Animal Assays Biological Methods

Unmodified lysine is, in principle, biologically available and will be determined as such by animal assays (21) and enzymatic methods (22, 23,24), or determined as reactive lysine by the Carpenter method (23,24,25, 26) and by guanidina-tion (24, 27). 

Biological samples from patients are generally not as useful for diagnosis of intoxications as they are for diagnosis of infectious diseases or chemical intoxications. The same is true of postmortem samples. Ricin can be identified with immunoassays in extracts of lung, liver, stomach, and intestines up to 24 hours after aerosol exposure. High doses of ricin can be identified in fixed lung tissue of aerosol-exposed laboratory animals by immunohistochemical methods. The staphylococcal enterotoxins can be detected by immunoassay in bronchial washes. Like blood and swab samples, postmortem tissue or fluid samples should be kept cold, preferably frozen, until they can be assayed. 

Purity for a small molecule is a relatively simple concept. Normally, an HPLC method is sufficient to measure the content and impurity levels of a small molecule drug. A macromolecule, such as a protein, has a much more complex behavior. Determining protein concentration by UV absorption spectroscopy can give a measure of the total protein in the product, but it will not necessarily differentiate between active protein and inactive protein (i.e., denatured or otherwise degraded). A validated method or methods to determine the biological activity of the molecule is needed. So, whereas protein concentration is usually tested as part of the specifications, it is also normally accompanied by one or more methods that measure or correlate to biological activity. This is the bioassay. These methods can be animal-based or cell-based, protein interaction assays, binding methods such as surface plas-mon resonance or ELISA (enzyme-linked immunosorbent assay) and immunoblot methods. 

Following the discovery of pantothenic acid in 1933, several biological methods were reported based on the growth response to pantothenic acid of chicks, yeast and bacteria such as Lactobacillus, Streptococcus and Proteus. Microbiological assays have been used more commonly than animal tests because they are cheaper, faster and simpler methods. Lactobacillus, in particular, responds well... 

Biological methods of assay for vitamin D were developed mostly during the period 1930-1945. Details of these basic techniques are well-documented in excellent reviews (Rosenberg, 1945 Bliss and Gyorgy, 1951). These assays are based upon measiu-ement of responses in D-deficient rats or chicks parameters such as prevention or cure of rickets are determined radiologically or by bone ash content. A salient feature about animal assays is the apparent species specificity exhibited by ergocalciferol and cholecalciferol. While the rat responds equally well to both vitamins, on the chick ergocalciferol is only about 3% as effective as cholecalciferol. 

Although riboflavin can be assayed more readily by chemical or microbiological methods than by animal methods, the latter are preferred for nutritional studies and as the basis of other techniques. Such assays depend upon a growth response the rat or chick is the preferred experimental animal. This method is particularly useful for assaying riboflavin derivatives, since the substituents frequently reduce or eliminate the biological activity. 

Mouse Bioassay. The mouse is the traditional animal of choice for detecting biological activity due to STX and TTX. Mice receive an intraperitoneal injection of sample and are observed for symptoms of intoxication, i.e., dypsnea, convulsions, and death. This method is effective for detecting biological activity of STX and TTX in numerous samples. For the standard STX assay, one mouse unit is defined as that quantity of STX injected i.p. in 1 ml solution that will... 

T. Ubuka, Assay methods and biological roles of labile sulfur in animal tissues. B. Analyt Technol. Biomed. Life Sci. 781, 227-249 (2002). 

---