In Vivo Diagnostics

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Chapter: Pharmaceutical Microbiology : The Manufacture And Quality Control Of Immunological Products

The most widely used of these are the tuberculins employed to detect sensitization by mycobacterial proteins and hence the possible presence of infection. These are prepared by growing approved strains of M. tuberculosis (or M. bovis or M. avium in preparations intended for veterinary use) in a protein-free medium for several weeks.


IN VIVO DIAGNOSTICS

 

A)                 Preparation

 

The most widely used of these are the tuberculins employed to detect sensitization by mycobacterial proteins and hence the possible presence of infection. These are prepared by growing approved strains of M. tuberculosis (or M. bovis or M. avium in preparations intended for veterinary use) in a protein-free medium for several weeks. The culture is then steamed for a prolonged period to kill surviving bacteria and to facilitate release of tuberculoproteins from the cells. The culture supernatant is recovered by centrifugation and further concentrated by evaporation and sterile filtered to make a product known as Old Tuberculin. The crude material may then be standardized against a reference preparation by titration in the skin of guinea-pigs sensitized to M. tuberculosis. In practice, further purification is usually performed by precipitation with trichloracetic acid or other protein precipitant to produce purified protein derivative, which is standardized by in vivo assay. Concentrated preparations containing 100 000 IU/ml are used to formulate working strengths such as 1000, 100 or 10 IU/ml. These have to be diluted in a medium containing a Tween surfactant to reduce adsorption to glass. The concentrated material can be used for intradermal testing by a multiprong device such as the Heaf or Tine method.

 

B)    Quality Control

 

Apart from standardization of potency, which also serves as an identity test, the material must be checked for sterility and for the absence of viable mycobacteria. Because of their slow growth the latter may not be detected by conventional sterility tests and it is usual to perform check tests by guinea pig inoculation, or by prolonged culture on Lowenstein–Jensen medium. The product is also checked for absence of reactogenicity in unsensitized guinea-pigs and if required by the regulatory authority, for abnormal toxicity.

 

Analogous intradermal test reagents such as mallein, histoplasmin and coccidioidin, are produced by similar methods. Their use has declined, however, as they, like the tuberculin test, detect previous exposure and sensitization to the antigens of the agent but not necessarily active infection.

 

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