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Keywords:

  • recombinant protein;
  • immunoagglutination test;
  • Chagas disease

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Experimental
  5. Results and discussion
  6. Conclusions
  7. Acknowledgements
  8. References

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.

Objectif

Déterminer les conditions sous lesquelles le test d'immunoagglutination pour la détection de la maladie de Chagas, obtenu à partir d'un nouveau complexe latex-(antigène recombinant chimérique), montre une plus grande discrimination entre les réponses d'un sérum contrôle positif et d'un contrôle négatif.

Méthodes

Les variables suivantes ont été déterminées: (i) le mécanisme de sensibilisation, (ii) l’émulsifiant utilisé pour la protéine de désorption, (iii) le temps de réaction, (iv) la force ionique du milieu réactionnel, (v) la concentration de particules, (vi) la présence d'agents bloquant, (vii) la présence de polyéthylène glycol comme potentialisateur de la réaction et (viii) les concentrations d'antigène et d'anticorps. La recherche des conditions optimales a été effectuée en faisant varier une variable à la fois. A cet effet, des particules de latex mono dispersés, sensibilisées avec une protéine chimérique recombinante (CP1) ont été soumises à différentes conditions. La réaction d'agglutination a été suivie par la mesure des variations de l'absorbance optique par turbidimétrie.

Résultats

La discrimination maximale entre les sérums négatifs et positifs a été obtenue à un temps de réaction de 5 min, lorsque les complexes latex à une concentration de protéine couplée de manière covalente de 2.90 mg/m2 ont été mis en contact avec le sérum non dilué dans un tampon borate pH 8 de 20 mm contenant de la glycine (0.1 m) et du polyéthylène glycol 8000 (3% v/v). Enfin, le complexe latex-protéine a été testé dans les conditions optimales obtenues, avec une série de sera positifs pour T. cruzi et la leishmaniose et de sera négatifs pour les deux parasites.

Conclusion

Le test d'immunoagglutination basé sur le complexe latex-CP1 pourrait être utilisé comme une méthode de dépistage permettant de détecter la maladie de Chagas. Ce test est rapide, facile à implémenter et pourrait être utilisé dans des conditions de terrain, mais ses résultats devraient être confirmés par des techniques de référence comme ELISA, HAI et IFI.

Objetivo

Determinar las condiciones bajo las cuales el ensayo de inmunoaglutinación para detectar la enfermedad de Chagas, mediante un nuevo complejo de látex – (antígeno quimérico recombinante, muestra una mayor discriminación entre las respuestas al suero control positivo y al suero control negativo.

Métodos

Se determinaron las siguientes variables: (i) el mecanismo de sensibilización, (ii) el agente emulsificador utilizado para la desabsorción de proteínas, (iii) el tiempo de reacción, (iv) la fuerza iónica del medio de reacción, (v) la concentración de partículas, (vi) la presencia de agentes bloqueantes (vii) la presencia de polietilenglicol como potenciador de la reacción, y (viii) las concentraciones de antígeno y anticuerpo. La búsqueda de condiciones óptimas se realizó cambiando una variable a la vez. Con este objetivo, las partículas monodispersas de látex, sensibilizadas con una proteína quimérica recombinante (CP1) fueron expuestas a diferentes condiciones. Se realizó un seguimiento a la reacción de aglutinación, midiendo los cambios en absorbancia óptica mediante turbidimetría.

Resultados

La discriminación máxima entre sueros negativos y positivos se obtuvo en un tiempo de reacción de 5 minutos cuando complejos de látex, con una concentración de 2.90 mg/m2 de proteína asociada covalentemente, se pusieron en contacto con suero sin diluir en una solución tampón de borato pH 8–20 mm con glicina (0.1 m) y polietileneglicol 8000 (3% w/v). Finalmente, el complejo látex-proteína se probó bajo las condiciones óptimas, con un panel de sueros T. cruzi-positivos, sueros positivos para leishmaniosis y sueros negativos para ambos parásitos.

Conclusión

La prueba de inmunoaglutinación basada en el complejo látex-CP1 podría utilizarse como un método de detección de la enfermedad de Chagas. Esta prueba es rápida, fácil de implementar y podría utilizarse bajo condiciones de campo; pero sus resultados deberían confirmarse mediante técnicas de referencia tales como ELISA, HAI e IFI.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Experimental
  5. Results and discussion
  6. Conclusions
  7. Acknowledgements
  8. References

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 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 enzyme-linked 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 visualisation or by instrumental methods (Santos & Forcada 2001; Peula-García 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

  1. Top of page
  2. Abstract
  3. Introduction
  4. Experimental
  5. Results and discussion
  6. Conclusions
  7. Acknowledgements
  8. References

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 (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-β-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 Magalhães, Fundação 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) = 5 min, (ii) = 15 min and (iii) = 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 (ΔA) 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

  1. Top of page
  2. Abstract
  3. Introduction
  4. Experimental
  5. Results and discussion
  6. Conclusions
  7. Acknowledgements
  8. References

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 ∆A(+)/∆A(−) = 5.09 , while the S2-CP1 latex–protein complex obtained by physical adsorption (PA) exhibited a ratio ∆A(+)/∆A(−) = 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).

Influence of the emulsifier employed for the protein desorption

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 ∆A(+)/∆A(−) = 5.09, while the same complex treated with SDS exhibited a ratio ∆A(+)/∆A(−) = 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.

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) = 5 min, (ii) = 15 min and (iii) = 25 min. When the reaction time was increased, the negative serum response increased, reducing the ∆A(+)/∆A(−) ratio and hindering the 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 = 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 ∆A 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.

image

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 ∆A(+)/∆A(−). Error bars indicate the SD for n = 3.

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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 ∆A(+)/∆A(−) ratios were obtained at low I (20 mm). Under these conditions, the polymeric chains which are 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 inline image 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.

image

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 ∆A(+)/∆A(−). Error bars indicate the SD for n = 3.

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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 × 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).

image

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 ∆A(+)/∆A(−). Error bars indicate the SD for n = 3.

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At high particle concentrations (6.53 × 109 particles/ml), the ∆A(+)/∆A(−) 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 non-specific agglutination, an intermediate particle concentration equal to 3.11 × 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 the presence of blocking agents

Blocking agents are normally employed to avoid non-specific 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 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 ∆A(+)/∆A(−) ratio.

image

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 ∆A(+)/∆A(−). Error bars indicate the SD for n = 3.

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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 ΔA(+)/ΔA(−) ratio was obtained with PEG of 8000 g/mol.

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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 ∆A(+)/∆A(−). Error bars indicate the SD for n = 3.

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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. ΔA(+)/ΔA(−) < 1]. However, such ratio was increased when PEG 8000 was added, giving place to a maximum ΔA(+)/ΔA(−) 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 ΔA(+)/ΔA(−).

Influence of the antigen and antibody concentrations

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 ∆A(+)/∆A(−) 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 ∆A(+)/∆A(−) 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.

image

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 ∆A(+)/∆A(−). Error bars indicate the SD for n = 3.

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Figure 7 shows the influence of the Ag concentration on the immunoagglutination assay using undiluted serum. The ∆A(+)/∆A(−) increases with the Ag concentration up to a density of bound protein of 2.90 mg/m2. However, higher Ag concentrations do not augment ∆A(+)/∆A(−). 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 ∆A(+)/∆A(−) 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 ∆A(+)/∆A(−) ratio reaches a maximum value, and the density of protein on 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.

image

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 ∆A(+)/∆A(−). Error bars indicate the SD for n = 3.

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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 (∆A/cut-off). Notice that (i) Chagas-positive sera clearly differed from the negative sera, and (ii) non-cross-reaction was detected.

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Figure 8. Relative optical distribution (∆A/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 (∆A/cut-off = 1).

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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

  1. Top of page
  2. Abstract
  3. Introduction
  4. Experimental
  5. Results and discussion
  6. Conclusions
  7. Acknowledgements
  8. References

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 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 × 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 Chagas-positive 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 non-cross-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.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Experimental
  5. Results and discussion
  6. Conclusions
  7. Acknowledgements
  8. References

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 Magalhães, Fundação Oswaldo Cruz, Recife (Brazil) and Dr. Miguel Hernán Vicco of Hospital J. B. Iturraspe, Santa Fe (Argentina) for the donation of serum samples.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Experimental
  5. Results and discussion
  6. Conclusions
  7. Acknowledgements
  8. References
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