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Gram negative bacteria frequently synthesize a great number of lipopolysaccharides (LPS) molecular species, the high LPS structural heterogeneity and its trend to conform molecular aggregates make the functioning of its molecular species extremely difficult. The application of chromatographic methods individually has allowed the obtainment of little homogeneous LPS fractions. With the combination of orthogonal chromatographic principles, only the purification of the simplest rough type LPS up to chemical homogeneity has been reached. This has determined that efforts aimed at more homogeneous LPS isolation is focused on the use of electrophoresis in smooth gel. Electrophoresis methods are capable of separating species of molecular masses next to the most complex smooth type LPS. Consequently upon this basis, a new methodology allowing the isolation of intact LPS of either rough or smooth types up to electrophoretic homogeneity has been developed. Under such methodology, sensible structural analysis through mass spectrometry and LAL (Limulus Amebocyte Lysate) activity of LPS individual species is feasible, composed of just a few chemical species. This methodology has proved the possible way of combining electrophoresis with other orthogonal separation principles, a further step which could guarantee the obtainment of chemically homogeneous preparations from complex LPS mixtures. [ABSTRACT FROM AUTHOR]

Requirements of regulatory organisations for the industry are necessaries in the first stages of the investigation and development processes of biotechnological products, about validation of analytical methods that demonstrate the identity, strength, quality and purity of the products. In this work, we standardised some physical, chemical and immunochemical methods for the characterisation of molecules as antigens, allergens and some fungicide molecules. Materials and methods. Chromatography methods: a) Gel filtration in Superose 12, Sephacryl S-100, Sephacryl S-1000, Sepharose CL-4B for the determination of size of outer membrane proteins (OMP) from Vibrio cholerae, Pseudomona, Shigella, Leptospira ssp and some antigens of vaccine conjugates. Thin Layer Chromatography (TLC) for the separation of chromophore compounds from Pseudomona and for the evaluation of metabolism carbohydrates. b) Electrophoresis method in polyacylamide gels in Phast System for the antigens identity of OMP and for the evaluation of identity and amount of the allergen proteins from Dermatophagoides siboney. c) Immunochemical method. Dot Blot for the identity of antigens in vaccine conjugates. Methods were classified according to the applications recommended by OMS: Specificity, Accuracy, Precision, Linearity, Range, Robustness. Results and Conclusions. Results are adequate according with the references for that methods. It will permits security and confidence for the characterisation of molecules. [ABSTRACT FROM AUTHOR]