ANALYTICAL
196, 61-68 (1991)
BIOCHEMISTRY
Detection Apparatus Chemiluminescence
for Multiple Heterogeneous Immunoassay Configurations
Omar S. Khalil,i Thomas F. Zurek, Curtis Pepe, Kevin Genger, Denise G. Huff, Charles Hanna, Roger Hu, Jim Mackowiack, and Larry Bennett Diagnostics
Received
Division,
January
Abbott
Laboratories,
Abbott Park, Illinois
Carole Coleman,
60064
7, 1991
We describe an apparatus for measuring signals emanated from two heterogeneous chemiluminescence immunoassay (CLIA) configurations: antibody-coated polystyrene beads, in reaction tray wells, and microparticles captured by a porous matrix. An optics and fluidics design which allows the use of a common detection head for these two different assay configurations is described. The detection head moves along three Cartesian coordinates to create a localized light-tight compartment around each individual disposable reaction vessel. Reproducibility of the light seal, trigger solution delivery, and mixing is achieved for acridinium-labeled CLIA. The coated polystyrene beads configuration is tested using @HCG, CEA, and TSH assays. The microparticle-capture configuration is tested using @HCG and HBsAg assays. The microparticle capture CLIA has shorter incubation times and the potential for ease of 0 1991 Academic Press. Inc. automation.
Chemiluminescence (CL)2 measurements offer a potential for detection limits lower than those achieved by fluorescence measurements (l-3). This alleviates problems associated with rejection of excitation light, fluctuation in light source intensity, heat dissipation from in-
i To whom correspondence should be addressed at Department 9YA, APlA, Transfusion Diagnostics R&D, Diagnostics Division, Abbott Laboratories, Abbott Park, IL 60064. ’ Abbreviations used: CL, chemiluminescence; IA, immunoassays; CB, coated bead; MPC, microparticle capture; pmt, photomultiplier tube; MEIA, microparticle enzyme immunoassay; MCV, microparticle capture vessel; HBsAg, hepatitis B surface antigen; PBS, phosphate-buffered saline; Chaps, 3-[(3-cholamidopropyl)dimethyl ammoniolpropanesulfonic acid, PHCG, human chorionic gonadotropin; CEA, carcinoembryonic antigen; TSH, thyroid-stimulating hormone; EDAC, 1-ethyl-3-(3dimethylaminopropyl)carbodiimide; Mes, 4-morpholineethanesulfonic acid; BSA, bovine serum albumin; LDS, lithium dodecyl sulfate. 0003.2697/91 53.00 Copyright 0 1991 by Academic Press, All rights of reproduction in any form
candescent lamps, and electronic noise generated by arc and flash lamps (3). Although CL is suggested as a preferred detection technique for a high sensitivity immunoassay (IA) (4-6), it has not been widely used. There are several reports on automating CLIA measurements (7,8). Two commercial CL systems have been recently introduced, a magnetizable microparticles/acridinium ester label system (9-11) and an EIA microtiter plate/ enhanced CL system (12). The emergence of these IA analyzers, manual luminometers (13), and new signal generating systems such as enzyme-activated dioxetane CL (14-17) indicates the growing interest in CL and the potential for its widespread use as a detection methodology for heterogeneous IAs. We describe an apparatus for measuring CL signals emanated in a variety of heterogeneous IA configurations. We also describe two methods for performing such assays. A novel design for CL collection optics and trigger solution injection is described. It allows the use of a common detection head for two assay configurations: coated polystyrene beads in reaction tray wells (CB-CLIA) and microparticles captured by a porous matrix (MPC-CLIA). The microprocessor-controlled apparatus can be used for either assay configuration by changing the carrier of the reaction vessels, the number of trigger solution injection ports, and the rate of trigger solution injection. The apparatus is best suited for detecting triggerable short-lived CL signals. EXPERIMENTAL
General Description of the Apparatus A block diagram of the apparatus is shown in Fig. 1. It consists of a light-tight enclosure containing a detection head that moves in the 2 and Y directions and a holder for an array of reaction vessels that moves in the X direction. The detection head is located in a “home position” where it is primed and purged and dark counts of 61
Inc. reserved.
62
KHALIL
c SYSTEM + 5 POWER + 12 -+ SUPPLY
BERTAN HIGHVOLTAGE SUPPLY
+
AMPLIFIER BOARD
COUNTER/ TIMER
+
PMT HAMMAMATSU R647-04 TRIGGER SOLUTION INJECTORS *=HAMILTON MICROLABM PIPETTER
X,YANDZ STEPPER MOTORS -
t
FIG.
1.
Block
MOTOR CONTROL BOARD diagram
COMPUTER IBM-XT
c -_ +
I
1 PRINTER
1
of the instrument.
the photon counting detector are determined. It moves during operation to predetermined “mapped” positions of the array of reaction vessels. Detection-Head Design and Construction Chemical excitation and collection of emitted light takes place within a light-tight compartment that is created when a shroud surrounding the detection head is positioned on the holder of the reaction vessels. A schematic diagram of the detection head is shown in Fig. 2. It consists of a light guide (3 mm diameter, 75 mm long) made of polished Pyrex glass and surrounded by equally spaced holes for trigger solution tubes. The tubes protrude 1 mm (0.04 in.) below the surface of the light guide. A machined polyacetal block holds a photomultiplier tube (pmt), socket, socket holder, and fluid-lines guide. A back plate holds these components and is fastened to the vertical movement slide.
ET
AL.
Coated bead CLIA is triggered in the following way: after incubation and washing, 100 ~1 phosphate buffer (pH 5.3) is added to each bead in the well. CL is triggered by injecting 200 ~1 of 0.03% alkaline peroxide at a linear speed of 1667 ~11sfrom four ports at 90” angles to each other. High-speed video tracing showed that the bead twirls in the well and complete mixing with the trigger solution is achieved at this injection speed. The use of four injection ports is needed to achieve this twirling and mixing action. After each trigger solution injection, the syringe pump is reversed and 100 ~1 is drawn back to prevent trigger solution siphoning. A schematic of the CB-CLIA injection and detection geometry is shown in Fig. 3. Microparticle-capture CLIA is triggered as follows: 100 ~1of 0.3% alkaline peroxide solution is injected from two collinear ports directed toward the walls of the funnel-shaped detection vessel at a rate of 500 pi/s. A smaller volume of trigger solution (100 ~1) at a lower injection speed was required for MPC-CLIA. Since it is difficult to equally distribute the fluid from the four ports, two ports are used. High-speed video tracing shows that the solution flows down the walls of the funnel and forms a puddle that evenly diffuses through the porous pad. The syringe pump is then reversed and 100 ~1 is drawn back to prevent trigger solution siphoning. In either configuration, the injection ports do not come in touch with the contents of the well or the microparticle capture vessel. Thus no contamination occurs as a result of drawing back 100 ~1of the trigger solution into the injection tubes after each injection step. A schematic of the MPC-CLIA injection and detection geometry is shown in Fig. 4. h
Fluid Delivery Trigger solution injection. A Hamilton Micro Lab M pipettor diluter (Hamilton, Inc., Reno, NV) controlled by an IBM PC-XT through an RS232C interface is used to inject the trigger solution through a 0.75-mm (0.03in.) i.d. Teflon tube to a Minstac multiport manifold. The O.&mm (0.02-in.) i.d. Teflon tubes (Anspec, Inc., Ann Arbor, MI) are connected to the manifold and their termini act as the injection ports in the detection head. Four injection ports at 90” angles to each other are used to trigger coated beads (CB-CLIA). Two injection ports are used for the captured microparticles (MPC-CLIA). The volume of the trigger solution, its rate of injection, and the peroxide concentration are varied from one configuration to the other. The signal collection and injection geometry remain the same.
FIG. 2. Schematic holder, (b) light guide, tioning the detection holder and fluid-lines (h) cover for fluid& socket holder, and (k)
diagram of the detection head: (a) light pipe (c) trigger solution ports, (d) shroud for posihead on the reaction vessel, (e) pmt, (fJ pmt guide, (g) back plate for holding components, and detector assembly, (i) pmt socket,6) pmt trigger solution delivery lines.
CHEMILUMINESCENCE
IMMUNOASSAY
CONFIGURATION
DETECTION
APPARATUS
63
Inc., Middlesex, NJ). It is powered to 960 V by a Bertan PMT-20-A-N high-voltage power supply (Bertan Associates, Hicksville, NY). The photon-counting amplifier is Model DM 102 (Spex Industries, Edison, NJ). The high-voltage supply is powered by 12 V dc and the amplifier and counter-timer boards are powered by 5 V dc. The photon-counting integration time is computer-selectable. In all the assays the full CL emission is integrated over a period of 6 s. Mechanism
FIG. 3. CL excitation and detection for coated-bead CLIA configuration showing: (a) light guide, (b) two of the trigger solution injection ports, (c) trigger solution injection lines, (d) black shroud, (e) white reflective reaction well, and (f) coated polystyrene bead.
Priming and purging. Trigger solution lines are primed with alkaline peroxide before sample processing. They are also purged with deionized water after each bath run. Priming and purging take place while the detection head is in the “home-position” which is a light-tight chamber with a zigzag-patterned drainage tube. The drainage chamber is machined of black polyacetal. When it is in the drainage chamber, the outer shroud of the detection head rests on the light sealing gasket (black Valcro)-padded rim; fluids are injected into an opening under this rim. A Gurman-Rubb pump (Coleman, Inc., Chicago, IL) is computer-activated as part of the assay sequence to pull the prime and purge fluids from this opening, through the zigzag-patterned tube, to a drainage reservoir. This prevents flooding of the detection head during the priming and purging steps. A cross section of the drainage chamber is shown in Fig. 5. The movement of the detector between the home position and the detection positions takes place within the apparatus’ light-tight enclosure. The operator is prompted by the computer screen to open this enclosure only when the detector is protected in the home position, to prevent its exposure. Detection
Electronics
The detector is a Hamamatsu 647-04 head-on photomultiplier tube and an E849-24 socket (Hamamatsu,
Reaction vessels are mounted in a holder that moved in the X direction. The adaptor for reaction vessels is mounted on a 30.48-cm (12-in.) ball slide (Del-Tron Precision, Inc., Brookfield, CT) and is driven by a stepper motor through a timing-belt reduction system. Resolution is set at 0.229 mm (0.0009 in.) per half step. The detection head is mounted on a 5.08-cm (2-in.) ball slide and moved in the 2 and X directions. It is kept in the home position for priming and for dark count determinations. A lifter mechanism, powered by a permanent magnet synchronous motor, moves the detection head along the 2 axis through a linkage. A full stroke positions the detection head in up or down positions. The
FIG. 4. CL excitation and detection for microparticle capture CLIA configuration showing: (a) light guide, (b) the trigger solution injection ports, (c) trigger solution injection lines, (d) black shroud, (e) microparticle capture vessel, (f) porous fibrous matrix, (g) absorbant material, (h) vent hole, and (i) holder for the microparticle capture vessels.
64
KHALIL
-a
ET
AL.
The X axis end-of-travel, Y axis end-of-travel, and lifter up and down movements are determined using a piece of machined aluminum as a flag which interrupts an optosensor (TRW Electronic Components, Carrolton, TX). All reaction vessel positions are mapped into a two-dimensional matrix with an X-Y format. This allows the detection head to randomly access any sample by calling out any absolute position. Coated Bead CLIA
e--J FIG. 6. A cross section of the light-tight drainage chamber showing: (a) CL detection head, (b) light-gasket-padded rim, (c) drainage opening, (d) zigzag patterned drainage tube, and (e) outlet to the pump through the base plate of the light-tight instrument enclosure.
vertical ball-slide assembly carrying the detection head is further mounted on a 15.4-cm (6-in.) horizontal slide for the Y movement. It is stepper motor driven along the Y axis to any predetermined location, with a step resolution 0.025 mm (0.001 in.) per half step, using a timing belt. The lifter mechanism is then lowered to the detection head to rest on the reaction tray or the carrier of the MPC-CLIA reaction vessels and creates a light-tight seal around each reaction vessel. Three position sensors are set on the 2 axis. The up detector sensor ensures that the detector head clears the top of the reaction vessels before its X-Y movement. The down sensor ensures that the detector clears the up sensor and is at a height that allows it to sit in the home position. The down/down sensor indicates that the detector cleared the reaction vessel during its downward movement and has settled on the reaction vessel holder. In this position a light-tight seal is generated between the detector head and the reaction vessel holder. If the detector fails to clear the down/down sensor, a seating error is flagged. The detector moves up and tries two more times then moves to the next position. This ensures low dark counts and prevents the possibility of assay false positive results as the particular position where a light-tight seal is not established will not be triggered and read. The 1.8” permanent magnet stepper motors (Model M061-FD-301, Superior Electronics, Bristol, CT) are used in the X and Y movements. They are controlled by an IBM PC-XT via a Tecmar stepper motor control board (Tecmar Inc., Solon, OH). A 60 Hz, 30 rpm, bidirectional 120 V ac motor is used for the 2 movement.
Vessels
Coated bead CLIA reaction trays are injectionmolded from polystyrene impregnated with 5% (by weight) titanium dioxide to prevent optical cross-talk between adjacent wells and to reflect the generated CL signal toward the detector. The trays have the same dimensions as the polystyrene reaction trays used with EIA kits. The solid phase is 6.35-mm (f-in.) polystyrene beads from the same kits (Abbott Laboratories, North Chicago, IL). After incubation with the samples (standards), they are washed with water using Abbott Penta Wash. A carriage is designed to mount the processed tray on the X-moving slide for CL detection in the described apparatus. Microparticle
Capture
Vessels
The reaction vessels are the capture matrix blotter part of the microparticle enzyme IA (MEIA) reaction cell (18). It consists of a black plastic funnel which is sonically welded to a barrel containing a cylindrical cellulose acetate blotter. A glass filter-paper disk (fibrous matrix) is wedged between the two plastic parts and is in intimate contact with the blotter to facilitate fluid removal. A vent-hole is located at the bottom of the blotter and is connected to a channel molded on the side of the barrel. This protruded molded feature is used to locate the microparticle capture vessel (MCV) in a holder that is subsequently mounted in the instrument. The MCV holder is a machined black polyacetal block 12.7 X 30.48 X 1.27 cm (5 X 12 X 0.5 in.) with 60 positions arranged in a 5 X 12 array. The center-to-center spacing of the MCV positions is 20.37 cm (0.8 in.). Each position has a hole with four key-ways which can mate with the molded feature on the MCV for reproducible positioning of the reaction vessels under the detection head. The 5 x 12 arrangement and the center-to-center spacing of the MCVs match those of the wells in the white polystyrene reaction tray. Data Reduction Once sample processing is completed, a data matrix is displayed presenting the total counts per integration time for each sample position. A statistics program allows choosing an individual coordinate, entire or partial row, entire or partial column, or any combination
CHEMILUMINESCENCE
IMMUNOASSAY
CONFIGURATION
thereof. It calculates the mean, standard deviation, and relative standard deviation for the selected group of samples. Dark counts are determined while the detection head is in its home position. Ten read cycles (total signal integration time) are stored and averaged. Dark counts are normally
196, 61-68 (1991)
BIOCHEMISTRY
Detection Apparatus Chemiluminescence
for Multiple Heterogeneous Immunoassay Configurations
Omar S. Khalil,i Thomas F. Zurek, Curtis Pepe, Kevin Genger, Denise G. Huff, Charles Hanna, Roger Hu, Jim Mackowiack, and Larry Bennett Diagnostics
Received
Division,
January
Abbott
Laboratories,
Abbott Park, Illinois
Carole Coleman,
60064
7, 1991
We describe an apparatus for measuring signals emanated from two heterogeneous chemiluminescence immunoassay (CLIA) configurations: antibody-coated polystyrene beads, in reaction tray wells, and microparticles captured by a porous matrix. An optics and fluidics design which allows the use of a common detection head for these two different assay configurations is described. The detection head moves along three Cartesian coordinates to create a localized light-tight compartment around each individual disposable reaction vessel. Reproducibility of the light seal, trigger solution delivery, and mixing is achieved for acridinium-labeled CLIA. The coated polystyrene beads configuration is tested using @HCG, CEA, and TSH assays. The microparticle-capture configuration is tested using @HCG and HBsAg assays. The microparticle capture CLIA has shorter incubation times and the potential for ease of 0 1991 Academic Press. Inc. automation.
Chemiluminescence (CL)2 measurements offer a potential for detection limits lower than those achieved by fluorescence measurements (l-3). This alleviates problems associated with rejection of excitation light, fluctuation in light source intensity, heat dissipation from in-
i To whom correspondence should be addressed at Department 9YA, APlA, Transfusion Diagnostics R&D, Diagnostics Division, Abbott Laboratories, Abbott Park, IL 60064. ’ Abbreviations used: CL, chemiluminescence; IA, immunoassays; CB, coated bead; MPC, microparticle capture; pmt, photomultiplier tube; MEIA, microparticle enzyme immunoassay; MCV, microparticle capture vessel; HBsAg, hepatitis B surface antigen; PBS, phosphate-buffered saline; Chaps, 3-[(3-cholamidopropyl)dimethyl ammoniolpropanesulfonic acid, PHCG, human chorionic gonadotropin; CEA, carcinoembryonic antigen; TSH, thyroid-stimulating hormone; EDAC, 1-ethyl-3-(3dimethylaminopropyl)carbodiimide; Mes, 4-morpholineethanesulfonic acid; BSA, bovine serum albumin; LDS, lithium dodecyl sulfate. 0003.2697/91 53.00 Copyright 0 1991 by Academic Press, All rights of reproduction in any form
candescent lamps, and electronic noise generated by arc and flash lamps (3). Although CL is suggested as a preferred detection technique for a high sensitivity immunoassay (IA) (4-6), it has not been widely used. There are several reports on automating CLIA measurements (7,8). Two commercial CL systems have been recently introduced, a magnetizable microparticles/acridinium ester label system (9-11) and an EIA microtiter plate/ enhanced CL system (12). The emergence of these IA analyzers, manual luminometers (13), and new signal generating systems such as enzyme-activated dioxetane CL (14-17) indicates the growing interest in CL and the potential for its widespread use as a detection methodology for heterogeneous IAs. We describe an apparatus for measuring CL signals emanated in a variety of heterogeneous IA configurations. We also describe two methods for performing such assays. A novel design for CL collection optics and trigger solution injection is described. It allows the use of a common detection head for two assay configurations: coated polystyrene beads in reaction tray wells (CB-CLIA) and microparticles captured by a porous matrix (MPC-CLIA). The microprocessor-controlled apparatus can be used for either assay configuration by changing the carrier of the reaction vessels, the number of trigger solution injection ports, and the rate of trigger solution injection. The apparatus is best suited for detecting triggerable short-lived CL signals. EXPERIMENTAL
General Description of the Apparatus A block diagram of the apparatus is shown in Fig. 1. It consists of a light-tight enclosure containing a detection head that moves in the 2 and Y directions and a holder for an array of reaction vessels that moves in the X direction. The detection head is located in a “home position” where it is primed and purged and dark counts of 61
Inc. reserved.
62
KHALIL
c SYSTEM + 5 POWER + 12 -+ SUPPLY
BERTAN HIGHVOLTAGE SUPPLY
+
AMPLIFIER BOARD
COUNTER/ TIMER
+
PMT HAMMAMATSU R647-04 TRIGGER SOLUTION INJECTORS *=HAMILTON MICROLABM PIPETTER
X,YANDZ STEPPER MOTORS -
t
FIG.
1.
Block
MOTOR CONTROL BOARD diagram
COMPUTER IBM-XT
c -_ +
I
1 PRINTER
1
of the instrument.
the photon counting detector are determined. It moves during operation to predetermined “mapped” positions of the array of reaction vessels. Detection-Head Design and Construction Chemical excitation and collection of emitted light takes place within a light-tight compartment that is created when a shroud surrounding the detection head is positioned on the holder of the reaction vessels. A schematic diagram of the detection head is shown in Fig. 2. It consists of a light guide (3 mm diameter, 75 mm long) made of polished Pyrex glass and surrounded by equally spaced holes for trigger solution tubes. The tubes protrude 1 mm (0.04 in.) below the surface of the light guide. A machined polyacetal block holds a photomultiplier tube (pmt), socket, socket holder, and fluid-lines guide. A back plate holds these components and is fastened to the vertical movement slide.
ET
AL.
Coated bead CLIA is triggered in the following way: after incubation and washing, 100 ~1 phosphate buffer (pH 5.3) is added to each bead in the well. CL is triggered by injecting 200 ~1 of 0.03% alkaline peroxide at a linear speed of 1667 ~11sfrom four ports at 90” angles to each other. High-speed video tracing showed that the bead twirls in the well and complete mixing with the trigger solution is achieved at this injection speed. The use of four injection ports is needed to achieve this twirling and mixing action. After each trigger solution injection, the syringe pump is reversed and 100 ~1 is drawn back to prevent trigger solution siphoning. A schematic of the CB-CLIA injection and detection geometry is shown in Fig. 3. Microparticle-capture CLIA is triggered as follows: 100 ~1of 0.3% alkaline peroxide solution is injected from two collinear ports directed toward the walls of the funnel-shaped detection vessel at a rate of 500 pi/s. A smaller volume of trigger solution (100 ~1) at a lower injection speed was required for MPC-CLIA. Since it is difficult to equally distribute the fluid from the four ports, two ports are used. High-speed video tracing shows that the solution flows down the walls of the funnel and forms a puddle that evenly diffuses through the porous pad. The syringe pump is then reversed and 100 ~1 is drawn back to prevent trigger solution siphoning. In either configuration, the injection ports do not come in touch with the contents of the well or the microparticle capture vessel. Thus no contamination occurs as a result of drawing back 100 ~1of the trigger solution into the injection tubes after each injection step. A schematic of the MPC-CLIA injection and detection geometry is shown in Fig. 4. h
Fluid Delivery Trigger solution injection. A Hamilton Micro Lab M pipettor diluter (Hamilton, Inc., Reno, NV) controlled by an IBM PC-XT through an RS232C interface is used to inject the trigger solution through a 0.75-mm (0.03in.) i.d. Teflon tube to a Minstac multiport manifold. The O.&mm (0.02-in.) i.d. Teflon tubes (Anspec, Inc., Ann Arbor, MI) are connected to the manifold and their termini act as the injection ports in the detection head. Four injection ports at 90” angles to each other are used to trigger coated beads (CB-CLIA). Two injection ports are used for the captured microparticles (MPC-CLIA). The volume of the trigger solution, its rate of injection, and the peroxide concentration are varied from one configuration to the other. The signal collection and injection geometry remain the same.
FIG. 2. Schematic holder, (b) light guide, tioning the detection holder and fluid-lines (h) cover for fluid& socket holder, and (k)
diagram of the detection head: (a) light pipe (c) trigger solution ports, (d) shroud for posihead on the reaction vessel, (e) pmt, (fJ pmt guide, (g) back plate for holding components, and detector assembly, (i) pmt socket,6) pmt trigger solution delivery lines.
CHEMILUMINESCENCE
IMMUNOASSAY
CONFIGURATION
DETECTION
APPARATUS
63
Inc., Middlesex, NJ). It is powered to 960 V by a Bertan PMT-20-A-N high-voltage power supply (Bertan Associates, Hicksville, NY). The photon-counting amplifier is Model DM 102 (Spex Industries, Edison, NJ). The high-voltage supply is powered by 12 V dc and the amplifier and counter-timer boards are powered by 5 V dc. The photon-counting integration time is computer-selectable. In all the assays the full CL emission is integrated over a period of 6 s. Mechanism
FIG. 3. CL excitation and detection for coated-bead CLIA configuration showing: (a) light guide, (b) two of the trigger solution injection ports, (c) trigger solution injection lines, (d) black shroud, (e) white reflective reaction well, and (f) coated polystyrene bead.
Priming and purging. Trigger solution lines are primed with alkaline peroxide before sample processing. They are also purged with deionized water after each bath run. Priming and purging take place while the detection head is in the “home-position” which is a light-tight chamber with a zigzag-patterned drainage tube. The drainage chamber is machined of black polyacetal. When it is in the drainage chamber, the outer shroud of the detection head rests on the light sealing gasket (black Valcro)-padded rim; fluids are injected into an opening under this rim. A Gurman-Rubb pump (Coleman, Inc., Chicago, IL) is computer-activated as part of the assay sequence to pull the prime and purge fluids from this opening, through the zigzag-patterned tube, to a drainage reservoir. This prevents flooding of the detection head during the priming and purging steps. A cross section of the drainage chamber is shown in Fig. 5. The movement of the detector between the home position and the detection positions takes place within the apparatus’ light-tight enclosure. The operator is prompted by the computer screen to open this enclosure only when the detector is protected in the home position, to prevent its exposure. Detection
Electronics
The detector is a Hamamatsu 647-04 head-on photomultiplier tube and an E849-24 socket (Hamamatsu,
Reaction vessels are mounted in a holder that moved in the X direction. The adaptor for reaction vessels is mounted on a 30.48-cm (12-in.) ball slide (Del-Tron Precision, Inc., Brookfield, CT) and is driven by a stepper motor through a timing-belt reduction system. Resolution is set at 0.229 mm (0.0009 in.) per half step. The detection head is mounted on a 5.08-cm (2-in.) ball slide and moved in the 2 and X directions. It is kept in the home position for priming and for dark count determinations. A lifter mechanism, powered by a permanent magnet synchronous motor, moves the detection head along the 2 axis through a linkage. A full stroke positions the detection head in up or down positions. The
FIG. 4. CL excitation and detection for microparticle capture CLIA configuration showing: (a) light guide, (b) the trigger solution injection ports, (c) trigger solution injection lines, (d) black shroud, (e) microparticle capture vessel, (f) porous fibrous matrix, (g) absorbant material, (h) vent hole, and (i) holder for the microparticle capture vessels.
64
KHALIL
-a
ET
AL.
The X axis end-of-travel, Y axis end-of-travel, and lifter up and down movements are determined using a piece of machined aluminum as a flag which interrupts an optosensor (TRW Electronic Components, Carrolton, TX). All reaction vessel positions are mapped into a two-dimensional matrix with an X-Y format. This allows the detection head to randomly access any sample by calling out any absolute position. Coated Bead CLIA
e--J FIG. 6. A cross section of the light-tight drainage chamber showing: (a) CL detection head, (b) light-gasket-padded rim, (c) drainage opening, (d) zigzag patterned drainage tube, and (e) outlet to the pump through the base plate of the light-tight instrument enclosure.
vertical ball-slide assembly carrying the detection head is further mounted on a 15.4-cm (6-in.) horizontal slide for the Y movement. It is stepper motor driven along the Y axis to any predetermined location, with a step resolution 0.025 mm (0.001 in.) per half step, using a timing belt. The lifter mechanism is then lowered to the detection head to rest on the reaction tray or the carrier of the MPC-CLIA reaction vessels and creates a light-tight seal around each reaction vessel. Three position sensors are set on the 2 axis. The up detector sensor ensures that the detector head clears the top of the reaction vessels before its X-Y movement. The down sensor ensures that the detector clears the up sensor and is at a height that allows it to sit in the home position. The down/down sensor indicates that the detector cleared the reaction vessel during its downward movement and has settled on the reaction vessel holder. In this position a light-tight seal is generated between the detector head and the reaction vessel holder. If the detector fails to clear the down/down sensor, a seating error is flagged. The detector moves up and tries two more times then moves to the next position. This ensures low dark counts and prevents the possibility of assay false positive results as the particular position where a light-tight seal is not established will not be triggered and read. The 1.8” permanent magnet stepper motors (Model M061-FD-301, Superior Electronics, Bristol, CT) are used in the X and Y movements. They are controlled by an IBM PC-XT via a Tecmar stepper motor control board (Tecmar Inc., Solon, OH). A 60 Hz, 30 rpm, bidirectional 120 V ac motor is used for the 2 movement.
Vessels
Coated bead CLIA reaction trays are injectionmolded from polystyrene impregnated with 5% (by weight) titanium dioxide to prevent optical cross-talk between adjacent wells and to reflect the generated CL signal toward the detector. The trays have the same dimensions as the polystyrene reaction trays used with EIA kits. The solid phase is 6.35-mm (f-in.) polystyrene beads from the same kits (Abbott Laboratories, North Chicago, IL). After incubation with the samples (standards), they are washed with water using Abbott Penta Wash. A carriage is designed to mount the processed tray on the X-moving slide for CL detection in the described apparatus. Microparticle
Capture
Vessels
The reaction vessels are the capture matrix blotter part of the microparticle enzyme IA (MEIA) reaction cell (18). It consists of a black plastic funnel which is sonically welded to a barrel containing a cylindrical cellulose acetate blotter. A glass filter-paper disk (fibrous matrix) is wedged between the two plastic parts and is in intimate contact with the blotter to facilitate fluid removal. A vent-hole is located at the bottom of the blotter and is connected to a channel molded on the side of the barrel. This protruded molded feature is used to locate the microparticle capture vessel (MCV) in a holder that is subsequently mounted in the instrument. The MCV holder is a machined black polyacetal block 12.7 X 30.48 X 1.27 cm (5 X 12 X 0.5 in.) with 60 positions arranged in a 5 X 12 array. The center-to-center spacing of the MCV positions is 20.37 cm (0.8 in.). Each position has a hole with four key-ways which can mate with the molded feature on the MCV for reproducible positioning of the reaction vessels under the detection head. The 5 x 12 arrangement and the center-to-center spacing of the MCVs match those of the wells in the white polystyrene reaction tray. Data Reduction Once sample processing is completed, a data matrix is displayed presenting the total counts per integration time for each sample position. A statistics program allows choosing an individual coordinate, entire or partial row, entire or partial column, or any combination
CHEMILUMINESCENCE
IMMUNOASSAY
CONFIGURATION
thereof. It calculates the mean, standard deviation, and relative standard deviation for the selected group of samples. Dark counts are determined while the detection head is in its home position. Ten read cycles (total signal integration time) are stored and averaged. Dark counts are normally
