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Rapid agglutination testing in an ultrasonic standing wave
Journal of Immunological Methods, 165 (1993) 47-57
47
© 1993 Elsevier Science Publishers B.V. All rights reserved 0022-1759/93/$06.00
JIM 06802
Rapid agglutination testing in an ultrasonic standing wave M.A. G r u n d y a, W . E . B o l e k b, W.T. C o a k l e y a a n d E. B e n e s b School of Pure and Applied Biology, University of Wales College of Cardiff, Cathays Park, Cardiff CF1 3TL, UK, and b Technische Universitiit, Institut fiir Allgemeine Physik, Wiedner Haupstrasse 8-12, 1040 Wien, Austria (Received 25 November 1992, revised received 12 May 1993, accepted 17 May 1993)
The time taken to perform diagnostic agglutination tests can be significantly reduced by applying an ultrasonic standing wave field to a droplet of reactants held in a capillary tube. Avian erythrocytes, bacteria and latex particles from commercially available test kits were agglutinated in 15 s, 5 min, and 1 min respectively. These times compare favourably with the times of 30 min, 4 h, and 8 min required for agglutination by the methods prescribed for the respective kits. No loss in sensitivity or specificity was observed with the ultrasonic method. A multi-test procedure is also described whereby a series of five droplets loaded in a single capillary can be tested in less than 4 min by drawing the capillary along the axis of the ultrasonic field of a ring transducer. Key words: Ultrasound; Ultrasonic standing wave field; Agglutination test; Ring transducer; Immunoprecipitation
Introduction
Agglutination reactions are employed in diagnostic laboratory testing for the presence of a wide variety of antibodies, infectious agents or chemical compounds in samples of patients' body fluids. These tests may (particularly in the case of weakly positive samples) require long incubation periods before a positive agglutination can be discerned. For example, the Weil-Felix (Proteus cell agglutination) test for anti-Rickettsial antibodies takes 12 h at room temperature or 4 h at 50°C (conditions recommended by Wellcome Diagnostics) and the development of diagnostic test patterns in microtitre plate haemagglutination assays employing antibody or antigen-coated avian Correspondence to: W.T. Coakley, School of Pure and Applied Biology, University of Wales College of Cardiff, Cathays Park, Cardiff CF1 3TL, UK. Tel.: (0222) 874000, ext. 4287; Fax: (0222) 874305.
erythrocytes occurs within 60 min (Cayzer et al., 1974). Particles or cells suspended in an ultrasonic standing wave (of a pressure amplitude which does not induce cavitation or acoustic streaming) can rapidly concentrate particles at positions of particle potential energy minima in the field (Whitworth and Coakley, 1992). These concentrated cells can also experience sound-induced attractive particle-particle interactions (Hager, 1991; Hager and Benes, 1991). The enhancement of the rate of immuno-agglutination of cells due to these particle concentrating interactions has been demonstrated for Legionella in the presence of antiserum (Jepras et al., 1989) and for antibody-coated turkey erythrocytes in the presence of viral particles (Grundy et al., 1989). These single-sample tests involved dipping the end of a narrow glass capillary tube into a reservoir containing a mixture of serum and cells such that about 30 /zl of reactants were drawn up into the
48 tube by capillarity. The reservoir and capillary were mounted above a plane transducer which generated a standing wave in the suspension along the capillary axis. Positive agglutination was rapidly detected by the adhesion of Legionella to the capillary wall at separations of half an acoustic wavelength in the axial direction. In the case of a reverse passive haemagglutination assay (RPHA) for the presence of hepatitis B virus surface antigen (HBsAg) the ultrasound-concentrated cells did not adhere to the capillary wall. The weak viral crosslinking of the ultrasonicated cells was readily disrupted in flow so that the sample could not be ejected for examination of agglutination. Instead, following ultrasonication, the capillary was removed from the reservoir and inverted so that the aggregates of cells produced in the ultrasonic field fell onto the curved surface of the reaction droplet's meniscus. Following a short delay, positive and negative agglutination reactions were distinguishable by characteristic patterns on the meniscus (Grundy et al., 1989). The total time of about 4 rain required to test a single serum sample for the presence of HBsAg was short compared with the 30 min required to conventionally perform the same test in microtitre wells. We report below on the development of a ring transducer apparatus in which the rates of a variety of immuno-agglutination reactions were enhanced without recourse to capillary inversion. Most agglutination assays employed in diagnostic laboratories involve either bacterial agglutination, haemagglutination or passive agglutination of an antibody or antigen-coated particle or cell (Kuby, 1992). We have examined Proteus agglutination, the HBsAg R P H A and passive agglutination of antigen-coated latex particles (as particular examples of bacterial, passive red cell and particle agglutinations, respectively) in the ring transducer system. The ring transducer device allows axial capillary movement which, unlike the situation for the standing wave generated by a plane transducer (Jepras et al., 1989; Grundy et al., 1989) facilitates the rapid testing of a series of reaction droplets held in a single capillary tube. The potential of this system for multiple sample testing has been demonstrated by ultrasonicating a series
of five droplets (including positive and negative samples) for each of the above agglutination reactions.
Materials and methods
Agglutination test reactants and their conventional test procedures Reverse passive haemagglutination assay for hepatitis B virus surface antigen. A suspension of lyophilized tanned turkey erythrocytes coated with purified horse antibody to HBsAg was prepared from a hepatitis B surface antigen H A screening kit (Wellcome Diagnostics, Dartford, UK). Turkey erythrocytes coated with normal horse globulin were used as control ceils. Heat inactivated diluted human serum containing HBsAg (Wellcome Diagnostics, Dartford, UK) was further diluted 1 / 8 with diluent buffer (sterile phosphate buffered saline, pH 7.2, containing normal turkey serum, normal horse serum, normal human serum and sodium azide), to serve as a positive control. A 1 / 8 dilution of normal human serum served as a negative control. The conventional R P H A method for the detection of HBsAg involves mixing equal (25 ~zl) volumes of serum and test cell suspensions in U-shaped microtitre wells. Following 30 min incubation at room temperature positive and negative agglutination reactions are discernible by their characteristic sedimentation patterns. Proteus cell agglutination. A suspension of stained killed Proteus OX19 cells preserved in formalin and thiomersal was agglutinated with rabbit anti-Proteus OX19 agglutinating serum. Normal rabbit serum served as a negative control (Wellcome Diagnostics, Dartford, UK). The titre of the Proteus agglutinating serum was estimated by adding one drop of the stained Proteus suspension to 1 ml of each of a series of doubling dilutions (from 1 / 2 0 to 1/1280) of serum held in test-tubes. The tubes were then incubated at 50°C for 4 h (the conventional incubation conditions for the Weft-Felix test) and examined for the highest dilution showing signs of cell agglutination. Passive latex agglutination test for the detection of antibodies to cytomegaloL,irus. A suspension
49
o f latex particles coated with disrupted cytomegalovirus (CMV) and human CMV agglutinating serum were obtained from a CMVScan latex agglutination kit (Becton Dickinson, Oxford, UK). Normal human serum served as a negative control. The recommended test procedure for the CMVScan kit involves adding one drop of CMVcoated latex particle suspension to 25 ~1 of serum sample on a black test card. The reactants are then mixed and the card rotated for 8 min before the reaction droplet is examined for evidence of agglutination. Using this method the titre of the CMV agglutinating serum was estimated by testing a series of doubling dilutions for the highest dilution giving positive latex agglutination.
Ultrasonic assay technique Fig. 1 illustrates the assembly used to ultrasonicate agglutination reaction droplets held in a capillary tube. The reaction chamber consisted of a 12.5 mm long, 31.5 mm inside diameter and 3.4 mm wall thickness PZT4 Sonox P4 tubular transducer (Hoechst Ceram Tec., Germany), sandwiched between two discs of perspex. Each of these discs had a central hole which was very slightly larger than the 2.8 mm outside diameter, glass capillary tube (of 2.0 mm inside diameter: Fisons Scientific Equipment, Loughborough, UK) so as to accurately locate the tube on the transducer axis and yet allow water to pass between the tube and the rim of the hole (as required below). A perspex tube was sealed at one end and attached to the lower face of the reaction chamber. The perspex tube, together with the reaction chamber and a small reservoir on top of the reaction chamber were filled with distilled water, taking care not to introduce any air bubbles. The water in the reaction chamber served as a coupling medium for ultrasound between the transducer and the capillary tube, whilst the reservoir of water allowed for insertion and vertical displacement of the capillary without the introduction of air bubbles which might have impaired the ultrasonic field. Prior to insertion into the apparatus the capillary tube was loaded with up to five reaction droplets separated by air spaces using a Pi Pump pipette filler (Fisons Scientific Equip-
capillary tube reservoir
RF
"eaction droplets ~
transduc
separated by ~ir spaces
perspex water
Fig. 1. Experimental assembly for the exposure of reaction droplets within a capillary tube to ultrasound. T h e dimensions of the tubular transducer were: 12.5 m m high, 31.5 m m inside diameter and 3.4 m m wall thickness. T h e transducer driving voltage (RF) is derived from a radio frequency amplifier. Represented diagrammatically is an ultrasonicated droplet showing concentration of cells into an annulus about the axis of the system, whilst the cells of a previously ultrasonicated droplet (above the reaction chamber) are shown sedimenting onto the lower meniscus of the droplet.
ment, Loughborough, UK). The reaction droplets consisted of 25/xl of antibody component plus 25 txl of antigen component. The test procedure involved placing a droplet (which had been drawn into the capillary) at the sensitive high sound pressure axial region of the reaction chamber (Fig. 1). The transducer was driven at 1.97 MHz (an odd multiple of its fundamental radial thickness resonance of 0.66 MHz). The impedance of the water filled ring at 1.97 MHz was 7.5 ~. A low amplitude signal obtained from a two channel
50 frequency synthesizer (Model 3326A Hewlett Packard) was amplified by a 100 W rapid frequency amplifier (Model 2100, Eni, New York, USA) and the required amplifier output voltage was applied to the tubular transducer for a selected time. A schematic diagram of the ultrasound generating assembly is shown in Fig. 2. In cases where multiple droplets were to be tested the sound was switched off when the caprilary was raised to bring the next droplet into position. The pipette filler remained connected to the capillary throughout the test to prevent movement of the reaction droplets within it.
Measurement of the acoustic pressure fieM of the ring transducer A radial ultrasonic pressure field plot (Fig. 3) was obtained, with the upper perspex disc of the reaction chamber removed, by moving the sensing end of a Dapco hydrophone mounted on a motorized stage at a constant speed from the inner wall to the axis of the ring transducer. The hydrophone consisted of a squared-off hypodermic needle of about 1.2 mm outer diameter and 34 mm in length with the sensing element at the tip of the needle. Fig. 3 shows the relative acoustic pressure field of the ring transducer (driven at its fundamental resonance frequency of 0.66 MHz) and illustrates the relatively very high ultrasound pressures generated at the axis as a result of the focusing effect
I generator RFsignalI
t
1
ring transducer Fig. 2. A schematic diagram of the ultrasound generating system. A radio frequency (RF) signal from a frequency synthesizer was amplified and applied to the tubular transducer via connections to its electroded inner and outer surfaces.
I I
I
16.5 mm Fig. 3. Relative radial pressure profile in water for the 31 mm inner diameter ring transducer when driven at its fundamental resonance frequency of 0.66 MHz with an applied transducer voltage of 3.0 Vp-p. of the transducer's geometry. The axial peak pressure is up to ten times greater than off-axis pressure peaks elsewhere in the field. A field plot was not made at 1.97 MHz (the operating frequency for the agglutination tests) because the tip of the hydrophone was too large to measure accurately the change in pressure between successive maxima or minima.
Results
Reverse passive haemagglutination assay When the ultrasonic field was turned on, the turkey cells in the HBsAg R P H A reaction
51
droplets concentrated into an annulus about the axis of the capillary tube. The exposure times required for the first signs of annular concentration were established for a range of transducer voltages and are shown in Fig. 4. The sound exposure regime then selected for the haemagglutination experiments was to apply 10 Vp-p to the transducer for 15 s (Fig. 4). When the ultrasound was turned off to end an exposure the concentrated cells within the reaction droplet sedimented rapidly onto the meniscus. Immediate examination with a × 8 magnifying glass showed small clumps of cells scattered over the meniscus for all combinations of serum and cells tested. However, within two minutes there was a clear difference (Table I) between the menisci of the test cell plus positive serum mixtures, where a typical granular appearance was maintained (Fig. 5a), and the other cell plus serum combinations where the clumps of cells produced in the ultrasonic field had disaggregated to produce a clear meniscus and a smooth ring of cells around the edge of the meniscus (as in Fig. 5b). The sensitivity of the ultrasonic technique was the same as the microtitre well method, i.e., the highest dilution of HBsAg positive serum which
302826242220A
~ 164.o 1 2 10" 86" 420
i
~
~
~,
~ ~
"~ ~
~ l'o
transducer voltage (vp-p)
Fig. 4. Time taken for initial detection of an annulus of concentrated test cells for a range of voltages applied to a ring transducer driven at 1.97 MHz.
TABLE I RESULTS OF E X P O S U R E OF DROPLETS OF D I F F E R ENT COMBINATIONS OF A V I A N E R Y T H R O C Y T E S A N D SERA TO U L T R A S O U N D Droplet no.
Composition of RPHA reaction droplet
Ultrasonic agglutination
Test cells + negative serum Test cells + positive serum Control cells + positive serum Test cells + positive serum Control cells + negative serum
No Yes No Yes No
agglutinated the test cells was 1 / 6 4 for both methods. Some experiments were carried out to establish the response of the ultrasonic R P H A test to the sound exposure conditions. To this end samples were exposed to ultrasound for 15 s periods with an applied transducer voltage of 100 Vp-p (i.e., ten times the voltage normally employed to produce rapid agglutination of test cells). With this higher voltage positive and negative test reactions continued to be distinguishable after sedimentation despite the fact that the acoustic inter-particle forces causing clumping of control cells were 100 times greater (Nyborg, 1978; Hager, 1991) than the forces on cells treated at 10 Vp-p, i.e., no false positive results were obtained at the higher sound pressures. It was found that using voltages at and just above the 1.2 Vp-p threshold for cell concentration into an annulus in 15 s (Fig. 4) positive haemagglutination reactions were not subsequently detected at the meniscus. This was because insufficient agglutination had occurred during the exposure to ultrasound. While, in the interest of magnification and at the cost of depth of focus, some resolution was lost photographically (Fig. 5a) positive and negative samples were always clearly distinguishable by eye following the 10 Vp-p 15 s exposure selected for routine use. The total time taken to process five R P H A reaction droplets held in one capillary tube and drawn along the axis of the transducer as described in the materials and methods section was less than 3.5 min. This time consisted of five 15 s exposures, post-exposure movements of the capillary and a final 2 min for the cells of the last
52 d r o p l e t to a d o p t t h e i r definitive c o n f i g u r a t i o n on the meniscus.
Bacterial agglutination F o l l o w i n g 4 h i n c u b a t i o n in test t u b e s at 50°C, t h e g r e a t e s t d i l u t i o n o f Proteus a g g l u t i n a t i n g s e r u m showing a g g l u t i n a t i o n o f Proteus ceils was 1 / 1 6 0 . This s a m e d i l u t i o n o f a n t i s e r u m s h o w e d
Proteus a g g l u t i n a t i o n following 5 min e x p o s u r e to u l t r a s o u n d with an a p p l i e d t r a n s d u c e r v o l t a g e of 30 Vp-p. T h e Proteus cell a g g l u t i n a t e s w e r e stable against flow stress a n d so the r e a c t i o n d r o p l e t s c o u l d be e x p e l l e d f r o m the c a p i l l a r y o n t o a cer a m i c tile a n d e x a m i n e d i m m e d i a t e l y following s o n i c a t i o n w i t h o u t waiting for p r e c i p i t a t i o n o n t o a meniscus. M o r e c o n c e n t r a t e d s a m p l e s o f anti-
Fig. 5. Test droplet meniscus in a capillary two minutes after a 15 s exposure to ultrasound showing (a) a characteristic granular distribution of HBsAg agglutinated turkey erythrocytes over the full surface of the meniscus and (b) control non-agglutinated turkey erythrocytes in a smooth ring at the edge of the meniscus.
53 T A B L E II DEGREE OF PROTEUS CELL AGGLUTINATION DILUTIONS OF AGGLUTINATING SERUM
WITH TIME OF EXPOSURE
TO ULTRASOUND
D i l u t i o n o f Proteus a g g l u t i n a t i n g s e r u m
Treatment
1 min ultrasound 5 min ultrasound 1 rain n o u l t r a s o u n d 10 rain n o u l t r a s o u n d
-ve control
1/20
1/40
1/80
1/160
+ + + + . +
+ + + + . -
+
+
-
-
.
FOR DIFFERENT
1/320
.
Key." - = n o a g g l u t i n a t i o n , + = a g g l u t i n a t i o n visible o n close i n s p e c t i o n , a n d + + = a g g l u t i n a t i o n c l e a r l y visible.
serum required a shorter sonication period (at the same transducer voltage) and produced a stronger agglutination of Proteus cells (Table II). Fig. 6 shows that the degree of Proteus cell agglutination with a 1 / 2 0 dilution of agglutinating serum is much greater following 1 min exposure to ultrasound than it is (for the same volumes of test components) after 4 h in an Eppendoff tube at 50°C (scaled down equivalent of the r e c o m m e n d e d test-tube agglutination method) or 10 min rotation on a tile.
In four experiments where the capillary was loaded with a series of 5 reaction droplets (3 × 1 / 2 0 dilution of agglutinating serum and 2 × negative controls separated by air spaces), and each droplet sonicated for 1 min, agglutination was observed in all of the droplets containing antiserum whilst the negative control droplets showed no signs of agglutination. No agglutination was observed following ultrasonic treatment (up to 15 min) with serum samples which were diluted more than 1/160, indi-
i,i~i~iiiil;,iiiiilL ~,~i!i!i~i,~i~i¸~' i~~!!!iii~ii~iiii{i{iiii!i!i!!!!!!i¸¸!!>i~!! i!i!iil i~ '~'?~ i!¸''~:¸~!ii~;!,iiiii~'¸'¸¸~'',;
Fig. 6. Proteus cell a g g l u t i n a t i o n w i t h 1 / 2 0 d i l u t i o n o f a n t i s e r u m f o l l o w i n g ( a ) 1 m i n e x p o s u r e in a c a p i l l a r y to u l t r a s o u n d a n d i m m e d i a t e e x p u l s i o n o n t o a tile, ( b ) 10 rain r o t a t i o n o n a tile w i t h o u t e x p o s u r e to u l t r a s o u n d , a n d ( c ) 4 h t e s t - t u b e i n c u b a t i o n in a n E p p e n d o r f t u b e at 50°C. d is a n e g a t i v e c o n t r o l o f u l t r a s o n i c a t e d Proteus cells plus n o r m a l s e r u m .
54
cating that although the ultrasonic treatment markedly reduced the test time it did not increase the sensitivity of this reaction.
Passive latex agglutination Following 8 min rotation on a test card the greatest dilution of agglutinating serum from the CMVScan kit showing agglutination of the CMV-coated latex test particles within the prescribed 8 min was 1/40. This dilution showed agglutinated particles when a sample was expelled onto a test card following exposure in the capillary to ultrasound with a transducer voltage of 30 Vp-p for 15 s. Increasing the sonication time resulted in a greater degree of agglutination (Table III). The degree of latex particle agglutination was greater following 1 rain ultrasonic treatment than was seen following 8 min rotation on the test card (Fig. 7). Table III shows that the sensitivity of the ultrasonic test is the same as the conventional test (limit of 1 / 4 0 dilution).
TABLE IlI D E G R E E OF C Y T O M E G A L O V I R U S - C O A T E D LATEX A G G L U T I N A T I O N W I T H TIME OF E X P O S U R E TO ULT R A S O U N D F O R D I F F E R E N T D I L U T I O N S OF AGG L U T I N A T I N G SERUM Time of exposure to ultrasound (s)
Dilution of CMV agglutinating serum 1/20
1/40
15 30 45 60
+ + + ++ ++
+ + ++ ++
1/80
- ve control 1/160
m
~ no agglutination, + = agglutination visible on close inspection, and + + = agglutination clearly visible.
Key:
In six experiments where the capillary was loaded with a series of five reaction droplets (3 × 1/20 serum dilution spaced by 2 × negative controls) agglutination was observed in all of the
5
Fig. 7. Agglutination of cytomegalovirus-coated latex with 1/20 dilution of agglutinating serum following (a) 1 min exposure to ultrasound and immediate expulsion onto a test card, (b) 1 rain rotation on a test card without ultrasound, and (c) 8 min rotation without ultrasound, d is a negative control, i.e., coated latex beads plus normal serum following ultrasonication for 1 min. The upper strip of tests shows the reaction droplets in their 'wet' state where, because of the optical properties of the droplets, the difference in the degree of latex particle agglutination is difficult to fully appreciate in a still photograph. When these reaction droplets are dried (lower strip) the different levels of agglutination are much more obvious. (N.B. drying the droplets is not a recommended diagnostic procedure.)
55 TABLE IV COMPARISON OF TIMES REQUIRED TO TEST A REACTION DROPLET WITH ULTRASOUND AND WITH A CONVENTIONAL TEST Ultrasound
Conventional agglutination
time (rain)
Technique
Antigen coated latex
0.25
Slide/card rotation
Proteus with
5.0
In test tube (50°C)
2.25
Microtitre well
Reaction
Time (rain) 8.0 240
antiserum Antibody coated erythrocytes with virus in serum
30
droplets containing antiserum whilst the negative control droplets showed no signs of agglutination.
Discussion
The times taken to agglutinate cells or particles for the three tests examined are summarised in Table IV and were significantly less than the times required when these reactions were performed using the conventional methods prescribed in the respective test kits. Results for positive test and negative control samples (Tables I-III; Figs. 5-7) show that the ultrasonic technique retains test selectivity. While the ultrasound did not increase the sensitivity of the reactions tested here, there was an enhancement in the degree of Proteus cell and latex particle agglutination, which made positive agglutinations easier to discern (Figs. 6 and 7). We will return in future work to the question of sensitivity enhancement with the ultrasonic system. The ultrasound was generated using an assembly of laboratory test equipment (materials and methods section). The experimental results show that the transducer voltages required for the test were 10 Vp-p for the large avian erythrocytes and 30 Vp-p for the smaller bacteria and latex beads. The power required to drive a resonant 8 J2 transducer at 30 Vp-p is 14 VA. Using either a high frequency transformer for impedance match-
ing or an amplifier with the same impedance as the transducer the total supply power needed is about 30 VA. This is less than the 100 VA available from the broad band amplifier described here. A dedicated device would consist of a simple narrow band linear amplifier optimised for operation at about 2 MHz. A frequency lock-in to maintain the resonant condition would dispense with the need for a frequency synthesizer. Such a device is currently being designed with a view to making the technique more widely accessible at reasonable cost. Fig. 3, which gives relative pressure distributions in a radial scan of the tubular transducer at its fundamental radial thickness resonance of 0.66 MHz, was presented to emphasise the relatively high pressure peak obtaining on the axis of such tubes. Absolute pressure values are not given partly because the hydrophone was not calibrated for such measurements but largely because the tubular transducer was driven at an odd harmonic (1.97 MHz) of its fundamental frequency under the test conditions. The hydrophone diameter (1.2 mm) was too large to resolve standing wave pressure peaks at this frequency (where the half wavelength of ultrasound in water is 0.38 mm). An estimate of the 'effective' pressure associated with a particular transducer voltage may be obtained utilising the following information. It has been shown that 9 /zm polystyrene beads concentrate at half wavelength separations in a 3 MHz standing wave in a wide container in 10 s at a pressure amplitude of 80 kPa (Gould, Coakley and Grundy, 1992). 10 p~m diameter polystyrene beads and human erythrocytes move at comparable rates (for the purposes of this comparison) in ultrasonic standing wave fields (Gould and Coakley, 1974). It has been found that fixed and unfixed human erythrocytes have similar acoustic properties (Weiser and Apfel, 1982). The fixed turkey erythrocytes used in the present work form an annulus in the capillary in 10 s at a transducer voltage of 1.4 V (Fig. 3). If (from the result with 9 /.Lm beads (Gould et al., 1992) above) the pressure is taken to be 80 kPa at 1.4 V (the voltage necessary to form an annulus of fixed erythrocytes in 10 s) then the 'effective' pressure in the tube under the test transducer voltage (10 V) condition is about 600 kPa.
56 The ring transducer described also makes it possible to treat multiple samples in the same capillary by drawing it stepwise along the axis of the transducer following the treatment of each droplet. Because ultrasound would not be transmitted through the interdroplet air spaces, multiple sample treatment was not possible using the plane transducer system previously described (Grundy et al., 1989; Jepras et al., 1989) where the standing wave was set up along the longitudinal axis of a capillary tube. The 2.25 rain required to test a single droplet in the reverse passive haemagglutination assay (Table IV) consisted of a 15 s ultrasound exposure followed by a 2 rain delay before examination of the cell distribution on the meniscus. The 3.5 min required to test five samples largely consisted of the 15 s exposure times for each of the drops and the 2 min delay before the distinctive definitive distribution of cells (Fig. 5) on the meniscus had occurred. A reaction test time of 3.5 min compared well with the 30 min required to discern positive agglutination when the reactants were mixed in microtitre wells (Table IV). Where a single test involved ejection of a sample onto a tile or test card (as for the Proteus or CMV test) the time to test multiple droplets with the system of Fig. 1 becomes linearly proportional to the number of droplets. It may be (provided that multiple samples can be accurately located and spaced in the capillary) that a stack of tubular transducers, spaced so as to coincide with the positions of the droplets, could reduce the total test time for a multi-test device to essentially the time for a single test. The ultrasonic test times given above do not include the time for preparation of the reaction droplets. The only essential difference between the preparation times of droplets for exposure to ultrasound and droplets for conventional testing was the few seconds required to load droplets into and in some cases expel droplets from the capillary. In view of the overall rapidity of the agglutination reactions using ultrasound this extra preparation time is negligible. In multiple sample tests a negative reaction droplet was often positioned above a positive reaction droplet such that when the droplets were expelled from the capillary (following sonication) the negative droplet passed through the region
which had held the positive droplet. In such cases no agglutination was observed in the negative droplets indicating that levels of cross-contamination were not significant. It has been demonstrated above that by exposing reaction droplets in a capillary to a radial standing wave field from a ring transducer, it was possible to rapidly screen samples for HBsAg, anti-Proteus antibodies, or anti-CMV antibodies. The forces acting on particles in suspension in an ultrasonic field are particle volume, density and compressibility dependent (Nyborg, 1978; Coakley et al,, 1989; Hager, 1991) nevertheless we have shown that the technique is sufficiently versatile to agglutinate the range of particle types typically employed in diagnostic agglutination assays (Kuby, 1992), The technique is thus of general applicability.
Acknowledgements M.A.G. was supported by SERC grant G R / F 26706. W.E.B. was supported by the Austrian Science Foundation Project P 7198-PHY and the Erwin Schroedinger Society for Microsciences.
References Cayzer, I., Dane, D.S., Cameron, C.H. and Denning, J.V. (1974) A rapid haemagglutination test for hepatitis B antigen. Lancet i, 747. Coakley, W.T., Bardsley, D.W., Grundy, M.A., Zamani, F. and Clarke, D.J. (1989) Cell manipulation in ultrasonic standing wave fields. J. Chem. Tech. Biotechnol. 44, 43. Gould, R.K. and Coakley, W.T. (1974) The effect of acoustic forces on small particles in suspensions. In: L. Bjorno (Ed.), Proceedings of the 1973 Symposiumon Finite Amplitude Wave Effects in Fluids. IPC Science and Technology Press, Guildford, p. 252. Gould, R.K., Coakley, W.T. and Grundy, M.A. (1992) Upper sound pressure limits on particle concentration in fields of ultrasonic standing-wave at megahertz frequencies. Ultrasonics 30, 239. Grundy, M.A., Coakley, W.T. and Clarke, D.J. (1989) Rapid detection of hepatitis B virus using a haemagglutination assay in an ultrasonic standing wave field. J. Clin. Lab. Immunol. 30, 93. Hager, F. and Benes, E. (1991) A summaryof all forces acting on spherical particles in a sound field. Ultrasonics International 91, Conf. Proc., pp. 283-286.
57 Jepras, R.I., Clarke, D.J. and Coakley, W.T. (1989) Agglutination of Legionella pneumophila by antiserum is accelerated in an ultrasonic standing wave. J. Immunol. Methods 120, 201. Kuby, J. (1992) Immunology. W.H. Freeman, New York. Nyborg, W.L. (1978) Radiation pressure and radiation forces. In: F.J. Fry (Ed.), Ultrasound: Its Applications In Medicine and Biology. Elsevier, Amsterdam, Part 1, p. 52. Weiser, M.A.H. and Apfel, R.E. (1982) Extension of acoustic
levitation to include the study of micron-size particles in a more compressible host fluid. J. Acoust. Soc. Am. 71, 1261. Whitworth, G. and Coakley, W.T. (1992) Particle column formation in a stationary ultrasonic field. J. Acoust. Soc. Am. 91, 79. Whitworth, G., Grundy, M.A. and Coakley, W.T. (1991) Transport and harvesting of suspended particles using modulated ultrasound. Ultrasonics 29, 439.
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© 1993 Elsevier Science Publishers B.V. All rights reserved 0022-1759/93/$06.00
JIM 06802
Rapid agglutination testing in an ultrasonic standing wave M.A. G r u n d y a, W . E . B o l e k b, W.T. C o a k l e y a a n d E. B e n e s b School of Pure and Applied Biology, University of Wales College of Cardiff, Cathays Park, Cardiff CF1 3TL, UK, and b Technische Universitiit, Institut fiir Allgemeine Physik, Wiedner Haupstrasse 8-12, 1040 Wien, Austria (Received 25 November 1992, revised received 12 May 1993, accepted 17 May 1993)
The time taken to perform diagnostic agglutination tests can be significantly reduced by applying an ultrasonic standing wave field to a droplet of reactants held in a capillary tube. Avian erythrocytes, bacteria and latex particles from commercially available test kits were agglutinated in 15 s, 5 min, and 1 min respectively. These times compare favourably with the times of 30 min, 4 h, and 8 min required for agglutination by the methods prescribed for the respective kits. No loss in sensitivity or specificity was observed with the ultrasonic method. A multi-test procedure is also described whereby a series of five droplets loaded in a single capillary can be tested in less than 4 min by drawing the capillary along the axis of the ultrasonic field of a ring transducer. Key words: Ultrasound; Ultrasonic standing wave field; Agglutination test; Ring transducer; Immunoprecipitation
Introduction
Agglutination reactions are employed in diagnostic laboratory testing for the presence of a wide variety of antibodies, infectious agents or chemical compounds in samples of patients' body fluids. These tests may (particularly in the case of weakly positive samples) require long incubation periods before a positive agglutination can be discerned. For example, the Weil-Felix (Proteus cell agglutination) test for anti-Rickettsial antibodies takes 12 h at room temperature or 4 h at 50°C (conditions recommended by Wellcome Diagnostics) and the development of diagnostic test patterns in microtitre plate haemagglutination assays employing antibody or antigen-coated avian Correspondence to: W.T. Coakley, School of Pure and Applied Biology, University of Wales College of Cardiff, Cathays Park, Cardiff CF1 3TL, UK. Tel.: (0222) 874000, ext. 4287; Fax: (0222) 874305.
erythrocytes occurs within 60 min (Cayzer et al., 1974). Particles or cells suspended in an ultrasonic standing wave (of a pressure amplitude which does not induce cavitation or acoustic streaming) can rapidly concentrate particles at positions of particle potential energy minima in the field (Whitworth and Coakley, 1992). These concentrated cells can also experience sound-induced attractive particle-particle interactions (Hager, 1991; Hager and Benes, 1991). The enhancement of the rate of immuno-agglutination of cells due to these particle concentrating interactions has been demonstrated for Legionella in the presence of antiserum (Jepras et al., 1989) and for antibody-coated turkey erythrocytes in the presence of viral particles (Grundy et al., 1989). These single-sample tests involved dipping the end of a narrow glass capillary tube into a reservoir containing a mixture of serum and cells such that about 30 /zl of reactants were drawn up into the
48 tube by capillarity. The reservoir and capillary were mounted above a plane transducer which generated a standing wave in the suspension along the capillary axis. Positive agglutination was rapidly detected by the adhesion of Legionella to the capillary wall at separations of half an acoustic wavelength in the axial direction. In the case of a reverse passive haemagglutination assay (RPHA) for the presence of hepatitis B virus surface antigen (HBsAg) the ultrasound-concentrated cells did not adhere to the capillary wall. The weak viral crosslinking of the ultrasonicated cells was readily disrupted in flow so that the sample could not be ejected for examination of agglutination. Instead, following ultrasonication, the capillary was removed from the reservoir and inverted so that the aggregates of cells produced in the ultrasonic field fell onto the curved surface of the reaction droplet's meniscus. Following a short delay, positive and negative agglutination reactions were distinguishable by characteristic patterns on the meniscus (Grundy et al., 1989). The total time of about 4 rain required to test a single serum sample for the presence of HBsAg was short compared with the 30 min required to conventionally perform the same test in microtitre wells. We report below on the development of a ring transducer apparatus in which the rates of a variety of immuno-agglutination reactions were enhanced without recourse to capillary inversion. Most agglutination assays employed in diagnostic laboratories involve either bacterial agglutination, haemagglutination or passive agglutination of an antibody or antigen-coated particle or cell (Kuby, 1992). We have examined Proteus agglutination, the HBsAg R P H A and passive agglutination of antigen-coated latex particles (as particular examples of bacterial, passive red cell and particle agglutinations, respectively) in the ring transducer system. The ring transducer device allows axial capillary movement which, unlike the situation for the standing wave generated by a plane transducer (Jepras et al., 1989; Grundy et al., 1989) facilitates the rapid testing of a series of reaction droplets held in a single capillary tube. The potential of this system for multiple sample testing has been demonstrated by ultrasonicating a series
of five droplets (including positive and negative samples) for each of the above agglutination reactions.
Materials and methods
Agglutination test reactants and their conventional test procedures Reverse passive haemagglutination assay for hepatitis B virus surface antigen. A suspension of lyophilized tanned turkey erythrocytes coated with purified horse antibody to HBsAg was prepared from a hepatitis B surface antigen H A screening kit (Wellcome Diagnostics, Dartford, UK). Turkey erythrocytes coated with normal horse globulin were used as control ceils. Heat inactivated diluted human serum containing HBsAg (Wellcome Diagnostics, Dartford, UK) was further diluted 1 / 8 with diluent buffer (sterile phosphate buffered saline, pH 7.2, containing normal turkey serum, normal horse serum, normal human serum and sodium azide), to serve as a positive control. A 1 / 8 dilution of normal human serum served as a negative control. The conventional R P H A method for the detection of HBsAg involves mixing equal (25 ~zl) volumes of serum and test cell suspensions in U-shaped microtitre wells. Following 30 min incubation at room temperature positive and negative agglutination reactions are discernible by their characteristic sedimentation patterns. Proteus cell agglutination. A suspension of stained killed Proteus OX19 cells preserved in formalin and thiomersal was agglutinated with rabbit anti-Proteus OX19 agglutinating serum. Normal rabbit serum served as a negative control (Wellcome Diagnostics, Dartford, UK). The titre of the Proteus agglutinating serum was estimated by adding one drop of the stained Proteus suspension to 1 ml of each of a series of doubling dilutions (from 1 / 2 0 to 1/1280) of serum held in test-tubes. The tubes were then incubated at 50°C for 4 h (the conventional incubation conditions for the Weft-Felix test) and examined for the highest dilution showing signs of cell agglutination. Passive latex agglutination test for the detection of antibodies to cytomegaloL,irus. A suspension
49
o f latex particles coated with disrupted cytomegalovirus (CMV) and human CMV agglutinating serum were obtained from a CMVScan latex agglutination kit (Becton Dickinson, Oxford, UK). Normal human serum served as a negative control. The recommended test procedure for the CMVScan kit involves adding one drop of CMVcoated latex particle suspension to 25 ~1 of serum sample on a black test card. The reactants are then mixed and the card rotated for 8 min before the reaction droplet is examined for evidence of agglutination. Using this method the titre of the CMV agglutinating serum was estimated by testing a series of doubling dilutions for the highest dilution giving positive latex agglutination.
Ultrasonic assay technique Fig. 1 illustrates the assembly used to ultrasonicate agglutination reaction droplets held in a capillary tube. The reaction chamber consisted of a 12.5 mm long, 31.5 mm inside diameter and 3.4 mm wall thickness PZT4 Sonox P4 tubular transducer (Hoechst Ceram Tec., Germany), sandwiched between two discs of perspex. Each of these discs had a central hole which was very slightly larger than the 2.8 mm outside diameter, glass capillary tube (of 2.0 mm inside diameter: Fisons Scientific Equipment, Loughborough, UK) so as to accurately locate the tube on the transducer axis and yet allow water to pass between the tube and the rim of the hole (as required below). A perspex tube was sealed at one end and attached to the lower face of the reaction chamber. The perspex tube, together with the reaction chamber and a small reservoir on top of the reaction chamber were filled with distilled water, taking care not to introduce any air bubbles. The water in the reaction chamber served as a coupling medium for ultrasound between the transducer and the capillary tube, whilst the reservoir of water allowed for insertion and vertical displacement of the capillary without the introduction of air bubbles which might have impaired the ultrasonic field. Prior to insertion into the apparatus the capillary tube was loaded with up to five reaction droplets separated by air spaces using a Pi Pump pipette filler (Fisons Scientific Equip-
capillary tube reservoir
RF
"eaction droplets ~
transduc
separated by ~ir spaces
perspex water
Fig. 1. Experimental assembly for the exposure of reaction droplets within a capillary tube to ultrasound. T h e dimensions of the tubular transducer were: 12.5 m m high, 31.5 m m inside diameter and 3.4 m m wall thickness. T h e transducer driving voltage (RF) is derived from a radio frequency amplifier. Represented diagrammatically is an ultrasonicated droplet showing concentration of cells into an annulus about the axis of the system, whilst the cells of a previously ultrasonicated droplet (above the reaction chamber) are shown sedimenting onto the lower meniscus of the droplet.
ment, Loughborough, UK). The reaction droplets consisted of 25/xl of antibody component plus 25 txl of antigen component. The test procedure involved placing a droplet (which had been drawn into the capillary) at the sensitive high sound pressure axial region of the reaction chamber (Fig. 1). The transducer was driven at 1.97 MHz (an odd multiple of its fundamental radial thickness resonance of 0.66 MHz). The impedance of the water filled ring at 1.97 MHz was 7.5 ~. A low amplitude signal obtained from a two channel
50 frequency synthesizer (Model 3326A Hewlett Packard) was amplified by a 100 W rapid frequency amplifier (Model 2100, Eni, New York, USA) and the required amplifier output voltage was applied to the tubular transducer for a selected time. A schematic diagram of the ultrasound generating assembly is shown in Fig. 2. In cases where multiple droplets were to be tested the sound was switched off when the caprilary was raised to bring the next droplet into position. The pipette filler remained connected to the capillary throughout the test to prevent movement of the reaction droplets within it.
Measurement of the acoustic pressure fieM of the ring transducer A radial ultrasonic pressure field plot (Fig. 3) was obtained, with the upper perspex disc of the reaction chamber removed, by moving the sensing end of a Dapco hydrophone mounted on a motorized stage at a constant speed from the inner wall to the axis of the ring transducer. The hydrophone consisted of a squared-off hypodermic needle of about 1.2 mm outer diameter and 34 mm in length with the sensing element at the tip of the needle. Fig. 3 shows the relative acoustic pressure field of the ring transducer (driven at its fundamental resonance frequency of 0.66 MHz) and illustrates the relatively very high ultrasound pressures generated at the axis as a result of the focusing effect
I generator RFsignalI
t
1
ring transducer Fig. 2. A schematic diagram of the ultrasound generating system. A radio frequency (RF) signal from a frequency synthesizer was amplified and applied to the tubular transducer via connections to its electroded inner and outer surfaces.
I I
I
16.5 mm Fig. 3. Relative radial pressure profile in water for the 31 mm inner diameter ring transducer when driven at its fundamental resonance frequency of 0.66 MHz with an applied transducer voltage of 3.0 Vp-p. of the transducer's geometry. The axial peak pressure is up to ten times greater than off-axis pressure peaks elsewhere in the field. A field plot was not made at 1.97 MHz (the operating frequency for the agglutination tests) because the tip of the hydrophone was too large to measure accurately the change in pressure between successive maxima or minima.
Results
Reverse passive haemagglutination assay When the ultrasonic field was turned on, the turkey cells in the HBsAg R P H A reaction
51
droplets concentrated into an annulus about the axis of the capillary tube. The exposure times required for the first signs of annular concentration were established for a range of transducer voltages and are shown in Fig. 4. The sound exposure regime then selected for the haemagglutination experiments was to apply 10 Vp-p to the transducer for 15 s (Fig. 4). When the ultrasound was turned off to end an exposure the concentrated cells within the reaction droplet sedimented rapidly onto the meniscus. Immediate examination with a × 8 magnifying glass showed small clumps of cells scattered over the meniscus for all combinations of serum and cells tested. However, within two minutes there was a clear difference (Table I) between the menisci of the test cell plus positive serum mixtures, where a typical granular appearance was maintained (Fig. 5a), and the other cell plus serum combinations where the clumps of cells produced in the ultrasonic field had disaggregated to produce a clear meniscus and a smooth ring of cells around the edge of the meniscus (as in Fig. 5b). The sensitivity of the ultrasonic technique was the same as the microtitre well method, i.e., the highest dilution of HBsAg positive serum which
302826242220A
~ 164.o 1 2 10" 86" 420
i
~
~
~,
~ ~
"~ ~
~ l'o
transducer voltage (vp-p)
Fig. 4. Time taken for initial detection of an annulus of concentrated test cells for a range of voltages applied to a ring transducer driven at 1.97 MHz.
TABLE I RESULTS OF E X P O S U R E OF DROPLETS OF D I F F E R ENT COMBINATIONS OF A V I A N E R Y T H R O C Y T E S A N D SERA TO U L T R A S O U N D Droplet no.
Composition of RPHA reaction droplet
Ultrasonic agglutination
Test cells + negative serum Test cells + positive serum Control cells + positive serum Test cells + positive serum Control cells + negative serum
No Yes No Yes No
agglutinated the test cells was 1 / 6 4 for both methods. Some experiments were carried out to establish the response of the ultrasonic R P H A test to the sound exposure conditions. To this end samples were exposed to ultrasound for 15 s periods with an applied transducer voltage of 100 Vp-p (i.e., ten times the voltage normally employed to produce rapid agglutination of test cells). With this higher voltage positive and negative test reactions continued to be distinguishable after sedimentation despite the fact that the acoustic inter-particle forces causing clumping of control cells were 100 times greater (Nyborg, 1978; Hager, 1991) than the forces on cells treated at 10 Vp-p, i.e., no false positive results were obtained at the higher sound pressures. It was found that using voltages at and just above the 1.2 Vp-p threshold for cell concentration into an annulus in 15 s (Fig. 4) positive haemagglutination reactions were not subsequently detected at the meniscus. This was because insufficient agglutination had occurred during the exposure to ultrasound. While, in the interest of magnification and at the cost of depth of focus, some resolution was lost photographically (Fig. 5a) positive and negative samples were always clearly distinguishable by eye following the 10 Vp-p 15 s exposure selected for routine use. The total time taken to process five R P H A reaction droplets held in one capillary tube and drawn along the axis of the transducer as described in the materials and methods section was less than 3.5 min. This time consisted of five 15 s exposures, post-exposure movements of the capillary and a final 2 min for the cells of the last
52 d r o p l e t to a d o p t t h e i r definitive c o n f i g u r a t i o n on the meniscus.
Bacterial agglutination F o l l o w i n g 4 h i n c u b a t i o n in test t u b e s at 50°C, t h e g r e a t e s t d i l u t i o n o f Proteus a g g l u t i n a t i n g s e r u m showing a g g l u t i n a t i o n o f Proteus ceils was 1 / 1 6 0 . This s a m e d i l u t i o n o f a n t i s e r u m s h o w e d
Proteus a g g l u t i n a t i o n following 5 min e x p o s u r e to u l t r a s o u n d with an a p p l i e d t r a n s d u c e r v o l t a g e of 30 Vp-p. T h e Proteus cell a g g l u t i n a t e s w e r e stable against flow stress a n d so the r e a c t i o n d r o p l e t s c o u l d be e x p e l l e d f r o m the c a p i l l a r y o n t o a cer a m i c tile a n d e x a m i n e d i m m e d i a t e l y following s o n i c a t i o n w i t h o u t waiting for p r e c i p i t a t i o n o n t o a meniscus. M o r e c o n c e n t r a t e d s a m p l e s o f anti-
Fig. 5. Test droplet meniscus in a capillary two minutes after a 15 s exposure to ultrasound showing (a) a characteristic granular distribution of HBsAg agglutinated turkey erythrocytes over the full surface of the meniscus and (b) control non-agglutinated turkey erythrocytes in a smooth ring at the edge of the meniscus.
53 T A B L E II DEGREE OF PROTEUS CELL AGGLUTINATION DILUTIONS OF AGGLUTINATING SERUM
WITH TIME OF EXPOSURE
TO ULTRASOUND
D i l u t i o n o f Proteus a g g l u t i n a t i n g s e r u m
Treatment
1 min ultrasound 5 min ultrasound 1 rain n o u l t r a s o u n d 10 rain n o u l t r a s o u n d
-ve control
1/20
1/40
1/80
1/160
+ + + + . +
+ + + + . -
+
+
-
-
.
FOR DIFFERENT
1/320
.
Key." - = n o a g g l u t i n a t i o n , + = a g g l u t i n a t i o n visible o n close i n s p e c t i o n , a n d + + = a g g l u t i n a t i o n c l e a r l y visible.
serum required a shorter sonication period (at the same transducer voltage) and produced a stronger agglutination of Proteus cells (Table II). Fig. 6 shows that the degree of Proteus cell agglutination with a 1 / 2 0 dilution of agglutinating serum is much greater following 1 min exposure to ultrasound than it is (for the same volumes of test components) after 4 h in an Eppendoff tube at 50°C (scaled down equivalent of the r e c o m m e n d e d test-tube agglutination method) or 10 min rotation on a tile.
In four experiments where the capillary was loaded with a series of 5 reaction droplets (3 × 1 / 2 0 dilution of agglutinating serum and 2 × negative controls separated by air spaces), and each droplet sonicated for 1 min, agglutination was observed in all of the droplets containing antiserum whilst the negative control droplets showed no signs of agglutination. No agglutination was observed following ultrasonic treatment (up to 15 min) with serum samples which were diluted more than 1/160, indi-
i,i~i~iiiil;,iiiiilL ~,~i!i!i~i,~i~i¸~' i~~!!!iii~ii~iiii{i{iiii!i!i!!!!!!i¸¸!!>i~!! i!i!iil i~ '~'?~ i!¸''~:¸~!ii~;!,iiiii~'¸'¸¸~'',;
Fig. 6. Proteus cell a g g l u t i n a t i o n w i t h 1 / 2 0 d i l u t i o n o f a n t i s e r u m f o l l o w i n g ( a ) 1 m i n e x p o s u r e in a c a p i l l a r y to u l t r a s o u n d a n d i m m e d i a t e e x p u l s i o n o n t o a tile, ( b ) 10 rain r o t a t i o n o n a tile w i t h o u t e x p o s u r e to u l t r a s o u n d , a n d ( c ) 4 h t e s t - t u b e i n c u b a t i o n in a n E p p e n d o r f t u b e at 50°C. d is a n e g a t i v e c o n t r o l o f u l t r a s o n i c a t e d Proteus cells plus n o r m a l s e r u m .
54
cating that although the ultrasonic treatment markedly reduced the test time it did not increase the sensitivity of this reaction.
Passive latex agglutination Following 8 min rotation on a test card the greatest dilution of agglutinating serum from the CMVScan kit showing agglutination of the CMV-coated latex test particles within the prescribed 8 min was 1/40. This dilution showed agglutinated particles when a sample was expelled onto a test card following exposure in the capillary to ultrasound with a transducer voltage of 30 Vp-p for 15 s. Increasing the sonication time resulted in a greater degree of agglutination (Table III). The degree of latex particle agglutination was greater following 1 rain ultrasonic treatment than was seen following 8 min rotation on the test card (Fig. 7). Table III shows that the sensitivity of the ultrasonic test is the same as the conventional test (limit of 1 / 4 0 dilution).
TABLE IlI D E G R E E OF C Y T O M E G A L O V I R U S - C O A T E D LATEX A G G L U T I N A T I O N W I T H TIME OF E X P O S U R E TO ULT R A S O U N D F O R D I F F E R E N T D I L U T I O N S OF AGG L U T I N A T I N G SERUM Time of exposure to ultrasound (s)
Dilution of CMV agglutinating serum 1/20
1/40
15 30 45 60
+ + + ++ ++
+ + ++ ++
1/80
- ve control 1/160
m
~ no agglutination, + = agglutination visible on close inspection, and + + = agglutination clearly visible.
Key:
In six experiments where the capillary was loaded with a series of five reaction droplets (3 × 1/20 serum dilution spaced by 2 × negative controls) agglutination was observed in all of the
5
Fig. 7. Agglutination of cytomegalovirus-coated latex with 1/20 dilution of agglutinating serum following (a) 1 min exposure to ultrasound and immediate expulsion onto a test card, (b) 1 rain rotation on a test card without ultrasound, and (c) 8 min rotation without ultrasound, d is a negative control, i.e., coated latex beads plus normal serum following ultrasonication for 1 min. The upper strip of tests shows the reaction droplets in their 'wet' state where, because of the optical properties of the droplets, the difference in the degree of latex particle agglutination is difficult to fully appreciate in a still photograph. When these reaction droplets are dried (lower strip) the different levels of agglutination are much more obvious. (N.B. drying the droplets is not a recommended diagnostic procedure.)
55 TABLE IV COMPARISON OF TIMES REQUIRED TO TEST A REACTION DROPLET WITH ULTRASOUND AND WITH A CONVENTIONAL TEST Ultrasound
Conventional agglutination
time (rain)
Technique
Antigen coated latex
0.25
Slide/card rotation
Proteus with
5.0
In test tube (50°C)
2.25
Microtitre well
Reaction
Time (rain) 8.0 240
antiserum Antibody coated erythrocytes with virus in serum
30
droplets containing antiserum whilst the negative control droplets showed no signs of agglutination.
Discussion
The times taken to agglutinate cells or particles for the three tests examined are summarised in Table IV and were significantly less than the times required when these reactions were performed using the conventional methods prescribed in the respective test kits. Results for positive test and negative control samples (Tables I-III; Figs. 5-7) show that the ultrasonic technique retains test selectivity. While the ultrasound did not increase the sensitivity of the reactions tested here, there was an enhancement in the degree of Proteus cell and latex particle agglutination, which made positive agglutinations easier to discern (Figs. 6 and 7). We will return in future work to the question of sensitivity enhancement with the ultrasonic system. The ultrasound was generated using an assembly of laboratory test equipment (materials and methods section). The experimental results show that the transducer voltages required for the test were 10 Vp-p for the large avian erythrocytes and 30 Vp-p for the smaller bacteria and latex beads. The power required to drive a resonant 8 J2 transducer at 30 Vp-p is 14 VA. Using either a high frequency transformer for impedance match-
ing or an amplifier with the same impedance as the transducer the total supply power needed is about 30 VA. This is less than the 100 VA available from the broad band amplifier described here. A dedicated device would consist of a simple narrow band linear amplifier optimised for operation at about 2 MHz. A frequency lock-in to maintain the resonant condition would dispense with the need for a frequency synthesizer. Such a device is currently being designed with a view to making the technique more widely accessible at reasonable cost. Fig. 3, which gives relative pressure distributions in a radial scan of the tubular transducer at its fundamental radial thickness resonance of 0.66 MHz, was presented to emphasise the relatively high pressure peak obtaining on the axis of such tubes. Absolute pressure values are not given partly because the hydrophone was not calibrated for such measurements but largely because the tubular transducer was driven at an odd harmonic (1.97 MHz) of its fundamental frequency under the test conditions. The hydrophone diameter (1.2 mm) was too large to resolve standing wave pressure peaks at this frequency (where the half wavelength of ultrasound in water is 0.38 mm). An estimate of the 'effective' pressure associated with a particular transducer voltage may be obtained utilising the following information. It has been shown that 9 /zm polystyrene beads concentrate at half wavelength separations in a 3 MHz standing wave in a wide container in 10 s at a pressure amplitude of 80 kPa (Gould, Coakley and Grundy, 1992). 10 p~m diameter polystyrene beads and human erythrocytes move at comparable rates (for the purposes of this comparison) in ultrasonic standing wave fields (Gould and Coakley, 1974). It has been found that fixed and unfixed human erythrocytes have similar acoustic properties (Weiser and Apfel, 1982). The fixed turkey erythrocytes used in the present work form an annulus in the capillary in 10 s at a transducer voltage of 1.4 V (Fig. 3). If (from the result with 9 /.Lm beads (Gould et al., 1992) above) the pressure is taken to be 80 kPa at 1.4 V (the voltage necessary to form an annulus of fixed erythrocytes in 10 s) then the 'effective' pressure in the tube under the test transducer voltage (10 V) condition is about 600 kPa.
56 The ring transducer described also makes it possible to treat multiple samples in the same capillary by drawing it stepwise along the axis of the transducer following the treatment of each droplet. Because ultrasound would not be transmitted through the interdroplet air spaces, multiple sample treatment was not possible using the plane transducer system previously described (Grundy et al., 1989; Jepras et al., 1989) where the standing wave was set up along the longitudinal axis of a capillary tube. The 2.25 rain required to test a single droplet in the reverse passive haemagglutination assay (Table IV) consisted of a 15 s ultrasound exposure followed by a 2 rain delay before examination of the cell distribution on the meniscus. The 3.5 min required to test five samples largely consisted of the 15 s exposure times for each of the drops and the 2 min delay before the distinctive definitive distribution of cells (Fig. 5) on the meniscus had occurred. A reaction test time of 3.5 min compared well with the 30 min required to discern positive agglutination when the reactants were mixed in microtitre wells (Table IV). Where a single test involved ejection of a sample onto a tile or test card (as for the Proteus or CMV test) the time to test multiple droplets with the system of Fig. 1 becomes linearly proportional to the number of droplets. It may be (provided that multiple samples can be accurately located and spaced in the capillary) that a stack of tubular transducers, spaced so as to coincide with the positions of the droplets, could reduce the total test time for a multi-test device to essentially the time for a single test. The ultrasonic test times given above do not include the time for preparation of the reaction droplets. The only essential difference between the preparation times of droplets for exposure to ultrasound and droplets for conventional testing was the few seconds required to load droplets into and in some cases expel droplets from the capillary. In view of the overall rapidity of the agglutination reactions using ultrasound this extra preparation time is negligible. In multiple sample tests a negative reaction droplet was often positioned above a positive reaction droplet such that when the droplets were expelled from the capillary (following sonication) the negative droplet passed through the region
which had held the positive droplet. In such cases no agglutination was observed in the negative droplets indicating that levels of cross-contamination were not significant. It has been demonstrated above that by exposing reaction droplets in a capillary to a radial standing wave field from a ring transducer, it was possible to rapidly screen samples for HBsAg, anti-Proteus antibodies, or anti-CMV antibodies. The forces acting on particles in suspension in an ultrasonic field are particle volume, density and compressibility dependent (Nyborg, 1978; Coakley et al,, 1989; Hager, 1991) nevertheless we have shown that the technique is sufficiently versatile to agglutinate the range of particle types typically employed in diagnostic agglutination assays (Kuby, 1992), The technique is thus of general applicability.
Acknowledgements M.A.G. was supported by SERC grant G R / F 26706. W.E.B. was supported by the Austrian Science Foundation Project P 7198-PHY and the Erwin Schroedinger Society for Microsciences.
References Cayzer, I., Dane, D.S., Cameron, C.H. and Denning, J.V. (1974) A rapid haemagglutination test for hepatitis B antigen. Lancet i, 747. Coakley, W.T., Bardsley, D.W., Grundy, M.A., Zamani, F. and Clarke, D.J. (1989) Cell manipulation in ultrasonic standing wave fields. J. Chem. Tech. Biotechnol. 44, 43. Gould, R.K. and Coakley, W.T. (1974) The effect of acoustic forces on small particles in suspensions. In: L. Bjorno (Ed.), Proceedings of the 1973 Symposiumon Finite Amplitude Wave Effects in Fluids. IPC Science and Technology Press, Guildford, p. 252. Gould, R.K., Coakley, W.T. and Grundy, M.A. (1992) Upper sound pressure limits on particle concentration in fields of ultrasonic standing-wave at megahertz frequencies. Ultrasonics 30, 239. Grundy, M.A., Coakley, W.T. and Clarke, D.J. (1989) Rapid detection of hepatitis B virus using a haemagglutination assay in an ultrasonic standing wave field. J. Clin. Lab. Immunol. 30, 93. Hager, F. and Benes, E. (1991) A summaryof all forces acting on spherical particles in a sound field. Ultrasonics International 91, Conf. Proc., pp. 283-286.
57 Jepras, R.I., Clarke, D.J. and Coakley, W.T. (1989) Agglutination of Legionella pneumophila by antiserum is accelerated in an ultrasonic standing wave. J. Immunol. Methods 120, 201. Kuby, J. (1992) Immunology. W.H. Freeman, New York. Nyborg, W.L. (1978) Radiation pressure and radiation forces. In: F.J. Fry (Ed.), Ultrasound: Its Applications In Medicine and Biology. Elsevier, Amsterdam, Part 1, p. 52. Weiser, M.A.H. and Apfel, R.E. (1982) Extension of acoustic
levitation to include the study of micron-size particles in a more compressible host fluid. J. Acoust. Soc. Am. 71, 1261. Whitworth, G. and Coakley, W.T. (1992) Particle column formation in a stationary ultrasonic field. J. Acoust. Soc. Am. 91, 79. Whitworth, G., Grundy, M.A. and Coakley, W.T. (1991) Transport and harvesting of suspended particles using modulated ultrasound. Ultrasonics 29, 439.