www.elsevier.com/locate/jim

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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