Alternative assays methods

G McKay, et al. Development and application of a radioimmunoassay for fluphenazine based on monoclonal antibodies and its comparison with alternate assay methods. J Pharm Sci 79 240, 1990. 

The use of the perchlorate anion to precipitate the cobalt complex to determine the yield [1] is deprecated on the grounds of potential hazard [2], though it was not found possible to cause the salt to detonate by pounding, but it will bum if ignited. A spectroscopic assay method is suggested as an alternative to precipitation [3],... 

Haneke, K.E, et al., ICCVAM evaluation of the murine local lymph node assay. III. Data analysis completed by the National Toxicology Program Interagency Center for the Evaluation of Alternative Toxicological Methods, Regul. Toxicol. Pharmacol., 34, 274, 2001. 

Besides, the aforesaid visual methods of assay i.e., observing the change in colour of indicators used, alternative instrumental methods such as potentiometric, amperometric, polarographic, conductometric methods are also employed in determining the end-point. 

If analytical methods are at the heart of biopharmaceutical development and manufacturing, then protein concentration methods are the workhorse assays. A time and motion study of the discovery, development, and manufacture of a protein-based product would probably confirm the most frequently performed assay to be protein concentration. In the 1940s Oliver H. Lowry developed the Lowry method while attempting to detect miniscule amounts of substances in blood. In 1951 his method was published in the Journal of Biological Chemistry. In 1996 the Institute for Scientific Information (ISI) reported that this article had been cited almost a quarter of a million times, making it the most cited research article in history. This statistic reveals the ubiquity of protein measurement assays and the resilience of an assay developed over 60 years ago. The Lowry method remains one of the most popular colorimetric protein assays in biopharmaceutical development, although many alternative assays now exist. 

Methods have successfully been transferred to various laboratories in inter-company cross-validation exercises for a chiral separation, for an assay of the main component in a formulation and for drug stoichiometry. Revalidation is an alternative to method transfer in case of changes in product composition or analytical procedure (cf. Section I.L). Although a method transfer in CE is not a major difficulty, some aspects have to be considered, especially if a method is transferred to an instrument of another manufacturer. 

It is required for quantitative purity assays, and it must be established across the specified range of the analytical procedure. This can be done, by establishing the recovery rate over the range of the method. Alternatively, a method comparison between a validated method and a new method can be performed. Accuracy can be determined by spiking degraded, aggregated, pure or impure material into a known amount of sample. A theoretical recovery would then be calculated and the spike material analyzed using the chosen method. The actual recovery should then be compared to the theoretical recovery to calculate the accuracy of the method. Accuracy in this case would be reported as percent recovery. 

The costs associated with hit identification using diverse assay systems and technologies as well as, alternatively, biophysical methods and approaches are regarded... 

A complementary approach is to conduct the assays under high-throughput automated conditions. This can be either through the miniaturization of assays, that is, 96-384 plates and if possible 1536, or through the use of alternative assay technologies (e.g., microfluidics). Both scenarios require studies of equivalency testing and backwards compatibility with previous methods and results. 

Three in vitro alternative assays are endorsed as scientifically validated by ECVAM in 2001 the EST, the MM, and WEC assays. The best results were obtained by combining the EST and WEC assays. The EST assay has the advantage of not using animals at all. As human cell culture technology improves, particularly regarding stem cells (8, 9), new methods will undoubtedly evolve that will enable a closer in vitro detection of in vivo human teratogenesis. 

The embryonic stem cell test is an animal-free alternative test method for developmental toxicity. Mouse embryonic stem cells are cultured in a hanging drop method to form embryoid bodies. These embryoid bodies, when plated on tissue culture dishes, differentiate to form contracting myocardial cell foci within 10 days. Inhibition of cardiomyocyte differentiation by test compounds serves as the end point of the assay, as monitored by cormting contracting muscle foci under the microscope. 

This unit describes four of the most commonly used total protein assay methods. Three of the four are copper-based assays to quantitate total protein the Lowry method (see Basic Protocol 1 and Alternate Protocols 1 and 2), the bicinchoninic acid assay (BCA see Basic Protocol 2 and Alternate Protocols 3 and 4), and the biuret method (see Basic Protocol 3 and Alternate Protocol 5). The fourth is the Coomassie dye binding or Bradford assay (see Basic Protocol 4 and Alternate Protocols 6 and 7), which is included as a simple and sensitive assay, although it sometimes gives a variable response depending on how well or how poorly the protein binds the dye in acidic pH. A protein assay method should be chosen based on the sensitivity and accuracy of method as well as the condition of the sample to be analyzed. 

The selection of a protein standard is potentially the greatest source of error in any protein assay. Of course, the best choice for a standard is a highly purified version of the predominate protein found in the samples. This is not always possible nor always necessary. In some cases, all that is needed is a rough estimate of the total protein concentration in the sample. For example, in the early stages of purifying a protein, identifying which fractions contain the most protein may be all that is required. If a highly purified version of the protein of interest is not available or it is too expensive to use as the standard, the alternative is to choose a protein that will produce a very similar color response curve with the selected protein assay method. 

An alternative colorimetric method relies on the reaction with vanillin under acidic conditions. A 2-mL aliquot of a freshly prepared solution of vanillin (1 g/100 mL) in 70% sulfuric acid is added to 1 mL of aqueous plant extract. The mixture is incubated in a 20°C-waterbath and after exactly 15 min. the absorbance at 500 nm read. The concentration of proanthocyanidins is expressed as (+)-catechin equivalents (used for the standard curve). This assay is specific for flavonols. As a consequence, when using this assay to determine the concentration of condensed tannins, widely distributed monomeric flavonols, such as catechin (1.39) and epicatechin (1.90), can interfere (Hagerman and Butler, 1989). 

Sailstad, D. M. Murine local lymph node assay An alternative test method for skin hypersensitivity testing. Lab. Animal 31 36-41, 2002. 

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