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Services for Identification and Assay Interferon researchers per se may require independent verification of sample specifications or may wish to decrease the overhead costs, and eliminate biohazard considerations in the preparation of virus stocks, in connection with occasional or repetitive bioassays of interferons. Although interferon reference standards are used widely, repeated assays in a given laboratory frequently vary by a factor of two to three, even after normalization to reference units. The results commonly depend on minute details of the assay method including cell strain, density, age, virus strain, multiplicity of infection and the viral function used to quantify interferon activity. In some cases the appropriate activity standards do not exist, or are only now becoming available, as for human and mouse gamma interferons as well as mouse alpha, mouse beta and rat interferons and several anti- interferon sera. Moreover, the ratio of sensitivities of a given cell line to these interferons (or their mixtures) frequently varies relative to available standards. Even when additional standards become available these pitfalls demand constant care in method and interpretation.Our manufacture of interferon and anti- interferon reagents requires frequent and repetitive bioassay of interferons by reliable methods [2]. Access BioMedical has twenty years of experience in improving the assay of interferons and other antiviral activities, and in devising novel bioassay methods in vitro and in vivo. Our laboratory was among those which participated in the 1985 international collaborative titration to assign potencies to four mouse and ten human interferon reference reagents recently made available by NIAID and NIBSC. The many years of experience, together with the economy of volume, allow higher quality results and technical support at lower cost than for any other laboratory. Moreover, Access BioMedical provides additional biological, biochemical and immunochemical characterization services, employing methods exemplified below and on pp. 22- 25. Our laboratory can also provide specialized bioassay techniques when sensitivity must be maximized, to demonstrate synergistic actions with other cytokines, or when interfering agents are present. Details of bioassay techniques and interpretation of results are described in ref. [2] (reprint available on request). Human, simian, rabbit, mouse, rat, avian, canine, bovine, caprine, ovine, equine and porcine interferons are assayed routinely in our laboratory by either of two methods: (1) The microtest assay is cost- effective when many samples are required. It has a small sample requirement (>= 0.2 mL). It is a duplicate serial endpoint dilution assay in 96- well multiplates. The resolution (i.e., difference which is experimentally significant) is ca. 30%. Prices for microtest assays are shown in Table 3 (p. 30) and typical results are illustrated in Fig. 6 (2) The vital dye uptake assay has increased resolution (ca. 10%) and reliability. It is useful for process or quality control purposes or when increased resolution is otherwise desired. It is a quadruplicate serial endpoint dilution assay in 96- well multiplates. Survival of cells is quantified spectrophotometrically after staining with a vital dye. Survival vs. log interferon dilution is plotted and subjected to regression analysis (correlation coefficients are generally >0.97). The titer, slope and correlation are derived. Prices are shown in Table 4 (p. 30). Results are illustrated in Fig. 7. Both methods quantify interferon activity by measurement of the protective effect against cytocidal infection with encephalomyocarditis (EMC) or vesicular stomatitis (VS) viruses. The cell/virus combinations for the most frequently requested bioassays are shown in the table below. Many other cell strains, viruses and assay techniques based upon other measures of virus expression are available on request. A complete listing of the cell strains currently maintained in our laboratory will be found on p. 99ff. Human, rabbit and mouse interferon samples are co- assayed with the appropriate NIH, or equivalent, reference reagents and the results are normalized to NIH reference units (IU/mL). [Standardization of rat interferon assays is further discussed on p. 25.] |
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