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Journal of Immunological Methods 287 (2004) 159–167
Research Paper
Amino, chloromethyl and acetal-functionalized latex particles for
immunoassays: a comparative study
M.P. Sanz Izquierdoa, A. Martın-Molinab, J. Ramosc, A. Rusa, L. Borquea,J. Forcadac, F. Galisteo-Gonzalezb,*
aLaboratorio de Analisis Clınicos, Complejo Hospitalario San Millan-San Pedro, Logrono (La Rioja), SpainbGrupo de Fısica de Fluidos y Biocoloides, Departamento de Fısica Aplicada, Facultad de Ciencias, Universidad de Granada,
18071 Granada, Spainc Institute for Polymer Materials POLYMAT and Grupo de Ingenierıa Quımica, Dpto. de Quımica Aplicada, Facultad de Ciencias Quımicas,
Universidad del Paıs Vasco/EHU, Apdo, 1072, 20080 San Sebastian, Spain
Received 31 July 2003; received in revised form 19 December 2003; accepted 27 January 2004
Abstract
Latex particles with different functionalized surface groups (amino, acetal and chloromethyl) for the covalent linking of
protein molecules were synthesized and characterized. Immunopurified anti-ferritin antibodies were then covalently coupled with
a mean efficiency rate (protein covalently bound to latex particles with respect to the total amount of protein added) of 60%. The
reagents developed were applied to the measurement of serum ferritin concentration in a turbidimetric procedure, showing a good
measuring range and a lowest detection limit of 3.5 ng/ml in the case of the amino-modified particles. These immunological
reagents were compared with a commercial nephelometric method, showing a good linear correlation in all cases but no
transferability in the acetal and chloromethyl latexes with additional carboxyl groups, probably due to interference with other
serum components. The differences among latexes found in this study indicate that it would be necessary to optimize the assay
conditions for each type of particle, in order to achieve a maximum immunoreactivity.
D 2004 Elsevier B.V. All rights reserved.
Keywords: Immunoassay; Ferritin; Turbidimetry; Method comparison
1. Introduction
Latex particles coated with a suitable antibody (or
antigen) have been utilized extensively in research and
diagnostics for the quantification of numerous biomo-
lecules in different biological fluids (Peula-Garcıa et
0022-1759/$ - see front matter D 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.jim.2004.01.020
* Corresponding author. Tel.: +34-958-24-32-12; fax: +34-958-
24-32-14.
E-mail address: galisteo@ugr.es (F. Galisteo-Gonzalez).
al., 2002; Borque et al., 2000; Kawaguchi, 2000).
They amplify the antigen–antibody reaction, enhanc-
ing immunoassays that show a good analytical sensi-
tivity (Caballero et al., 1999). Covalent binding of
antigens or antibodies to latex particles improves the
test performance compared to physical adsorption,
because the reagents formed are more stable over time
(Ortega-Vinuesa et al., 1998). Moreover, particles with
specific functional groups on their surface can bind
proteins covalently in the appropriate orientation,
directly or by additional activation, giving more sen-
M.P.S. Izquierdo et al. / Journal of Immunological Methods 287 (2004) 159–167160
sitive and specific immunoreagents. Some of the sur-
face groups able to covalently bond (directly or with
preactivation) amino acid protein residues are amino,
chloromethyl and acetal groups.
In previous papers (Sarobe et al., 1998; Miraballes-
Martinez and Forcada, 2000; Miraballes-Martinez et
al., 2001; Santos and Forcada, 2001), we have de-
scribed the synthesis and characterizations of these
functionalized latexes, as well as the preparation of a
new reagent based on latexes with amino surface
groups for the measurement of human ferritin (Ramos
et al., 2003). In the present study, different immunore-
agents with amino, chloromethyl, and acetal-function-
alized latexes were prepared and compared using a
fully automated turbidimetric procedure developed
and optimized in a common clinical chemistry analyz-
er, achieving the high sensitivity required for serum
ferritin quantification. Results of a turbidimetric assay
with these reagents were compared with the immuno-
reactivity obtained by a nephelometric assay using
commercially available reagents.
This approach would be generally applicable in the
development of other turbidimetric or nephelometric
tests for the automated measurement of numerous
haptens or proteins.
Table 1
Preparation of seed particles for the chloromethyl and acetal-
functionalized latexes
Seed Water
(g)
Styrene
(g)
MA-80
(g)
NaHCO3
(g)
K2S2O8
(g)
PSA7 625 263 8.45 1.00 1.00
T= 85 jC; rpm= 250.
2. Materials and methods
2.1. Materials
The polymerization reagents used to produce latex-
es with amino surface groups have been reported in a
previous paper (Ramos et al., 2003).
In the case of polymerizations yielding chloro-
methyl and acetal surface groups, chloromethylstyrene
(CMS, Fluka), acrolein diethyl acetal (ADEA,
Aldrich) and methacrylic acid (MAA, Fluka), were
the functional monomers and comonomer, respective-
ly. Sodium dihexylsulfosuccinate (Aerosol MA-80)
was used as an emulsifier in the first step; potassium
persulfate (Fluka) and sodium methabisulfite (Merck)
were used as the components of the redox initiator
system in the second step. Sodium hydrogen carbonate
(Fluka) was used as the buffer.
Bovine serum albumin (BSA), Tween 20 (polyoxy-
ethylene-20-sorbitan monolaurate), sodium borohy-
dride and glutaraldehyde (25% aqueous solution)
were obtained from Sigma. All chemicals used were
of analytical grade and were used without further
purification. Doubly deionized water was used
throughout the work.
2.2. Synthesis of seed particles
Monodisperse core polystyrene particles were syn-
thesized by batch emulsion polymerization. The seed
(PSA7) was synthesized in a single step using the
preparation and reaction conditions given in Table 1
for the chloromethyl (CMS1 and CMS2) and acetal-
functionalized particles (ACET1 and ACET2). In the
case of the amino-functionalized latex (MH4), details
of the synthesis of the cationic seed have been reported
previously (Ramos et al., 2003).
Reactions were carried out in 1 or 2 l thermostated
reactors fitted with a reflux condenser, stainless steel
stirrer, sampling device, and nitrogen inlet tube. The
stirring rate was 250 rpm and the reaction temperature
(85 jC) was controlled. The reaction time was at least
12 h.
2.3. Synthesis of final functionalized latex particles
The reagents and reaction conditions used to obtain
the final latex particles are described in Table 2. The
reaction mixture was stirred at a rate of 200 rpm for 1
h at room temperature, under an atmosphere of N2.
Once the reaction temperature (60 jC) was reached,
the initiator solution was added. The final latex was
removed from the reactor and polymerization was
quenched with hydroquinone.
2.4. Latex characterizations
The mean particle diameters (dp) of the seeds and
final latexes were determined by Photon Correlation
Spectroscopy (PCS, Coulter N4 Plus). The particle
size distributions (PSD) were determined by Trans-
Table 3
Characteristics of the functionalized latexes
Latex X (%) dp(nm)
PDI r (AC/cm2) Functional group
(10� 7 mEq/cm2)
MH4 100 295 1.008 6.61F 0.62 0.69F 0.06
CMS1 91.8 283 1.024 3.44F 0.03 3.45F 0.24
CMS2 91.0 279 1.015 32.01F 0.06 3.00F 0.14
ACET1 64.1 280 1.060 2.14F 0.15 1.22F 0.11
ACET2 79.0 278 1.010 32.0F 1.5 1.20F 0.10
X%: conversion; dp: mean particle diameter; PDI: particle
distribution index; r: charge distribution.
Table 2
Reagents used for the preparation of the chloromethyl and acetal-
functionalized latexes
CMS1 CMS2 ACET1 ACET2
Water (g) 400 400 400 400
Seed PSA7 (g) 150 150 150 150
Styrene (g) 30 30 30 35
CMS (g) 15 15 – –
ADEA (g) – – 15 10
MAA (g) – 2.0 – 2.0
NaHCO3 (g) 0.793 0.793 0.793 0.793
K2S2O8 (g) 0.793 0.793 0.793 0.793
Na2S2O5 (g) 0.557 0.557 0.557 0.557
T= 60 jC; rpm= 200.
M.P.S. Izquierdo et al. / Journal of Immunological Methods 287 (2004) 159–167 161
mission Electron Microscopy (TEM, Hitachi H-7000
FA) on representative samples and analyzed using
Bolero software (AQ Systems). A polydispersity index
(PDI) was calculated from the PSD (Sarobe and
Forcada, 1996). After filtration of the latexes with
glass wool to eliminate coagulum formed in the
reaction, if any, the conversion (X%) was determined
gravimetrically as the ratio of the polymer formed to
the total monomer used. A latex sample was removed
from the reactor and weighed before an aliquot of
hydroquinone solution of known concentration was
added to the sample in order to stop polymerization.
The sample was dried in a vacuum oven until a
constant weight was achieved. The weight of the dried
sample is the weight of the polymer formed (subtract-
ing the weight of non-polymeric solids such as the
emulsifier and the different salts).
The surface density of charged groups (amino,
sulfate and carboxyl) was determined by automatic
conductimetric titrations. Determination of chloro-
methyl groups was based on the nucleophilic attack
of glycine in alkaline medium (Sarobe et al., 1998),
and acetal groups were measured by acidification and
reaction with hydroxylamine (Santos and Forcada,
2001).
2.5. Antibodies, samples and calibrators
Microparticles were coated with a specific immu-
nopurified rabbit polyclonal IgG antibody directed
against human ferritin (Borque et al., 1999). Anti-
ferritin antibody was immunopurified from rabbit
hyperimmune serum on a human ferritin-Sepharose 4
Fast Flow column (Amersham Pharmacia Biotech,
Piscataway, NJ) using standard procedures (Vaitukai-
tis, 1981; Cham et al., 1985). The IgG antibody was
eluted with 0.1 M Glycine/HCl buffer, pH 2.5, imme-
diately neutralized and dialyzed against 20 mM phos-
phate buffer, pH 8.0.
Serum specimens from 45 patients with ferritin
concentrations from 0 to 500 ng/ml were analyzed.
As calibrator we used a ferritin solution (human
liver ferritin in a 0.1 M Tris Buffer pH = 7.4, contain-
ing 60 g/l BSA and 40 AM NaN3 as preservative)
calibrated against the N-ferritin standard (Dade Behr-
ing, Marburg Germany) on the BNII analyzer system
and with the NA-Latex Ferrritin reagents (Dade Behr-
ing, Ref OQDD 10/11). This standard was calibrated
with reference to the WHO international reference
preparation (80/578).
2.6. Covalent coupling protocols
Five latexes, MH4, CMS1, CMS2, ACET1, and
ACET2 (Table 3) were used to prepare the immunore-
agents with a final particle concentration of 5 mg/ml.
Immunopurified anti-ferritin antibody (0.9 mg/m2)
was added to the latexes in order to ensure a high
covalent efficiency rate (protein covalently bound with
respect to the total amount of protein added) and the
best analytical response, following previous studies
(Ramos et al., 2003).
Phosphate 0.01 M (pH 6.8) and carbonate 0.1 M
(pH 9.5) buffers were used in the immunoreagent
preparation. The wash buffer was phosphate buffer
0.1 M saline (pH 7.4)/Tween20 1% solution and the
reaction/storage buffer (pH 7.4) was glycine-buffered
saline-bovine serum albumin (GBS-BSA) containing
0.17 M of NaCl, 0.1 M of glycine, BSA 1 g/l, Tween20
1%, N3Na 40 mg/ml.
M.P.S. Izquierdo et al. / Journal of Immuno162
(a) Amino-functionalized latex particles. Particles
were covalently coated with antibodies according
to a previously described procedure (Ramos et al.,
2003) based on the activation of amino groups of
the particles using glutaraldehyde (0.125%) and
the reduction of imine with sodium borohydride
(10 mg/ml).
(b) Chloromethyl-functionalized latex particles. Anti-
ferritin IgG antibodies were incubated with the
latex particles in 10 mM pH 7.2 phosphate buffer
for 3 h at 37 jCwith stirring, and covalent coupling
was achieved by a direct reaction between chloro-
methyl functionalized latex and antibody amino
groups (Sarobe et al., 1998). After centrifugation,
the particles were resuspended in GBS pH 8.2 and
incubated for 10 min to block any excess of
unreacted chloromethyl groups. The coated par-
ticles were centrifuged and washed with the wash
buffer and finally, the sensitized microparticles
were resuspended in the reaction buffer and kept at
4 jC.(c) Acetal-functionalized latex particles. The first step
was the activation of acetal groups to aldehyde by
acidification of the medium with HCl (pH 2),
incubating with continuous mixing for 30 min at
room temperature, and neutralizing and buffering
at pH 7.2. The anti-human ferritin IgG solution
was added and gently mixed for 2 h at room
temperature. The bound imine formed was
reduced by incubating with 10 mg/ml sodium
borohydride for 1 h at room temperature with
continuous mixing. After removal of the reducing
agent by repeated centrifugation and washing, the
latex particles were washed repeatedly with the
wash buffer to elute antibodies not covalently
attached to the particles. Finally, the coated
microparticles were resuspended in the reaction
buffer, kept at 4 jC and briefly subjected to
sonication before use to provide a working latex
reagent.
In all the latex preparation procedures, the degree of
coverage was calculated by measuring the affinity-
purified antibody coupled to the latex particles using
the copper reduction/bicinchoninic acid reaction (BCA
method, Pierce) and by measurement of the absor-
bance at 560 nm in a UV–visible spectrophotometer
(Pharmacia Biotech, Ultrospec 1000).
2.7. Immunoassay procedure
The immunoreagents were adapted to the measure-
ment of human ferritin in serum. The immunoassay
was originally designed and optimized for the sensi-
tized amino modified latex (Ramos et al., 2003) and
used without modification.
The immunoagglutination reaction was evaluated
by a turbidimetric assay using a clinical chemistry
analyzer (Abx Diagnostic, CobasRMira Plus). In es-
sence the procedure was as follows: 90 Al of reactionlatex reagent were pipetted with 200 Al of assay buffer
into a cuvette. After 75 s of incubation, the agglutina-
tion reaction was started by the addition of 25 Al serumsample or calibrator and the reaction progress was
monitored at a wavelength of 600 nm for 5 min at 37
jC. The absorbance change was calculated with the
kinetic rate mode of the Cobas Mira Plus Analyzer as
absorbance change per minute.
2.8. Prozone, detection limit and initial slopes
In order to study the prozone effect, rate values of
serial dilutions with Tris-BSA buffer of a sample
containing a high ferritin concentration (>10,000 ng/
ml) were measured.
We expressed analytical sensitivity as the lowest
detection limit and it was calculated from the mean
plus three times the standard deviation (S.D.) of the
zero calibrator (absorbance of the ferritin free
calibrator).
Initial slopes were calculated using a quadratic
correction to estimate the slope at the origin (ferritin
concentration = 0) with the SIMFIT Program (Com-
puter Software Version: 5.4 W. G. Bardsley, University
of Manchester).
2.9. Comparison study
The immunoreactivity of the prepared latex rea-
gents measured in the clinical chemistry analyzer by
turbidimetry was compared with the reactivity ob-
served with a commercial reagent in a BNII nephelo-
metric analyzer. The relationships between procedures
were studied using nonparametric Passing-Bablok
regression and Pearson’s correlation coefficient. The
regression line was y =mx + b, where x was the ferritin
concentration (ng/ml) measured in BNII, and y the
logical Methods 287 (2004) 159–167
M.P.S. Izquierdo et al. / Journal of Immunological Methods 287 (2004) 159–167 163
ferritin concentration (ng/ml) measured in the Co-
basRMira Plus.
Table 4
Covalent efficiency and initial slopes of immunoresponse curves
Latex Covalent
extent (%)
Initial slopes
MH4 60 0.237F 0.010
CMS1 60 0.097F 0.003
CMS2 35 0.109F 0.005
ACET1 85 0.130F 0.005
ACET2 61 0.206F 0.010
3. Results and discussion
All latexes were characterized by measuring the
conversions, the particle size distributions and the
surface densities of charged and functionalized groups.
In Table 3, the conversion (X%), the mean diameter
of the particles obtained by PCS (dp) and the PDI are
shown. The PDI determinations showed that the final
latex particles could be considered as monodisperse
(PDI lower than 1.05) (Tsaur and Fitch, 1987).
The main objective of this work from the point of
view of the syntheses was to obtain stable latex
preparations with functional amino, chloromethyl
and acetal surface groups. The cationic nature of the
amino latexes suggests that the best option for initia-
tors and emulsifiers, if high stability is required, is to
ensure that both are of the same type, either cationic or
nonionic (Miraballes-Martinez et al., 2001; Ramos et
al., 2003). The amino latex (MH4) was obtained using
the cationic initiator AIBA (2,2V-azobisisobutyrami-
dine dihydrochloride) and the emulsifiers HDTAB
(hexadecyltrimethylammonium bromide, Aldrich)
and DTAB (dodecyltrimethylammonium bromide,
Aldrich) (Ramos et al., 2003). In the case of chlor-
omethyl and acetal latexes, the initiator was potassium
persulfate and the second step was carried out without
emulsifier. The only emulsifier used, MA-80, was
added to obtain the seed particles in the first step.
The comonomer MAAwas used in latexes ACET2
and CMS2 with the aim of giving additional electro-
static stabilization to the particles (Yeliseyeva, 1982).
The charge provided by this comonomer is not depen-
dent on desorption phenomena, unlike the charge
coming from the emulsifiers. The conversions of the
final latexes formed on the previously synthesized
seeds were 91% or higher for all the reactions, except
for the acetal latexes (ACET1 and ACET2). In these
two reactions, the conversions were relatively lower
than those obtained in the rest of the polymerizations.
This result has been reported previously (Santos and
Forcada, 1999).
In all cases, an initial amount of 0.9 mg/m2 of anti-
ferritin IgG was added to the different latexes, follow-
ing the corresponding binding protocols. With this
value, all the protein is ensured to be initially adsorbed,
at least physically (data from adsorption studies not
shown). Following this initial adsorption step and the
subsequent protocol for covalency, the different sam-
ples were treated with tensioactive reagents to deter-
mine the extent of the covalent bonding. The mean
covalent coupling efficiency rate obtained was between
35% and 85%, the acetal reagent showing the highest
value (see Table 4). Additional carboxyl groups on the
acetal and chloromethyl latexes decreased the efficien-
cy, giving a lower amount of antibody covalently
coupled. It is possible that the highest surface electro-
static repulsion in these particles prevents the approach
of antibody molecules to the particle surface. In the
case of the CMS2 latex, which shows the lowest level
of antibody covalently coupled (35%), it can be also
argued that the additional negative charge may catalyze
the dehydrohalogenation reaction of surface chloro-
methyl groups, losing active covalency points and
giving less antibody coupling.
However, a larger amount of covalently coupled
antibody does not always imply a higher immunoreac-
tivity for the sensitized latex particles, since antibody
conformation and orientation at the particle surface are
also fundamental parameters. In this way, we can
observe in Fig. 1 the immunoresponse of the different
sensitized latexes as a function of the concentration of
ferritin. As can be seen, in the case of both acetal and
chloromethyl latexes, the particles with additional
carboxyl groups (ACET2 and CMS2) showed a higher
immunoreactivity than that of the corresponding par-
ticles without carboxyl groups (ACET1 and CMS1),
although the percentage of covalency was higher in
these reagents. In these cases, the bond formed must be
highly effective in terms of conformation and/or ori-
entation. It is evident that the presence of these addi-
tional negatively charged carboxyl groups helps to
provide an appropriate three-dimensional arrangement
Fig. 1. Calibration curves.n, Acetal-carboxyl latex (ACET2); o, amino latex (MH4);5, acetal latex (ACET1);E, chloromethyl-carboxyl latex
(CMS2); D, chloromethyl latex (CMS1).
M.P.S. Izquierdo et al. / Journal of Immunological Methods 287 (2004) 159–167164
of the antibody molecules on the latex particle surface,
which are exposing their immunologically active
regions with greater effectiveness for the antigen–
antibody reaction. On the other hand, it can be also
argued that a high density of antibody molecules at the
surface may result in a lower response due to steric
effects which reduce or hide the number of reactive
groups that the antigen molecule can access. Neverthe-
less, this argument does not seem very plausible in our
case where the initial amount of antibody added to the
surface is relatively small (0.9 mg/m2, corresponding
approximately to 25% of the surface coverage).
The similarity in both acetal and chloromethyl
latexes, on which additional carboxyl groups reduce
the number of covalent bonds but increase immuno-
reactivity, suggests that antibody coupling is hin-
dered by the excess of negative charge, which
hinders the approach or impedes the chemical reac-
tion. However, due to the repulsion provoked by this
excess of negative charge, the external surface
groups and associated antibody molecules seem to
be more extended into the solution, and they are
thereby more accessible to the corresponding antigen
molecules. This extreme is somewhat confirmed by
the good immunological results obtained with the
other kind of reagent presented here, that prepared
with the amino functionalized latex. In this case, it
can be assumed that the glutaraldehyde employed to
chemically attach the antibody molecule to the
surface acts as a spacer arm which allows the
coupled protein to separate from the surface, increas-
ing accessibility and immunoreactivity.
In Fig. 2, the immunoresponse of the reagents
prepared with the different particles is plotted as a
function of a wider range of ferritin concentrations. In
this way, it is possible to observe the phenomenon of
the prozone, that is, the existence of an antigen con-
centration with a maximum response; above this value,
the immunoresponse did not increase, but actually
decreased due to saturation of the antibody sites with
antigen molecules. This implies that two different
concentration values can be ascribed to the same
analytical signal, and this provides the upper limits of
the immunoassay. The useful measuring interval
extends up to this maximum value. As can be observed,
these values ranged up to 500 or 1500 ng/ml, depend-
ing on the type of reagent studied. The amino latex
reagent (MH4) showed an equivalence point of 1000
ng/ml of ferritin. The other important parameter that
characterizes these reagents is the analytical sensitiv-
ity, which determines the minimum value of antigen
concentration that can be accurately measured and
Fig. 2. Prozone study. o, amino latex (MH4); n, acetal-carboxyl latex (ACET2); 5, acetal latex (ACET1); D, chloromethyl latex (CMS1); E,
chloromethyl-carboxyl latex (CMS2).
M.P.S. Izquierdo et al. / Journal of Immunological Methods 287 (2004) 159–167 165
distinguished from background noise. The five
reagents showed detection limits below 10 ng/ml, the
amino functionalized latex being the best of all, with
an analytical sensitivity of 3.5 ng/ml.
Fig. 3. Comparison of serum ferritin turbidimetric assay in the CobasRMir
the same specimens in the nephelometric BNII analyzer using commercia
Sensitivity was related with the initial slope of the
immunological response curve (Edwards and Leather-
boarrow, 1997; Kondo et al., 1990; Quesada et al.,
1998). As can be seen qualitatively in Fig. 1, and
a analyzer using the MH4 latex reagent with the results obtained on
l reagents.
Table 5
Regression parameters of different latexes compared using the BNII
method
Latex Regression
slopes
Regression
y-intercepts
Regression
coefficient (r)
MH4 0.98F 0.04 � 1F 2 0.99
CMS1 1.08F 0.10 � 0.3F 5 0.95
CMS2 0.17F 0.24 � 3.8F 2.3 0.95
ACET1 0.95F 0.05 0.3F 3 0.99
ACET2 0.72F 0.32 14F 3 0.98
M.P.S. Izquierdo et al. / Journal of Immunological Methods 287 (2004) 159–167166
quantitatively in Table 4, the highest slopes were
obtained for the amino and the acetal-carboxyl latexes,
those with the lowest measuring concentration interval.
Fig. 3 shows the correlation of amino (MH4) latex
reagent with the nephelometric test. The regression
analysis with a 0.95 confidence interval gives the
statistical data of slopes and y-intercept shown in Table
5. The correlation coefficients obtained were between
0.95 and 0.99. Latexes CMS1 and CMS2 showed the
worst correlation (lower values of r), while the immu-
noreagent obtained from amino-modified particles
gave the best comparative results.
The data presented in Table 5 indicate that the
results obtained by both methods correlated well.
However, the turbidimetric method using reagents
CMS2 and ACET2 (latexes with the higher surface
charge and extra carboxyl groups) showed significant
differences compared to the nephelometric procedure.
The regression parameters indicated the existence of
proportional and constant systematic errors. Since both
methods were calibrated with the same set of calibra-
tors, these differences could be ascribed to interactions
with other components of the serum samples (matrix
effect). These may interact in some way with the
carboxyl groups present on the surface of these par-
ticles, reducing the colloidal stability of the protein
covered particles or the accessibility of the antibody
molecules to the corresponding antigen in the sample.
4. Conclusions
The microparticle reagents described in the present
study provide an adequate particle-enhanced turbidi-
metric immunoassay for serum ferritin. In the initial
step of adsorption and covalent linking of the antibody,
a clear reduction in the amount of antibody bound was
observed when extra charge was provided by carboxyl
groups in the case of acetal and chloromethyl latexes.
Nevertheless, a higher immunoreactivity was detected
using these latexes, suggesting a more favourable
conformation and/or orientation of the antibody mol-
ecules for the immunological reaction with the antigen.
In all cases, the measuring interval of ferritin con-
centrations was quite wide, up to 500 or 1500 ng/ml,
and the five reagents prepared showed detection limits
which were below 10 ng/ml, the amino functionalized
latex being the best of all, with an analytical sensitivity
of 3.5 ng/ml.
When this turbidimetric assay was compared with a
commercial nephelometric immunoassay, a good line-
ar correlation was observed. However, the important
differences between latexes found in this study indi-
cate that each different type of particle needs to be
optimized for the assay conditions in order to achieve a
good immunoreactivity and agreement with other
analytical immunoassays for human serum ferritin.
Acknowledgements
This work was supported by the Spanish ‘‘Tech-
nology and Science Ministry’’ (projects MAT 99-0662
and AGL 2001-3843-C02-02). J.F. acknowledges the
financial support from the Basque Government
ETORTEK 2002-04, under project MAAB.
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