Alternative assays

Because of such difficulties alternative assays have been investigated, and sometimes are used in conjunction with, or instead of, bioassays. The most popular alternative assay system is the immunoassay. 

An alternative assay format entails the use of virus-specific DNA probes. These can be used to screen the biopharmaceutical product for the presence of viral DNA. The assay strategy is similar to the dot blot assays used to detect host-cell-derived DNA contaminants, as discussed earlier. 

Immunogold assays are now widely used to localize proteins within cells and, in particular, to achieve the detail and resolution that cannot be obtained with alternative assays such as immunofluoresence or bio-... 

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. 

Not only is this expensive but we believe that it is very innacurate at low levels of toxicity. In addition, there are vast shellfish beds in remote areas of coastline that cannot be opened up for commercial exploration both because of the remoteness from mouse monitoring laboratories, and because of the expense. There is a recognized need for a alternative assay, but in reading the title of the communication a first reaction must be "why a Bioassay " and then "why a fly ". 

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. 

Dunlap Has anyone looked for melanopsin in the somatic cells by an alternative assay ... 

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. 

In 2007, the DART committee held a workshop on alternative assays, which was followed up by a workshop held at the European Teratology Society Annual Meeting in 2009. These workshops focused on three alternative assays (1) whole embryo culture (WEC), (2) mouse embryonic stem cell tests (mESC), and (3) zebrafish. Each assay was presented and data from users were shared, and strengths and limitations were discussed. It should be noted that the WEC and mESC are validated by ECVAM as alternative embryotoxicity assays. Still, there are numerous research needs before even validated tests can achieve regulatory acceptance. The discussions, conclusions, and recommendations of the 2007 workshop were published by Chapin et al. (14). Bullet lists of next steps to move forward were defined for each assay (14) and are briefly summarized here ... 

In the 1990s, ECVAM held a forum to vet and evaluate new alternative assays, and developed a list of compounds for testing (24). The key driver for this activity was the fact that DART studies require large numbers of animals. The primary focus of this activity was embryo-fetal toxicity. The list generated from this forum was tested in three assays (later validated by ECVAM) (1) the micromass assay, (2) the rat WEC assay, and (3) the embryonic stem cell test (25). Compounds on the Brown list were classified as either strong, weak, or non-teratogens. The three assays successfully predicted the compound classification about 80% of the time. However, the embryonic stem cell test later performed poorly on a different group of chemicals with known in vivo activities (26). 

In summary, the DART committee has been greatly involved in alternative or in vitro assays because of the recognition that investments have to be made now in anticipation of future benefit. It is also expected that other HESI committees will be providing support towards alternative assay development as the DART committee has. 

As noted earlier, there are several agencies or centers for alternative assay validation. Their processes are briefly summarized below. 

According to de Jong et al. [16] and Piraud et al. [55] urinary protein suppresses DMB-GAG complex formation and results in lower total GAG concentrations. An alternative assay has been developed that avoids problems with GAG-protein interactions. The main difference is that measurements are carried out at basic pH. 

Maurer, J.K., Parker, R.D., Jester, J.V. Extent of initial comeal injury as the mechanistic basis for ocular irritation key findings and recommendations for the development of alternative assays. Regul Xoxicol Pharmacol 36(1), 106-117 (2002)... 

Fig. 1. A high-throughput platform of the carbohydrate-based microarrays. A high-precision robot designed to produce cDNA microarrays was utilized to spot carbohydrate antigens onto a chemically modified glass slide. The microspotting capacity of this system is approximately 20,000 spots per chip. The antibody-stained slides were then scanned for fluorescent signals with a Biochip Scanner that was developed for cDNA microarrays. The microarray results were subsequently confirmed by at least one of the conventional alternative assays.

Correlation of in-vivo assays with alternative assays... 

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