Eduardo José Alécio-Oliveira1*, Petrus Santa Cruz2, Severino Alves Junior2, Antonio Teixeira4, Carla Nunes4,Elizabete Malageño5, Renato J. R. Molica3, José Luiz de Lima Filho5
1
Centro Federal de Educação Tecnológica de Pernambuco (CEFET-PE), 50740-540, Recife-PE, Brazil.
2
Departamento de Química Fundamental (DQF) da Universidade Federal de Pernambuco (UFPE), Recife-PE, Brazil.
3
Instituto de Tecnológico do Estado de Pernambuco (ITEP), Recife-PE, Brazil. 4
Laboratório Multidisciplinar de Pesquisa em Doença de Chagas da Universidade de Brasília (UnB), Brazil.
5
Laboratório de Imunopatologia Keizo Asami (LIKA), Universidade Federal de Pernambuco (UFPE), Recife-PE, Brazil.
*
Corresponding author: Centro Federal de Educação Tecnológica de Pernambuco (CEFET-PE), Av. Profº Luiz Freire, 500, Cidade Universitária, Recife-PE, 50740-540, Brazil. e.mail: [email protected]
ABSTRACT
Lanthanide cryptates are usually stable complexes and their solubility in aqueous solutions allows to biological applications. In the present work, the Eu3+ ion was used as a probe locally with microcystin-LR antibody neighborhood in the following system [Eu ⊂ bpy.bpy.py(CONH2CH2CH2NH2)2]3+-Anti-Microcystin-LR-KLH IgG immobilized in a super-absorbent polyacrylamide gel (SAPG). The 5D0→7Fn transitions were monitored by electric dipole transitions highly dependent of the site symmetry and magnetic dipole ones, they were used as reference to evaluate the interaction antigen-antibody through hypersensitive transitions.The spectra for 10-5 M free antibody, 10-5 M cryptate and 2:1 toxin concentration showed a specific change in the 5D0→7F2 transition in the presence of free IgG. The conjugated antibody-Europium cryptate was carried out and the 5D0→ 7
INTRODUCTION
Lanthanide ion complexes could be used as light conversion molecular devices – LCMD’s. The absorption and energy-transfer emission sequence involve distinct absorbing (the ligand) and emitting (the lanthanide ion) components (de Sá et al., 2000).
The lanthanides complexes has been an important research goal and different class of ligand have been proposed. The Eu (III), Tb (III) and Sm (III) ions have excellent luminescence properties when chelated with ligands that have broad intense absorption bands. The chelating agents used with Eu (III) and Tb(III) ion include cryptands, β-diketonates tridentate pyridine dicarbolylic acids, multidentate pyridine, bipyridine, terpyridine and their structural analogues as polyaminopolycarboxylates, cyclodextrins and crown-ether derivatives (Lemmetyinen et al., 2000; de Sá et al., 2000).
Cryptates are coordination compounds with exeptional thermodynamic and kinetic stability and are extremely stable in aqueous media (Gasow et al., 1977; Prat et al., 1991) and have the advantage to the conventional europium chelates which are subject to dissociation at low concentration and to exchange with other ionic species, despite the europium ion that cannot be displaced from a cryptate by others metal ions present in samples or reagents (Prat et al., 1991).
To suitable bioaffinity assays is desired for the chelates of Ln(III) a long excitation wavelength to avoid background fluorescence, high chemical, photochemical and kinetic stability, good solubility in water, long luminescence life-time, high luminescence quantum yield and minor interference in the affinity of the biomolecules (Lemmetyinen et al., 2000).
Because of its characteristics lanthanide compounds have been utilized in fluorometric assay (Karsilayan et al., 1997; Prat et al., 1991; Mukkala et al., 1995) and as probes in time-resolved fluorometry (Prat et al., 1991).
Absorbent gel presents the capacity to absorb great amounts of water and ionic solutions of low osmolaridade, what it makes possible its use as support for active biological substances. The polymer net of the absorbent gel has the advantage of its biocompatibilidade, being compatible with diverse specialized pharmaceutical and biological applications as: Preparation of pharmaceutical controlled release drugs, manufacture of catheters and artificial skin, construction of biosensors, beyond several other commercial applications as in absorbents of personal hygiene as infantile diapers and products of feminine hygiene (Bucholz & Peppas, 1994).
We report here the use of Eu3+ ion to probe locally the microcystin-LR antibody neighborhood in the system [Eu ⊂ bpy.bpy.py(CONH2CH2CH2NH2)2]3+-anti-microcystin- LR-KLH IgG immobilized in a commercial super-absorbent polyacrylamide gel - SAPG.
MATERIALS AND METHODS
All reagents when not specified were analytical grade; Europium cryptate [Eu ⊂ bpy.bpy.py(CONH2CH2CH2NH2)2] was prepared according to Rodriguez-Ubis et al. (1984) and Lehn & Roth (1991); The pyridine group was reacted with ethylenediamine to forming the amide groups and purified by liquid reversed-phase chromatography; Acrylic acid/Polyacrylamide hydrogel was a commercial product; Microcystin-LR toxin was purchased from Sigma and sulfo-SMCC from Pierce.
Antibody production and purification
Polyclonal antibodies were produced by conventional methods as described for Karnikowski (2001). Using Microcystin-LR (Sigma) and keyhole-limpet hemocyanin - KLH (Sigma) rabbit was immunized with a solution of 500 µg of microcystin-LR-KLH conjugated in 500 µl sodium phosphate buffer (pH 7.4). The conjugated was mixed with 500 µl Freund’s complete adjuvant (Sigma) and injected subcutaneously at three dorsal sites. Booster injections consisting of 500 µg antigen were administered intravenously into the marginal ear vein at days 28 and 56, with collection of 20 ml of blood at days 35, 63 and 93. The blood was clot at room temperature and antisera were colected and centrifuged at 2,000xg (Micronal centrifuge). The colected antisera were purified in an
affinity column of protein G Pharmacia (HiTrap, Amersham, London, UK) stabilized with 5 ml of 20 mM phosphate buffer (pH 7.0). Portions of 0.5 ml were applied in the column washed with 5.0 ml of the same phosphate buffer and the fraction of interest was extracted with 3.0 ml of 0.1M glicina-HCl (pH 2.7) buffer directly in a tube with 0.3 ml of 0.1M tris-HCl buffer (pH 9.0). The antibody solution was reduced to a volume of approximately 200 µl using a 30 KDa Centricon centrifugal filter unit (Millipore).
Antibodies Activity
Indirect enzyme immunoassay (Karnikowski, 2001) utilizing a 96 well microtiter plate (Maxisorp Nunc, Roskilde, Denmark) coated with microcystin-LR-chicken ovoalbumin was utilized to verify the activity of conjugated IgG antimicrocystin-LR-KLH- Europium cryptate. Pure IgG antimicrocystin-LR-LKH was used as control. The Dilution for determination of the titres had been carried through from solution with 50 µg of protein / 50 µl.
Super-Absorbent Polyacrylamide gel Properties
The used polymer was a commercial gel granules with 2 at 5 mm diameter. The infrared spectrum was carried out in KBr tablets pressed under vacuum and read into the region of 4000 at 500 cm-1 with resolution of 2 cm-1 in a Bruker model IF66 spectometer.
Gel particle with about 20 mg had been left in contact with 10 ml of the solutions of 0.1 M NaCl pH 2.9, 7.1 and 10.8 for a period of 2 hours. After this time the solution was drained and the excess in the surface absorbed with filter paper. Gel masses were weighed in analytical balance with sensitivity 0.0001g (Ohaus Corporation, USA). The results were expressed in mg of absorbed solution/mg of gel.
To verify the capacity of SAPG to immobilize protein, were used bovine serum albumin - BSA and glucose oxidase – GOD as a model. Granules with approximately 10 mg were deeping in 2 ml of bovine serum albumin solution 0.034 mg/ml prepared in 0.1M sodium phosphate buffer (pH 6.5). As control a triplicate of SAPG was tested in
ultrapure water (resistivity > 18,3 MOhm). To each 20 min. one SAPG granule were removed of the solution and washed with 0.9% (w/v) NaCl solution. The supernatant and washing waters were pooled and analyzed to the protein concentration utilizing the method of Bradford (1976) and the immobilized percentual calculated.
With glucose oxidase an granule with approximately 10 mg were immersed in 0.045 mg/ml glucose oxidase - GOD solution prepared in 0.1 M sodium phosphate buffer (pH 5.9) and left in contact for up to 180 minutes. To each 20 minutes three SAPG had been removed of the solution washed with 0,9% (w/v) NaCl solution and cooled at -80ºC. The granules were lyophilized and stored at -18ºC before activity enzimatic test.
The enzymatic activity of GOD was determined using a glucose kit (Laborlab, Brazil) with peroxidase in the concentration of 440 U/l. The SAPG were placed in acrylic cuvets of 3.2 ml, added 2 ml of the color reagent, incubated under soft agitation at 37ºC. To each 20 min. the samples were read in an digital spectrophotometer (Micronal mod. B442, Brazil) in the wavelength of 505 nm. The activity of the free enzyme was determined using 100 mg/dl glucose standard solution.
Super-Absorbent Polyacrylamide gel antibody neighborhood: Test 1
Two hundred and fifty milliliters of solutions a) Europium cryptate (1x10-5 M) b) Europium cryptate (1x10-5 M) + IgG antimicrocystin-LR-KLH (1x10-5 M) and c) Europium cryptate (1x10-5 M) + IgG antimicrocystin-LR-KLH (1x10-5 M) + Microcystin-LR (2x10-5 M) were placed in contact by 60 minutes at ambiente temperature and left in contact with the SAPG granules (10 mg each).
Spectroscopic measurements
After liquid absorption the SAPG granules were analyzed using emission spectroscopy in the range of 5000 at 7200 Aº. The luminescence spectra were obtained by scanning with a 1m double-grating Jobin-Yvon U-1000 monochromator. The
excitation wavelength (310 nm) was selected by a 0.25 m Jobin-Yvon H-10 monochromator, using a 150 W Xe-Hg lamp as the excitation source. The light detection was performed by a water-cooled RCA C31034 photomultiplier tube, the photocurrent signal being acquired through an EG & G discriminator model 1182 and digitally stored by a Jobin-Yvon Spectralink interface and a personal computer. This set- up allows for measurements at room temperature (300 K).
The 5D0→7Fn transitions (J = 0-4) were monitored - electric dipole transitions highly dependent of the site symmetry and magnetic dipole ones (5D0→7F1) - used as reference to evaluate the interaction antigen-antibody through hypersensitive transitions.
Europium Cryptate - IgG Conjugation (Maleimide Method)
Conjugation of the anti-microcystin-LR-KLH IgG to the europium cryptate understood three main stages: a) Linking of the cryptate to sulfo-SMCC (Sulfo- succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate), b) Reduction of dissulfide groups of the IgG to form –SH group and c) Linking of the cryptate-SMCC to activated IgG.
Firstly, 8 mg of the europium cryptate had been dissolved in 5 ml of 20 mM sodium phosphate buffer (pH 7.4) and mixed with 0.5 ml of 30 mM sulfo-SMCC. The mixture was kept in reaction for 30 minutes protected of the light. To eliminate the sulfo- SMCC excess reagent the solution was purified in Fast Performance Liquid Chromatography -FPLC using a strong anionic Mono-Q HR 5/5 Pharmacia column (Prat et al., 1991). The column was equilibrated with 5.0 ml of 20 mM sodium phosphate buffer (pH 7.0) in flow of 0.5 ml/min. The mixture in analysis was injected in volumes of 1.5 mL and the column eluted with 20 mM sodium phosphate, 1M NaCl, 10% (v/v) dimethyl sulphoxide (pH 7.0). The fractions (0.5 mL) were collected and the absorbance read at 307 nm.
In the second stage dissulfide groups of the IgG were reduced with dithiothreitol – DTT. Using eppendorf tube 450 µl of a 6.53 mg/ml IgG solution was added of 5.0 µL of a 1.0 M DTT solution and kept for 30 min. protected by the light without agitation.
The DTT excess was eliminated by FPLC in a fast desalting column HR 10/10 Pharmacia. The column was stabilized with 50 ml of 20 mM sodium phosphate and 150 mM NaCl (pH 7.0) in a flux of 2 ml/min. Volumes of 200 µL was injected and volumes of 0.5 ml recovered each 15 seconds. The samples were analyzed by protein presence using the Bradford reaction. Positive protein fractions were pooled and concentrated centrifugation in 30 kD Centricon centrifugal filter unit (Millipore). After, the volumes were measured and the protein concentration quantitatively analyzed in a microplate protein assay.
The third and last stage was the process of linking the europium cryptate activated with carbodiimide group (SMCC) to the IgG whose sulfhydryl groups were reduced with DTT. A volume of 2.2 ml of 0.21 mg/ml protein concentration antibodies was mixed a 1 ml solution contend 553 µg of activated cryptate. The mixture was kept in reaction for 14 hours at 4ºC under light protected and gently agitation. After that the sample was purified in HR 10/10 desalt column and the fractions collected analyzed by spectrophotometry in 238 nm and 307 nm.
Super-Absorbent Polyacrylamide gel antibody-Europium cryptate neighborhood: Test 2
Two hundred and fifty milliliters of solutions with a) Europium cryptate-IgG antimicrocystin-LR-KLH (1x10-5 M) and b) Europium cryptate-IgG antimicrocystin-LR- KLH (1x10-5 M) + Microcystin-LR (2x10-5 M) were placed in contact and the fluorescence analyzed as described above in the Test 1.