Specific bioassay

Incubation periods in excess of 2 h were required before this activity was detected in cell-free supernatants. More recently, the use of cDNA probing of Northern transfers (to detect specific mRNA levels), the use of ELISA techniques (to detect protein levels immunologically) and the development of more specific bioassays (culture techniques in which a biomolecule stimulates proliferation in a particular cell line) have resulted in a more thorough analysis of IL-1 production by neutrophils. IL-1 is only poorly expressed in blood neutrophils because mRNA for this cytokine is detectable only at very low levels (if at all), and protein production is usually below the level of detection of most assays. However, exposure of neutrophils to lipopolysaccharide (LPS), or to cytokines such as GM-CSF, TNF or IL-1 itself, results in a rapid but transient increase in IL-1 expression. 

As pointed out above, the bioassay design depends on the objective(s) of the study. A bioassay to determine allelopathic interactions in the field or in an ecological setting may have a quite different design than one used to determine PGR activity of a compound or to determine its molecular mode of action. Specific bioassays can be used to follow the isolation/purification of allelochemicals, evaluate their phytotoxic (or growth simulation) effects (i.e., visual effects), determine their host range/selectivity, evaluate allelopathic action of volatile compounds, or examine physiological/biochemical effects, such as photodynamic and membrane effects, effects on photosynthesis, specific enzyme sites, and effects at the ultrastructural level to locate receptor sites or sites of injury. Several examples of useful bioassays will be presented later. 

Bains, P. S. 1990. Purification, chemical characterization, host-specificity, bioassay, mode of action, and herbicidal use of the toxin produced by Alternaria brassicae. Dissertation Abst. Internat. 50, 2708-B... 

Tchan, Y. T., Roseby, J. E., and Funnell, G. R. 1975. A new rapid specific bioassay method for photosynthesis inhibiting herbicides. Soil Sci. Biochem. 7, 39-44... 

BCEPD. 1996. Part C, Section 4. Specific bioassay requirements, Microtox . British Columbia Field Sampling Manual For Continuous Monitoring Plus the Collection of Air, Air-Emission, Water, Wastewater, Soil, Sediment, and Biological Samples, 1996 ed. Laboratory and Systems Management, Environmental Protection Department, Ministry of Environment, Lands and Parks, Province of British Columbia, B.C., Canada, pp. 91, 98,99. 

The CRP work plan was proposed at the project s first Research Coordination Meeting (RCM), held in Bucharest in October 2002. The evaluation of a new therapeutic radiopharmaceutical depends on several analytical techniques to establish the product s stability and chemical, radiochemical and pharmaceutical purity. In addition, specific bioassays must be developed to evaluate its biological efficacy. These bioassays are product specific and thus need to be worked out separately for each radiopharmaceutical. Participants also identified potential lead molecules and isotopes to be used during the CRP for the development of therapeutic radiopharmaceuticals. 

The Rice Lamina Inclination as a Specific Bioassay for Brassinosteroids... 

Development of bioassays for the isolation of bioactive compounds from natural sources has played an important role in recent natural products chemistry. For the isolation and purification of BRs from plant sources, highly sensitive and specific bioassays are indispensable, because of the very low concentration of BRs in plants. The following three bioassays have been employed for the BRs purification procedure to guide the fractionation. 

Highly sensitive and specific bioassays are indispensable for the studies of isolation of BRs from plant sources. The above-described three bioassays have greatly contributed to the study of BRs and will continue to do so in the future. 

Specificity. Using the first member of the family, brassinolide (BR), an extensive survey of its effects in 17 bioassays, which varied in their responses to gibberellins, auxins and cytokinins, showed that BR did not behave exclusively as any one of those hormones. In some supposedly specific bioassays BR was as effective, or more so, as the hormone the assay was supposed to detect (9,10). This also applies to the rice lamina inclination assay (11), which is now frequently used. 

Another major problem that has plagued the field of thymic hormone research is that specific bioassays for thymic hormones are not available. Indeed, the thymic hormones have been aptly termed hormones in search of a bioassay (Bach and Carnaud, 1976). It is quite simple to add a thymic factor to a routine immune assay and look for a positive or negative influence. However, unless the target cell population for such studies is well defined, the results are likely to be difficult to interpret in terms of normal physiological mechanisms. This latter point no doubt explains many of the inconsistencies reported in the thymic hormone field. 

Specificity (Bioassays and CLB Assays) Inclusion of an approach for establishing specificity is useful in determining the presence of interfering substances and for assurance that the inhibitory response is attributed to specific... 

The multiple actions of individual cytokines mean that cell lines rarely respond only to one cytokine. This is well illustrated by the efforts to develop specific bioassays for lL-1. Although the original mouse thymocyte proliferation assay was thought to be IL-1 specific, it is now clear that many cytokines influence the assay. IL-6 and TNF can replace IL-1 (G7, U4), and IL-2 and IL-4 can sygerize with... 

H29. Hopkins, S. J., and Humphreys, M. Simple, sensitive and specific bioassay of interleukin-1. J. Immunol Methods 120, 271-276 (1989). 

In the 1990 s scientists at Harbor Branch Oceanographic Institution continued to study the secondary metabolites of deep water sponges. They introduced new, highly specific bioassays into their... 

Assay Generally HPLC is sufficient for assay, identification, and impurities Generally overall protein concentration, plus at least one specific bioassay and one or more assays showing binding correlated with clinical experience during development... 

Miscellaneous Glycoproteins. Bovine lutrophin, bovine thyrotrophin, and human chorionic gonadotrophin preparations with carbodi-imide-mediated covalently cross-linked sub-units have been isolated in high yield and characterized by c.d. spectroscopy, radioligand receptor assays, and hormone-specific bioassays. The c.d. spectra of the native and cross-linked hormones did not differ much, suggesting that there is little conformational change on cross-linking. 

Some lines of evidence point to other, non-indolic, natural products that may also act as auxins in plant tissues. Some are as yet unidentified chemically, others (like phenylacetic acid) act as auxins in bioassays, occur in plant tissues, but have not been shown to play a role in natural growth regulation. Also some other hormones, such as the gibberellins, have at least weak auxin activity. Interpretation of the importance of substances such as these will have to await the development of specific chemical methods of analysis which are at least as sensitive as the relatively non-specific bioassays now employed out of necessity. 

An important point to consider in the design of bioassays is the complexity of the behavior being observed. In a complex process such as the identification and acceptance of a host for feeding or oviposition, several different behaviors must be released in sequence by unique stimuli. Visual or volatile chemical cues may be used to bring the insect in contact with the potential host, after which tactile or nonvolatile chemical cues may be used to actually identify the host and assess its quality for utilization. In such cases, it may be necessary to dissect the overall response into its components and develop specific bioassays for each stimulus separately, rather than to attempt to develop a consistent, discriminating bioassay that utilizes the whole behavioral process. 

The method of statistical analysis of an oviposition bioassay depends on the specific bioassay and the parameters measured. In general, these assays will involve counts or frequencies (e.g., the number of eggs produced, or the number of ovipositions attempted), and analysis by goodness-of-fit G-tests will be most appropriate. General guidelines for the analysis of these data are presented in section 5.1.3. 

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