Analytical techniques for determining biotin.

Biotin is a vitamin of the B-complex, which plays an important biochemical role in every living cell. In the recent years, the interest in this vitamin has been rekindled, mainly due to its association with serious human disorders, such as the inherited syndrome multiple carboxylase deficiency, which can be successfully treated with biotin administration. Diagnosis of biotin deficiency as well as monitoring of biotin levels in biological fluids of patients receiving biotin treatment is crucial. Equally important is the determination of biotin levels in pharmaceutical preparations as well as in food and food supplement products, which constitute the main source of biotin in humans. Several analytical methods for measuring biotin in various samples, e.g. human fluids, pharmaceutical formulations, food material etc., have been reported in the literature. In this review, the most representative of these methods are presented, and their characteristics are evaluated.

Investigation of the epitopic structure of thymosin beta10 by epitope mapping experiments.

We present here a study on the epitopic structure and the immunochemical characteristics of thymosin beta10 (Tbeta10), a 43 aminoacid peptide involved in important cellular mechanisms, by using the epitope mapping Multipin method. Octapeptides overlapping by one amino acid so as to represent the whole sequence of Tbeta10 were synthesized on polystyrene pins and screened, using an ELISA method, with a polyclonal antiserum raised against intact recombinant Tbeta10. The octapeptides were also tested with anti-peptide oligoclonal antisera raised against the synthetic fragments Tbeta10[1-16] and Tbeta10[31-43], with polyclonal antisera raised against natural thymosin gamma4 (Tbeta4) or thymosin beta9 (Tbeta9), and with anti-peptide oligoclonal antisera raised against various fragments of Tbeta4 (i.e. Tbeta4[1-11], Tbeta4[30-43] and Tbeta4[16-38]). Four distinct epitopic fragments were revealed, namely the sequences 1-13, 19-30, 29-40 and 36-43. Among them, the sequence 36-43 appears to offer unique immunochemical characteristics to the Tbeta10 molecule.

Development of specific anti-thymosin beta 10 antipeptide antibodies for application in immunochemical techniques.

We present theoretical and experimental data necessary for raising specific antibodies for thymosin beta 10, a 43-amino acid residues peptide occurring in human tissues. We postulate that thymosin beta 10 contains three major antigenic determinants (residues 2-8, 17-25, and 35-41). For antibody development, we synthesized the N-terminal fragment thymosin beta 10(1-16) as well as the C-terminal fragments thymosin beta 10(31-43) and thymosin beta 10(38-43), due to their putative antigenic properties and minimal structural similarity with the homologous peptide thymosin beta 4, which also occurs in humans. The putative antigenic determinant 17-25 is present in all beta-thymosins and was therefore not synthesized. All antisera raised against the above peptide fragments or the intact molecule of thymosin beta 10 were found capable of recognizing and binding synthetic or natural thymosin beta 10 with high specificity, showing minimal cross-reactivity with thymosin beta 4 isolated from bovine tissues or synthetic thymosin alpha 1. Due to its easy preparation and the highly specific affinity of the antibody raised against it for the intact peptide, the fragment thymosin beta 10(38-43) may be considered the antigen of choice for developing anti-thymosin beta 10 antibodies, which can eventually be applied to immunochemical studies.