lolm~m¢)chem~tl ~, 1975. Vol 12 pp 349 3;51
Pergamon Press
Printed In Great Britain
COMMUNICATION TO THE EDITORS I M M U N O A S S A Y BY LIGHT SCATTERING S P E C T R O S C O P Y RICHARD
J. C O H E N
and GEORGE
B. B E N E D E K
Department of Physics, Center for Materials Science and Engineering and Harvard-M.I.T. Program in Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, U S.A. (Received 23 October 1974}
Abstract--Quasi-elastic light scattering spectroscopy is found to prowde a precise and sensitive means of studying the very early stages of aggregation in agglutination reactions, and thereby can serve as a very sensitive and accurate means of measuring antibody or anngen levels. In this investigation polystyrene mlcrospheres are coated with bovine serum albumin. Rabbit antibody to bovine serum albumin is introduced into the solunon containing these coated polystyrene spheres in order to agglutinate them. The degree of agglutination, as a function of antibody concentranon, is determined by measuring the spectral width of laser light scattered from solutions of the m~crospheres.
Agglutmanon reacnons are widely used in biology and the light scattered from a polydisperse solution of particles medicine to detect small quantities of antibody or antigen IS determined by the distribunon of diffusion constants of molecules (Kabat and Mayer, t961) Agglutination reac- the particles. By analysis of the spectrum (or correlation tions involve the m vitro aggregation of microscopic carrier function), we may obtain D which is the average of the diffuparticles which bear on their surface antigenic molecules. sion constants of the particles weighted by the intensity of the light scattered by each particle (Hocker et al, 1973). The Aggregation occurs when antibody molecules correspondlng to the antigen are introduced into the solution of the appearance of even a few aggregates in a previously carrier particles The converse procedure of agglutinating monodisperse solution of particles causes a marked drop in antibody-coated particles with the appropriate polyhapbecause the aggregates scatter light more strongly than do tenlc antigen molecules is also possible (Bozlcevlch et al., the single particles Quasi-elastic light scattering is a par1963). Some of the carrier particles which have been used ticularly suitable means of following agglutination in that it are red blood cells, bacteria and polystyrene spheres. At low measures only the relative distribution of particle sizes, and concentrations of antibody small aggregates consisting of is independent of absolute particle concentrations. Hence, only a few carrier particles are formed At higher con- quasi-elastic light scattering measurements are insensitive to centratlons of antibody the aggregates grow so large as to the anomahes of precipitation and adsorption of particles form visible clumps on to foreign surfaces. Conventionally, the appearance of this visible agglutnlate In this study we have applied quasi-elastic light scattering has been taken as the criterion for the presence of the anti- spectroscopy to studying agglutination in a model system in body. Clearly this detection criterion suffers from several order to demonstrate the feaslbihty of this technique as a defects. First, the formation of the grossly visible agglutinate method of immunoassay. As our model system we chose to requires a much larger concentration of antibody than examine the agglutination of polystyrene spheres needed to form small microscopic aggregates. Moreover, (0.357/~m dial coated with bovine serum albumin (BSA) whereas the reversible formation of small aggregates is a The agglutinating agent used was rabbit antiserum to BSA. specific and reproducible process, the appearance of macro- The results of a typical light scattering immunoassay perscopic agglutinates is subject to many poorly controlled informed on this system are shown in Fig. 1. Here we plot the fluences, such as the presence of foreign surfaces. In addlmean diffusion constant D obtained from the correlation non, the appearance of a grossly visible agglutinate is so fu nctlon of the scattered light as a function of antibody conqualitative a criterion that it ~s difficult experimentally to centration C. The concentration of antibody was varied in quantitatively determine the associated antibody titer At steps of a factor of 2 by serial dilution while the conbest, the antibody titer can only be determined to w~thln a centration of the antigen-coated polystyrene spheres was factor of two by this method. held constant. The striking decrease in D with increasing We report here an investigation which, while prehmmary, concentration of antibody results from the formation of clearly indicates that the techmque of quasi-elastic hght aggregates of the coated polystyrene spheres. Below is an scattering spectroscopy (Benedek. 1969) provides a quantioutline of the general experimental procedure used to tative, sensitive and accurately reproducible means for meaobtain the D vs C curves. suring the degree of aggregation in the early microscopic Polystyrene spheres (0.357/~m dial obtained from Dow phase. By using this spectroscopic technique in place of Chemical Company were dialyzed for 48 hours against disusing the usual visual criterion, the agglutlnanon reacnon tilled water to remove surfactant. A solution 1% in polycan serve as an accurate, quantitative and much more sensi- styrene spheres and 3 mg/ml in BSA was made up in 0.02 tive immunoassay. M pH 8"0 sodium phosphate buffer. This solution was Quasi-elastic light scattering spectroscopy is a laser tech- allowed to incubate overnight at 4°C. In order to remove nique used to study the Brownian motion of particles m BSA molecules that had not adhered to the polystyrene solution. The Brownian mot)on of a parUcle in solution is spheres, the solution was centrifuged at 15,000 rev/mln for characterized by its diffusion constant D, which is a mono40 min, the supernatant was discarded, and the pellet resustomcally decreasing function of particle size. This Brownlan pended in buffer This centrlfugation procedure was then motion broadens the spectral linewidth of the laser hght repeated. The concentration of the resultant polystyrene scattered by the particle in direct proportion to D The specBSA stock solution was determined by weighing the solid trum (or its Fourier transform, the correlation function) of residue obtained by drying a measured volume of solution. 349
~5
C o m m u n i c a t i o n to lhc Editol ,,
lz F LIGHT SCATTERING SPECTROSCOPY
-,,.
"&" o 8
,
d"
\
O o4
\ \o
O2
o *~1 -~o -tO
I
I
I
-8
L
I
-6
I -4
I
I -2
\°~o I
I 0
Iog~ (C/Co)
Fig I Plot el m e a n dlfftlslon c o n s t a n t D `is log 2 IC (',,) '`;here (" is the a n t i b o d y c o n c e n t n m o n and C,, = 5 ,ug a n t i b o d y , ml
TURBIDIMETRY
3 IE 0
I ,-- illl ON el
O ~ O__ll,,~. i _ _ I ~O
.2
1
_ii0 -i~
I
I -8
I
i -6
i
I -4
i
l -2
l
i 0
Iogz (C/Co) Fig 2 Plot Of tile r e l a m c lurbldit.\ T is log2 ( C Co) ivherc C is the a n t i b o d y c o n c e n t r a t i o n and (',, -= 5 /zg a n t l b o d ? m[
The data for Figs I and 2 wcre o b t a i n e d ul the lbllov, lng m a n n e r Serial d i l u t i o n s of a n t i s e r u m to BSA were prep a r e d m a series of test tubes, and BSA-coated polystyrene ,,pheres (0-357/xm dial ,+','ere a d d e d m the m a n n e r described Ill the text All a h q u o t of each ~alnplc was d r a w n off a n d placed in scparate s p e c t r o p h o t o m e t e r cuvettes. Both the test tube s o l u t i o n s a n d the c u l e t t e s o l u t i o n s were a l l o w e d to m c u b a t e at r o o m l e m p e r a t u r e for 24 hr Then the s a m p l e s in the tcst tubes '`'`ere used for h g h t scattering spectro.,,coplc m e a s u r e m e n t s in the lashlon described m the tcxt a n d the s a m p l e s m the cuvettes were used for t u r b l d i m c t r i c m e a s u r e m e n t s at a /'`a'`clcngth of 340 n m A S p e c l r a - P h y s i c s a r g o n - i o n laser was used for the light scattering m e a s u r e m e n t s and a Gilford 240 spectrep h o t o m e t e r was used R~r the turbM]metric m e a s u r e m e n t s Final p o l y s t y r e n e BSA reagent c o n c c n t r a i l o n ill the samples was 11 IIg'ml No visible a g g l n t u m t i o n was observed tor any of thc a n t i b o d y c o n c e n t r a t i o n s used Samples wcrc p r e p a r e d lor D mea.',ulements in the lollowlng m a n n e r Serial d l h l t l o n s of r a b b i l anll.,,erum to BSA o b t a i n e d from C a l b l o c h e m C o m p a n y '`'`'ere p r e p a r e d in a series of te,,t tubes. To each tube ~ e r e a d d e d identical ahq u o t s of a fixed d l l u l i o n of the p o l ) s t y r c n c BSA stock solution prcfiltcied t h r o u g h a 1 /lm N u c l e o p o r e filter In most e x p e r i m e n t s the final fluid '`olume per tube was 2 ml, and Ihe final polystyrene BSA c o n c e n t r a l l o n in each tube was chosen lo c o m e o u t to be a p p r o x i m a t e l y l ( ) / l g ' m l The mix* In some e x p e r i m e n t s IIO m a c r o s c o p i c a g g l u t i n a t e was observed e`+en tit 5 tzg ml el antfl'~ody, tile highest conc e n t r a t i o n of a n t i b o d y used "I As might be expected, the s t a n d a r d D / t C curve '`'aries slightly for different p r c p a r a h o n s of the stock p o l y s t y r e n e BSA reagent
tures were alh)'`led to Incubate ai r o o m l e i n p e r a t u f c Ior 24 hr Then the s a m p l e s were liltered l l n o u g h a 3 lea Nuclco pore filtm rote a light scatterHlg cell Llghl s c a t t c r m g m e a s u r e m e n t s were p e r l o r l n e d tit 25 ( and at a ~calterHig angle of 9() A Spectra Ptl\slc.. hchtinl-ilcon la~el el algon-lOll lase~ run tit 10 nl~lv' o u t p u t g d~ tlscd Ill conlLlnclion wlth a double-scaled a u t o c o r r c l a t o r D a l a l i c i t aim l_wcd by. tile m e t h o d o f c t u n u l a n t ~ IKoppel I()72t Ab.>ohHc antibod_~ c o n c e n t r a t i o n was d e t e r m i n e d ht q u a n l l t d I l l c pl c clp]tln analysis b 5 C a l b i o c h e m ( o m p a n y Tile purpose o[ tills study '`~,a', tO den)onSlla[c the IedMbihi} and p o l e n t m l of this technique .is a m e t h o d t,t nn IllUnOass~.l'`' a n d lie effort/'`sJble to m a r k e d b lllClCaac the a b s o l u t e scnslhvlly of the system In lact ~ e expect theoict~ call) that the m l n l l n a l i i d e l e c l a b l e antd~od\ c o n c c n t r a t , m should ~ar 3 i n i e r s e h as the ,,tlrtacc dicul lac.e areas man'` hmes larger thail tho,,c ti,~cd hole ai-c coin nlerclalli available, hi Pulurc i~ oi-k it o l/ill dcicl mule expel im e n i a l l y l'`hcther such till Illmieasff In ~,t.'nMtli'lly cdn Ill lac[ be a c h l e l e d \l~e tll~,o perlc}rlned t;;o COlltlol CX[)Cllil/cil[~, Ill OIIC C\ pclnllcl/I tic p r e p a r e d serlaI dihl[iOiV, el l a b b l t alltisci-ulli to BSA and a d d e d p o l y s t y l c n c ~phelc,, CO
Pergamon Press
Printed In Great Britain
COMMUNICATION TO THE EDITORS I M M U N O A S S A Y BY LIGHT SCATTERING S P E C T R O S C O P Y RICHARD
J. C O H E N
and GEORGE
B. B E N E D E K
Department of Physics, Center for Materials Science and Engineering and Harvard-M.I.T. Program in Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, U S.A. (Received 23 October 1974}
Abstract--Quasi-elastic light scattering spectroscopy is found to prowde a precise and sensitive means of studying the very early stages of aggregation in agglutination reactions, and thereby can serve as a very sensitive and accurate means of measuring antibody or anngen levels. In this investigation polystyrene mlcrospheres are coated with bovine serum albumin. Rabbit antibody to bovine serum albumin is introduced into the solunon containing these coated polystyrene spheres in order to agglutinate them. The degree of agglutination, as a function of antibody concentranon, is determined by measuring the spectral width of laser light scattered from solutions of the m~crospheres.
Agglutmanon reacnons are widely used in biology and the light scattered from a polydisperse solution of particles medicine to detect small quantities of antibody or antigen IS determined by the distribunon of diffusion constants of molecules (Kabat and Mayer, t961) Agglutination reac- the particles. By analysis of the spectrum (or correlation tions involve the m vitro aggregation of microscopic carrier function), we may obtain D which is the average of the diffuparticles which bear on their surface antigenic molecules. sion constants of the particles weighted by the intensity of the light scattered by each particle (Hocker et al, 1973). The Aggregation occurs when antibody molecules correspondlng to the antigen are introduced into the solution of the appearance of even a few aggregates in a previously carrier particles The converse procedure of agglutinating monodisperse solution of particles causes a marked drop in antibody-coated particles with the appropriate polyhapbecause the aggregates scatter light more strongly than do tenlc antigen molecules is also possible (Bozlcevlch et al., the single particles Quasi-elastic light scattering is a par1963). Some of the carrier particles which have been used ticularly suitable means of following agglutination in that it are red blood cells, bacteria and polystyrene spheres. At low measures only the relative distribution of particle sizes, and concentrations of antibody small aggregates consisting of is independent of absolute particle concentrations. Hence, only a few carrier particles are formed At higher con- quasi-elastic light scattering measurements are insensitive to centratlons of antibody the aggregates grow so large as to the anomahes of precipitation and adsorption of particles form visible clumps on to foreign surfaces. Conventionally, the appearance of this visible agglutnlate In this study we have applied quasi-elastic light scattering has been taken as the criterion for the presence of the anti- spectroscopy to studying agglutination in a model system in body. Clearly this detection criterion suffers from several order to demonstrate the feaslbihty of this technique as a defects. First, the formation of the grossly visible agglutinate method of immunoassay. As our model system we chose to requires a much larger concentration of antibody than examine the agglutination of polystyrene spheres needed to form small microscopic aggregates. Moreover, (0.357/~m dial coated with bovine serum albumin (BSA) whereas the reversible formation of small aggregates is a The agglutinating agent used was rabbit antiserum to BSA. specific and reproducible process, the appearance of macro- The results of a typical light scattering immunoassay perscopic agglutinates is subject to many poorly controlled informed on this system are shown in Fig. 1. Here we plot the fluences, such as the presence of foreign surfaces. In addlmean diffusion constant D obtained from the correlation non, the appearance of a grossly visible agglutinate is so fu nctlon of the scattered light as a function of antibody conqualitative a criterion that it ~s difficult experimentally to centration C. The concentration of antibody was varied in quantitatively determine the associated antibody titer At steps of a factor of 2 by serial dilution while the conbest, the antibody titer can only be determined to w~thln a centration of the antigen-coated polystyrene spheres was factor of two by this method. held constant. The striking decrease in D with increasing We report here an investigation which, while prehmmary, concentration of antibody results from the formation of clearly indicates that the techmque of quasi-elastic hght aggregates of the coated polystyrene spheres. Below is an scattering spectroscopy (Benedek. 1969) provides a quantioutline of the general experimental procedure used to tative, sensitive and accurately reproducible means for meaobtain the D vs C curves. suring the degree of aggregation in the early microscopic Polystyrene spheres (0.357/~m dial obtained from Dow phase. By using this spectroscopic technique in place of Chemical Company were dialyzed for 48 hours against disusing the usual visual criterion, the agglutlnanon reacnon tilled water to remove surfactant. A solution 1% in polycan serve as an accurate, quantitative and much more sensi- styrene spheres and 3 mg/ml in BSA was made up in 0.02 tive immunoassay. M pH 8"0 sodium phosphate buffer. This solution was Quasi-elastic light scattering spectroscopy is a laser tech- allowed to incubate overnight at 4°C. In order to remove nique used to study the Brownian motion of particles m BSA molecules that had not adhered to the polystyrene solution. The Brownian mot)on of a parUcle in solution is spheres, the solution was centrifuged at 15,000 rev/mln for characterized by its diffusion constant D, which is a mono40 min, the supernatant was discarded, and the pellet resustomcally decreasing function of particle size. This Brownlan pended in buffer This centrlfugation procedure was then motion broadens the spectral linewidth of the laser hght repeated. The concentration of the resultant polystyrene scattered by the particle in direct proportion to D The specBSA stock solution was determined by weighing the solid trum (or its Fourier transform, the correlation function) of residue obtained by drying a measured volume of solution. 349
~5
C o m m u n i c a t i o n to lhc Editol ,,
lz F LIGHT SCATTERING SPECTROSCOPY
-,,.
"&" o 8
,
d"
\
O o4
\ \o
O2
o *~1 -~o -tO
I
I
I
-8
L
I
-6
I -4
I
I -2
\°~o I
I 0
Iog~ (C/Co)
Fig I Plot el m e a n dlfftlslon c o n s t a n t D `is log 2 IC (',,) '`;here (" is the a n t i b o d y c o n c e n t n m o n and C,, = 5 ,ug a n t i b o d y , ml
TURBIDIMETRY
3 IE 0
I ,-- illl ON el
O ~ O__ll,,~. i _ _ I ~O
.2
1
_ii0 -i~
I
I -8
I
i -6
i
I -4
i
l -2
l
i 0
Iogz (C/Co) Fig 2 Plot Of tile r e l a m c lurbldit.\ T is log2 ( C Co) ivherc C is the a n t i b o d y c o n c e n t r a t i o n and (',, -= 5 /zg a n t l b o d ? m[
The data for Figs I and 2 wcre o b t a i n e d ul the lbllov, lng m a n n e r Serial d i l u t i o n s of a n t i s e r u m to BSA were prep a r e d m a series of test tubes, and BSA-coated polystyrene ,,pheres (0-357/xm dial ,+','ere a d d e d m the m a n n e r described Ill the text All a h q u o t of each ~alnplc was d r a w n off a n d placed in scparate s p e c t r o p h o t o m e t e r cuvettes. Both the test tube s o l u t i o n s a n d the c u l e t t e s o l u t i o n s were a l l o w e d to m c u b a t e at r o o m l e m p e r a t u r e for 24 hr Then the s a m p l e s in the tcst tubes '`'`ere used for h g h t scattering spectro.,,coplc m e a s u r e m e n t s in the lashlon described m the tcxt a n d the s a m p l e s m the cuvettes were used for t u r b l d i m c t r i c m e a s u r e m e n t s at a /'`a'`clcngth of 340 n m A S p e c l r a - P h y s i c s a r g o n - i o n laser was used for the light scattering m e a s u r e m e n t s and a Gilford 240 spectrep h o t o m e t e r was used R~r the turbM]metric m e a s u r e m e n t s Final p o l y s t y r e n e BSA reagent c o n c c n t r a i l o n ill the samples was 11 IIg'ml No visible a g g l n t u m t i o n was observed tor any of thc a n t i b o d y c o n c e n t r a t i o n s used Samples wcrc p r e p a r e d lor D mea.',ulements in the lollowlng m a n n e r Serial d l h l t l o n s of r a b b i l anll.,,erum to BSA o b t a i n e d from C a l b l o c h e m C o m p a n y '`'`'ere p r e p a r e d in a series of te,,t tubes. To each tube ~ e r e a d d e d identical ahq u o t s of a fixed d l l u l i o n of the p o l ) s t y r c n c BSA stock solution prcfiltcied t h r o u g h a 1 /lm N u c l e o p o r e filter In most e x p e r i m e n t s the final fluid '`olume per tube was 2 ml, and Ihe final polystyrene BSA c o n c e n t r a l l o n in each tube was chosen lo c o m e o u t to be a p p r o x i m a t e l y l ( ) / l g ' m l The mix* In some e x p e r i m e n t s IIO m a c r o s c o p i c a g g l u t i n a t e was observed e`+en tit 5 tzg ml el antfl'~ody, tile highest conc e n t r a t i o n of a n t i b o d y used "I As might be expected, the s t a n d a r d D / t C curve '`'aries slightly for different p r c p a r a h o n s of the stock p o l y s t y r e n e BSA reagent
tures were alh)'`led to Incubate ai r o o m l e i n p e r a t u f c Ior 24 hr Then the s a m p l e s were liltered l l n o u g h a 3 lea Nuclco pore filtm rote a light scatterHlg cell Llghl s c a t t c r m g m e a s u r e m e n t s were p e r l o r l n e d tit 25 ( and at a ~calterHig angle of 9() A Spectra Ptl\slc.. hchtinl-ilcon la~el el algon-lOll lase~ run tit 10 nl~lv' o u t p u t g d~ tlscd Ill conlLlnclion wlth a double-scaled a u t o c o r r c l a t o r D a l a l i c i t aim l_wcd by. tile m e t h o d o f c t u n u l a n t ~ IKoppel I()72t Ab.>ohHc antibod_~ c o n c e n t r a t i o n was d e t e r m i n e d ht q u a n l l t d I l l c pl c clp]tln analysis b 5 C a l b i o c h e m ( o m p a n y Tile purpose o[ tills study '`~,a', tO den)onSlla[c the IedMbihi} and p o l e n t m l of this technique .is a m e t h o d t,t nn IllUnOass~.l'`' a n d lie effort/'`sJble to m a r k e d b lllClCaac the a b s o l u t e scnslhvlly of the system In lact ~ e expect theoict~ call) that the m l n l l n a l i i d e l e c l a b l e antd~od\ c o n c c n t r a t , m should ~ar 3 i n i e r s e h as the ,,tlrtacc dicul lac.e areas man'` hmes larger thail tho,,c ti,~cd hole ai-c coin nlerclalli available, hi Pulurc i~ oi-k it o l/ill dcicl mule expel im e n i a l l y l'`hcther such till Illmieasff In ~,t.'nMtli'lly cdn Ill lac[ be a c h l e l e d \l~e tll~,o perlc}rlned t;;o COlltlol CX[)Cllil/cil[~, Ill OIIC C\ pclnllcl/I tic p r e p a r e d serlaI dihl[iOiV, el l a b b l t alltisci-ulli to BSA and a d d e d p o l y s t y l c n c ~phelc,, CO
