RESUMEN
Current methodology to determine absence of live mycobacteria in tuberculin purified protein derivative (PPD) takes up to 8 weeks to perform and may also involve testing on animals. In this paper we describe an in vitro test utilising the tetrazolium salt, 2,3-bis-(2-methoxy-4-nitro-5-sulphenyl)-(2H)-tetrazolium-5-carboxanilide (XTT) to monitor the absence of live Mycobacterium tuberculosis (Mtb) in PPD. In the presence of live cells XTT is converted to a coloured formazan product that can be measured spectrophotometrically. Live mycobacteria present in spiked PPD were detected by a marked change in optical density above background levels. This test is easy to perform and is complete in just 48 hr.
Asunto(s)
Mycobacterium/aislamiento & purificación , Sales de Tetrazolio , Tuberculina/química , Estudios de Evaluación como Asunto , Sensibilidad y EspecificidadRESUMEN
Current methods for determining the identity of substrains of Mycobacterium bovis BCG (BCG) vaccine are labour intensive, or provide only limited substrain differentiation. In this paper we describe a multiplex PCR that distinguishes between M. tuberculosis (TB) and M. bovis and the non-pathogenic BCG strain, and also subdivides the BCG vaccine substrains investigated into seven distinct fingerprints based on six target regions in the DNA. This test is specific, rapid, reproducible and portable and is proposed as a novel test for BCG vaccine control. It offers substantial advantages over the methods currently in use. Using this test we have characterised a number of commercial BCG vaccines.
Asunto(s)
Vacuna BCG/genética , Reacción en Cadena de la Polimerasa/métodos , Secuencia de Bases , Dermatoglifia del ADN , Cartilla de ADN/genética , ADN Bacteriano/genética , ADN Bacteriano/aislamiento & purificación , Humanos , Mycobacterium bovis/clasificación , Mycobacterium bovis/genética , Mycobacterium bovis/inmunología , Mycobacterium tuberculosis/genética , Reacción en Cadena de la Polimerasa/estadística & datos numéricos , Especificidad de la EspecieRESUMEN
Recent developments in peptide technology enable the use of random peptide libraries for identifying linear amino acid sequences (mimotopes) which can mimic conformational epitopes without necessarily exhibiting amino acid sequence homology with the native linear sequence. In this study a 15-mer random peptide library displayed on the surface of a filamentous phage has been used to characterise the conformational epitopes recognised by a monoclonal antibody raised against the envelope protein gp120 of feline immunodeficiency virus (FIV). Three mimotopes were identified that reacted with the selecting antibody in an immunoblot assay. Sequence analysis revealed that, whereas the three mimotopes had several amino acids in common, there was no significant homology with the primary amino acid sequence of gp120 although some amino acids were shared between the variable region (V3) and the three mimotopes. Petide mimtopes of complex retroviral glycoproteins may have potential uses as novel vaccines and for the serological diagnosis of FIV.
Asunto(s)
Anticuerpos Monoclonales , Anticuerpos Antivirales , Bacteriófagos/inmunología , Proteína gp120 de Envoltorio del VIH/inmunología , Virus de la Inmunodeficiencia Felina/inmunología , Biblioteca de Péptidos , Secuencia de Aminoácidos , Animales , Especificidad de Anticuerpos , Gatos , Immunoblotting , Datos de Secuencia MolecularRESUMEN
Standardisation and control of the live Mycobacterium bovis BCG (BCG) vaccine is performed as specified by the World Health Organisation (WHO) and the European Pharmacopoeia (EP). The conventional viable count for control of potency of BCG vaccine is performed by culturing on solid medium. This assay method is not only time consuming but may give variable results. A tetrazolium salt assay has been developed and evaluated as a potential additional, or replacement, test for determining number of viable organisms. The tetrazolium salts 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 2,3-bis-(2-methoxy-4-nitro-5-sulphenyl)-(2H)-tetrazolium-5-carb oxanilide (XTT) used as alternative substrates in the assay both gave more rapid and reproducible results than the conventional viable count. XTT showed greater sensitivity than MTT with a lower detection limit of about 7x10(4) colony forming units (c.f.u.) ml(-1). The XTT assay has proven effective for determining viability of suspensions prepared from several BCG vaccine substrains, covering a range of viable units, without the need for modification. This assay is easily performed and takes just 48 h to produce an estimate of viable cell content compared with 3 weeks for the conventional method.