Optimisation and standardisation of an immunoagglutination assay for the diagnosis of Trypanosoma cruzi infection based on latex-(recombinant antigen) complexes.

Tropical Medicine and International Health

doi:10.1111/tmi.12225

volume 19 no 1 pp 37–46 january 2014

Optimisation and standardisation of an immunoagglutination assay for the diagnosis of Trypanosoma cruzi infection based on latex-(recombinant antigen) complexes Valeria S. Garcia1, Ver onica D. G. Gonzalez1, Ivan S. Marcipar2, Jorge R. Vega1 and Luis M. Gugliotta1 1 INTEC, Universidad Nacional del Litoral and CONICET, Santa Fe, Argentina 2 Laboratorio de Tecnologıa Inmunolo´gica, Universidad Nacional del Litoral and CONICET, Santa Fe, Argentina

Abstract

objective To determine the conditions under which the immunoagglutination assay to detect Chagas disease, obtained from a novel latex-(chimeric recombinant antigen) complex, shows greater discrimination between the responses of a positive control serum and a negative control serum. methods The following variables were determined: (i) the sensitisation mechanism, (ii) the emulsifier employed for protein desorption, (iii) the reaction time, (iv) the ionic strength of the reaction medium, (v) the particle concentration, (vi) the presence of blocking agents, (vii) the presence of polyethyleneglycol as potentiator of reaction and (viii) the antigen and antibody concentrations. The search of optimal conditions was investigated by varying one variable at a time. To this effect, monodisperse latex particles sensitised with a recombinant chimeric protein (CP1) were subjected to different conditions. The agglutination reaction was followed by measuring the changes in the optical absorbance by turbidimetry. results The maximum discrimination between negative and positive sera was obtained at a reaction time of 5 min, when latex complexes with a concentration of covalently coupled protein of 2.90 mg/m2 were put in contact with undiluted sera in buffer borate pH 8–20 mM containing glycine (0.1 M) and polyethyleneglycol 8000 (3% w/v). Finally, the latex–protein complex was tested under the obtained optimal conditions, with a panel of Trypanosoma cruzi-positive sera, leishmaniasis-positive sera and -negative sera for both parasites. conclusion The immunoagglutination test based on the latex-CP1 complex could be used as a screening method for detecting Chagas disease. This test is rapid, easy to implement and could be used under field conditions; but its results should be confirmed by reference techniques like ELISA, HAI, and IFI. keywords recombinant protein, immunoagglutination test, Chagas disease

Introduction Chagas is a tropical parasitic disease caused by the flagellate protozoan Trypanosoma cruzi. It is estimated that 8– 11 million people in Mexico, Central America, and South America have Chagas disease, most of whom do not know they are infected. Annually, 41 200 new cases occur in endemic countries, and 14 400 infants are born with congenital Chagas disease. About 20 000 deaths per year are attributed to this illness (Kirchhoff 2010; Rassi et al. 2010). Control strategies have mostly focused on detecting the parasite and preventing transmission. Chagasic infection is mostly diagnosed when specific antibodies (Ab) against T. cruzi antigens (Ag) are detected in patient’s blood by direct or indirect parasitological methods (in the acute

© 2013 John Wiley & Sons Ltd

phase), or when specific Ab against T. cruzi Ag are detected in patient′s blood, by use of conventional serological methods (in the chronic phase) such as enzymelinked immunosorbent assay (ELISA), indirect immunofluorescence (IFI) or indirect hemagglutination (HAI) (DPDx 2010). An alternative detection method is the immunoagglutination test. Typical immunoagglutination assays are based on latex microspheres with Ag molecules bound to their surface. An aqueous dispersion of these microspheres is mixed with a sample containing Ab molecules from whole blood, serum, etc. The Ab molecules normally bind two Ag molecules situated on different microspheres and cause agglutination of latex microspheres. Main advantages of this method are its rapidness, simplicity and convenient determination by direct 37



V. S. Garcia et al. Optimising an assay to diagnose T. cruzi

visualisation or by instrumental methods (Santos & Forcada 2001; Peula-Garcıa et al. 2002; Lucas et al. 2006; Polpanich et al. 2007; Garcia et al. 2012, 2013). Our previous works (Gonzalez et al. 2005, 2008a,b, 2010; Garcia et al. 2012, 2013) aimed at synthesising, characterising and sensitising polystyrene, carboxylated and acetal latexes for producing latex-(antigenic protein) complexes from the total homogenate of the parasite or recombinant proteins. Then, the produced latex–protein complexes were characterised and employed in immunoagglutination tests for detecting Chagas disease (Garcia et al. 2012, 2013). As the composition of the buffer may play an important role in controlling the physicochemical processes of immunological recognition and agglutination in latex particle assays (Holownia et al. 2001; Perez-Amodio et al. 2001; Wang et al. 2004), we investigated the composition of the immunoagglutination reaction medium that maximises the discrimination between positive and negative sera. The effects of different variables on the Ag–Ab reaction in the immunoassay were analysed, with the aim of reaching the conditions able to maximise such reaction. To this effect, the following variables were considered: (i) particle concentration, (ii) Ag and Ab concentrations, (iii) buffer ionic strength, (iv) surface blocking agents of the latex particles (e.g. amino acids such as glycine and emulsifiers such as Tween-20) and (v) molecular weight and concentration of polyethyleneglycol, which acts as an immunoagglutination rate and sensitivity enhancer. Other factors, such as sensitisation mechanism, emulsifier employed for producing the latex– protein complex and reaction time, were also investigated. An adequate selection of such variables/factors would allow optimising the immunoassay behaviour in the sense of minimising non-specific binding, increasing the reaction rate and enhancing its sensitivity, because they determine the success of the diagnosis method (Andreotti et al. 2003). Finally, the latex–protein complex was tested under optimal conditions with sera from Chagas- and Leishmaniasis-infected patients and sera from non-infected patients.

Experimental Materials and methods Reagents. All chemicals employed in this study were of analytical grade and were used without further purification. Double-distilled and deionised water was utilised in all experiments. Salt concentrations were calculated to obtain a final ionic strength of 20 mM and 150 mM. Higher ionic strength values were reached with NaCl 38

(Cicarelli). Tween-20 (Sigma) and glycine (Sigma) were used as additives for blocking surface hydrophobic sites and surface functional groups that remain free of Ags, respectively. The emulsifiers employed for the protein desorption from the particle surface were sodium dodecylsulfate (SDS) (Cicarelli) and Triton X-100 (Sigma). Polyethyleneglycol (PEG) (Sigma) of different molecular weights (between 1000 and 20 000 g/mol) was used as accelerator of the immunoagglutination. The latex– protein complexes were resuspended in borate buffer 2 mM, pH 8 (Anedra) for their storage and subsequent use in the immunoassays. Polymer latexes. Two monodisperse latexes were employed. The polystyrene (PS; indicated by S2) latex of particle diameter 300 nm was synthesised through an emulsifier-free and unseeded emulsion polymerisation of styrene. The carboxylated latex (indicated by C2) of particle diameter 418 nm was synthesised through a semibatch copolymerisation of styrene and methacrylic acid onto the uniform S2 latex seed. The polymerisation reaction conditions for the synthesis of the S2 and C2 latexes and their colloidal characteristics were previously reported (Gonzalez et al. 2008a,b; Garcia et al. 2012). After the end of polymerisations, the unreacted comonomers and initiator were eliminated by serum replacement. Recombinant proteins. The employed recombinant Ag of T. cruzi was the chimeric protein CP1 (a unique macromolecule built as the tandem expression of 2 highly antigenic peptides, RP1 and RP2). E. coli BL21 (DE3) cells bearing the plasmidic construction, pET-32a/CP1, was grown overnight in Luria Broth medium, supplemented with ampicillin at 37 °C, with agitation. The protein expression was induced for 3 h with isopropyl-b-D-thiogalactopyranoside (Camussone et al. 2009). Finally, the protein was purified by nickel affinity chromatography. The purity of the recombinant protein CP1 was analysed by 15% polyacrylamide gel electrophoresis and stained with Coomassie brilliant blue, according to the method described by Laemmli (Laemmli 1970). Latex–protein complexes. They were obtained by physical adsorption or by covalent coupling of the recombinant protein CP1 onto the latex particles. For the physical adsorption, increasing concentrations of proteins (0.1–1.2 mg/ml) were added to the S2 latex (0.2 m2 of latex particles surface) in 1.5-ml microcentrifuge tubes and were gently shaken during 5 h at room temperature. Finally, the latex–protein complexes were isolated by ultracentrifugation and were redispersed in borate buffer of pH 8. The surface densities of physically adsorpted





proteins were calculated from the total added protein and the protein that remains in solution. For the covalent coupling, increasing concentrations of antigenic proteins (0.3– 1.2 mg/ml) were added to the C2 latex samples (0.2 m2 of latex particles surface) in the presence of N-N-(3-dimethylamine propyl) N’-ethyl carbodiimide activator and in 1.5-ml microcentrifuge tubes, and they were shaken during 5 h at room temperature. The latex–protein complexes were isolated by ultracentrifugation, and the non-covalently coupling protein was desorbed with Triton X-100 (or SDS) during 24 h. Finally, the resulting latex–protein complexes were redispersed in borate buffer at pH 8.0 (Gonzalez et al. 2010). The surface densities of covalently bound proteins were calculated from the total linked protein and the desorbed protein that remains in solution. In all cases, the concentrations of dissolved protein were determined through the copper reduction/bicinchoninic acid (BCA) method (Ortega-Vinuesa et al. 1995). Serum samples. The positive serum sample, with a high titre of Ab, was obtained from T. cruzi infected patients from an endemic region located in northeast Argentina. The negative serum sample was obtained from a healthy blood donor from the same Argentine region. The T. cruzi infection status of the patients was determined by using two different conventional test, namely commercial Enzyme Chagatest ELISA and Chagatest HAI, both from Wiener Lab (Argentina). The serological condition was ascertained when concordant results were obtained with both conventional tests, according to recommendations given by the World Health Organization (Cura et al. 1994). Sera from individuals infected with Leishmania were obtained from patients of the Centro de Pesquisas Aggeu Magalh~aes, Fundacß~ao Oswaldo Cruz, Recife PE, Brazil. Immunoagglutination assay. It was performed according to Garcia et al. 2013. However, when the effect of reaction time was analysed, three different times were used: (i) t = 5 min, (ii) t = 15 min and (iii) t = 25 min. In all cases, the agglutination reaction was detected by turbidimetry, measuring the optical absorbance (A) at 570 nm in an UV/vis spectrophotometer (Perkin Elmer Lambda 25), and the increment in A (DA) was determined by subtracting the absorbance of a blank (the complex without serum) to the absorbance measured for the (complex + serum) sample. Results and discussion The search of optimal conditions was carried out by varying only one variable at a time, and keeping all the


other variables constant. As the immunoagglutination mainly occurs by a mechanism of an initial immunorecognition (Perez-Amodio et al. 2001; Thanh & Rosenzweig 2002), and as interaction processes are pH and temperature dependent, it is expected that both the pH and temperature of the reaction medium affect the immunoagglutination performance. Buffer borate (pH 8.0) was used in all the assays to work at a pH near the physiological value, and the experiments were carried out at ambient temperature to simulate the field conditions in which the immunoagglutination reactives will be employed. Influence of the sensitisation mechanism The fixation between the Ag and the particle surface can be physical or chemical. The effect of the sensitisation mechanism was considered by comparing the results obtained in immunoagglutination assays after 5 min of reaction when the Ag was physically adsorbed or covalently coupled to the particle surface. For the C2-CP1 latex–protein complex obtained by covalent coupling (CC), the ratio between the response to positive and negative sera was ΔA(+)/ΔA( ) = 5.09 , while the S2-CP1 latex–protein complex obtained by physical adsorption (PA) exhibited a ratio ΔA(+)/ΔA( ) = 0.67. The S2-CP1 latex–protein complex has a limited applicability in immunodiagnosis due to the partial desorption of Ag that normally occurs during its storage. Also, the PA of Ags onto PS particles can give place to unspecific reactions due to the hydrophobic character of the PS surface that allows adsorbing proteins (in an unspecific way) on parts of the surface free of Ags, thus resulting in false diagnosis. The covalent coupling of Ags to functional groups on the particle surface provides certain advantages from the point of view of its application in the development of immunodiagnostic test because: (i) the Ags immobilised by CC retain a maximum of their antigenicity, while the Ags immobilised by PA retain only a small fraction or even lose completely their binding capacity to Ab due to the protein denaturation on the surface (Hidalgo-Alvarez & Galisteo-Gonzalez 1995), (ii) CC is permanent, and it may prevent elution of bound protein during storage, thus increasing the shelf life, (iii) if the correct coupling chemistry is chosen, covalent attachment can orient the protein molecule properly, thus improving the activity of the bound proteins and (iv) CC avoids the proteins desorption from the particle surface in the presence of surfactants. Thus, CC improves the specific character of the test, the reactivity and the stability of the immunoassay (Seradyn Inc. 1988). 39




During the sensitisation by covalent coupling, the protein binds to the particle surface both physically and covalently. To minimise the problems associated with the presence of physically absorbed proteins during the immunoassay, the latexes sensitised by CC were treated with an emulsifier to remove non-covalently bound proteins, so as to ensure that all proteins on the particle surface were chemically attached (Peula et al. 1995; Ortega-Vinuesa et al. 1996). Although similar covalently bound results were observed when the final redissolution operations, to induce protein desorption, were carried out with the anionic emulsifier SDS instead of the non-ionic Triton X-100 (91% covalently linked CP1 with SDS and 92% covalently linked CP1 with Triton X-100), the results of the immunoagglutination assays using Triton X-100 or SDS were clearly different. For the C2-CP1 latex–protein complex treated with Triton X-100, the ratio between the response to positive and negative sera was ΔA(+)/ΔA( ) = 5.09, while the same complex treated with SDS exhibited a ratio ΔA(+)/ΔA( ) = 1.06. The SDS binds to non-polar regions of polypeptides providing negative charge and causing the loose of their native conformation (Otzen 2002). Due to protein denaturation, complexes obtained by using SDS are unable to differentiate between sera from infected and non-infected patients. For this reason, the positive serum response was close to the negative serum response when SDS was employed. This last behaviour could also be related to non-specific agglutination reactions caused by the interaction of negatively charged SDS with several positively charged residues on human serum albumin (HSA), which is the most abundant protein in human blood plasma and constitutes about half of the blood serum protein. For previous reasons, the complexes treated with SDS are not suitable for application in immunoagglutination tests.

discrimination between positive and negative sera (Figure 1). At time >5 min, the negative serum response increases due to the presence in the serum of others Ab or intrinsic interferences of serum such as proteins (fibrinogens, globulins and albumins), glucose and mineral ions, which eventually deposit on the particles surface, or simply because the stability of the latex–protein complex decreases under the assay conditions. All this leads to increase the number of false positives, thus decreasing the specificity of the assay (Selby 1999). However, when t = 5 min, the response of the negative serum was low at room temperature, it reduces the frequency of false positives, and a good discrimination between positive and negative sera was obtained. Even though not shown, when analysing the evolution of ΔA during the first 5 min of reaction, it was observed that the latex–protein complex was stable in the absence of Ab (blank) and that the positive serum response was clearly better than the negative serum response from the beginning of the reaction, suggesting that the Ag–Ab reaction occurred rapidly. Influence of the ionic strength The influence of ionic strength (I) of the reaction medium (in which the immunoassay is performed) on the discrimination between positive control serum and negative control serum was analysed. Figure 2 shows that the greater ΔA(+)/ΔA( ) ratios were obtained at low I (20 mM). Under these conditions, the polymeric chains which are

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Influence of the emulsifier employed for the protein desorption

1.55

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15 t [min]

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Influence of reaction time The reaction time (t) is defined as the time interval between the mixture of the serum with the latex–protein complex and the absorbance reading. Because the immunoagglutination assay does not reach an end point, reaction time analysis is an important factor to consider when optimising an assay. To this effect, three different situations were analysed: (i) t = 5 min, (ii) t = 15 min and (iii) t = 25 min. When the reaction time was increased, the negative serum response increased, reducing the ΔA(+)/ΔA( ) ratio and hindering the

40

0.0

5

Figure 1 Influence of reaction time on the immunoagglutination assay in buffer borate pH = 8–20 mM containing glycine 0.1 M and PEG 8000 3% and using a concentration of covalently coupled protein equal to 3.90 mg/m2 and undiluted serum. The black bars represent the positive serum, and the grey bars the negative serum. Numbers on bars indicate the ratio between positive and negative sera responses, in terms of absorbance changes ΔA(+)/ΔA( ). Error bars indicate the SD for n = 3.





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ΔA

1.28 0.2

5.09

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150 I [mM]

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1.56 3.11 6.53 [Particles 109/ml]

Figure 2 Influence of the ionic strength of the reaction medium on the immunoagglutination assay in buffer borate pH = 8 containing glycine 0.1 M and PEG 8000 3% and using a concentration of covalently coupled protein equal to 3.90 mg/m2 and undiluted serum. The black bars represent the response to positive serum, and the grey bars the response to negative serum. Numbers on bars indicate the ratio between positive and negative sera ΔA(+)/ΔA( ). Error bars indicate the SD for n = 3.

Figure 3 Effect of the concentration of latex-Ag particles on the immunoagglutination assay in buffer borate pH = 8–20 mM containing glycine 0.1 M and PEG 8000 3% and using a concentration of covalently coupled protein equal to 3.90 mg/m2 and undiluted serum. The black bars represent the positive serum response, and the grey bars the negative serum response. Numbers on bars indicate the ratio between positive and negative sera responses ΔA(+)/ΔA( ). Error bars indicate the SD for n = 3.

covalently coupled to the Ags are extended into the solution, and the Ags are more exposed to interact with the Abs present in the serum. On the other hand, at low I, the stability of the latex–protein complexes is high because both protein chains and particle surface (due to the SO24 and COOH groups) are negatively charged, and there is a repulsion between particles, thus preventing non-specific agglutination. In contrast, at high I (150 mM), the polymeric chains covalently coupled to the Ags are closer to the particle surface, and this could obstruct the access of the Ab molecules to the immunological recognition sites by steric impediment or occlusion of the active site of Ag, thus becoming difficult the Ag– Ab interaction (Nance & Garratty 1987; Ghourchian & Kamo 1994; Gibbs 2001; Perez-Amodio et al. 2001). Also, at high I, the latex–protein complexes may be less stable due to a charge shielding by counterions generating non-specific agglutination.

At high particle concentrations (6.53 9 109 particles/ ml), the ΔA(+)/ΔA( ) ratio was also low. There are two possible explanations to this result. The first might be related to a decreased formation of immunocomplexes due to imbalance between Ag and Ab concentrations (Atassi et al. 1984). The second may be attributed to the higher particles concentration possibly resulting in an increased steric hindrance or non-specific agglutination (Hidalgo-Alvarez & Galisteo-Gonzalez 1995). To avoid the labile background interference or nonspecific agglutination, an intermediate particle concentration equal to 3.11 9 109 particles/ml was used. This ‘optimal’ concentration of particles allows the formation of a network of molecules characteristic of the agglutination processes.

Influence of particle concentration The effect of the concentration of Ag coated particles on the immunoagglutination reaction was investigated (Figure 3). At low particle concentration (1.56 9 109 particles/ml), the interference caused by the (complex) composition of the serum samples becomes significant. This is because the naturally occurring proteins found in serum samples can interfere with immunoassays. As previously mentioned, some well-known interfering substances in human sera are albumins, complement factors, lysozymes, lipids and fibrinogen (MacBeath 2002).


Influence of the presence of blocking agents Blocking agents are normally employed to avoid nonspecific interactions during the immunoreaction (to improve the response in the immunoassay). A review of the literature shows that for every detection method, several hundred protocols exist based on different blocking agents. Unfortunately, the optimum reagent has to be determined for each new assay, as they all have certain restrictions when used with real samples such as blood, serum, cell lysates or tissue sections. In this work, two different blocking agents were incorporated (glycine and Tween-20) to study their effect on the immunoagglutination assay. The Tween-20 emulsifier was used to block the surface hydrophobic sites of the latex–protein 41




complex, and the glycine was added to block the free functional groups (i.e. those which have not reacted with Ags during the sensitisation). As reflected in Figure 4, a greater discrimination between control sera was obtained in the presence of glycine or Tween-20 in the reaction buffer, with respect to results obtained in absence of blocking agents. Without blocking agents, some areas on the particle surface are available for non-specific binding, thus resulting in a reduction in the ΔA(+)/ΔA( ) ratio. 0.4

ΔA

5.09 6.05

0.2

1.14 0.0 Non additive Glycine Tween 20 Figure 4 Influence of blocking agents (glycine and Tween-20) on the immunoagglutination assay in buffer borate pH = 8– 20 mM containing PEG 8000 3% and using a concentration of covalently coupled protein equal to 3.90 mg/m2 and undiluted serum. The black bars represent the response to positive serum, and the grey bars the response to negative serum. Numbers on bars indicate the ratio between positive and negative sera ΔA (+)/ΔA( ). Error bars indicate the SD for n = 3.

0.4

Tween-20 is considered to be a temporary blocker, and its blocking ability can be simply removed by washing with water or aqueous buffer. Unlike Tween-20, glycine is a permanent blocker, and it only needs to be added once to the medium, after the particle surface has been sensitised with the Ag (Seradyn Inc. 1988; Gibbs 2001). Influence of the presence of polyethyleneglycol The addition of PEG to buffer reaction facilitates the formation of immune complexes, helps in amplification of signals and improves the assay system sensitivity (Creighton et al. 1973; Nance & Garratty 1987; Holownia et al. 2001; Wang et al. 2004). The PEG is an inert molecule that does not directly interact with the protein; however, it has shown to be a potentiator of Ag–Ab reactivity (Laurent 1963). This phenomenon is a result of a steric exclusion where large macromolecules, such as Ab and Ag, are excluded by the volume occupied by the PEG being more exposed in the dispersion medium and favouring the interaction Ag–Ab (Laurent 1963). The mechanism through which the PEG affects protein interactions depends on its molecular weight and its concentration. In this work, the influence of these two variables was analysed by fixing one of the variables and modifying the other. First, the effect of molecular weight was considered at a 3% w/v PEG concentration. Figure 5a shows that the highest DA(+)/DA( ) ratio was obtained with PEG of 8000 g/mol.

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4000 6000 8000 10 000 20 000 Molecular weight [Da]

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Figure 5 Influence of PEG on the response in the immunoagglutination assay in buffer borate pH = 8–20 mM containing glycine 0.1 M and using a concentration of covalently coupled protein equal to 3.90 mg/m2 and undiluted serum. (a) Effect of molecular weight for a concentration of 3% w/v, (b) influence of the concentration of PEG 8000. The black bars represent the response to the positive serum, and the grey bars the response to the negative serum. Numbers on bars indicate the ratio between the responses to positive and negative sera ΔA(+)/ΔA( ). Error bars indicate the SD for n = 3.

42





During the immunoassays, Ag and Ab react specifically causing the agglutination of the complexes. The degree of immunoagglutination was determined as a function of the concentration of Ag or Ab added. In the immunoagglutination assays developed here, the concentration of reactants can be modified in two ways: a) by varying the concentration of Ab in the serum sample by dilution and/ or b) by varying the concentration of Ag (or amount of bound protein per unit of particle surface). In any case, a change in ΔA(+)/ΔA( ) should be observed (Reverberi & Reverberi 2007). Figure 6 shows the influence of the Ab concentration on the immunoagglutination assay for a density of bound protein of 3.90 mg/m2. The ΔA(+)/ΔA( ) monotonically decreases to 1, as the serum is diluted, not having an adequate discrimination between positive and negative serum for higher dilutions. This response can be explained considering that an Ab molecule acts as a bridge to agglutinate two sensitised particles. Thus, a lower probability of particle agglutination is expected when decreasing the Ab concentration. Figure 7 shows the influence of the Ag concentration on the immunoagglutination assay using undiluted serum. The ΔA(+)/ΔA( ) increases with the Ag concentration up to a density of bound protein of 2.90 mg/m2. However, higher Ag concentrations do not augment ΔA(+)/ΔA( ). Notice that in the regions of Ag excess (3.90 and 4.93 mg/m2) and Ab excess (1.57 mg/m2), a lower discrimination between positive and negative serum is achieved and the system seems to lose reactivity because ΔA(+)/ΔA( ) diminishes. When Ag is in excess, all Ab are complexed to individual Ag molecules, so no aggregation occurs. When Ab is in excess, there is insufficient Ag to form an aggregate. This results in the formation of small antigen-antibody complexes. However, for a density of bound protein of 2.90 mg/m2, the ΔA(+)/ΔA( ) ratio reaches a maximum value, and the density of protein on


5.09 4.57 0.2 4.07 3.98 3.46

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Figure 6 Influence of the antibody concentrations on the immunoagglutination assay in buffer borate pH = 8–20 mM containing glycine 0.1 M and PEG 8000 3% and using a concentration of covalently coupled protein equal to 3.90 mg/m2. The black bars represent the response to positive serum, and the grey bars the response to negative serum. Numbers on bars indicate the ratio between positive and negative sera ΔA(+)/ΔA( ). Error bars indicate the SD for n = 3.

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Influence of the antigen and antibody concentrations

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Finally, the effect of varying the PEG 8000 concentration was analysed, and the results are presented in Figure 5b. Notice that conflicting results were obtained in the absence of PEG, in the sense that a higher response was obtained with the negative serum than with the positive serum [i.e. DA(+)/DA( ) < 1]. However, such ratio was increased when PEG 8000 was added, giving place to a maximum DA(+)/DA( ) of 5.09 at a concentration of 3% w/v. At lower PEG concentrations, the response ratio is not noticeable, and at higher PEG concentrations, a destabilising effect may occur, thus generating non-specific agglutination and lower values of DA(+)/DA( ).

5.09

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Bound protein [mg/m2] Figure 7 Influence of the antigen concentrations on the immunoagglutination assay in buffer borate pH = 8–20 mM containing glycine 0.1 M and PEG 8000 3% and using undiluted serum. The black bars represent the response to positive serum, and the grey bars the response to negative serum. Numbers on bars indicate the ratio between positive and negative sera ΔA(+)/ΔA( ). Error bars indicate the SD for n = 3.

the particle surface and the Ab concentration in the serum are in their optimum values. Here, the binding sites available for the Ag are proportionale to the Ab concentration, and the probability of cross-linking is more likely resulting in formation of large immune complexes. 43




Previous results are consistent with those of the precipitine curve proposed by Heidelberger and Kendall (Heidelberger & Kendall 1935), where agglutination is maximal when the ratio between Ag and Ab is optimal (zone of equivalence), but decreases when there is an excess of either Ab (antibody excess zone) or Ag (antigen excess zone) (Abbas et al. 1999). Test of the latex-CP1 complex at optimal conditions A panel of 15 Chagas-positive sera, 12 Leishmania-positive sera and 15 negative sera for both parasites was assayed, using the optimal reaction-medium conditions previously obtained. Figure 8 shows the results in terms of relative optical distributions (ΔA/cut-off). Notice that (i) Chagas-positive sera clearly differed from the negative sera, and (ii) non-cross-reaction was detected. In a future communication, different latex–protein complexes obtained either from the homogenate of the total parasite or from different recombinant proteins (simple and chimerics) will be evaluated under the conditions here found, against a panel of positive and negative sera in immunoagglutination assays for detecting Chagas disease. Conclusions We studied the immunoagglutination-assay conditions to detect Chagas disease, based on a novel latex-(chimeric recombinant antigen) complex. Main results were as follows: (i) the best discrimination between positive and 4

Acknowledgements

ΔA/cut-off

3

To CONICET, ANPCyT and Universidad Nacional del Litoral for the financial supports. We are also grateful to Dra. Maria Edileuza Felinto de Brito of Centro de Pesquisas Aggeu Magalh~ aes, Fundacß~ ao Oswaldo Cruz, Recife (Brazil) and Dr. Miguel Hern an Vicco of Hospital J. B. Iturraspe, Santa Fe (Argentina) for the donation of serum samples.

2

1

0 Negative

Chagas (+)

Leishmania (+)

Figure 8 Relative optical distribution (ΔA/cut-off) obtained for a panel of 15 Chagas-positive sera; 12 Leishmania-positive sera and 15 Chagas- and Leishmania-negative sera. The dashed lines show the relative mean values for each assay, and the continuous line indicates the relative cut-off value (ΔA/cut-off = 1).

44

negative sera was reached with latex–protein complexes obtained by CC, (ii) Triton X-100 was more suitable than SDS for removing loosely bound proteins from the particle surface, because Triton X-100 reduced protein denaturation and non-specific agglutination, (iii) a reaction time of 5 min proved to be adequate for the immunoassays, while higher times increased the response to negative sera, (iv) low ionic strength (20 mM) improved the Ag–Ab interaction and complex stability, (v) the particle concentration of 3.11 9 109 particles/ml proved to be adequate, because it increased the positive to negative response ratio and reduced the negative serum response, (vi) the use of undiluted serum and complexes with a density of bound protein of 2.90 mg/m2 produced the highest discrimination between positive and negative sera, (vii) the use of glycine at 0.1 M (as blocking agent) avoided non-specific interactions and improved the discrimination between positive and negative sera, (viii) PEG 8000 (3% w/v) proved to be adequate as a potentiator of the Ab–Ag reaction and increased the positive to negative response ratio with respect to PEG of other molecular weights and concentrations. Under such optimal conditions, a panel of 15 Chagaspositive sera, 12 Leishmania-positive sera (to determine the cross-reactivity) and 15 negative sera for both parasites was assayed. The negative sera response was clearly different from that of the Chagas-positive sera, and noncross-reaction was detected. Consequently, the immunoagglutination test based on the latex-CP1 complex could be used as a screening method for detecting Chagas disease. But, even though this test is rapid, easy to implement and could be used under field conditions, its results should be confirmed by reference techniques like ELISA, HAI and IFI.

References Abbas AK, Lichtman AH & Pober JS (1999) Cell and Molecular Immunology. Mc Graw Hill, Spain. Andreotti PE, Ludwig GV, Peruski AH, Tuite JJ, Morse SS & Peruski LF (2003) Immunoassay of infectious agents. BioTechniques 35, 850–859.





Atassi MZ, Van-Oss CJ & Absolom DR (1984) Molecular Immunology: A Textbook. Marcel Dekker, New York, NY. Camussone C, Gonzalez VDG, Belluzo MS et al. (2009) Comparison of recombinant Trypanosoma cruzi peptide mixtures versus multiepitope chimeric proteins as sensitizing antigens for immunodiagnosis. Journal of Clinical and Laboratory Immunology 16, 899–905. Creighton W, Lambert P & Miescher P (1973) Detection of antibodies and soluble antigen-antibody complexes by precipitation with polyethylene glycol. Journal of Immunology 4, 1219–1227. Cura E, Wendel S, Pinheiro FP & Weinserbacher M (1994) Manual de procedimientos de control de calidad para laboratorios de serologıas de los bancos de sangre. Organizaci on Panamericana de la Salud, Washington DC. DPDx (2010) Trypanosomiasis, American. Fact Sheet. Centers for Disease Control (CDC). http://www.dpd.cdc.gov/dpdx/ HTML/TrypanosomiasisAmerican.htm. Garcia VS, Gonzalez VDG, Vega JR, Marcipar IS & Gugliotta LM (2012) Synthesis of carboxylated and acetal latexes by emulsion polymerization. Application to the production of immunoagglutination test for detecting Chagas′disease. Latin American Applied Research 42, 405–412. Garcia VS, Gonzalez VDG, Caudana PC, Vega JR, Marcipar IS & Gugliotta LM (2013) Synthesis of latex-antigen complexes from single and multiepitope recombinant proteins. Application in immunoagglutination assays for the diagnosis of Trypanosoma cruzi infection. Colloids and Surfaces. B, Biointerfaces 101, 384–391. Ghourchian HO & Kamo N (1994) Improvement of latex piezoelectric immunoassay: detection of rheumatoid factor. Talanta 41, 401–406. Gibbs J (2001) Effective blocking procedures. ELISA Technical bulletin. catalog2.corning.com/Lifesciences/media/pdf/elisa3.pdf. 2001. Gonzalez VDG, Gugliotta LM, Vega JR & Meira GR (2005) Contamination by larger particles of two almost-uniform latices: analysis by combined dynamic light scattering and turbidimetry. Journal of Colloid and Interface Science 285, 581–589. Gonzalez VDG, Gugliotta LM & Meira GR (2008a) Latex of immunodiagnosis for detecting the Chagas disease. I. Synthesis of the base carboxylated latex. Journal of Materials Science. Materials in Medicine 19, 777–788. Gonzalez VDG, Gugliotta LM, Giacomelli CE & Meira GR (2008b) Latex of immunodiagnosis for detecting the Chagas disease: II. Chemical coupling of antigen Ag36 onto carboxylated latexes. Journal of Materials Science. Materials in Medicine 19, 789–795. Gonzalez VDG, Garcia VS, Vega JR, Marcipar IS, Meira GR & Gugliotta LM (2010) Immunodiagnosis of Chagas disease: synthesis of three latex–protein complexes containing different antigens of Trypanosoma cruzi. Colloids and Surfaces. B, Biointerfaces 77, 12–17. Heidelberger M & Kendall FE (1935) A quantitative theory of the precipitin reaction: II. a study of anazoprotein-antibody system. Journal of Experimental Medicine 62, 467–483.


Hidalgo-Alvarez R & Galisteo-Gonzalez F (1995) The adsorption characteristics of immunoglobulins. Heterogeneous Chemistry Reviews 2, 249–268. Holownia P, Perez-Amodio S & Price CP (2001) Effect of poly (ethylene glycol), tetramethylammonium hydroxide, and other surfactants on enhancing performance in a latex particle immunoassay of C-reactive protein. Analytical Chemistry 73, 3426–3431. Kirchhoff LV (2010) Chagas disease (American Trypanosomiasis). http://emedicine.medscape.com/article/214581-overview. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680– 685. Laurent TC (1963) The interaction between polysaccharides and other macromolecules. 5. The solubility of proteins in the presence of dextran. The Biochemical Journal 89, 253–260. Lucas LJ, Han JH & Yoon JY (2006) Using highly carboxylated microspheres to simplify immunoassays and enhance diffusional mixing in a microfluidic device. Colloids and Surfaces. B, Biointerfaces 49, 106–111. MacBeath G (2002) Protein microarrays and proteomics. Nature Genetics 32, 526–532. Nance SJ & Garratty G (1987) A new potentiator of red blood cell antigen-antibody reactions. American Journal of Clinical Pathology 87, 633–635. Ortega-Vinuesa JL, Bastos-Gonzalez D & Hidalgo-Alvarez R (1995) Comparative studies on physically absorbed and chemically bound IgG to carboxylated latexes, II. Journal of Colloid and Interface Science 176, 240–247. ´ lvarez R Ortega-Vinuesa LJ, Molina-Bolıvar JA & Hidalgo-A (1996) Particle enhanced immunoaggregation of F(ab′)2 molecules. Journal of Immunological Methods 190, 29–38. Otzen DE (2002) Protein unfolding in detergents: effect of micelle structure, ionic strength, pH, and temperature. Biophysical Journal 83, 2219–2230. Perez-Amodio S, Holownia P, Davey CL & Price CP (2001) Effects of the ionic environment, charge and particle surface chemistry for enhancing a latex homogeneous immunoassay of C-reactive protein. Analytical Chemistry 73, 3417–3425. Peula JM, Hidalgo-Alvarez R, Santos R, Forcada J & De Las Nieves FJ (1995) Covalent coupling of antibodies to aldehyde groups on polymer carriers. Journal of Materials Science. Materials in Medicine 6, 779–785. Peula-Garcıa JM, Molina-Bolivar JA, Velasco J, Rojas A & Galisteo-Gonzalez F (2002) Interaction of bacterial endotoxine (lipopolysaccharide) with latex particles: application to latex agglutination immunoassays. Journal of Colloid and Interface Science 245, 230–236. Polpanich D, Tangboriboonrat P, Elaissari A & Udomsangpetch R (2007) Detection of malaria infection via latex agglutination assay. Analytical Chemistry 79, 4690–4695. Rassi A Jr, Rassi A & Marin-Neto JA (2010) Chagas disease. Lancet 375, 1388–1402. Reverberi R & Reverberi L (2007) Factors affecting the antigenantibody reaction. Blood Transfusion 5, 227–240. doi:10. 2450/2007.0047-07.

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Santos RM & Forcada J (2001) Acetal functionalized polymer particles useful for immunoassays. III: preparation of latexprotein complexes and their applications. Journal of Materials Science. Materials in Medicine 12, 173–180. Selby C (1999) Interference in immunoassay. Annals of Clinical Biochemistry 36, 704–721. Seradyn Inc. (1988) Microparticle Immunoassay Techniques, 2nd edn. Seradyn Inc., Indianapolis, USA.

Thanh NTK & Rosenzweig Z (2002) Development of an aggregation based immunoassay for anti-protein a using gold nanoparticles. Analytical Chemistry 74, 1624–1628. Wang H, Lei CX, Li JS, Wu ZY, Shen GL & Yu RQ (2004) A piezoelectric immunoagglutination assay for Toxoplasma gondii antibodies using gold nanoparticles. Biosensors & Bioelectronics 19, 701–709.

Corresponding Author: Luis M. Gugliotta, INTEC, G€ uemes 3450, (3000) Santa Fe, Argentina. Tel.: +54-342-455-8450/1; Fax: +54-342-455-0944; E-mail: [email protected]

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An immunocytotoxic assay for Trypanosoma cruzi.

Performance Assessment of Four Chimeric Trypanosoma cruzi Antigens Based on Antigen-Antibody Detection for Diagnosis of Chronic Chagas Disease.

Regulation of immunity in Trypanosoma cruzi infection.

Trypanosoma cruzi infection and host lipid metabolism.

Diagnosis of congenital Trypanosoma cruzi infection: A serologic test using Shed Acute Phase Antigen (SAPA) in mother-child binomial samples.

Predictive role of polymerase chain reaction in the early diagnosis of congenital Trypanosoma cruzi infection.

Characterization of a candidate gene for GP72, an insect stage-specific antigen of Trypanosoma cruzi.

Immobilization of NTPDase-1 from Trypanosoma cruzi and Development of an Online Label-Free Assay.

Trypanosoma cruzi infection in immunosuppressed mice.

Trypanocidal drugs for chronic asymptomatic Trypanosoma cruzi infection.

Trypanosoma cruzi: immunoperoxidase antibody test for serologic diagnosis.

Effects of infection by Trypanosoma cruzi and Trypanosoma rangeli on the reproductive performance of the vector Rhodnius prolixus.

cure models on the prospects for vaccines for Trypanosoma cruzi infection.

[Diagnosis of Trypanosoma cruzi infection in opossums, naturally infected, using indirect immunofluorescence reaction].

An approach to the management of Trypanosoma cruzi infection (Chagas' disease) in immunocompromised patients.

Trypanosoma cruzi: course of infection in platelets-depleted mice.

Multicentre evaluation of an antigen-detection ELISA for the diagnosis of Trypanosoma brucei rhodesiense sleeping sickness.

Studies on the selective lysis and purification of Trypanosoma cruzi.

Interferon-gamma promotes infection of astrocytes by Trypanosoma cruzi.

Trypanosoma lewisi and T. cruzi: effect of infection on gestation in the rat.

Trypanosoma cruzi: modification of macrophage function during infection.

Antigen targeting to dendritic cells allows the identification of a CD4 T-cell epitope within an immunodominant Trypanosoma cruzi antigen.

Domestic Pig (Sus scrofa) as an Animal Model for Experimental Trypanosoma cruzi Infection.

Immunoagglutination test to diagnose Chagas disease: comparison of different latex-antigen complexes.