Cell ELISA systems

The involvement of several tyrosine kinases in various cancers requires efficient screening methodologies for the inhibitory compounds. Screening is divided into three steps (1) primary screening against the pure isolated PTK in a cell-free system. The objective is always an ELISA format. The compounds are screened against a battery of PTKs and Ser/Ther kinases in order that the pattern of selectivity can be established quickly [2]. 

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

FIGURE 9.11 An example of a cellular system designed to study inflammatory processes related to asthma and arthritis. Multiple readouts (ELISA measurements) from each of four cell types are obtained under conditions of four contexts (mixture of stimulating agents). This results in a complex heat map of basal cellular activities that can be affected by compounds. The changes in the heat map (measured as ratios of basal to compound-altered activity) are analyzed statistically to yield associations and differences. 

The rapid turnover rate of some enzymes allows ELISAs to be designed that surpass the sensitivity of radiolabeling techniques. In addition, substrates can be chosen to produce soluble products that can be accurately quantified by their absorbance or fluorescence. Alternatively, substrates are available which form insoluble, highly colored precipitates, excellent for localizing antigens in blots, cells, or tissue sections. The flexibility of enzyme-based assay systems makes the chemistry of enzyme conjugation one of the most important application areas in bioconjugate techniques. 

Fig. 12. Effect of spontaneous apoptosis on the activation of caspase-3 in human eosinophils. Eosinophils (2 x 10 /ml) were cultured for 3 h. Cells were lysed and caspase-3 activity was measured by caspase-3 colorimetric assay kit (R D Systems). Enzymatic products were measured at 405 nm with BIOTEK EL340 ELISA microplate reader (BIO-TEK Instrument Inc., Vermont). Human recombinant caspase-3 (5 U) (Calbiochem, California) was used as a positive control. The stimulation index was determined by direct comparison to the level of the normal control. Background readings from cell lysates and buffers were substracted from the readings of both induced and uninduced samples samples before calculating the stimulation index in caspase-3 activity. The differences between control and treated groups were assessed by Student s i-test. P < 0.05 P < 0.001 (Z2).

Test wells exhibiting growth for the presence of monoclonal antibodies when the hybridoma growth covers approx 25% of the base of the cell well. The assay system used should mimic the final test format required. Commonly, Triple Antibody Sandwich (TAS) (5) enzyme-linked immunosorbent assay (ELISA) is use for screening hybridomas for specific antibody. It is important when testing hybri-... 

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