Agglutination of Legionella pneumophila by antiserum is accelerated in an ultrasonic standing wave

Journal of Immunological Methods, 120 (1989) 201-205

201

Elsevier JIM 05195

Agglutination of Legionellapneumophila by antiserum is accelerated in an ultrasonic standing wave R.I. Jepras 1, D.J. Clarke 1 and W.T. Coakley 2 1 Sensor Group, Division of Biotechnology, PHLS Centrefor Applied Microbiology and Research, Porton Down, Salisbury SP40JG, U.K., and 2 School of Pure and Applied Biology, University of Wales Collegeof Cardiff, Newport Road, Cardiff CF2 1TA, U.K.

(Received8 November1988,revisedreceived20 January1989, accepted30 January1989)

The agglutination of Legionella pneumophila (LP) by diluted anti-LP whole rabbit serum has been compared in conventional microwell plates and in capillary containers where the suspension was exposed to a 1 MHz ultrasonic standing wave field. A positive reaction in the standing wave field was detected as a series of cell agglutinates, separated by half an acoustic wavelength (0.75 ram), distributed along the length of the capillary. Agglutination occurred in 60 s or less with ultrasound, while the incubation period for a positive microwell test was often of the order of hours. At a given antiserum concentration, ultrasound-induced agglutination occurred at LP concentrations two-fold lower than those giving a positive result in the microwell plate assays. At cell concentrations near the lower limit for detection of a positive result in the microwell plates a positive reaction was detected in the standing wave field at antiserum concentrations up to 500-fold lower than those forming visible precipitates in the conventional assay. Key words: Ultrasound; Standingwave; Accousticradiationforce; Agglutination;Legionellapneumophila

Introduction

Particles in an acoustic standing wave field experience radiation forces which tend to move them close to pressure nodes separated by half an acoustic wavelength (Kundt and Lehmann, 1874), which may ultimately lead to the aggregation of the particles. The radiation forces depend on some or all of the following properties: the frequency of the applied sound, the square of the sound pressure amplitude, the size of the particle and the difference between the density and compressibility of the particles and those of the suspending phase (Gould and Coakley, 1974; Nyborg, 1978; Coak-

Correspondence to: W.T. Coaldey,Schoolof Pure and Applied Biology,Universityof WalesCollegeof Cardiff,Newport Road, CardiffCF2 1TA, U.K.

ley et al., 1989). Biological cells can be concentrated in suspension by these forces (Dyson et al., 1971; Baker, 1972; Gould and Coakley, 1974; Coakley et al., 1989) and cell contact can be enhanced (Tllley et al., 1987). In the present study, we have investigated whether acoustic radiation forces could usefully accelerate the rate of agglutination of LP by antiserum. Agglutination in an ultrasonic field was compared with agglutination of LP in a conventional microwell plate assay.

Materials and methods Legionella pneumophila (strain Corby), inactivated by exposure to formaldehyde (0.1% v/v) for reasons of safety, and rabbit anti-LP serum were kindly provided by Dr. R. Fitzgeorge (PHLS CAMR, Porton Down). The antiserum was passed

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through a 0.22/,m filter (SLGV 025 BS, Millipore, Millex-GV, Molsheirn, France) and diluted as required in physiological saline. LP suspensions were washed and resuspended in physiological saline, LP concentrations were determined from phase contrast microscopy counts in a bacterial counting chamber (Thomas Weber, Middlesex, England). Portions (50/,1) of serial dilutions of cell suspension were added to samples (50/,1) of dilutions of antiserum preparation in the wells of a microwell plate (Nunc, Kamstrup, D K 4000 Roskilde, Denmark). The microwell plates were gently agitated manually after addition of the samples and were then left to stand at room temperature for 2 h, before being placed in a refrigerator at 4 ° C for 24 h. The time taken for bacterial cell aggregation detection by eye was recorded during the first 2 h after incubation commenced. In the case of ultrasound-treated samples, agglutination was compared in three different containers of 1 mm, 2 mm and 5 mm internal diameters. When working with 50/,1 volumes, 1 mm i.d. cylindrical capillaries (Drumond Scientific Company, Broomall, PA, U.S.A.) were used. A 150/,1 portion of diluted antiserum preparation was added to an equal volume of bacterial cell suspension in a small perspex container with a thin (12 # m thick) Cling Film base. The resulting 300 ~tl suspension was mixed briefly by gentle manual agitation. The capillary tubes were supported vertically in the suspension as shown in Fig. 1 and 50 /,1 of suspension were drawn by capillary action into the tube. Sealing the top of the tube with plasticine protected against the possibility of any fine ultrasonic aerosol formation (Lang, 1962) at the air-water interface. When working with 100/,1 volumes, 2 mm i.d. capillary tubes were used and a 100 /LI volume of reaction mixture was drawn into the tube using a syringe and silicone rubber tube attachment. When working with a 5 mm i.d. cylindrical glass container which had a Cling Film base 300/,1 of reaction mixture were pipetted into the tube. Ultrasound was generated by driving a 30 mm diameter lead zirconate PZT5 disc transducer (Vernatron, Southampton, U.K.) at its thickness resonant frequency of 1 MHz, using a sine wave generating oscillator and a 12 W radiofrequency power amplifier (Model RC 102-12, Wessex Electronics, Bristol, U.K.) at an output

CAPILLARY

P

RF

Fig. 1. Experimental arrangement for exposure of LP and antiserum mixtures to ultrasound. 1 MHz ultrasound from a radio-frequency generator (R_F) was coupled through degassed water to the small perspex container (P). Loading the LP and antiserum into the capillary is described in the text.

amplitude of 14.5 V (zero to peak). The sound was coupled through degassed water to a small perspex cylindrical container and the capillary as shown in Fig. 1. The coupling water was de-gassed under vacuum before loading (in order to avoid acoustic cavitation). The acoustic power output (measured by a radiation pressure balance) when 14.5 V was applied to similar transducers working (in the absence of sound reflection) into a large tank of degassed water was 120 mW (Coakley, 1971). Such power output can be readily generated by commercially available equipment such as physiotherapy ultrasonic units. For technical reasons it was not possible to carry out peak standing wave pressure amplitude measurements within, or close to the base of, the capillary. The results of replicate agglutination experiments were quite reproducible despite working in what, for a travelling wave, would be 'near field' distances from the transducer. The times for cell agglutination, at a fixed transducer voltage, did not appear to be critically dependent on variations in the location of the capillary from one experiment to another or

203 TABLE I TIME TAKEN FOR VISIBLE ULTRASOUND AGGLUTINATION IN A 1 mm (i.d.) CAPILLARY TUBE Organisms (LP) were mixed with diluted antiserum at the indicated concentrations and were exposed to ultrasound (see text for details). NR indicates no reaction after 15 min and ND indicates not done. no. of LP/ml

Time (min) taken for agglutination at the following dilutions of antiserum:

(>(lOS)

1/2

1/8

1/32

1/64

1/128

1/512

1/1024

1/2048

1/4096

1/8192

5 2.5 1.25 0.625

0.08 0.08 0.08 NR

0.08 2 2

0.08 2 2

0.08 ND NR

0.08 4

0.08 4

0.08 ND

ND 4

0.08 NR

0.08 NR

TABLE II TIME TAKEN FOR VISIBLE ULTRASOUND AGGLUTINATION IN A 2 mm (i.d.) CAPILLARY TUBE Other details as for Table I no. of LP/ml

Time (rain) taken for agglutination at the following dilutions of antiserum:

(×108)

1/2

1/4

1/8

1/32

1/64

1/256

1/512

1/1024

1/2048

1/4096

1/8192

5 2.5

0.08 1 ND 5

ND ND

0.08 1

ND ND

ND 4

0.08 NR

0.08

0.17

2

2

ND 2 NR

0.08 2

1

0.08 2 NR

1.25

0.625

NR

s h o w n in Fig. 2 for L P e x p o s e d to u l t r a s o u n d in d i l u t e d a n t i s e r u m . T h e b a n d s f o r m e d in less t h a n 1 rain a n d r e m a i n e d stable for m a n y m i n u t e s in the u l t r a s o n i c field. T h e a g g l u t i n a t i o n d i d n o t s e p a r a t e w h e n the s o u n d was switched off, i n d i c a t ing that the a g g l u t i n a t e s h a d b e c o m e a t t a c h e d to the wall o f the capillary. T h e s o u n d e x p o s u r e times r e q u i r e d for the form a t i o n o f visible a g g l u t i n a t e s are shown for the 1 m m i.d. a n d 2 m m i.d. capillaries in T a b l e s I a n d I I respectively. W e o b s e r v e d t h a t w h e n agglutination occurred, it was o f t e n d e t e c t a b l e after 5 s of

o n e x p e r i m e n t a l v a r i a t i o n s in the height o f the c o l u m n of liquid in the capillary.

Results

I n the a b s e n c e o f antiserum, there was n o d e tectable a g g r e g a t i o n w h e n b a c t e r i a l cells in the capillaries were e x p o s e d to u l t r a s o u n d , as described a b o v e ( d a t a n o t shown). A t y p i c a l e x a m p l e o f b a c t e r i a l a g g l u t i n a t i o n (in b a n d s s e p a r a t e d b y h a l f a w a v e l e n g t h along a 1 m m i.d. c a p i l l a r y ) is

TABLE III TIME TAKEN FOR VISIBLE AGGLUTINATION IN A CONVENTIONAL MICROWELL PLATE ASSAY Other details as for Table I. no. of LP/ml

Time (rain) taken for agglutination at the following dilutions of antiserum:

(>(lOS)

1/2

1/4

1/8

1/16

1/32

1 / 6 4 1/128

1/256

1/512

1/1024

1/2048

1/4032

10 5 2.5 1.25 0.625

10 20 22 60 NR

10 20 120 NR

10 26 NR

10 26

10 26

10 26

12 26

12 26

18 26

18 26

NR NR

10 26

204

Fig. 2. Photograph of LP in a 1 mm (i.d.) capillary containing antiserum, 20 s after exposure to ultrasound. The agglutinates are regularly distributed along the tube and appears to adhere to the wall of the capillary.

incubation in the ultrasound field and never took more than 4 min. Three measurements of time for band formation were made for every combination of LP and antiserum concentration tested in each experimental series. Very similar results were obtained on twice repeating each experimental series (data not shown). The sensitivity of detection of agglutination was slightly less in the 100/~1 (2 m m i.d.) capillaries than in the 50 /~1 (1 m m i.d.) capillaries. Agglutination was detected more readily in capillary tubes than when LP and antiserum were exposed in a 5 m m internal diameter glass tube, where agglutination only occurred after 5 s with 5 x l 0 s L P / m l and dilutions of antiserum less than 1/16. The times taken for the detection of visible agglutination in the conventional microwell plate assays are shown for different LP concentrations and antiserum dilutions in Table III. Very similar results were obtained in three separate experiments (data not shown).

Discussion

Examination of the results presented in Table I show that the time taken for a visible agglutinate to appear was almost independent of antiserum concentration for cell concentrations of 1 0 9 and 5 × 108 cells/ml up to the limiting antiserum dilution of 1/2048. This result implied that the c o n -

trolling factor leading to the formation of an agglutinate was the probability of cell-to-cell contact, that is, if the ceils came into contact, then the serum concentration was sufficient to bring about agglutination. When the LP concentration was decreased to 2.5 x 108 cells/ml and 1.25 x 108 no visible agglutinate was formed at antiserum dilutions less than 1 / 4 and 1 / 2 respectively. The 512-fold increase in antiserum concentration required (Table III) to produce a precipitate for a two-fold fall in concentration from 5 x 108 cells/ ml suggested that cell concentration had become a limiting factor. The lower limit of bacterial concentration for the detection of ultrasonic banding visible by eye (Table I) was the same for the test performed using cells in microwell plates for the 1 m m diameter capillaries, but was lower than the microwell test system by a factor of two for cells in 2 m m diameter tubes. Since cell bands were distributed every half wavelength (0.75 mm) along the capillaries the total volume of suspension from which cells entered each band was 0.75A m m 3, where A is the tube cross sectional area. The volume of cells from which each band was drawn was therefore 2.4 /~1 for the 2 mrn diameter capillary tube. At the lowest cell concentration giving a reaction (Table II), the m a x i m u m number of cells which could be drawn from such a volume is 1.5 X 105. This lower limit contrasts with a cell number of 1.25 x 107 from a comparable calculation for the 100 /~1 well samples in microwell plates assays (Table III). Agglutination of cells suspended in a 5 m m diameter tube was examined in the sonic case to determine whether the increased volume of suspension (0.75A m m 3) capable of contributing to a band would increase the sensitivity of the test. However, clear banding was not detected under the conditions which gave bands in the narrower tubes. Comparison of agglutination in the microwell plates (Table III) and in the 2 m m i.d. diameter tubes at a cell concentration (2.5 x 108), close to the lower limit of detection, shows that visible agglutinates (bands) occur in the ultrasonic test system at antiserum concentrations 512 times more dilute than for similar microwell assays. It can be seen from Tables I, II and I I I that under conditions where agglutination is detectable the time

205

required for a positive response after sonic treatment is typically two orders of magnitude less than the detection time in similar microwell plate assays. The above results point to the potential value of ultrasound treatment in immunoagglutination assays. We are now optimising the ultrasonic parameters and testing the agglutination of a range of organisms and cell types, including viable and motile cells.

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Clarke, D.J. (1989) Cell manipulation in ultrasonic standing wave fields. J. Chem. Technol. Biotechnol., in press. Dyson, M., Woodward, B. and Pond, J.B. (1971) Flow of red blood cells stopped by ultrasound. Nature 232, 572. Gould, R.K. and Coakley, W.T. (1974) The effects of acoustic forces on small particles in suspension. In: L. Bjrno (Ed.), Proceedings of the 1973 Symposium on Finite Amplitude Wave Effects in Fluids. Pergamon, Guildford, p. 252. Kundt, A. and Lehmann, O. (1874) Ueber longitudinale Schwingungen und Klangfiguren in cylindrischen Flussigkeitss~iulen. Ann. Phys. Chem. 153, 1. Lang, R.J. (1962) Ultrasonic atomization of liquids. J Acoust. Soc. Am. 34, 6. Nyborg, W.L. (1978) Physical principles of ultrasound. In: F.J. Fry (Ed.), Ultrasound: Its Applications in Medicine and Biology, Part 1. Elsevier, New York, p. 1. Tilley, D., Coakley, W.T., Gould, R.K., Payne, S.E. and Hewison, L.A. (1987) Real time observations of polylysine, dextran and polyethylene glycol induced mutual adhesion of erythrocytes held in suspension in an ultrasonic standing wave field. Eur. Biophys. J. 14, 499.