| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Articles |
1
Dade International, Glasgow Business Community, Newark, DE 19714-6101.
2
The Department of Clinical Biochemistry, St.
Bartholomew's and the Royal London School of Medicine and Dentistry,
Turner St., London E1 2AD.
a Author for correspondence. Fax 44 171 377 1544; e-mail c.p.price{at}mds.qmw.ac.uk
 |
Abstract |
|---|
 Top Abstract IntroductionMaterialsExperimental Procedures and...DiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterialsExperimental Procedures and...DiscussionReferences |
|---|
The reaction between a polyvalent antigen and a bivalent antibody is generally assumed to lead to aggregate formation through bridging by the antibody between antigen molecules, the amount of light scattering reflecting aggregate formation and the amount of antigen and (or) antibody present. In the case of an immunoinhibition assay for a hapten, the polyvalent hapten latex particles form an immunoaggregate in the presence of antibody, quantification of sample analyte being achieved by the amount of inhibition of aggregate formation by free antigen.
The development and routine use of these assays has advanced with the production of more uniform latex particles, the ability to conjugate hapten (or antibodies) to particles, and a greater knowledge of the chemistry of the particle surface as well as the antigen–antibody reaction.
In 1961 Oreskes and Singer characterized the binding of human
-globulins (hGG) to polystyrene latex particles
(6).1
They proposed that the mechanism of agglutination of hGG-coated
particles in certain serological assays for macroglobulins could be due
to either cross-linking by multivalent globulins or destabilization of
the particle colloidal suspensions upon binding of globulins to hGG on
particle surfaces. Later Singer et al. (7) and von
Schulthess et al. (8) investigated an assay in which
antibody-modified particles were agglutinated by a protein to which the
antibody was directed. These workers developed mathematical
descriptions of a proposed bridging mechanism and showed agreement of
observed behavior with their theories.
A colloidal suspension of latex particles is maintained predominantly by electrostatic repulsion between highly charged surfaces (9)(10). Many workers have studied influences of protein adsorption, pH, ionic strength, and detergents on the stability and surface charge of latex particles (9)(10)(11)(12). Reduction of the surface charge or zeta potential induces aggregation, and this can be achieved by increasing ionic strength, increasing protein concentration, and decreasing detergent concentration. Thus, to develop viable diagnostic assays with latex particles, paying careful attention to factors altering colloidal stability is essential.
While many analysts and educators would claim that all light-scattering immunoassays involving latex particles are based on bridging between polyvalent antigen molecules to achieve aggregate formation, the possibility of colloidal instability as the likely mechanism for aggregation still exists. In 1984 Craig proposed that the behavior of the reactions of certain diagnostic tests under development was consistent with particle destabilization upon immunospecific binding of antibodies to the particles (13).
We now report on studies of a latex particle immunoinhibition assay for phenytoin (diphenyl hydantoin) designed to elucidate the mechanism of the reaction. The assay involves hapten-labeled particles and a monoclonal antibody. Analytical performance of this assay, now in use on a commercial system, has been reported previously (14). Using a monovalent F(ab) antibody fragment, we report evidence that a bridging mechanism is not necessarily involved in the aggregation. Rather, we propose that under the specific conditions used, a rapid highly reversible immunospecific reaction of antibody with particle-bound analog is followed by a relatively slow colloidal aggregation that is not a result of immunoreactions, but results from the changed colloidal nature of the particle surfaces bearing protein.
|
Materials |
|---|
|
TopAbstractIntroductionMaterialsExperimental Procedures and...DiscussionReferences |
|---|
A monoclonal antiphenytoin antibody (IgG) and the F(ab')2 fragment from this antibody were also from Dade. The F(ab')2 and particle reagent are part of the aforementioned diagnostic product. The antibody was derived from a hybridoma cell line resulting from fusion of spleen cells of Balb/c mice immunized with keyhole limpet hemocyanin (KLH)-conjugated phenytoin and mouse myeloma cells, with standard techniques (15). The F(ab')2 preparation was stored in 9.6 mmol/L PBS, pH 7.4. From the same whole antibody, F(ab) fragments were prepared by standard procedures (16), with papain, D-cysteine, and EDTA from Sigma. The digestion mixture was dialyzed against PBS and then purified with a column containing protein A–Sepharose fast flow resin from Pharmacia equilibrated with 3 mol/L NaCl and 1 mol/L glycine, pH 8.8. This process removed Fc fragments and undigested IgG. After further dialysis of the eluted F(ab) against PBS, a final purification was performed by HPLC by using gel filtration Zorbax, GF-250 from DuPont, and a mobile phase of 0.2 mol/L sodium phosphate, pH 7.0. Analysis of the product was performed with the HPLC system and column described above and Wavescan/Nelson integrator analysis software from Nelson Analytical. This gave approximate molecular masses of 150 000, 90 000, and 45 000 Da for the IgG, F(ab')2, and F(ab) fractions, respectively. Preparations of the F(ab) fraction were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (nonreducing conditions) along with molecular mass calibrators with a Mini 2-Gel Device from Integrated Separation Systems with a precast 4–20% gradient gel run at 30 mA for 2 h. Gels stained with Coomassie blue showed only one band, which migrated to the same position as a 39 000-Da calibrator.
Poly(oxy-1,2-ethanediyl), 
-(nonylphenyl)-
-hydroxyphosphate, trade
named Rhodofac (previously Gafac, and herein referred to as such) was
obtained from Rhone-Poulenc. Sodium and potassium phosphate salts,
NaOH, and NaCl were from VWR Scientific. Phenytoin serum-based
calibrators were from Dade. Water used throughout the studies was
purified and deionized with an activated-carbon and mixed-bed resin
treatment system from U.S. Filter.
|
Experimental Procedures and Results |
|---|
|
TopAbstractIntroductionMaterialsExperimental Procedures and...DiscussionReferences |
|---|
agglutination reaction with f(ab')2 and f(ab)
antibodies
A 3-µL aliquot of human serum-based calibrator was mixed with
reaction buffer and particle reagent to a total volume of 194 µL;
after temperature equilibration for 30 s, the agglutination
reaction was initiated by adding 18 µL of either F(ab')2
or F(ab) at final concentrations of 0.15 and 0.50 µmol/L
respectively, followed by 20 µL of water as flush. The final particle
concentration was 6.2 nmol/L, equivalent to 11 µmol/L analog
phenytoin in the final reaction mixture, assuming that all of the label
was available for reaction. The progress curves are shown in Fig. 1
. No agglutination was observed when the phenytoin antibody
fragments were replaced by a nonspecific F(ab) at the same
concentration.
|
determination of rate dependence on antibody concentration
These experiments were undertaken with the protocol outlined
above, without the addition of sample and varying the
F(ab')2 concentration in the cuvette between 0 and 0.19
µmol/L. The initial rates were determined from the progress curves by
extrapolating the d(absorbance)/dt vs t plot
to the initiation point and plotting the initial rate obtained against
antibody concentration as shown in Fig. 2
.
|
determination of rate dependence on particle (antigen)
concentration
A volume of buffered particle reagent (150 µL) was diluted with
water and temperature-equilibrated for 30 s, and then
F(ab')2 antibody was added to a final volume of 231 µL.
The effect of varying the particle concentration between 0.5 and 4.0
nmol/L was studied and the first-, second-, and third-order plots of
the initial rates (determined as above) are shown in Fig. 3
.
|
effect of high antibody/particle antigen ratio
To study agglutination rates at very high concentration ratios of
antibody binding sites, a volume of buffered particle reagent (150
µL) was diluted with water and temperature-equilibrated for 30
s, and then F(ab')2 antibody was added to give a particle
concentration of 0.81 nmol/L in the final reaction mixture; a range of
F(ab')2 concentrations between 0.05 and 1.5 µmol/L was
studied (selected so that the influence of a large excess of antibody
over particle antigen reagent could be explored). The initial rates
were plotted against antibody concentration and are shown in Fig. 4
.
|
inhibition of in-progress agglutination by free phenytoin
These experiments were performed on a Dimension®
clinical chemistry system (Dade International) (17); this
analyzer can perform the particle-enhanced phenytoin assay used in this
study with the additional facility (albeit nonstandard) for manual
addition of a reagent during the progress of a reaction. In this case,
buffered particle reagent (6.2 nmol/L in the final mixture) and water
diluent were temperature-equilibrated for 65 s, after which the
agglutination reaction was initiated by addition of F(ab')2
antibody (0.15 µmol/L) to give a total volume of 500 µL. The
sample, in this case phenytoin serum calibrator, was added (4 µL)
with a 20-µL adjustable pipette exactly 20 s after antibody
initiation; mixing was effected by rapid in-and-out flow from an
adjustable 100-µL pipette. The pathlength was 0.5 cm. The progress
curves from a phenytoin-free serum calibrator and a 40 mg/L phenytoin
calibrator (final concentration 1.3 µmol/L) are shown in Fig. 5
.
|
determination of rate constants for binding of antibody to
phenytoin
Measurements were made on a BIAcoreTM surface plasmon
resonance detector from Biacore. The carboxymethyldextran-modified gold
sensor chips (18) used with this instrument were modified
to contain phenytoin analog on their surfaces as follows, with a
running buffer of 10 mmol/L HEPES-buffered saline (150 mmol/L NaCl, 3.4
mmol/L EDTA, 0.05 mL/L P-20 nonionic surfactant) at 5 µL/min flow
rate: (a) The gold sensor chip (certified CM 5) was
preactivated with 35 µL of a solution of 0.2 mol/L
N-ethyl-N'-(dimethylaminopropyl)-carbodiimide
(EDC) and 0.05 mol/L N-hydroxysuccinimide (NHS);
(b) thirty-five microliters of 100 mmol/L human serum
albumin (HSA) in 10 mmol/L sodium formate, pH 3.6, was injected;
(c) thirty-five microliters of 1 mol/L ethanolamine
hydrochloride, pH 8.5, was injected (to block remaining NHS ester
groups); (d) thirty-five microliters of 2 µmol/L
1-(4-carboxybutyl) 4,4-diphenylhydantoin succinimidyl ester in 100
mmol/L sodium borate, pH 8.3, was injected and allowed to react with
the immobilized HSA to create conjugated phenytoin; (e) ten
microliters of 50 mmol/L NaOH/200 mL/L acetonitrile was injected 20
times to condition the sensor chip.
Association rate constants were determined at 37 °C in duplicate in 100 mmol/L sodium phosphate, 1.3 g/L Gafac, pH 7.0. Five concentrations of F(ab')2 from 84 to 840 nmol/L were injected at a rate of 10 µL/min. Regeneration of the phenytoin-labeled sensor chip was accomplished after each run by injection of 10 µL of 50 mmol/L NaOH/200 mL/L acetonitrile, followed by equilibration with the above buffer solution. Dissociation rate constants were determined by injection of 45 µL of 10 mmol/L phenytoin in the above buffer solution after injection of 20 µL of 2.6 µmol/L F(ab')2. Analysis was carried out by using a biphasic fit to the integrated rate equation (19). This enabled the whole of the binding curve to be analyzed and enabled identification of separate binding components. The association rate constant analysis showed a monophasic binding with a bimolecular rate constant of (7.4 ± 3.0) x 104 (mol/L)-1 s-1. Dissociation rate analysis gave a predominantly monophasic dissociation with a calculated rate constant of (6.5 ± 2.2) x 10-3 s-1. The association equilibrium constant was thus 1.1 x 107 (mol/L)-1.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterialsExperimental Procedures and...DiscussionReferences |
|---|
is the basis of the
immunoinhibition assay principle, namely, in this case, inhibition of
reaction between antibody with phenytoin-modified particles to produce
initially dimers and then aggregates of increasing size containing
several antigen and antibody molecules. Early work by Singer et al.
(7) involved the study of agglutination of polystyrene
particles induced by protein adsorption, and von Schulthess et al.
(8) examined a system in which anti-IgA antibody-coated
polystyrene particles were agglutinated by IgA. Both studies led to
observations that a maximum occurred in the curve relation of
agglutination vs antibody concentration. This is analogous to the
relation observed by Heidelberger and Kendall (20)
relating agglutination to antigen concentration, illustrating the
typical bell-shaped curve. These authors and many others subsequently
have interpreted these findings with a proposed mechanism of
immunospecific bridging between particles by the bivalent antibody
molecules. Binding partner concentrations were found that were high
enough to approach saturation of half the binding sites on particles
during a rapid equilibration with antibody, with the result that
further increases in concentration led to less aggregation.
The results described in Fig. 4
do not show this behavior. The
concentration of latex particles was 0.81 nmol/L; for the 64-nm
diameter particle, the average surface area per particle is calculated
to be ~12 900 nm2, and, assuming a surface coverage
by each antibody molecule of 110 nm2, the maximum density
of antibody parking sites (i.e., coupled antigen molecules) is
estimated to be 120 (i.e., without steric hindrance existing). The 0.81
nmol/L particle concentration thus corresponds to a concentration of
available binding sites of ~97 nmol/L; the highest antibody
concentration used was 15 times this figure (1.5 µmol/L). Despite
this excess of antibody, no maximum of agglutination signal (the
Heidelberger–Kendall bell-shaped relation) was reached; this suggests
some other mechanism than bridging by bivalent antibody.
This proposal is substantiated in the data of Fig. 1
(right), showing
that the agglutination reaction proceeds with monovalent F(ab)
fragments, which offer no possibility of bridging. We used somewhat
higher concentrations of F(ab) than F(ab')2 to achieve
comparable agglutination rates in the experiments; this requirement
might be expected from (a) the need to use a concentration
of monovalent F(ab) twice that of the divalent F(ab')2 to
ensure the same concentration of antibody binding sites and
(b) the well-known alteration of affinity constants of
parent antibody molecules by enzymatic fragmentation. It is also
evident from Fig. 1
that the agglutination reaction with the monovalent
antibody is inhibitable by free (sample) phenytoin, just as in the case
with the bivalent antibody. Other experiments have shown that
nonspecific F(ab) and F(ab')2 fragments did not produce any
aggregation of phenytoin particles. Clearly, an immunospecific reaction
is taking place in the case of both F(ab) and F(ab')2
antibody fragments.
Observation of the reaction kinetics sheds further light on the
processes involved. We could not detect any lag phase after inhibition
of the reaction. The first step of the reaction, the binding of
antibody with antigen sites on particles, thus appears to be much
faster than the ensuing agglutination, which can be taken to be the
rate-limiting step. This behavior might be expected since the frequency
of collision of particles would be slower than collision of faster
diffusing antibody molecules with particles. The influence of reactant
concentrations on agglutination rate shown in Figs. 2
and 3
reveals
that the initial reaction rates are first order in antibody and
approximately second order in particle reagent. This is consistent with
the formation of dimer during the initial reaction phase, as induced by
prior binding of an antibody molecule to phenytoin analog. We used the
initial reaction rate as the basis for these determinations to avoid
the interpretive problems associated with changes in relative light
scattering of higher order of aggregates than dimers, absorbance
outside the range of photometric linearity, and the tendency of such
systems not to reach a clear equilibrium condition but to proceed to
full flocculation of immunoprecipitate.
The association equilibrium constant for the F(ab')2
was found to be >107 (mol/L)-1 and an
association rate constant of 7.4 x 104
(mol/L)-1 s-1, indicating an antibody with
acceptable affinity for hapten (21). However, the data in
Fig. 5
show that agglutination was arrested by addition of free
phenytoin, but without the reduction of light-scattering signal
associated with the breakdown of aggregate. We interpret this to mean
that binding between particle and antibody is readily replaced by free
drug, with the result that no further agglutination occurs. Since the
turbidity does not decrease after introduction of phenytoin, even at
very high concentration (which would have been expected with a
dissociation constant indicating a t0.5 of
100 s), the formation of aggregate is deemed not to be reversible,
indicating that aggregate formation is not solely due to
antigen–antibody binding and involves some other mechanism that is not
inhibited by free antigen. This stage of the reaction is proposed to
reflect a deterioration in the colloidal stability of the particle as a
consequence of binding of protein that induces agglutination and
eventually precipitation as suggested by Nakamura et al.
(9) and Hidalgo-Alvares and Galisteo-Gonzalez
(10)
Colloidal instability of protein-coated latex particles is a well-known phenomenon (7)(11)(12). As proteins are coupled or adsorbed to the surface, the surface charge (assessed as the zeta potential) decreases and with sufficient protein, the repulsive charge collapses and the particles agglutinate. Thus, the binding of increasing amounts of antibody to phenytoin, through a specific immunological mechanism, could cause sufficient alteration in the surface charge to induce agglutination. While the initial binding would be inhibitable by free antigen, the colloidal instability phase would not. The degree of change in the colloidal state is presumed to be slight, otherwise the agglutination progress would not be arrested by mopping up free antibody binding sites after initiation. The specific binding may induce a metastable colloidal mixture where hydrophobic interactions predominate, the relatively high detergent concentration (9 g/L) presumably playing an important role in regulating this reaction. Further experiments with nonionic and other anionic detergents might allow this hypothesis to be explored.
In summary, the observations reported here indicate that in this particular system, the formation of an immunoaggregate takes place in two stages, a first immunospecific reaction followed by a slower agglutination of particles that is induced by their altered colloidal state, thereby not requiring antibody bridging. All particle-enhanced immunoassay reactions are not expected to operate in this way, although one can reasonably expect that all particle-enhanced immunoprecipitation/agglutination reactions will depend to a certain degree on colloidal instability.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
|
References |
|---|
|
TopAbstractIntroductionMaterialsExperimental Procedures and...DiscussionReferences |
|---|
The following articles in journals at HighWire Press have cited this article:
|
|
 |
|
  Y. Umebayashi, Y. Miyamoto, M. Wakita, A. Kobayashi, and T. Nishisaka Elevation of Plasma Membrane Permeability on Laser Irradiation of Extracellular Latex Particles J. Biochem., August 1, 2003; 134(2): 219 - 224. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Englebienne, A. Van Hoonacker, and J. Valsamis Rapid Homogeneous Immunoassay for Human Ferritin in the Cobas Mira Using Colloidal Gold as the Reporter Reagent Clin. Chem., December 1, 2000; 46(12): 2000 - 2003. [Full Text] [PDF]
|
|
|
|
|
|
S. Guaita, J. M. Simo, N. Ferre, J. Joven, and J. Camps Evaluation of a Particle-enhanced Turbidimetric Immunoassay for the Measurement of Immunoglobulin E in an ILab 900 Analyzer Clin. Chem., September 1, 1999; 45(9): 1557 - 1561. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Q. Wei, V. P. Chu, A. R. Craig, J. E. Duffy, D. M. Obzansky, D. Kilgore, I. S. Masulli, C. M. Sanders, and J. C. Thompson Automated Homogeneous Immunoassay for Gentamicin on the Dimension Clinical Chemistry System Clin. Chem., March 1, 1999; 45(3): 388 - 393. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), 0.15 (
), 0.31 (
), 0.62 (
), and 1.24
µmol/L (•). Particle concentration was 6.2 nmol/L. Total volume was
234 µL with a pathlength of 1 cm.
