells 
of a disposable reaction tray, using an automated pipettor. 50 .mu.L of 
acridinium labeled anti-PCP antibodies was dispensed into each incubation 
well. The mixture was incubated for 9.6 minutes in a heated tunnel at 
32.degree. C. with the disposable device moving into the tunnel by the 
timing belt in steps at the rate of 0.8 inches per minute, the reaction 
tray being stationary for 36 seconds after each step for a reaction step 
to take place. After 9.6 minutes incubation on the moving timing belt, 50 
.mu.L a solution containing PCP-PGA capture reagent at a concentration of 
1.9 mg PGA/mL was dispensed into the incubation well through a tip 
centered on the well. The capture reagent solution was dispensed by an 
FMI-RH pump and controlled by the 310 Development system through a triac 
interface board. The reaction mixture was further incubated for 9.6 
minutes. The quaternary ammonium polymer-treated glass fiber matrices were 
rinsed with 100 .mu.L of the IMx buffer before reaction mixture transfer. 
As the disposable tray was positioned under the transfer device as 
described herein, the reaction mixture was transferred and washed from the 
shallow incubation well onto the pre-treated glass fiber matrix in the 
detection well. The disposable tray was moved on the timing belt to allow 
subsequent well pairs to be located under the transfer device and to 
affect transfer of the reaction mixture. The disposable device was then 
moved to a read position, where the chemiluminescence detection head of 
the present invention was lowered to mate with the surface feature on the 
first two wells on the disposable to create a light-tight seal. The 
retained and immobilized immune complex on the glass fiber matrix was 
triggered using 0.3% alkaline peroxide solution in 0.25M NaOH and the 
signal from each PMT/amplifier was controlled by a counter/timer board and 
each side was triggered with an independent pump. The photon counter 
signal was integrated for eight seconds. The measured signal for each well 
was considered to correspond to the amount of acridinium labeled conjugate 
attached to the glass fiber matrix surface by ionic forces. The data is 
shown in Table 5 and is expressed as the number of counts and as % 
inhibition. The last column shows the change in degree of polarization of 
a PCP analog labeled with fluorescent as it binds to anti-PCP antibody in 
a competitive binding assay in solution. The trend in the 
chemiluminescence signal of the entrapped product on the glass fiber 
matrix parallels that of prior art fluorescence polarization in solution 
using a commercially available fluorescence polarization analyzer (TDx 
Analyzer, Abbott Laboratories, North Chicago, Ill.) and commercially 
available fluorescence polarization kits (Abbott Laboratories, North 
Chicago, Ill.). 
The cut-off if this assay was considered to be 25 ng/mL. The data (Table 5) 
indicate that all controls containing 25 ng/mL PCP or higher were well 
differentiated from the negative control which indicates the validity of 
the present invention. 
TABLE 5 
______________________________________ 
Ion-Capture Competitive Binding Assay for 
Phenylcyclidine (PCP) in Urine 
PCP 
[ng/mL] Signal Counts 
% Inhibition 
Net Polarization 
______________________________________ 
0 188329 0.00 198 
25 50347 73.3 173 
60 30839 83.6 144 
120 23977 87.3 112 
250 20379 89.2 82 
500 19759 89.5 65 
______________________________________ 
Although the present invention has been described in terms of a prefered 
embodiment, it is anticipated that various modifications and improvements 
will occur to those skilled in the art upon consideration of the present 
invention. Thus shape, material and color of the vessel, material of the 
porous element, material and shape of the absorbent material, shape and 
design of the light guide, type of detector and method of detection, type 
of peroxide used as urea peroxide or similar compounds, wash solution 
composition, prewet solution composition, and method of treating porous 
elements to decrease nonspecific binding and provide the necessary 
interactions between the immobilizable complex and the porous element 
according to the present invention, can all be optimized by those skilled 
in the art. Although examples were shown for one step sandwich and 
competitive assays, two and more step assays can be performed. 
Microparticles used to perform the solid phase immunoassay are preferably 
selected to have an average diameter smaller than the average effective 
pore size of the porous element. Although the examples were given using 
carboxylated polystyrene particles, other particulate material can be 
used, such as polystyrene, polymethyl acylate, derivatized cellulose 
fibers, polyacylamide and the like. 
The ion capture procedures were described using polyglutamic acid as the 
polyanion acid derivatized polycationic material and other methods of 
attachment of these compounds to the assay components or the porous 
element can be used. 
Moreover, the assay method of this invention may be extended to smaller 
molecules or to nucleic acid probe assays. Furthermore, although the 
invention has been described using acridinium sulfonamide-labeled and 
alkaline phosphatase-labeled tracers, it may be extended to other acridin 
ium compounds or their analogs or even other luminescent compounds. For 
example, the read head design as described above may accommodate luminol 
type chemiluminescent immunoassays by using two ports for injecting 
trigger solution and two other ports for injecting a catalyst solution. It 
can also be extended to phenol-enhanced chemiluminescence assay. 
Apparatus and methods according to the present invention may be employed in 
assays for the detection of viral particles, such as HBsAg or HIV 
particles, or fragments thereof. Macromolecular disease state markers, 
such as carcinoembryonic antigen ("CEA") and alphafetoprotein ("AFP") may 
also be detected, as may nutritional status markers, such as vitamin B12, 
folate and ferritin. Also usefully detected by the apparatus and according 
to the methods of the present invention are hormones (e.g., B-HCG, TSH, LH 
and FSH). bacteria (e.g. streptococci) nuclei acid species (e.g. DNA or 
RNA). The present invention is also useful in small molecular competitive 
binding assays such as those for T3, T4, free T4 and digoxin. Substances 
of abuse may be detected using the methods and apparatus according to the 
present invention. 
Allergy testing may be carried out by attaching allergen extracts to 
microparticles forming protein-coated microparticles, which may be 
incubated with body fluid sample to capture specific IgE. In such an assay 
acridinium-labeled goat anti-human IgE may be employed as a conjugate 
which may be reacted with bound IgE, followed by washing, activating and 
reading of the result.