Journal o f Immunological Methods, 10 (1976) 85--96
85
@)North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands
A L A S E R D O P P L E R ASSAY F O R THE A N T I G E N - - A N T I B O D Y
REACTION
E.E. UZGIRIS
General Electric Research and Development Center, Schenectady, New York 12301, U.S.A. (Received 27 May 1 9 7 5 ; a c c e p t e d 15 August 1975)
The specific combination of antigen with antibody was detected rapidly and with high sensitivity by an assay based on particle electrophoresis and laser light scattering. Antibody attachment to dilute suspensions of submicroscopic polystyrene spheres coated with antigen was detected as changes in the Doppler shift of scattered laser light when the suspended particles were subjected to an electric field. Antibody or antigen concentrations in the range of 5--10 nanograms per ml can be detected in 30 min. The principal experimental features of the technique are described and include: a ) c h o i c e of particles;b) coating procedures; c) treatment for non-specificity; d) choice of measurement medium.
INTRODUCTION
Among the many methods already available for the detection of the antigen--antibody reaction, a new technique would be interesting only if it displayed potential for high sensitivity with high speed and/or simplicity. One such technique is based on the visual detection of proteins on metal slides (Giaever, 1973). I describe an assay based on electrokinetic effects, certainly an old principle extensively used in immunology (Ouchterlony and Nilsson, 1973), combined with a new, fast, and accurate particle mobility measuring m e t h o d , laser Doppler spectroscopy (Uzgiris, 1974; Uzgiris and Kaplan, 1974). The combination of the old and the new and the procedures I have devised yields an assay which is extremely fast and sensitive. The procedures and measurements are straightforward. The principal features of the assay are as follows: 1) Submicroscopic particles are coated with antigen; 2) these are then used to probe for the presence of antibodies specific to this chosen antigen in the media of interest; 3) antibodies in solution attach themselves to antigen sites on particles; 4) the formation of the second antibody layer on the particle's surface even if not completely covering the surface, causes a substantial decrease in the particle electrophoretic mobility. I will show below that the use of a dilute suspension of submicroscopic particles to probe for free antibodies can be a very sensitive probe because of the large mobility changes associated with antibody attachment. The mobility change is due n o t only to the low inherent charge of the antibody molecules
86 (they after all are mainly found in the 7 fraction of the serum proteins) but also due to the expansion of the h y d r o d y n a m i c shear plane away from the particle surface. High sensitivity may not necessarily be useful if it is also accompanied by a susceptibility to non-specific effects. In the present assay, washing of the particles by centrifugation is sufficient to remove non-specific mobility changes produced by exposure of the particles to concentrated serum or protein solutions. Thus the penalty for high sensitivity in this case is the necessity for centrifugation steps prior to measurement, a process encompassing from 15 to 30 min in the experiments described below. The attachment of free antibody molecules in solution to the antigenic particles is fast if one uses a dilute suspension of small particles, and if one is satisfied with a fractional coverage of the total particle surface area. This situation obtains in the present experiments, and I show below that in solutions containing nanogram/ml levels of antibody substantial mobility changes may be observed in as short a time as 20 to 30 min. To match the fast kinetics available in the present assay and not to add to the burden of the washing procedures, a fast, accurate, and simple readout of particle mobility is required. Laser Doppler spectroscopy provides.such a readout (Uzgiris, 1974; Uzgiris and Kaplan, 1974). Particles placed in an electric field scatter laser light with a Doppler shifted frequency due to their electrophoretic motion. The Doppler frequency shift is proportional to their mobility. By heterodyne detection, the power spectrum of the scattered laser light will describe the mobility distribution of particle suspensions in a time as short as 1 min. Thus the measurement of particle mobilities is fast compared to other procedures required in the assay. A further advantage of laser light scattering techniques, important for ultimate sensitivity, is that the mobility distribution of dilute suspensions of even very small particles can be readily measured. MATERIALS AND METHODS
Polystyrene latex spheres (PLS) I have chosen PLS as the probe particles because of their high charge and the demonstrated capability of coating such spheres with protein by simple adsorption (Brash and L y m a n , 1971). I have used Difco 0.81 pm PLS that have been extensively dialyzed against distilled water. I have also obtained satisfactory results with PLS purified with a mixed-bed-resin ion exchange technique.
Antigen coating A 100 #1 of PLS (1% by weight) was added to 1 ml of 0.154 N NaC1 containing 200 pl of 10% BSA solution (Miles Laboratories). This mixture was
87
at about pH 5.3. For successful adsorption, the important factors appear to be the use of reasonably clean PLS and coating under conditions of low pH, i.e., approximately in the range near the isoelectric point of the BSA. At neutral pH it appears that less surface coverage is attained than at pH 5.3, which is in agreement with observations of Bull for BSA on silica for example (Bull, 1956). After incubating for 30 min, the PLS are washed twice (5000 g, 20 min) with 10 ml 0.154 N NaC1 and suspended in 0.2 ml saline. Using these procedures and solution conditions successful adsorption was also accomplished with other proteins that were investigated: human serum albumin and rabbit serum.
Electrophoretic mobility measurements All mobility measurements were done in 0.005 N NaC1 using the Doppler shift of scattered laser light as a measure of the particle mobility in an applied electric field as shown schematically in fig. 1. A typical spectrum of light scattered from a BSA coated PLS suspension is shown in fig. 2. The average mobility of this particle population is deduced from the distance from the frequency origin on the abscissa to the center of the spectral peak
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Fig. 1. Schematic o f laser Doppler spectrometer. Laser light (Spectra Physics 124A) that is scattered by an angle 0s, defined by a pair of 0.5 mm slits, is detected by a photomultiplier (RCA7265). Part of the light scattered at this angle is due to the particles moving in the applied electric field. Consequently, this light will be Doppler shifted in frequency. The other component o f the detected light arises from stationary scattering centers o n t h e glass w i n d o w s o f t h e c u v e t t e ( L u m i n o n Inc. 2 × 10 X 4 5 r a m , o p t i c a l cell, T y p e 26), a n d t h i s light is n o t s h i f t e d in f r e q u e n c y . T h e s e t w o s c a t t e r e d light c o m p o n e n t s b e a t w i t h e a c h o t h e r in t h e p r o c e s s o f p h o t o d e t e c t i o n giving rise to a p h o t o c u r r e n t w h i c h is m o d u l a t e d a t t h e D o p p l e r f r e q u e n c y . T h e s p e c t r u m o f t h e p h o t o c u r r e n t will t h u s have a p e a k s h i f t e d f r o m t h e origin b y an a m o u n t e q u a l t o t h e D o p p l e r shift. We n o r m a l l y u s e a 10 c m focal l e n g t h lens t o f o c u s t h e i n c i d e n t laser b e a m . T h e ~ 2 m m wide a n d 1 0 i n t o l o n g e l e c t r o d e s o f a 1 m m gap spacing were i m m e r s e d i n t o t h e c u v e t t e so t h a t t h e electric field ( W a v e t e k 1 1 2 s q u a r e wave g e n e r a t o r s o u r c e ) was p e r p e n d i c u l a r t o t h e s h o r t 2 m m axis o f t h e c u v e t t e as s h o w n in t h e diagram. T h e p h o t o m u l t i p l i e r was a p p r o x i m a t e l y 40 c m f r o m t h e s c a t t e r i n g region. F o r f u r t h e r details see Uzgiris, 1 9 7 4 .
88
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20
40
60
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120
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FREQUENCY(Hz)
Fig. 2. O b s e r v e d D o p p l e r s p e c t r a for various a d d i t i o n s o f r a b b i t a n t i b o v i n e a l b u m i n s e r u m ( R A B A S ) t o s u s p e n s i o n s o f b o v i n e s e r u m a l b u m i n ( B S A ) c o a t e d particles (1.5 × 106 p a r t i c l e s / c c ) : a) n o a d d i t i o n s 53 V / c m ; b) 5 × 10 -4 R A B A S , c o n c e n t r a t i o n , 50 V / c m ; c) 10 -s R A B A S , 50 V / c m ; d) 3.5 × 10 -s R A B A S , 50 V / c m . S u f f i c i e n t t i m e was allowed a f t e r e a c h a d d i t i o n to r e a c h c o m p l e t i o n o f t h e a n t i g e n - - a n t i b o d y r e a c t i o n (see fig. 8 for m o r e details).
89 by the use of the relation M-
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where VD is the frequency shift; E the electric field along the direction of the scattering vector; X the wavelength; n the index of refraction of the medium; and 0 the scattering angle in the medium. The detected optical signals were integrated for 2 min by the spectrum analyzer to produce the spectrum shown in fig. 2. Sera and a n t i b o d i e s
Rabbit antibovine albumin serum (Miles Laboratories) was used as a source of antibodies for the BSA coated PLS. This serum was diluted just II0
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Fig. 3. Normalized Doppler frequency shifts (corrected to a constant electric field amplitude) and mobility corrected to 25°C for various serum additions: e, RABAS, data from fig. 2, no washing; A, RABAS plus 3 washes; ©, Rabbit control serum (RCS); D, RCS plus 3 washes.
90
prior to experiments 1 / 1 0 0 in saline and added in requisite amounts either to saline or to 0 . 0 0 5 N NaC1, or to other sera depending on the particular experiments. Rabbit control serum (Miles Laboratories), fetal calf serum, and h u m a n control serum (Miles Laboratories), were also used to test for nonspecific effects. Care was taken to keep the solutions from exceeding pH 7.5 when exposing the antigenic particles to concentrated protein solutions. Goat antirabbit serum (Miles Laboratories) and rabbit antihuman serum (Miles Laboratories) were used as sources of antibodies for rabbit serum coated PLS and h u m a n albumin coated PLS, respectively.
Probing for antibodies in solution For dilute solutions of rabbit control serum, fetal calf serum, or rabbit antibovine albumin serum (dilutions of I : 5 0 , 0 0 0 or greater), the attachment of antibodies to the particles can be observed directly in the cuvette itself by the changes in the particle mobility. Fig. 2 shows the observed Doppler freq u e n c y for various concentrations of rabbit antibovine albumin serum added to the cuvette. For these dilute solutions of rabbit serum or rabbit antibovine albumin serum, most of the observed changes are due to antibody attachment as indicated in fig. 3. Washing of the particles after exposure to either of these sera has minor effects o n the observed particle mobility. Fig.
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Fig. 4. Doppler frequency shift for constant electric field as a function of time after addit i o n o f R A B A S : o, 10 -s R A B A S a n d 106 p a r t i c l e s / m l ; I , 2 × 10 -6 R A B A S a n d 6 x l 0 s particles/ml.
91 4 shows a decreasing Doppler frequency shift with time elapsed after the addition of antiserum to the cuvette (2 X 10 -6 final dilution) containing the antigenic PLS. Exposure of the antigen-coated PLS to either concentrated protein solutions or concentrated sera requires washing of the particles in order to retain high particle mobility in the absence of specific antibody reactions. For example, exposure to concentrated h u m a n control serum reduces the BSA coated particle mobility by more than a factor of two. However, after three washes the mobility of the particles is within 10% of the starting value, i.e. the value measured for BSA coated spheres w i t h o u t exposure to any other protein solutions.
Washing of PLS For most experiments requiring centrifugation of the PLS particles after exposure to various sera, a Fischer Model 59 centrifuge with 1 ml polystyrene tubes was found to be very convenient. Five min at 5000 g was quite sufficient for a single wash. Typically from 2 to 3 such washes were performed when washing procedures were dictated. RESULTS
Antibody attachment and mobility changes I have already indicated in figs. 2--4 the mobility decreases concomitant with a n t i b o d y attachment. Were ionic conditions or pH changes responsible for these sort of results? Even w i t h o u t washing and resuspension in a standard solution, we can see in fig. 5 that the nature of the change is associated with a surface adsorption. After adding antiserum to 10 -S final dilution and allowing 1 h for completion of the reaction, a second group of antigenic particles was added, and their mobility remained largely unaffected 30 min later. Addition of rabbit control serum in dilutions of 10 -S also caused only a slight mobility depression as shown in fig. 3. The use of human albumin coated PLS and rabbit antihuman albumin serum gave similar results to those shown in figs. 2--4 for bovine albumin and rabbit antibovine albumin serum. Mobility reductions due to antibody a t t a c h m e n t were also observed for rabbit serum coated PLS and goat antirabbit serum. In this case the initial mobility of the antigenic particles, although lower than that of either HSA or BSA coated spheres, was reduced by 40% for a final goat antiserum titer of (1 : 50,000). The decrease in mobility as a function of antiserum added shows a steep decline, followed by a flattening and a saturation of the effect. Evidently, the initial molecular a t t a c h m e n t on the surface has much more important consequences for particle mobility than subsequent molecules which help to fill in the remaining open sites and complete the surface coverage. The departure of the mobility response from a response linear in area coverage, as indicated in fig. 4, is the source of the high sensitivity and high speed of the as-
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Fig. 5. Observed Doppler spectrum from a solution to which were added ~106 particles] ml plus 10 -s RABAS and to which, one hour later, another batch of new particles was added at 106 particles/ml. Measurement was made 20 min after last particle addition. say: Large m o b i l i t y changes a c c o m p a n y f r a c t i o n a l a n t i b o d y coverage o f the particle surface. O f course, the steepness of the response d e p e n d s on particle c o n c e n t r a t i o n and particle size: T h e m o r e dilute and t h e smaller t h e particles, t h e s t e e p e r t h e r e s p o n s e and the m o r e sensitive the test b e c o m e s . This d e p e n d e n c e was o b s e r v e d e x p e r i m e n t a l l y .
Kinetics T h e t i m e e v o l u t i o n o f t h e a n t i b o d y a t t a c h m e n t is displayed in fig. 4 f o r t w o d i f f e r e n t a n t i b o d y c o n c e n t r a t i o n s . F o r the very dilute c o n d i t i o n , 2 × 10 -6 a n t i s e r u m d i l u t i o n , 75% o f t h e r e a c t i o n is c o m p l e t e w i t h i n 30 min. Since such dilutions c o r r e s p o n d t o n g / m l a n t i b o d y levels (3 m g / m l stated a n t i b o d y level in t h e sera gives 6 n g / m l a f t e r d i l u t i o n b y 2 × 10-6), the results r e i n f o r c e t h e claim o f fast kinetics f o r this system. As fig. 4 also shows, f o r 10 -s dilutions, 20 min is sufficient t i m e t o run t h e r e a c t i o n t o 90% comp l e t i o n and p r o d u c e substantial particle m o b i l i t y changes. This f a c t was used to carry o u t i n h i b i t i o n studies d e s c r i b e d in the paragraphs b e l o w .
Inhibition of the antibody attachment It is clear t h a t t h e p r e s e n t s y s t e m is a sensitive p r o b e o f free a n t i b o d i e s and n o t necessarily a sensitive p r o b e o f free antigens. T h e r e f o r e , it is o f great
93 100,
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Fig. 6. I n h i b i t i o n o f a n t i b o d y a t t a c h m e n t to particle surface antigens by free antigen in solution.
practical interest to ascertain the performance of the assay in the inhibition mode. Such tests were performed for a variety of antiserum concentrations, with and w i t h o u t subsequent particle washing, and for particle-serum incubation times from 15--30 min to several hours. Some examples of the inhibition of the antibody a t t a c h m e n t to antigenic particles by the presence of free antigen molecules are shown in fig. 6. These data were obtained by adding varying amounts of antigen to the antiserum solutions, incubating these mixtures from 30 min to 1 h, adding the antigenic particles, waiting at least 15 min, and then measuring the particle mobility. As can be seen in the figure, 5 ng/ml BSA can substantially inhibit the antibody a t t a c h m e n t from antisera diluted 10 s times. A slight rise in inhibition with increasing BSA concentration may be indicative of antibody affinity constant variation. This point will be the subject of further study. Except for the 5 × 10 -s antiserum concentration levels, the particles were not washed prior to measurement in order to provide as direct an observation of the particle surface changes as possible. As a result, for those experiments omitting washing, some decrease in mobility is observed even for high antigen concentrations, presumably due to non-specific effects. On the other hand, for the 5 × 10 -5 antiserum samples, the washing restored the initial particle mobility, provided sufficient antigen was in solution to tie up the antibody molecules.
Effects of pH Measurements were done in a range of pH from 6.5--6.9. There were no i m p o r t a n t variations of particle mobility as a result of pH variation in this
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Fig. 7. Particle electrophoretic mobility versus pH: o, BSA coated PLS; ©, same plus 10 -s RABAS.
range. T h e n o r m a l i z e d D o p p l e r f r e q u e n c y shift as a f u n c t i o n o f p H , equival e n t t o m o b i l i t y versus p H , is s h o w n in fig. 7, b o t h f o r B S A c o a t e d PLS a n d f o r such particles a f t e r r e a c t i n g w i t h a n t i s e r u m . I, IONICSTRENGTH 0.02 O.OI
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Fig. 8. Particle electrophoretic m o b i l i t y versus ionic strength: o, B S A coated PLS; i , same plus 4 × 10 -6 R A B A S ; o, plus 10 -s R A B A S ; G, plus 5 × 10 -s R A B A S .
95
Effects of ionic strength We now consider the important question of the effects of ionic strength on the sensitivity of the assay. At what ionic strength can we expect to attain the largest mobility changes as a result of antibody attachment? If the ionic strength is very low, the Debye shielding length is large, and we may expect to see minor changes from fractional particle surface coverage by the antibodies. The potential at the shear plane would remain high because of the effectiveness of the charges on the particle surface. On the other hand, at high ionic strength, with a small Debye shielding distance, we would have a low particle mobility to start with. Hence, the magnitude of the mobility change upon antibody a t t a c h m e n t would not be large either. The data of fig. 8 support these expectations. The magnitude of the mobility change as a result of exposure to antiserum of 10 -s dilution is largest in the region of 0.005 N NaC1, which is the salt c o n t e n t of the standard measurement medium and which is a region of ionic strength of most convenience experimentally (Uzgiris, 1974; Uzgiris and Kaplan, 1974). DISCUSSION I have demonstrated that particle mobility can be used as a very sensitive probe of antibodies in free solution if certain conditions are satisfied. Of these, the most important seem to be the following: 1) High initial charge state for the test particle; 2) Succesful antigen attachment to most of the available binding sites on the particle surface; 3) Choice of dilute salt concentration in the measuring medium which allows accurate probing of particle mobility and which results in a Debye shielding distance which is neither too short nor too long. The smaller the particle size and the smaller their concentration, the more sensitive the assay becomes. This improvement must be balanced by increasing susceptibility to 'background noise' as well as increased difficulty and length of washing procedures as the test particles become smaller and less numerous. The data of fig. 4 show a very fast response. Since the antibody molecules arrive at the particle surface by diffusion, the surface coverage resulting from a particular incubation time, particle size, and antibody concentration can be calculated by using the solutions to the diffusion equation. One can predict that for 1 ng/ml antibody concentration and 0.80 pm PLS a monolayer will be formed after something like 6 h {using D = 4 × 10-7cm 2/sec, and 3 × 10-Tgm/cm 2 for monolayer coverage). However, through binding studies of labeled BSA and light scattering measurements, the BSA is known not to cover the PLS surface completely; probably only 25% coverage is achieved in these experiments. Also the response is not simply linear with surface coverage; we have seen the saturation-like behavior of fig. 3. Thus for antibody levels of a few ng/ml and 30 min incubation times, we may expect a large mobility response, y e t have a small particle surface coverage by the
96 antibodies. Th e sensitivity and the speed of the assay are not i n d e p e n d e n t quantities b u t are related by the molecular diffusion processes and the electrokinetic consequences of molecular adsorption. The elimentation of non-specificity by washing is n o t perfect because extra procedures must be i m pl e m ent ed and because a perfect recovery is not always attained; however, the procedures work. A ny m e t h o d based on the electrokinetic effect will always be susceptible to changing ionic and solution conditions, and some washing procedures to attain standardized conditions will always be necessary. For p o l y s t y r e n e there is additional non-specific a t t a c h m e n t of ions a n d / o r molecules to u n b o u n d sites on the surface of spheres. F o r t u n a t e l y , most of these loosely attached entities can be washed away. It may be t hat o t h e r test particles, with different surface properties, m a y prove to be superior to PLS, requiring only minimal washing. In any case, it appears that this laser D oppl er assay is now ready to be applied to real practical situations in i m m unol ogy. ACKNOWLEDGEMENTS I wish to th ank C.P. Bean, Ivar Giaever and H.P.M. F r o m a g e o t for helpful discussions and suggestions. REFERENCES Brash, J.L. and D.J. Lyman (1971) in: The Chemistry of Biosurfaces, Vol. 1, ed. M.L. Hair (Marcel Dekker, New York) p. 177. Bull, H.R. (1956) Biochim. Biophys. Acta 19,464. Giaever, Ivar (1973) J. Immunol. 110, 1424. Ouchterlony, 0. and L.fl,. Nilsson (1973) in: Handbook of Experimental Immunology, ed. D.M. Weir, 2nd ed. (Blackwell, Oxford) p. 19.1. Uzgiris, E.E. (1974) Rev. Sci. Instruments 45, 74. Uzgiris, E.E. and J.H. Kaplan (1974) Anal. Biochem. 60,455.
85
@)North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands
A L A S E R D O P P L E R ASSAY F O R THE A N T I G E N - - A N T I B O D Y
REACTION
E.E. UZGIRIS
General Electric Research and Development Center, Schenectady, New York 12301, U.S.A. (Received 27 May 1 9 7 5 ; a c c e p t e d 15 August 1975)
The specific combination of antigen with antibody was detected rapidly and with high sensitivity by an assay based on particle electrophoresis and laser light scattering. Antibody attachment to dilute suspensions of submicroscopic polystyrene spheres coated with antigen was detected as changes in the Doppler shift of scattered laser light when the suspended particles were subjected to an electric field. Antibody or antigen concentrations in the range of 5--10 nanograms per ml can be detected in 30 min. The principal experimental features of the technique are described and include: a ) c h o i c e of particles;b) coating procedures; c) treatment for non-specificity; d) choice of measurement medium.
INTRODUCTION
Among the many methods already available for the detection of the antigen--antibody reaction, a new technique would be interesting only if it displayed potential for high sensitivity with high speed and/or simplicity. One such technique is based on the visual detection of proteins on metal slides (Giaever, 1973). I describe an assay based on electrokinetic effects, certainly an old principle extensively used in immunology (Ouchterlony and Nilsson, 1973), combined with a new, fast, and accurate particle mobility measuring m e t h o d , laser Doppler spectroscopy (Uzgiris, 1974; Uzgiris and Kaplan, 1974). The combination of the old and the new and the procedures I have devised yields an assay which is extremely fast and sensitive. The procedures and measurements are straightforward. The principal features of the assay are as follows: 1) Submicroscopic particles are coated with antigen; 2) these are then used to probe for the presence of antibodies specific to this chosen antigen in the media of interest; 3) antibodies in solution attach themselves to antigen sites on particles; 4) the formation of the second antibody layer on the particle's surface even if not completely covering the surface, causes a substantial decrease in the particle electrophoretic mobility. I will show below that the use of a dilute suspension of submicroscopic particles to probe for free antibodies can be a very sensitive probe because of the large mobility changes associated with antibody attachment. The mobility change is due n o t only to the low inherent charge of the antibody molecules
86 (they after all are mainly found in the 7 fraction of the serum proteins) but also due to the expansion of the h y d r o d y n a m i c shear plane away from the particle surface. High sensitivity may not necessarily be useful if it is also accompanied by a susceptibility to non-specific effects. In the present assay, washing of the particles by centrifugation is sufficient to remove non-specific mobility changes produced by exposure of the particles to concentrated serum or protein solutions. Thus the penalty for high sensitivity in this case is the necessity for centrifugation steps prior to measurement, a process encompassing from 15 to 30 min in the experiments described below. The attachment of free antibody molecules in solution to the antigenic particles is fast if one uses a dilute suspension of small particles, and if one is satisfied with a fractional coverage of the total particle surface area. This situation obtains in the present experiments, and I show below that in solutions containing nanogram/ml levels of antibody substantial mobility changes may be observed in as short a time as 20 to 30 min. To match the fast kinetics available in the present assay and not to add to the burden of the washing procedures, a fast, accurate, and simple readout of particle mobility is required. Laser Doppler spectroscopy provides.such a readout (Uzgiris, 1974; Uzgiris and Kaplan, 1974). Particles placed in an electric field scatter laser light with a Doppler shifted frequency due to their electrophoretic motion. The Doppler frequency shift is proportional to their mobility. By heterodyne detection, the power spectrum of the scattered laser light will describe the mobility distribution of particle suspensions in a time as short as 1 min. Thus the measurement of particle mobilities is fast compared to other procedures required in the assay. A further advantage of laser light scattering techniques, important for ultimate sensitivity, is that the mobility distribution of dilute suspensions of even very small particles can be readily measured. MATERIALS AND METHODS
Polystyrene latex spheres (PLS) I have chosen PLS as the probe particles because of their high charge and the demonstrated capability of coating such spheres with protein by simple adsorption (Brash and L y m a n , 1971). I have used Difco 0.81 pm PLS that have been extensively dialyzed against distilled water. I have also obtained satisfactory results with PLS purified with a mixed-bed-resin ion exchange technique.
Antigen coating A 100 #1 of PLS (1% by weight) was added to 1 ml of 0.154 N NaC1 containing 200 pl of 10% BSA solution (Miles Laboratories). This mixture was
87
at about pH 5.3. For successful adsorption, the important factors appear to be the use of reasonably clean PLS and coating under conditions of low pH, i.e., approximately in the range near the isoelectric point of the BSA. At neutral pH it appears that less surface coverage is attained than at pH 5.3, which is in agreement with observations of Bull for BSA on silica for example (Bull, 1956). After incubating for 30 min, the PLS are washed twice (5000 g, 20 min) with 10 ml 0.154 N NaC1 and suspended in 0.2 ml saline. Using these procedures and solution conditions successful adsorption was also accomplished with other proteins that were investigated: human serum albumin and rabbit serum.
Electrophoretic mobility measurements All mobility measurements were done in 0.005 N NaC1 using the Doppler shift of scattered laser light as a measure of the particle mobility in an applied electric field as shown schematically in fig. 1. A typical spectrum of light scattered from a BSA coated PLS suspension is shown in fig. 2. The average mobility of this particle population is deduced from the distance from the frequency origin on the abscissa to the center of the spectral peak
_ELECTRODES
\
"SCATTERING CELL
PHOTOMULTIPLIER
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I
r s,,coR I ]~ v
RE,L-,,,E I
SPECTRU"l
( ANALYZER I
Fig. 1. Schematic o f laser Doppler spectrometer. Laser light (Spectra Physics 124A) that is scattered by an angle 0s, defined by a pair of 0.5 mm slits, is detected by a photomultiplier (RCA7265). Part of the light scattered at this angle is due to the particles moving in the applied electric field. Consequently, this light will be Doppler shifted in frequency. The other component o f the detected light arises from stationary scattering centers o n t h e glass w i n d o w s o f t h e c u v e t t e ( L u m i n o n Inc. 2 × 10 X 4 5 r a m , o p t i c a l cell, T y p e 26), a n d t h i s light is n o t s h i f t e d in f r e q u e n c y . T h e s e t w o s c a t t e r e d light c o m p o n e n t s b e a t w i t h e a c h o t h e r in t h e p r o c e s s o f p h o t o d e t e c t i o n giving rise to a p h o t o c u r r e n t w h i c h is m o d u l a t e d a t t h e D o p p l e r f r e q u e n c y . T h e s p e c t r u m o f t h e p h o t o c u r r e n t will t h u s have a p e a k s h i f t e d f r o m t h e origin b y an a m o u n t e q u a l t o t h e D o p p l e r shift. We n o r m a l l y u s e a 10 c m focal l e n g t h lens t o f o c u s t h e i n c i d e n t laser b e a m . T h e ~ 2 m m wide a n d 1 0 i n t o l o n g e l e c t r o d e s o f a 1 m m gap spacing were i m m e r s e d i n t o t h e c u v e t t e so t h a t t h e electric field ( W a v e t e k 1 1 2 s q u a r e wave g e n e r a t o r s o u r c e ) was p e r p e n d i c u l a r t o t h e s h o r t 2 m m axis o f t h e c u v e t t e as s h o w n in t h e diagram. T h e p h o t o m u l t i p l i e r was a p p r o x i m a t e l y 40 c m f r o m t h e s c a t t e r i n g region. F o r f u r t h e r details see Uzgiris, 1 9 7 4 .
88
I
I
-
I
i
l
i
(c}
0
20
40
60
80
I00
120
140
FREQUENCY(Hz)
Fig. 2. O b s e r v e d D o p p l e r s p e c t r a for various a d d i t i o n s o f r a b b i t a n t i b o v i n e a l b u m i n s e r u m ( R A B A S ) t o s u s p e n s i o n s o f b o v i n e s e r u m a l b u m i n ( B S A ) c o a t e d particles (1.5 × 106 p a r t i c l e s / c c ) : a) n o a d d i t i o n s 53 V / c m ; b) 5 × 10 -4 R A B A S , c o n c e n t r a t i o n , 50 V / c m ; c) 10 -s R A B A S , 50 V / c m ; d) 3.5 × 10 -s R A B A S , 50 V / c m . S u f f i c i e n t t i m e was allowed a f t e r e a c h a d d i t i o n to r e a c h c o m p l e t i o n o f t h e a n t i g e n - - a n t i b o d y r e a c t i o n (see fig. 8 for m o r e details).
89 by the use of the relation M-
UD
E~
" ~ 0 sin
where VD is the frequency shift; E the electric field along the direction of the scattering vector; X the wavelength; n the index of refraction of the medium; and 0 the scattering angle in the medium. The detected optical signals were integrated for 2 min by the spectrum analyzer to produce the spectrum shown in fig. 2. Sera and a n t i b o d i e s
Rabbit antibovine albumin serum (Miles Laboratories) was used as a source of antibodies for the BSA coated PLS. This serum was diluted just II0
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SERUM CONCENTRATION (PARTS PER IO5)
Fig. 3. Normalized Doppler frequency shifts (corrected to a constant electric field amplitude) and mobility corrected to 25°C for various serum additions: e, RABAS, data from fig. 2, no washing; A, RABAS plus 3 washes; ©, Rabbit control serum (RCS); D, RCS plus 3 washes.
90
prior to experiments 1 / 1 0 0 in saline and added in requisite amounts either to saline or to 0 . 0 0 5 N NaC1, or to other sera depending on the particular experiments. Rabbit control serum (Miles Laboratories), fetal calf serum, and h u m a n control serum (Miles Laboratories), were also used to test for nonspecific effects. Care was taken to keep the solutions from exceeding pH 7.5 when exposing the antigenic particles to concentrated protein solutions. Goat antirabbit serum (Miles Laboratories) and rabbit antihuman serum (Miles Laboratories) were used as sources of antibodies for rabbit serum coated PLS and h u m a n albumin coated PLS, respectively.
Probing for antibodies in solution For dilute solutions of rabbit control serum, fetal calf serum, or rabbit antibovine albumin serum (dilutions of I : 5 0 , 0 0 0 or greater), the attachment of antibodies to the particles can be observed directly in the cuvette itself by the changes in the particle mobility. Fig. 2 shows the observed Doppler freq u e n c y for various concentrations of rabbit antibovine albumin serum added to the cuvette. For these dilute solutions of rabbit serum or rabbit antibovine albumin serum, most of the observed changes are due to antibody attachment as indicated in fig. 3. Washing of the particles after exposure to either of these sera has minor effects o n the observed particle mobility. Fig.
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Fig. 4. Doppler frequency shift for constant electric field as a function of time after addit i o n o f R A B A S : o, 10 -s R A B A S a n d 106 p a r t i c l e s / m l ; I , 2 × 10 -6 R A B A S a n d 6 x l 0 s particles/ml.
91 4 shows a decreasing Doppler frequency shift with time elapsed after the addition of antiserum to the cuvette (2 X 10 -6 final dilution) containing the antigenic PLS. Exposure of the antigen-coated PLS to either concentrated protein solutions or concentrated sera requires washing of the particles in order to retain high particle mobility in the absence of specific antibody reactions. For example, exposure to concentrated h u m a n control serum reduces the BSA coated particle mobility by more than a factor of two. However, after three washes the mobility of the particles is within 10% of the starting value, i.e. the value measured for BSA coated spheres w i t h o u t exposure to any other protein solutions.
Washing of PLS For most experiments requiring centrifugation of the PLS particles after exposure to various sera, a Fischer Model 59 centrifuge with 1 ml polystyrene tubes was found to be very convenient. Five min at 5000 g was quite sufficient for a single wash. Typically from 2 to 3 such washes were performed when washing procedures were dictated. RESULTS
Antibody attachment and mobility changes I have already indicated in figs. 2--4 the mobility decreases concomitant with a n t i b o d y attachment. Were ionic conditions or pH changes responsible for these sort of results? Even w i t h o u t washing and resuspension in a standard solution, we can see in fig. 5 that the nature of the change is associated with a surface adsorption. After adding antiserum to 10 -S final dilution and allowing 1 h for completion of the reaction, a second group of antigenic particles was added, and their mobility remained largely unaffected 30 min later. Addition of rabbit control serum in dilutions of 10 -S also caused only a slight mobility depression as shown in fig. 3. The use of human albumin coated PLS and rabbit antihuman albumin serum gave similar results to those shown in figs. 2--4 for bovine albumin and rabbit antibovine albumin serum. Mobility reductions due to antibody a t t a c h m e n t were also observed for rabbit serum coated PLS and goat antirabbit serum. In this case the initial mobility of the antigenic particles, although lower than that of either HSA or BSA coated spheres, was reduced by 40% for a final goat antiserum titer of (1 : 50,000). The decrease in mobility as a function of antiserum added shows a steep decline, followed by a flattening and a saturation of the effect. Evidently, the initial molecular a t t a c h m e n t on the surface has much more important consequences for particle mobility than subsequent molecules which help to fill in the remaining open sites and complete the surface coverage. The departure of the mobility response from a response linear in area coverage, as indicated in fig. 4, is the source of the high sensitivity and high speed of the as-
92 I
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80
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120
FREQUENCY (Hz)
Fig. 5. Observed Doppler spectrum from a solution to which were added ~106 particles] ml plus 10 -s RABAS and to which, one hour later, another batch of new particles was added at 106 particles/ml. Measurement was made 20 min after last particle addition. say: Large m o b i l i t y changes a c c o m p a n y f r a c t i o n a l a n t i b o d y coverage o f the particle surface. O f course, the steepness of the response d e p e n d s on particle c o n c e n t r a t i o n and particle size: T h e m o r e dilute and t h e smaller t h e particles, t h e s t e e p e r t h e r e s p o n s e and the m o r e sensitive the test b e c o m e s . This d e p e n d e n c e was o b s e r v e d e x p e r i m e n t a l l y .
Kinetics T h e t i m e e v o l u t i o n o f t h e a n t i b o d y a t t a c h m e n t is displayed in fig. 4 f o r t w o d i f f e r e n t a n t i b o d y c o n c e n t r a t i o n s . F o r the very dilute c o n d i t i o n , 2 × 10 -6 a n t i s e r u m d i l u t i o n , 75% o f t h e r e a c t i o n is c o m p l e t e w i t h i n 30 min. Since such dilutions c o r r e s p o n d t o n g / m l a n t i b o d y levels (3 m g / m l stated a n t i b o d y level in t h e sera gives 6 n g / m l a f t e r d i l u t i o n b y 2 × 10-6), the results r e i n f o r c e t h e claim o f fast kinetics f o r this system. As fig. 4 also shows, f o r 10 -s dilutions, 20 min is sufficient t i m e t o run t h e r e a c t i o n t o 90% comp l e t i o n and p r o d u c e substantial particle m o b i l i t y changes. This f a c t was used to carry o u t i n h i b i t i o n studies d e s c r i b e d in the paragraphs b e l o w .
Inhibition of the antibody attachment It is clear t h a t t h e p r e s e n t s y s t e m is a sensitive p r o b e o f free a n t i b o d i e s and n o t necessarily a sensitive p r o b e o f free antigens. T h e r e f o r e , it is o f great
93 100,
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NOSERUM ADDITIONS ~(5X I0-5 RABASa WASHING)
(10-~ RABAS)
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Fig. 6. I n h i b i t i o n o f a n t i b o d y a t t a c h m e n t to particle surface antigens by free antigen in solution.
practical interest to ascertain the performance of the assay in the inhibition mode. Such tests were performed for a variety of antiserum concentrations, with and w i t h o u t subsequent particle washing, and for particle-serum incubation times from 15--30 min to several hours. Some examples of the inhibition of the antibody a t t a c h m e n t to antigenic particles by the presence of free antigen molecules are shown in fig. 6. These data were obtained by adding varying amounts of antigen to the antiserum solutions, incubating these mixtures from 30 min to 1 h, adding the antigenic particles, waiting at least 15 min, and then measuring the particle mobility. As can be seen in the figure, 5 ng/ml BSA can substantially inhibit the antibody a t t a c h m e n t from antisera diluted 10 s times. A slight rise in inhibition with increasing BSA concentration may be indicative of antibody affinity constant variation. This point will be the subject of further study. Except for the 5 × 10 -s antiserum concentration levels, the particles were not washed prior to measurement in order to provide as direct an observation of the particle surface changes as possible. As a result, for those experiments omitting washing, some decrease in mobility is observed even for high antigen concentrations, presumably due to non-specific effects. On the other hand, for the 5 × 10 -5 antiserum samples, the washing restored the initial particle mobility, provided sufficient antigen was in solution to tie up the antibody molecules.
Effects of pH Measurements were done in a range of pH from 6.5--6.9. There were no i m p o r t a n t variations of particle mobility as a result of pH variation in this
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Fig. 7. Particle electrophoretic mobility versus pH: o, BSA coated PLS; ©, same plus 10 -s RABAS.
range. T h e n o r m a l i z e d D o p p l e r f r e q u e n c y shift as a f u n c t i o n o f p H , equival e n t t o m o b i l i t y versus p H , is s h o w n in fig. 7, b o t h f o r B S A c o a t e d PLS a n d f o r such particles a f t e r r e a c t i n g w i t h a n t i s e r u m . I, IONICSTRENGTH 0.02 O.OI
0.1 ]
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Fig. 8. Particle electrophoretic m o b i l i t y versus ionic strength: o, B S A coated PLS; i , same plus 4 × 10 -6 R A B A S ; o, plus 10 -s R A B A S ; G, plus 5 × 10 -s R A B A S .
95
Effects of ionic strength We now consider the important question of the effects of ionic strength on the sensitivity of the assay. At what ionic strength can we expect to attain the largest mobility changes as a result of antibody attachment? If the ionic strength is very low, the Debye shielding length is large, and we may expect to see minor changes from fractional particle surface coverage by the antibodies. The potential at the shear plane would remain high because of the effectiveness of the charges on the particle surface. On the other hand, at high ionic strength, with a small Debye shielding distance, we would have a low particle mobility to start with. Hence, the magnitude of the mobility change upon antibody a t t a c h m e n t would not be large either. The data of fig. 8 support these expectations. The magnitude of the mobility change as a result of exposure to antiserum of 10 -s dilution is largest in the region of 0.005 N NaC1, which is the salt c o n t e n t of the standard measurement medium and which is a region of ionic strength of most convenience experimentally (Uzgiris, 1974; Uzgiris and Kaplan, 1974). DISCUSSION I have demonstrated that particle mobility can be used as a very sensitive probe of antibodies in free solution if certain conditions are satisfied. Of these, the most important seem to be the following: 1) High initial charge state for the test particle; 2) Succesful antigen attachment to most of the available binding sites on the particle surface; 3) Choice of dilute salt concentration in the measuring medium which allows accurate probing of particle mobility and which results in a Debye shielding distance which is neither too short nor too long. The smaller the particle size and the smaller their concentration, the more sensitive the assay becomes. This improvement must be balanced by increasing susceptibility to 'background noise' as well as increased difficulty and length of washing procedures as the test particles become smaller and less numerous. The data of fig. 4 show a very fast response. Since the antibody molecules arrive at the particle surface by diffusion, the surface coverage resulting from a particular incubation time, particle size, and antibody concentration can be calculated by using the solutions to the diffusion equation. One can predict that for 1 ng/ml antibody concentration and 0.80 pm PLS a monolayer will be formed after something like 6 h {using D = 4 × 10-7cm 2/sec, and 3 × 10-Tgm/cm 2 for monolayer coverage). However, through binding studies of labeled BSA and light scattering measurements, the BSA is known not to cover the PLS surface completely; probably only 25% coverage is achieved in these experiments. Also the response is not simply linear with surface coverage; we have seen the saturation-like behavior of fig. 3. Thus for antibody levels of a few ng/ml and 30 min incubation times, we may expect a large mobility response, y e t have a small particle surface coverage by the
96 antibodies. Th e sensitivity and the speed of the assay are not i n d e p e n d e n t quantities b u t are related by the molecular diffusion processes and the electrokinetic consequences of molecular adsorption. The elimentation of non-specificity by washing is n o t perfect because extra procedures must be i m pl e m ent ed and because a perfect recovery is not always attained; however, the procedures work. A ny m e t h o d based on the electrokinetic effect will always be susceptible to changing ionic and solution conditions, and some washing procedures to attain standardized conditions will always be necessary. For p o l y s t y r e n e there is additional non-specific a t t a c h m e n t of ions a n d / o r molecules to u n b o u n d sites on the surface of spheres. F o r t u n a t e l y , most of these loosely attached entities can be washed away. It may be t hat o t h e r test particles, with different surface properties, m a y prove to be superior to PLS, requiring only minimal washing. In any case, it appears that this laser D oppl er assay is now ready to be applied to real practical situations in i m m unol ogy. ACKNOWLEDGEMENTS I wish to th ank C.P. Bean, Ivar Giaever and H.P.M. F r o m a g e o t for helpful discussions and suggestions. REFERENCES Brash, J.L. and D.J. Lyman (1971) in: The Chemistry of Biosurfaces, Vol. 1, ed. M.L. Hair (Marcel Dekker, New York) p. 177. Bull, H.R. (1956) Biochim. Biophys. Acta 19,464. Giaever, Ivar (1973) J. Immunol. 110, 1424. Ouchterlony, 0. and L.fl,. Nilsson (1973) in: Handbook of Experimental Immunology, ed. D.M. Weir, 2nd ed. (Blackwell, Oxford) p. 19.1. Uzgiris, E.E. (1974) Rev. Sci. Instruments 45, 74. Uzgiris, E.E. and J.H. Kaplan (1974) Anal. Biochem. 60,455.
