Immunodetection using polysiloxane / PVA-glutaraldehyde Polysiloxane / PVA-glutaraldehyde hybrid composite as solid phase for immunodetections by ELISA

! "#$% ! & ' () * + , '-.+/// '-(// 0 + ! 1 0 ! 2 + 2 ) * 3 $ 3 + Correspondence L.B. Carvalho Jr. Laboratório de Imunopatologia Keizo Asami, UFPE 50670-420 Recife, PE Brasil Fax: +55-81-271-8485 E-mail: lbcj@npd.ufpe.br Research supported by CNPq/ CTPETRO (No. 463655/001) and the Japan International Cooperation Agency (JICA). Received May 22, 2001 Accepted February 6, 2002

The alkoxide sol-gel process is an efficient method to prepare silica glass by the hydrolysis of alkoxysilane precursors and by subsequent condensation of the remaining silanols, followed by aging and drying under ambient atmospheres and sintering.The hydrolysis and polycondensation of tetraethoxysilane is as follows: Si(OEt) 4 Hydrolysis n(EtO)xSi(OH)y-x Polycondensation (SiO 2 )n The advantages of this technique are the homogeneity and purity of the gels and a relatively low sintering temperature.Such advantages are important to prepare single, massive and unchangeable glasses, thin films and optic fibers (1,2).Understanding the chemical reactions in the various steps of the sol-gel process leads to a better control of the process and therefore to an improvement in the reproducibility of the final product (3)(4)(5).The sol-gel technique also is an excellent method to prepare hybrid material.The low temperature synthesis enables organic or inorganic species to be incorporated into rigid silicon oxide matrices without degradation.The resulting composite combines the chemical and physical properties of the guest with an excellent optic, thermal, and chemical stability of the host silicon oxide matrices.
Hybrid inorganic-organic composites derived by sol-gel processing are materials that can potentially be designed for a wide range of applications.Structural diversity is achieved by controlling the relative ratio of organic versus inorganic content, the level of structural complexity of the organic component and its chemical nature, the chemical composition of the inorganic precursor molecule, and the reaction condition used to synthesize the composite.Thus, this synthetic framework offers the promise of engineered multicomponent composite material designed for specific desired applications.Since the surprising discovery of Braun et al. (6) that active protein can be entrapped in a sol-gel matrix without losing its activity, solgel-derived glass has emerged as a new class of material well suited for the immobilization of biomolecules.This inorganic material is particularly attractive for the fabrication of biosensors since it possesses physical rigidity, chemical inertness and high photochemical and thermal stability and experiences negligible swelling in aqueous and organic solvents.However, the wettability of some glass materials is not high, and depends on the silica-forming monomers.It is well known that neither highly hydrophobic nor totally hydrophilic matrices are desirable for sensing applications.In the former matrix, the analyte cannot approach the embedded enzyme, and the latter exhibits a large background current in amperometric biosensors.When chemical modifiers such as metal dispersion, water-soluble polymers, and proteins were added to the material, the resulting composite became more hydrophilic.
This paper describes the synthesis of small discs of a polysiloxane-polyvinyl alcohol (PVA) composite prepared by the sol-gel technique in order to provide a more biocompatible microenvironment and a more suitable surface for covalent immobiliza-tion.A chemical arm was later introduced through the glutaraldehyde molecule by acid catalysis (7).Free chemical groups were still available by which proteins could be covalently fixed.Antigen from Yersinia pestis was used as a model to evaluate the biological properties of retention of these immobilized proteins.Therefore, this antigen-composite disc derivative was used in ELISA to detect anti-F1 IgG in serum from immunized rabbit.
Polysiloxane-PVA disc preparation.The hybrid composite was synthesized by preparing a mixture of 5 ml tetraethoxysilane (Aldrich, Milwaukee, WI, USA), 5 ml ethanol (Aldrich) and 6 ml of a 2% (w/v) solution of PVA (MW 72,000, Reagen, Rio de Janeiro, RJ, Brazil).The reagents were carefully added and two or three drops of concentrated HCl were added in order to catalyze the reaction.The solution was stirred for 5 min at 60ºC and aliquots (10 µl) of the mixture were then transferred to microwells of tissue culture plates (disc shape) to complete the reaction.The molds remained closed for 10 days and were then opened until they no longer exhibited weight loss due to evaporation of residual water and solvent.The polysiloxane-PVA discs were then kept in 0.067 M sodium phosphate buffer, pH 7.2, at 4-10ºC until use.
Polysiloxane-PVA sensitization.The polysiloxane-PVA discs were introduced into microwells of tissue culture plates and treated with 2.5% (w/v) glutaraldehyde (Riedel) and 10 mM H 2 SO 4 at 28°C for 1 h and washed thrice with deionized water.
ELISA.The blocked antigen disc was incubated at 37ºC for 1 h in 100 µl of PBSdiluted serum (1:200) from immunized and non-immunized rabbits against Y. pestis produced at the Centro de Pesquisas Aggeu Magalhães, FIOCRUZ.The preparation was then washed 5 times with PBS/Tween and incubated in 100 µl of goat anti-rabbit IgG conjugated with horseradish peroxidase (Sigma), diluted 4,000 times in PBS at 37ºC for 1 h.Five washings with PBS/Tween were then carried out.The substrate solution (200 µl), consisting of 0.325% (w/v) orthophenylenediamine dihydrochloride (OPD, Sigma) and 0.085% (v/v) H 2 O 2 in 0.3 M Tris-citrate buffer, pH 6.0, was added and incubated at room temperature (28ºC) for 15 min in the dark.The reaction was stopped with 2.5 M H 2 SO 4 (25 µl).The discs were removed and the absorbance of the supernatant was read at 492 nm with a Microplate reader (model 550; BioRad Laboratories, Hercules, CA, USA).The above procedure was previously standardized by determining optimal conditions with respect to antigen, serum and second antibody conjugate concentrations.Samples (100 µl) of antigenic solution containing 0.6 to 5.0 µg protein were incubated with the activated discs and the antigen discs obtained were incubated with immunized and non-immunized serum (100 µl) diluted 100 to 800 times.ELISA was then applied to these discs containing increasing amounts of antigen and serum and using conjugate diluted 1:4,000.ELISA using 1.25 µg antigen and serum dilutions of 1:200 established the optimum dilution of goat anti-rabbit IgG conjugated with horseradish peroxidase.
Titration of goat anti-rabbit IgG conjugated with horseradish peroxidase using 1.25 µg of F1 antigen, immunized and non-immunized sera diluted 1:200 times and PBS as control, showed that the best dilution was attained at serum dilution 1:4,000.
Figure 1 shows the relationship between the concentration of F1 antigen from Y. pestis immobilized onto the polysiloxane-PVAglutaraldehyde composite discs using different serum dilutions and the absorbance obtained with ELISA, using goat anti-rabbit IgG conjugated with horseradish peroxidase diluted 1:4,000 times.Hyperbolic curves were obtained as expected by the nature of antigen-antibody complex formation, which follows the Michaelis-Menten equation for enzyme-substrate complex formation (9).Also, a significant difference in absorbance values was observed when comparing immunized and non-immunized serum, while values close to zero were detected for the control (PBS).From these results one can conclude that 1.25 µg of antigen per composite disc is sufficient to standardize the methodology.
Therefore, ELISA using this matrix can be set up as follows: 1.25 µg of F1 antigen for each composite-glutaraldehyde disc, 1:4,000 conjugate dilution, and 1:200 serum dilution.A comparison between this method and conventional ELISA, based on antigen adsorption onto polyvinyl chloride (PVC) plates, is shown in Table 1.Firstly, regarding the sera collected from immunized rabbits (N = 8), mean values (± SD) of 1.505 ± 0.262 and 1.184 ± 0.254 were calculated for ELISA absorbance using the composite discs and the conventional procedure, respectively.The independent t-test applied to these mean values showed no significant difference at the 0.05 level.A linear correlation was observed when the absorbance values for ELISA using composite discs were plotted against those obtained for the conventional procedure (r = 0.92662 and P = 0.00093).For the sera collected from non-immunized rabbits (N = 8) mean values of 0.311 ± 0.023 and 0.190 ± 0.044 were estimated for the composite discs and conventional ELISA procedures, respectively.Briefly, the performance of ELISA with F1 antigen covalently fixed on the polysiloxane-PVA-glutaraldehyde composite discs is comparable to that based on the adsorption of the antigen on PVC plates in the conventional procedure.Furthermore, the amount of antigen is one fourth that employed in the conventional PVC plates (5 µg/well).
Lehtonen and Viljanen (10), studying the concentration of antigen in ELISA for the detection of chicken anti-bovine serum albumin, used three polymers as solid phase and two immobilization procedures: polystyrene and nylon (adsorption) and cyanogen bromide-activated paper (covalent link).There was a noticeable release of the adsorbed antigen during the assay from both polystyrene (30%) and nylon (60%).Desorption of antigen from activated paper during the assay was lower than with polystyrene or nylon.It is well known that protein release is more likely to occur from adsorbed antigens than from covalently linked preparations, particularly under extreme conditions of pH, ionic strength and temperature.
In addition, the performance of the polysiloxane-PVA-glutaraldehyde disc as a matrix for immunodetection, its easy synthesis and the low cost are additional advantages for commercial application.More than 1,000 discs can be synthesized at a cost of US$ 0.50.The preservation of the biological properties of this immobilized antigenic preparation, employing the sol-gel discs as the solid phase, presents promising applications to other proteins such as enzymes, hormones and receptors.This further use of the present support is currently under investigation in our laboratories.

Table 1 .
Comparison of ELISA using polysiloxane/polyvinyl alcohol-glutaraldehyde discs and polyvinyl chloride (PVC) as solid phases.
*The independent t-test indicated that both mean values were significantly different (P<0.05).