Cell-based ELISA

Cell-based ELISA assay pGSK3 3 IC50 = 1.9 pM... 

Molecular weight of heavy and light chains Peptide mapping Amino acid analysis Intrinsic fluorescence spectroscopy Thermal denaturation monitored by fluorescence Fourier transfrome infrared spectroscopy Binding (e.g., ELISA, BiaCore, etc) Potency (e.g., cell based, ELISA)... 

Reporter gene assay (RGA), e.gv lucHerase, SEAP Secondary m essengers, e.g., Ca24(FLIPR), cAMP, IP3 J Cell-based ELISA e.g.,QytoBlDt Cell based physiological assay... 

Cell-Based Cell-based ELISA are indirect or direct ELISA systems that use intact cells... 

Table 2. Kinase selectivity assays and cell-based ELISA IC50s for antiphosphorylation of kinases.

In addition to its use in quantitating the product, ELISA can also be used as a readout tool for cell-based bioassays or other assays that measure a biological activity conferred by the drug product. The end point of the bioassay may be a... 

Host-cell protein ELISA have the advantage of quantitating host protein impurities. The disadvantage, however, is that the quantitation is of a group of impurities. Western blot analysis, on the other hand, provides the analyst with a relative level of an individual impurity compared to other impurities. If the level of one or more host protein impurities appears to be excessive based on the intended use of the drug product then it may be necessary to identify those impurities. This can provide assurance that the impurity is innocuous and it can also define the physicochemical properties of the impurity such that the process can be modified to reduce its presence in future production lots. The identification can also lead to the development of a quantitative assay for monitoring the individual impurity in every lot. 

Fluorescent microvolume assay technology (FMAT ) is a bead-based or cell-based fluorescent technology for homogeneous ELISA-like assays. In FMAT, a laser beam is focused on the bottom of the assay well and the localised fluorescence intensity bound to beads (or cells) is detected as an area of intense fluorescence over the unbound and background fluorescence in solution. Different analytes can be detected with appropriate fluorophores and, by using different sized beads, the assay can be multiplexed to monitor multiple analytes.13... 

Because of the destabilizing effect of the mutations on the structure of the mutant proteins, the NA mutant and some of the HA mutants display a marked thermo-lability. The NA mutant loses both enzyme activity as well as reactivity to the NC-10 mAb, which only recognizes the native NA (7). The HA mutants lose the ability to bind to the receptor on red blood cells, resulting in a decrease in the hemagglutination titer, or to the sialic acids on fetuin in a fetuin-based ELISA assay. 

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. 

It would seem from the above studies that the ideal test would be for the lymphokines without the use of a viable indicator cell. These substances are produced in low levels, and thus the cell based assays such as LTT and LIE or MIF are required. Although ELISA based or RIA based assays can be used to detect and quantitate cytokines, reagents are not yet available for these human lymphokines (LIF, MIF, LTT) specifically. 

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