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`v. NUSSENZWEIG
`
`
`
`M.W. TURNER
`
`Page 1 of 12
`
`ILMN EXHIBIT 1023
`
`
`
`Page 1 of 12
`
`ILMN EXHIBIT 1023
`
`

`

`J®flD’M. ®JF
`EEflE[ETUE\‘H’®Ea®(EE@@E
`E"xEE@[$]®E)S
`
`editors: V. NUSSENZWEIG
`
`Department of Parhoragy. New York University Medical Center, School of Medicine,
`New York, NY 10016, U.S.A.
`
`M.W. TURNER
`
`Department of immunology, institute of Child Health, University of London, 30 Guflford
`Street, London WC1N TEH, U.K.
`
`Volume 101, 1987
`
`
`
`ELSEVIEH SCIENCE PUBLISHERS B,V. — AMSTERDAM
`
`Page 2 of12
`
`
`
`Page 2 of 12
`
`

`

`© 1987 Elsevier Science Publishers B.V. (Biomedical Division)
`All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any
`means, electronic. mechanical, photocopying, recording or otherwise without the prior written permission of the publisher. Elsevier
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`journal entails the author's irrevocable and exclusive authorization of the publisher to collect any sums or considerations for copying
`or reproduction payable by third parties (as mentioned in article 17 paragraph 2 of the Dutch Copyright Act of 1912 and in the
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`Page 3 of 12
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`

`

`Journal of Immun0logit'al' Methods. 101 H937} 63—7l
`Elsevier
`
`JIM 04395
`
`63
`
`Kinetics of antibody binding to solid—phase—immobilised antigen
`
`Effect of diffusion rate limitation and steric interaction
`
`l-lakan Nygren ‘, Maria Werthen * and Marine Stenberg 1
`‘' Departmertt offlistology, Unit‘:-rsitji‘ of Giirebarg, Giitehorg, am} 3 Research Luborutwfr of Electronics,
`Chafnrers Urtit-'er.rit_t' of Tewhriolagv, Giitebarg. Sweden
`
`(Received 20 January 1987', revised received 2 March 1987. accepted 6 March 1987)
`
`The binding of monoclonal antibodies to surface-immobilised antigen was studied. Antibodies against
`dinitrophenyl—benzene and 0°-ethyl-2’—de0xyguanosine with a known affinity for the antigen were used.
`The amount of bound antibodies was measured by ellipsometry with an accuracy of 10.15 pmol/cmz. and
`a sensitivity of 0.1] pmol/cmz.
`The binding rate of the initial antibody binding could become diffusion rate limited, and the binding
`rate at surface concentrations above 1 pmot/cmi was affected by steric interaction between bound
`thntibodies. Bound antibodies did not dissociate when rinsed with saline for up to 20 h. but dissociated in
`the presence of antigen (0.1 mM). The dissociation rate did not follow any identifiable rate constant. The
`results are discussed in relation to theoretical models of the kinetics of antigen-antibody reactions at
`solid—liquid interfaces.
`
`Key words: Antibody affinity: Kinetics: lmmunoassay: Monoclonal antibody: Solid phase
`
`Introduction
`
`Antigen-antibody reactions in a homogeneous
`liquid phase is a well studied process and the
`kinetics of the reaction has been described by
`several authors {for review. see Karush. 1978). In
`recent years. irnmunoassays based on liquid—pt-iase
`reactions have to a large extent been replaced by
`methods based on antigen-antibody reactions with
`one of the reactants immobilised on a solid phase.
`The interpretation of results of solid-phase assays
`is based on the assumption that the kinetics of the
`antigen-antibody reaction at a solid phase is equal
`to the reaction in solution.
`
`Measurements of antibody binding to solid-
`
`Corre.rpumfem‘c 10.‘ H. Nygren. Department of Histology.
`University of Goteborg. P.0.B. 3303]. S-400 33 Goteborg.
`Sweden.
`
`phase immobilised antigen have revealed that the
`kinetics of the reaction differ from the kinetics of
`
`the corresponding liquid phase reaction in several
`respects. The initial
`forward reaction often be-
`comes diffusion rate limited at plane surfaces
`(Stenberg et aI.. l932‘. Nygren and Stenherg. 1985)
`due to the high surface concentration of immobi-
`lised antigen together with the slow diffusion of
`antibodies (Stenberg et al.. 1985). The initial diffu-
`sion rate-limited binding is followed by a reaction
`rate-limited association with an unexpectedly low
`association rate constant (Nygren et al.. 1986).
`The dissociation rate of bound antibody is slower
`at an interphase than in a solution (Nygren et al..
`1985] and the binding of antibody reaches a con-
`centration-dependent saturation level
`that
`is not
`caused by a dynamic equilibrium (Nygren and
`Stenberg. 1985).
`The present study was undertaken in order to
`
`W21-l?S9/S7/$03.50 '." 1937 Elsevier Science Publishers B.V. (Biomedical Division)
`
`Page 4 of12
`
`
`
`Page 4 of 12
`
`

`

`64
`
`further elucidate the mechanisms behind the kinet-
`
`TABLE I
`
`ics of antigen-antibody reactions at a solid-liquid
`inter-phase.
`
`AFFINITY OF MONOCLONAL ANTIBODIES AGAINST
`DNP AND Of‘-EtdGu0 MEASURED IN SOLUTION “
`
`Monoclonal antibody
`41
`47
`49
`51
`53
`57
`ER-6
`
`Equilibrium constant
`4.3 x10“
`0.9 x10"
`4.1 x10’
`1.1 x10’
`1.5 x11)’
`0.35x10“
`2.0 x10”
`
`“ Data from Rajewsky et al. (1980) and Stanley et al. (1933).
`
`Materials and methods
`
`Antigen and antibodies
`Monoclonal antibodies directed against di-
`nitrophenyl-benzene (DNP antibodies. Mal) 41,
`47, 49, 51, 53 and 57) were a generous gift from
`Prof. M. Steward, London. A high affinity mono-
`clonal antibody (ER-6) directed against 0"—ethyl—
`2'-deoxyguanosine (0“-EtdGuO) was a gift from
`Prof. M. Rajewsky and Dr. J. Adamkiewicz. Uni-
`versity of Essen. F.R.G. The characteristics of the
`antibodies used have been described previously by
`the suppliers (Rajewsky et al.. 1980: Stanley et al..
`1983). The equilibrium constants of the antibodies
`used. measured in solution. are shown in Table I.
`
`The DNP was coupled to bovine serum al-
`bumin (BSA, Sigma) by mixing dinitrobenzene
`sulphonic acid and bovine serum albumin in
`carbonatezbicarbonate buffer
`(0.1 M pH 9.5).
`After 24 h. free DNP was removed by dialysis
`against phosphate—buffered saline (PBS, 0.05 M
`phosphate pH 7.2). The substitution grade was
`determined by light absorbance at 280 and 405
`nm and an epitope density of 33 determinants/
`protein molecule was used for the experiments.
`The 0°-ethylguanosine (0"—EtGuO) was used cou-
`pled to keyhole limpet haemocyanin at an epitope
`density of 80 determinants/protein molecule. The
`0°-EtGuO preparation was supplied by Prof. M.
`Rajewsky.
`
`Preparation of Fab fragments
`Fab fragments were prepared by digestion of
`Mab 49 (IgG1) with papain in 0.1 M phosphate
`buffer pH 7.0, with 0.01 M cysteine and 0.02 M
`EDTA for 16 h at 37°C. The fragments were
`isolated by gel filtration on a Sephacryl S-200
`column as described previously (Nygren. 1932). Fe
`fragments were removed by affinity chromatogra-
`phy on a Sephadex column with immobilised pro-
`tein A (Pharmacia Fine Chemicals. Uppsala,
`Sweden).
`
`Page 5 of12
`
`Experiments
`Methylised silicon wafers were used as sub-
`strate (Nygren et al., 1986). The antigen was ad-
`sorbed to the surface by immersion of the silicon
`wafers into antigen-containing PBS (10 pg pro-
`tein/ ml) overnight. The wafers were then rinsed in
`water, blown dry and placed in a humidified
`chamber. Drops of PBS with different antibody
`content were placed on the wafers for various
`periods of time. The dissociation of bound anti-
`bodies was measured by rinsing for differing time
`periods in PBS with or without antigen in the
`solution. In some experiments the rinsing solution
`was vigorously stirred with a plastic Cylinder rotat- 1
`ing at 1500 rpm over the silicon plates which were
`immobilised in a layer of paraffin.
`The reactions were stopped by rapid rinsing
`with PBS followed by a short
`rinse in distilled
`water and drying in an air current. Control
`in-
`cubations of antibodies on wafers coated with
`
`BSA without hapten was performed in every ex-
`periment. The amount of antibody bound to the
`control plates was subtracted from the values ob-
`tained by incubation on the hapten-coated wafers.
`All of the values subsequently presented are ad-
`justed with respect
`to any background seen in
`controls.
`
`Effipsometry
`The measurement of thin organic films by el-
`Iipsometry is based on a physical characteristic of
`reflected polarized light. When light
`linearly
`polarized in a plane impinges on a reflecting
`surface, the reflected light is elliptically polarised.
`The shape and the orientation of the ellipse de-
`
`
`
`
`
`Page 5 of 12
`
`

`

`65
`
`A
`
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`t
`
`1. The amount of bound antibody (pmol/cm: -_t0.i5
`Fig.
`pmol/cmzerror of measurement) measured by ellipsometry in
`relation to reaction time. a; Anti-DNP zn-It'tbndies(
`, Mala 49
`and I. Mab 53') at 30 pg/ml and (A. Mab 49 and O. Mab 57)
`at 100 _ug/mi. b: Anti-DNP antibodies (A. Mab 4]‘. A. Mab
`47: +. Mai: 51; and X. Mab S3) at 30 pg/ml (lower curves)
`and 100 pg/ml (upper curves}. c: Anti—O°-EtdGu() antibody
`Mab ER-6 at
`three different concentrations (100 pg/ml. 10
`pug/ml and 5 pg/ml].
`
`pend on the angle of incidence and the optical
`constants of the surface material. If a thin trans-
`
`parent film covers the surface, the parameters of
`the ellipse are altered and the magnitude of the
`changes are well within the realm of direct mea-
`surement_
`
`The wafers were examined in a comparison
`ellipsometer
`(SagaX 125, Sagax, Goteborg,
`Sweden) and the amount of bound antigen and
`bound antibody was determined as described pre-
`viously (Stenberg and Nygren, 1983). The varia-
`tion of the results of triplicate samples were the
`same as triplicate readings of one sample. Thus,
`the variation of
`the measurement
`is
`the main
`
`source of uncertainty. All of the values presented
`have an uncertainty of 10.15 pmol/cm2.
`The theorethical considerations behind the
`
`analysis of data have been described previously
`{Nygren et al. 1986). The equation used for calcu-
`lation of diffusion limitation is the solution of
`
`Fick's law of diffusion for plane surfaces:
`
`I 5‘-‘(Z/IH)Co1/E
`
`(1)
`
`where S is the surface-concentration of bound
`
`antibody, CU is the concentration of antibody in
`solution, D is the diffusion constant of the anti-
`bodies (4 X 1D‘7cm2/s), and I is time (5).
`
`Negative staining
`ex-
`The
`antigen—antibody complexes were
`amined in the electron microscope after negative
`staining with uranyl acetate. Sample-supporting
`grids were made by silicon etching of oxidised
`silicon wafers as described previously (Stenberg et
`al., 1987). The resulting quartz membrane was
`made hydrophobic by incubation in vaporized
`hexamethyldisilazane (Dow Coming). The grids
`were immersed in DNP-BSA (10 ug/ml) for 30
`min. rinsed in PBS and placed in a moist chamber.
`Drops of freshly diluted antibody solutions were
`placed on the grids and were allowed to react for
`various periods of time. The reaction was stopped
`by rinsing with saline followed by 2% uranyl
`acetate. The excess of uranyl acetate was blotted
`off with a filter paper and the grids were dried in
`air.
`
`Electron microscopy
`The samples were examined in a Jeol 100 CX
`
`Page 6 of12
`
`
`
`Page 6 of 12
`
`

`

`615
`
`electron microscope at an accelerating voltage of
`80 kV. Photographs were taken at 50000 X
`magnification and were further enlarged to 300000
`times for evaluation and quantitative measure-
`ments of the number of antibody molecules hound
`per unit area.
`
`above 1-1.5 pmol/cmz for all monoclonal anti»
`bodies (Mab). The initial binding rate of Mabs 4],
`47. 51 and 53 (Fig. 1b) is lower than that of Mabs
`49, 57 and ER-6 (Figs. 1a and c). The amount of
`bound antibody plotted in relation to the maxi-
`
`4
`
`F.
`~“
`r
`E
`I
`-“L TE
`E
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`C‘
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`Data from "Assdotci 41- 57 “
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`2-.
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`Results
`
`Association reaction
`
`The amount of bound antibody in relation to
`association time and antibody concentration is
`plotted in Fig. 1a—c. There is a rapid initial anti-
`body binding followed by a decrease in binding
`rate at surface concentrations of bound antibodies
`
`5
`
`AT
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`theor. amount bound tprnol /cm’)
`
`
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`Amountboundiprnol
`
`
`
`Tm.-or. amount bound tnmol /cmzl
`
`logtll
`
`(51
`
`Fig. 2. The amount of bound antibody. measured by ellipsomc-
`try. in relation to the calculated amount of antibody that could
`reach the surface by diffusion.
`at: Mabs 49 (Cl). 57 (I) and
`ER-6 (X). Data from Fig. la and c. b: Mabs 41 (A). 4T(¢.)_
`S1t_+) and 53(x}. Data from Fig. lb.
`
`Page 7 of12
`
`Fig. 3. The amount 0|’ bound antibody (error = ;|;D.I
`pmol/cmz) ‘In relation to the logarithm of reaction time.
`diffusion rate limited reaction is indicated by a line. a: Mab
`(El) and 5?(I) at 100 pg/ml. b: Mab 41 (A), 47 (1), S]{ +)
`530:) at I00 pg/m1.t':Mab ER-6 at 100 pg/rnl.
`
`
`
`;.
`
`
`
`Page 7 of 12
`
`

`

`67
`
`mum amount of antibody that could reach the
`surface by diffusion is shown in Figs. 2a and b.
`As can be seen the initial binding of Mabs 49, 57
`and ER-6 is diffusion-rate limited (Fig. 2a) while
`the binding of Mabs 41, 47, 51 and 53 is not
`strictly limited by diffusion (Fig. 2b).
`In Fig. 3 the amount of antibody bound is
`plotted versus the logarithm of time for up to 72
`h. For the anti-DNP antibodies (Figs. 3:: and (3)
`
`the amount of bound antibodies shows a linear
`
`relation to log(r) in this time interval. which indi-
`cates that
`the binding of antibodies continues
`slowly without reaching a certain saturation level.
`The association rate of Maths 4], 47, and 51
`decreases at an amount of bound antibodies of
`
`about 1.5 pmol/cmz. A time delay is seen as a
`plateau in the curve of the time dependence of
`antibody binding (Figs. 1b and 3.6). After about
`
`*1‘!
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`Fig. 4. Dissociation of bound antibodies. The amount of bound antibody (10.15 pmol/cm’) has been plotted against time of rinsing
`in buffer with or without antigen. a—d: Dissociation with PBS (D). DNP-lysine (O. 0.1 mM DNP) and DNP-BSA (I, 0.1 mM DNP).
`:1: Mai) 41. b = Mab 4')‘. c= Math 51. d= Mab 53. e: Dissociation in the presence of DNP-lysine (0.1 mM DNP) of M31: 49 (CI).
`Mal: 5? (I) and Math ER—6(O). f: Dissociation of Fab fragment of Mab 49 in PBS (Cl). PBS with stirring at 1500 rpm (I) and in the
`presence of DNP-lysine (O. 0.1 mM DNP).
`
`Page 8 of12
`
`
`
`Page 8 of 12
`
`

`

`A L
`
`a
`
`l'\J
`
`63
`
`50 s delay the binding increases, but with a lower
`association rate than the initial. The time delay of
`antibody binding is also seen with Mabs 49 and S7
`in Figs. la, 2:: and 30 at an amount bound of
`1.0-1.5 pmol/cmz. The binding of ER-6 stops at
`an amount of bound antibodies of 1.2 pmol/cm:
`(Figs. It and 3c).
`
`Dissociation
`
`The dissociation of bound antibody, after rins-
`ing in different solutions.
`is shown in Figs. 451-].
`With PBS as rinsing solution the bound antibodies
`do not dissociate within 20 h dissociation time
`
`(Figs. 4rr—d). Attempts were made to compare
`dissociation in PBS with and without stirring and
`there was no change in dissociation rate when the
`solution was stirred at 1500 rpm. Fig. 4f shows
`the dissociation of bound Fab-fragments prepared
`from Mab 49.
`In unstirred PBS no dissociation
`could be detected. In stirred PBS the bound Fab
`
`fragments dissociate initially for up to I h. but for
`longer rinsing times the dissociation rates decreases
`markedly. In the presence of antigen (Fig. 4f ) the
`bound Fab fragments are completely dissociated
`after 5 h of rinsing. (Detection limits in el|ipsome-
`try of bound antibodies and Fab fragments are
`0.11 pmol/cm’ and 0.44 prnol/cm: respectively.)
`In Figs. 4a—e dissociation of bound antibody
`in the presence of antigen is shown for all six
`anti-DNP antibodies. After 20 it of dissociation
`
`78% of the initial amount of antibody 51 is still
`bound at
`the surface. For Mabs 4i and 57 less
`
`than 5% of the initial amount of antibody (<1 0.11
`pmol/cm!) is left at the surface. Antibodies 47. 59
`and 53 have dissociation rates which are inter-
`mediate to these, with an amount bound of 8%.
`
`TABLE II
`
`.0 O
`
`Ch.
`
`log (cl (M)
`
`‘J4
`
`m_
`
`Fig. 3. Amount of bound antibody (i0.1S pmol/cm") in
`relation to antibody concentration (log scale) after 24 h reac-
`tion time.
`
`29% and 2096 respectively of the initial amount of
`antibodies at the surface.
`The dissociation of Mab ER-6 starts at a lower
`
`surface density as the association stops at about
`1.2 pmol/crnz. After 20 h of rinsing in the pres-
`ence of antigen 35% of
`the initial amount of
`antibodies is still bound at the surface.
`
`Association as. concenrratian
`
`The amount of bound antibody in relation to
`antibody concentration after 24 h of association is
`shown in Fig. 5. At antibody concentrations of
`2.56 x10“’ M to 2.30 x 10-3 M all six anti-DNP
`antibodies have a constant amount of bound anti-
`
`bodies of 0.45-0.67 pmol/cm’ at the surface. At
`an increased antibody concentration the amount
`of bound antibodies is proportional
`to the loga-
`rithm of antibody concentration in the solution.
`At antibody concentrations higher than l.88><
`10'? M the concentration dependence of
`the
`
`
`
`SURFACE CONCENTRATION OF BOUND ANTIBODIES MEASURED BY ELLIPSOMETRY AND ELECTRON MI"
`CROSCOPY
`
`Monoclonal
`
`Concentration
`
`Reaction
`
`Surface concentration of Mab
`
`antibody
`
`57
`47
`57
`
`oi antibodies
`(#3/mi)
`100
`100
`30
`
`time (s)
`
`50
`15
`50
`
`Ellipsomew
`(prnol/crnz)
`1.9
`0.63
`1.0
`
`Eimmn micro?
`copy (prnol/cm’)
`2.0?
`0.57
`0.70
`
`Intermolecular
`
`distance “ (nm)
`
`9
`17
`16
`
`' Value calculated from the surface concentration measured by electron microscopy.
`
`
`
`Page 9 of12
`
`
`
` 4 Amountbound(omor/cm?)
`
`
`
`
`
`I
`
`I
`
`.
`
`
`
`Page 9 of 12
`
`

`

`69
`
`Fig. 6. Electron micrograph of monoclonal antibodies (Math 57) bound to surface-immobilized DNP-BSA. Negative staining with 2%
`uranyl acetate. a: Control grid incubated in DNP-BSA (100 pg/ml for 2 h). The BSA molecules adsorbs to the surface in pattern of
`spheric structures (X 200 000). b: Grid incubated with DNP-BSA as in 0. followed by incubation with antibodies (100 pg/ml for 50
`s) { X20000(}). The insertion (XSOODDG) is a detail showing the structure of bound antibodies (arrows),
`
`bound antibody is lower. but still proportional to
`the logarithm of antibody concentration and no
`certain saturation level can be identified.
`
`with the picture seen in the electron microscope
`(Fig. 6}), insert).
`
`Electron microscopy
`Antibodies bound to surface immobilised anti-
`
`gen were examined in the electron microsope by
`negative staining with uranyl acetate (Figs. 6a and
`b}. The adsorbed carrier protein (BSA)
`forms
`spheric aggregates at
`the hydrophobic silicon
`surface (Fig. 6a). The bound antibodies could be
`seen as Y—shaped molecules (arrows) with a length
`of 8 nm. width of 7 nm and thickness of 2 nm
`
`(Fig. 6b. insert). The number of bound antibodies
`was counted and the numbers of antibodies seen
`
`in the electron microscope were compared to the
`optical mass of antibodies measured by ellipsome-
`try (Table II). As can be seen. there is an agree-
`ment between these results. The calculated dis-
`
`tance between molecules (Table II) is consistent
`
`Discussion
`
`In the present study it has been shown that the
`initial binding of antibodies to solid-phase im-
`mobilised antigen is a reaction that often becomes
`diffusion rate limited. There was no correlation
`
`between the antibody affinity for the antigen and
`the diffusion rate limitation of
`the association
`
`reaction. Diffusion rate limitations of biospecific
`reactions at
`solid surfaces have been shown
`
`reactions
`enzyme—substrate
`experimentally for
`(Trurnit, 1954), binding of cholera toxin to gang-
`lioside GM1 (Stenberg and Nygren, 1982). protein
`adsorption (De Feijter, 1978; Wojcieshowskij et
`al.. 1986) and binding of polyclonal antibodies to
`protein antigen (Stenberg et al_, 1982; Nygren and
`
`Page10of12
`
`
`
`Page 10 of 12
`
`

`

`of bound ER-6, either due to its higher affinity or
`due to the higher epitope density. An equilibrium
`is the least probable alternative since we cannot
`measure any dissociation of bound antibodies in
`the absence of antigen in solution.
`The increased binding strength of antibodies
`due to their bivalcnce and the slow diffusion of
`
`dissociated antibodies have been suggested as a
`possible mechanism behind the stability of anti-
`gen-antibody complexes at a surface (Berzowslty
`and Berkower, 1984). We here show that stirring
`of the rinsing buffer increases the dissociation rate
`of Fab fragments in PBS, indicating that the diffu-
`sion of dissociated ligand may limit the dissocia-
`tion rate.
`
`The finding that antibodies dissociated only in
`the presence of antigen could be interpreted in
`two ways. either as blocking of a reassociation or
`as an induced dissociation of bound antibodies.
`
`The dissociation of antibodies in the presence of
`antigen did not follow a simple and identifiable
`rate constant and it is not possible to interpret the
`data from the dissociation experiments as a result
`of a local equilibrium at the surface. It should be '_
`noted that the dissociation of bound antibodies in .
`
`our experiments is qualitatively similar to the dis--
`sociation of adsorbed proteins which has been
`shown not to proceed spontaneously in buffer. but
`proceed rapidly as an exchange reaction with other
`proteins (Bosco and Brash. 1.931; Vroman et al.,
`1980).
`
`The relationship between the amount of bound
`antibodies to the logarithm of the concentration of
`antibodies could be interpreted as a result of a
`dynamic equilibrium with a Kd at the antibody-
`concentration that gives a binding half of the
`maximum. The experimental data could then be
`plotted as a Scatchard plot (Nygren et al., 1936).
`However.
`the amount of bound antibodies con-
`
`
`
`tinues to increase during '72 h reaction time and
`the reaction could then not be at equilibrium after
`24 h. We also conclude from the results of the
`
`present study that antibody binding at levels above
`1 pmol/cm: is influenced by steric interactions to
`a degree where this interaction rather than the
`intrinsic antibody binding rate is rate determining.
`Furthermore,
`in the experimental data shown '
`Fig. 5, no correlation could be found between t
`concentration dependence of binding and anti
`
`70
`
`Stenberg, 1985). The mechanisms behind the dif-
`fusion rate iimitation of biospecific reactions has
`been described theoretically (Stenberg et al.. 1986).
`The binding of the anti—DNP antibodies showed
`a sudden rate decrease at a surface concentration
`of 1.04.5 pmol/cm: and then continued at a rate
`that was linear with the logarithm of time. without
`reaching an identified saturation level. An amount
`of bound antibody of 1.5 pmol/cmz, is equal to a
`molecule density of 9 X 10" lgG molecules/cmz.
`At this antibody density the average distance be-
`tween the molecules is about 10 nm which corre-
`
`sponds to the molecular diameter of IgG. The
`time delay of antibody binding could indicate that
`further binding needs a reorganization of the anti-
`bodies at
`the surface. which would also explain
`the slow rate of binding at surface concentrations
`above 1 pmol/cmz.
`In a previous study (Nygren et al., 1986), the
`slow non—diffusion rate-limited binding of anti-
`bodies was interpreted as a reaction with an iden-
`tified forward rate constant. The rate constant was
`
`surprisingly low and did not relate to the antibody
`affinity. Similar calculations of the forward rate
`constant of the data presented in Figs. 3a and b
`give values that correspond to those presented
`previously. It seems reasonable to conclude that
`the rate of antibody binding above a critical
`surface concentration of bound antibodies
`is
`
`litnited by a reorganization of the layer of bound
`antibodies.
`
`The binding of the high affinity antibody ER-6
`was shown to cease at a surface concentration of
`1.2 pmol/cmz after the initial diffusion rate limited
`reaction. The saturation level of the binding of
`Mab ER-6 could be theoretically explained in
`alternative ways. (i) saturation of the binding sites
`according to a simple Langmuir isotherm (Lang-
`muir, 1918) (ii) steric blocking of available antigen
`by bound antibodies (Nygren et al.. 1986} or (iii)
`equilibrium according to the law of mass action.
`Considering the fact
`that
`the epitope density of
`the 0“-Et-Guo was higher than that of the DNP it
`is not
`likely that cessation of binding is due to
`saturation of sites. More probably.
`the bound
`antibodies block further binding. The difference
`compared to the anti-DNP antibodies which con-
`tinue to bind slowly at higher surface concentra-
`tions could be a lack of reorganisation of the layer
`
`Page11of12
`
`
`
`Page 11 of 12
`
`

`

`body affinity measured in solution. Thus it could
`be concluded that the relation between amount of
`
`bound antibody and antibody_ concentration that
`is demonstrated in the present study and in a
`previous study (Nygren et al., 1986) is not merely
`a reflection of the binding strength of the antibod-
`ies.
`the for-
`In conclusion we have found that
`malism used to describe the kinetics of the anti-
`
`gen-antibody reaction in solution is not satisfac-
`tory for the description of the corresponding reac-
`tion at a solid phase.
`
`Acknowledgements
`
`The present study was supported by grants
`from the Swedish Medical Research Council Pro-
`
`-ject no. 06235 and from_the Research Council of
`the Swedish Board of Technical Development Pro-
`ject no. 85-3222.
`
`References
`
`l
`
`Berzowslty. LA. and Berkower. LJ. (1934) Antigen-antibody
`interaction. In: W.E. Paul (Ed.), Fundamental Immunology
`(Raven Press. New York) p. 595.
`B-osco. M.C. and Brash. J.L. (1931) Adsorption of Fibrinogcn
`on glass: reversibility aspects. J. Colloid interface Sci. 82.
`217,
`De Feijter. .l.A.. Benjamins. I. and Veer. FA. (1978) E!lipsom-
`etry as a tool to study the adsorption behaviour of synthetic
`and biopolymers at the air-water interface. Biopolymers 1?.
`I159.
`Karush. F.. (1978) The affinity of antibody: range. variability
`and the role of multivalence. In: G.W. Litmann and RA.
`Good (Eds). Immunoglobulins (Plenum. New York) p. 85.
`Langmuir,
`l. (1918) The adsorption of gases on plane surfaces
`of glass. mica and platinum. J. Am. Chem. Soc. 40. 13§_i.
`Nygren. H. (1982) Conjugation of horseradish peroxidase to
`Fab fragments with different homobifunctional and hetero-
`
`71
`
`J. Histochem Cyto-
`
`bifunctional cross-linking reagents.
`chem. 30. 407.
`(1985) Kinetics of antibody
`Nygren. H. and Stenberg. M.
`binding to surface immobilized antigen: effect of mass
`transport on the ELISA. J. Colloid interface Sci. 107. 560.
`Nygren. H., Czerltinsky, C. and Stenberg. M. (1985) Dissocia-
`tion of antibodies bound to surface immobilized antigen. J.
`lmmunol. Methods 35. 87.
`(I986) De-
`Nygren.
`I-1.. Kaartinen. M. and Stenberg. M.
`termination by ellipsometry of the affinity of monoclonal
`antibodies. J. lmmtinol_ Methods 92. 219.
`Rajewslty. M., Muller. R... Adamkiewicz, J. and Drozcliok. W.
`(1980) Immunological detection and quantification of DNA
`components structurally modified by alltylating carcino-
`gens. In: B. Pullman. P.O.P. Ts‘o and H. Gelboin (Eds.).
`Carcinogenesis: Fundamental Mechanisms and Environ-
`mental Effects (Reidel. F.R.G.) p. 207.
`Stanley. C.. Lew, AM. and Steward. M. (1983) The measure-
`ment of antibody affinity: a comparison of five techniques
`utilizing a panel of monoclonal anti-DNP antibodies and
`the effect of high affinity antibody on the measurement of
`low affinity antibody. 1. immunol. Methods 64. 119.
`Stenberg. M. and Nygren. H. (1982) A receptor ligand reaction
`studied by a novel analytical tool ~ the isc-scope ellipsome-
`ter. Anal. Biochem. 121'. 183.
`Stenberg. M. and Nygren. H. (1983) The use of the isoscope
`ellipsometer in the study of adsorbed proteins and bio-
`specific reactions. J. Phys. (Paris). Colloq_ 10. 12.
`_ Stenberg. M.. Elwing. H. and Nygren. H. (1982) Kinetics of
`reaction zone formation with radial diffusion of ligands
`over a receptor-coated surface. J. Theor. Biol. 93. 307.
`Stenberg. M.. Stiblert, L. and Nygren. H.
`(1986) External
`diffusion in solid-phase immunoassays. J. Theor. Biol. 120,
`129.
`
`Steriberg, M., Sternme, S. and Nygren, H. (198?) An improved
`negative staining technique using a thin quartz membrane
`as sample support. I. Staining TochrttJ[.. in press.
`Trurnit. HJ. (1954) Studies on enzyme systems at a solid-liquid
`interface. II. The kinetics of adsorption and reaction. Arch.
`Biochem. 51. 176.
`Vroman, L., Adams, A.L., Fischer. G.C. and Munoz, P.C.
`(I930) interaction of high molecular weight kininogen. Fac-
`tor Xll and fibrinogen in plasma at interfaces. Blood 55.
`156.
`
`Wojcieshowskij. P.. Ten Hove. P. and Brash. I.L. (1986) Phe-
`nomenology and mechanisms of the transient adsorption of
`fibrinogen from plasma. J. Colloid Interface Sci. 111. 455.
`
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