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Bidimensional electroforesis is the method more employed for the analysis of complex mixtures of extracted proteins of cells, tissue or other biological samples. Using this technique the proteins are separated according to two independent properties in two steps: a first dimension where these are fractioned on the base of their isoelectric point through isoelectric focusing and a second dimension in which the previously separate proteins are fractioned based on their dimensions through SDS poliacrylamide gel electrophoresis. Isoelectric focusing is generally carried out using expensive commercial gels (e.g. Immobiline DryStrip gel). A home mademethod was developed to produce gels useful for IEF. 30% T Acrylamide and 8M Urea solutions were deionized using mixed bed ion exchangers. The quality of the deionization processes was evaluated through the variations of solutions´ conductivities of both reagents. Acrylamide deionization was also evaluated through variations in the pH and the Absorbance at 290 nm. The obtained data were processed statistically with STATISTIC 6.0.Two electrophoresis formats, circular gels and horizontal gels were tested. The applied sample was human plasma delipidated with dithiothreitol and Tween-80 for 15 min. There were significant differences in the values of conductivity before and after deionización, (p <0.05), for both solutions. The pattern of bands was obtained satisfactorily for the two systems when deioniozed solutions were used. These results allow to conclude that gels prepared with the developed system substitute satisfactorily the commercial gels for identical purposes. By this way we contribute to decrease the 10/90 gap (only 10% of the world funds for health research is used in the investigation of 90% of the world problems of health), where the reduction of the costs of the analytic methods is among the strategies outlined by the Global Forum for Investigations in Health for teh 2003-2005 period. [ABSTRACT FROM AUTHOR]

Bidimensional electroforesis is the method more employed for the analysis of complex mixtures of extracted proteins of cells, tissue or other biological samples. Using this technique the proteins are separated according to two independent properties in two steps: a first dimension where these are fractioned on the base of their isoelectric point through isoelectric focusing and a second dimension in which the previously separate proteins are fractioned based on their dimensions through SDS poliacrylamide gel electrophoresis. Isoelectric focusing is generally carried out using expensive commercial gels (e.g. Immobiline DryStrip gel). A home made method was developed to produce gels useful for IEF. 30% T Acrylamide and 8M Urea solutions were deionized using mixed bed ion exchangers. The quality of the deionization processes was evaluated through the variations of solutions' conductivities of both reagents. Acrylamide deionization was also evaluated through variations in the pH and the Absorbance at 290 nm. The obtained data were processed statistically with STATISTIC 6.0. Two electrophoresis formats, circular gels and horizontal gels were tested. The applied sample was human plasma delipidated with dithiothreitol and Tween-80 for 15 min . There were significant differences in the values of conductivity before and after deionización, (p <0.05), for both solutions. The pattern of bands was obtained satisfactorily for the two systems when deioniozed solutions were used. These results allow to conclude that gels prepared with the developed system substitute satisfactorily the commercial gels for identical purposes. By this way we contribute to decrease the 10/90 gap (only 10% of the world funds for health research is used in the investigation of 90% of the world problems of health), where the reduction of the costs of the analytic methods is among the strategies outlined by the Global Forum for Investigations in Health for teh 2003-2005 period. [ABSTRACT FROM AUTHOR]

The detection of antibodies against the transmembrane glycoprotein (gp21) is very important for the diagnosis of Human T-Cell Leukemia Virus (HTLV) infection, because it has been demonstrated that this protein is a predominant viral antigen, the one that stimulates the antibody production in HTLV-infected individuals. The recombinant proteins and the synthetic peptides corresponding to this glycoprotein are broadly utilized, as antigens, in the HTLV immunodiagnosis. The aim of this work was to obtain, by means of chemical synthesis in solid phase, two peptides: gp21 (13) (361-404) and gp21 (M-1) (377-400) of the gp21 HTLV-I transmembrane protein. The yield obtained in the gp21 (M-1) peptide synthesis was 85 % and for gp21 (13) peptide was 80 %. The two peptides were purified by High Performance Liquid Chromatography Reverse-Phase, and characterized by Electrospray Ionization Mass Spectrometry. Both synthetic peptides were evaluated in an UltramicroELISA assay, with characterized HTLV-I-positive samples, obtaining a reactivity of 92 and 62 % for peptides gp21 (13) and gp21 (M-1), respectively. The specificity for the two synthetic peptides was 100 %. This study demonstrated the possibility to obtain peptides of gp21 protein, through chemical synthesis in solid phase, and their utility as antigens in the HTLV immunodiagnosis. [ABSTRACT FROM AUTHOR]

The human immunodeficiency virus type 2 (HIV-2) was detected in countries of Western Africa and it is another cause of the Acquired Immunodeficiency Syndrome (AIDS). The env gene encodes the synthesis of the precursor gp140 protein that is cleaved by the virus protease into two proteins: gp36 and gp105. The synthetic peptides and recombinant proteins of the transmembrane gp36 glycoprotein show a high reactivity and specificity in the detection of antibodies in samples of infected patients, for what they are broadly used in the HIV-2 immunodiagnosis. In the present work the reactivity of a synthetic peptide gp36 (5) (585-610) and the recombinant protein gp36 from transmembrane region of HIV-2 were tested with 15 positive samples of HIV-2. In order to assess specificity of those antigens, 74 sera from seropositive Cuban people to HIV-1 and 30 samples from healthy blood donors were tested. UMELISA HIV 1+2 Recombinant assay (TecnoSUMA International S.A., Cuba) was used as a reference for the detection of antibodies to HIV 1 and 2. All the samples used in the study were confirmed by Western Blot assay. Sensitivity for both antigens was 100 %. The synthetic peptide gp36 (5) didn't detect the positive samples of the HIV-1 evaluated, contrary to the recombinant protein that detected 46 % of the samples. Specificity for both antigens was 100 % with the samples from healthy blood donors. This study demonstrated the high reactivity of the recombinant protein and the synthetic peptide, and the high specificity of this last one with HIV-2 positive samples. [ABSTRACT FROM AUTHOR]

Human T-cell Leukemia virus type I (HTLV-I) was the first human lymphotropic retrovirus isolated from human patients, in 1980. The env gene encodes the synthesis of the precursor protein, gp62 which is cleaved by the virus protease into two proteins: gp21 and gp46, both expressed in the cell surface. The transmembrane gp21 protein is one the most antigenic proteins of HTLV virus. This protein, obtained by the recombinant DNA technology or by chemical synthesis, is very useful as an antigen in HTLV-I immunodiagnosis. In the present work, the antigenicity of two synthetic peptides gp21 (M-1) (377-400) and gp21 (13) (361-404), and a recombinant protein of the transmembrane (gp21) region of the HTLV-I was evaluated, with 23 positive samples of the Panel PRP-205 (Boston Biomedica Inc), and five samples of HTLV-I seropositive Cuban patients. All the samples used in the study were confirmed by Western Blot assay. In order to assess specificity of those antigens, 30 serum samples from healthy blood donors were tested. There was a reactivity of 62 % and 92 % for the synthetic peptides gp21 (M-1), gp21 (13), respectively, and 85 % for the recombinant protein. Specificity for antigens was 100%. This study showed the high reactivity of the synthetic peptides compared with the recombinant protein. [ABSTRACT FROM AUTHOR]

Since 1996, the European Centre for Validation of Alternative Methods recommended the use of IgY for substitution of mammalian IgG to minimize harm situations in animal's experimentation for the production of polyclonal antibodies. In 1999, IgY technology was accepted by Federal Veterinarian Office of Swiss Government as an alternative method to support the care and healthy status of animals. Many publications show that IgY takes advantage over IgG to avoid interferences in several immunological assays for the fact that immune complexes that contain avian antibodies do not bind Fc or complement receptors on cells and IgY could replace a similar IgG. This knowledge has been the basis of this work that has consisted of the conjugation of highly pure IgY anti mouse IgG with FITC by covalent junction via primary free amino groups of immunoglobulins. The conjugate obtained showed a good molar ratio FITC/protein for a recommended performance. The optimal working dilution obtained for the particular conjugate was rational demonstrating to be useful as secondary antibody in enumeration of human cellular surface antigens. The cellular enumeration was assayed simultaneously with similar reagents from mammalian source and results showed no significant differences by statistical analysis. [ABSTRACT FROM AUTHOR]

As early as 1893, an experiment was described in which the immunisation of a hen results in the transfer of specific antibodies (Abs) into the egg yolk was demonstrated. Taking in mind this knowledge, this work has been focused on the immunisation of hens with commercial mouse IgG to prepare IgY polyclonal antibodies and label them with horse radish peroxidase (HRP) to use as alternative similar mammal's antibodies in ELISA tests to determine murine monoclonal antibodies. Immunisation schedules were carried out in rabbits and hens simultaneously with different dosages of commercial preparation of mouse IgG. Rational titres of antibodies were obtained from egg yolk in comparison to rabbit's antibodies. Here, it is demonstrated that sodium m-periodate method is also useful to offer IgY enzymatic conjugates with characteristics similar to those obtained from mammal's source. The performance of ELISA assay by egg yolk's conjugate showed several advantages in comparison to similar one with rabbit's conjugate in parameters like linearity and precision. Finally, it is demonstrated that IgY Technology is an alternative to produce enzymatic conjugates at lower cost and can be accepted as an attractive way for traditional mammalian polyclonal antibodies technology. [ABSTRACT FROM AUTHOR]

Various enzymatic and chemical methods for the preparation of non-radioactive nucleic acid probes have been developed. Bisulfite-catalyzed transamination and acylation reactions into DNA are employed, as a chemical method, to obtain labeled DNA probes. If the reactions are carried out under DNA denaturalization condition, the labelling could be increased. Likewise, the label detection could be increased by means of the use of an appropriated spacer arm and also, by performing the label detection in single-stranded DNA. In this work, the procedure for obtainment labelled DNA probes, by chemical synthesis, using the p-bromobenzoyl radical as label was carried out. We developed a method for separating single-stranded DNA (s.s.DNA) from double stranded DNA, through Hydroxylapatite (HA3) chromatography. The sensitivity in the label detection was evaluated by chromogenic dot immunobinding assays using an anti-BLC monoclonal antibody (5H11E5), previously obtained. [ABSTRACT FROM AUTHOR]