Calcium mobilization assay

Fig. 3. Comparison between IP-One kit versus calcium mobilization assay (384-well format). Human embryonic kidney (HEK) 293 cells expressing a chemokine receptor were evaluated on HTRF IP-One kit (CisBio, Bedford, MA) and fluroescent imaging plate reader (FLIPR) with Calcium 3 kit (Molecular Devices, Mountain View, CA).

To assess activity at the insect ryanodine receptor, pyridyl pyrazoles of Table II were tested in a calcium mobilization assay, using neurons from the American cockroach, Periplaneta americana. These studies have confirmed the mode of action to be RyR activation. Compounds D11-D17 showed exceptional potency in this assay with activity in the range of0.03-0.30 pM. The data shows the ability of anthranilic diamides to release internal calcium stores while failing to activate voltage-gated calcium channels. Furthermore, calcium mobilization induced by anthranilic diamides is blocked following treatment with 1 pM ryanodine, consistent with action at the ryanodine receptor. 

Several other techniques for have evolved for biochemical assays. In chapter 2 of this book, Omann and Sklar report on a method of fluoroimmunoassay where the bound and unbound antigen are separated by the quenching of fluorescence that accompanies antibody binding. Then, in chapter 3, Holl and Webb show how they achieved a sensitive measurement of nucleic acids by the enhancement in fluorescence that accompanies the binding of fluorescent dyes to nucleic acids. Chandler et al, also used fluorescence enhancement to monitor calcium mobility in neutrophil cells. 

In contrast, functional assays that look at post-receptor events, such as cAMP stimulation (Gs), cAMP inhibition (G ), inositol triphosphate (IP3)/mono-phosphate (IP1) increase (Gq), or intracellular calcium mobilization (Gq), are homogeneous, for the most part nonradioactive, and easy to automate (with... 

Measure the fluorescence emission at 505 nm using 340 nm excitation. Monitor the baseline. When a stable baseline is obtained for 10-30 s, add the chemokine to be tested. After the peak of calcium mobilization has subsided (about 1 min), calibrate the assay by adding 20 pL of minimum buffer, allow for stabilization, then add 20 pL of maximum buffer to dissolve cell membranes in order to obtain full complexation of fura-2 with Ca2+. Record the baseline, the peak, the minimum and the maximum values (see Fig. 1). The concentration of Ca2+ mobilized is calculated according to the following modified formula (6) ... 

The introduction of phenol extraction by Aurbach (A4) has enabled Rasmussen (R3) to produce a much more active principle with a potency of about 2500 units (USP)/mg dry weight when assayed by the method of Munson (Mil). This material possesses both phosphaturic and calcium-mobilizing activities. Its minimum molecular weight, calculated from its amino acid composition, is about 8500. Rasmussen interprets his latest... 

Fluo-4 Method for FLIPR Assay to Detect Calcium Mobilization Proprietary GPCR... 

The choice of the mobile phase is very important, as fluorescence is sensitive to fluorescence quenchers. Highly polar solvents, buffers, and halide ions quench fluorescence. The pH of the mobile phase is also important to fluorescence efficiency for example, quinine and quinidine only display fluorescence in strongly acidic conditions, whereas oxybarbiturates are only fluorescent in a strongly alkaline solution [67,68]. Due to the stability of the chromatographic sorbents, the use of very acidic or basic mobile phase may not be possible. One alternative is to alter the effluent pH postcolumn. Postcolumn addition of sulfuric acid has been used for the assay of ethynodiol diacetate and mestranol in tablets [69]. Another example is the determination of tetracycline antibiotics in capsules and syrup where EDTA and calcium chloride were added to enhance fluorescence [70]. 

One of the most widely used calcium channel blockers is verapamil. Verapamil hydrochloride was assayed from pharmaceutical preparations by ElGhany et al. [133]. The separations were performed on 200 x 100 mm and 100 x 100 mm silica gel 60 F254 plates using an ethyl acetate/methanol/water mobile phase and scanning the plates at 278 nm. The accuracy and precision data for the assay of spiked verapamil samples and the recovery data of verapamil from commercial preparations are shown in Tables 10.20 and 10.21. 

Different resin columns (Ca++, Pb++, H+, Ag+, etc.) are used for different sample types, with calcium being the most common form and water as the mobile phase at 85°C8 (see Figure 7.1). The basis of separation is mixed-mode, which includes size-exclusion, ion-exclusion, ligand exchange, and hydrophobic interaction with the polystyrene support. This reliable assay methodology has excellent precision, sensitivity (10-20ng), linearity (0.050-800 pg), and column lifetime.8 This is the preferred approach for most routine assays. An amino column with acetonitrile/water (80/20) (AOAC method) can also be used, however, sensitivity is lower and column lifetime is limited.7... 

Assay for intestinal calcium transport and in vivo bone mobilization... 

Assessments of in vivo activity are based primarily on two assays the stimulation of intestinal calcium transport and the mobilization of calcium and phosphate from bone in animals (rats or chicks) maintained on a low calcium or low phosphorus diet. Many of the relevant results in this area have been reviewed previously and are also mentioned in preceding sections of this review... 

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