FITC insulin

The dissociation constant (Kd) of a monoclonal antibody with fluorescein isothiocyanate- (FITC)-labeled insulin and unlabeled insulins from several species were measured using CE with laser-induced fluorescence detection (CE-LIF) (9). Kd determinations were made by separating free FITC-labeled insulin and its complex with the antibody in equilibrated solutions in 6 s or less (Fig. 3). Dissociation and association rates for insulin, FITC-insulin, and the antibody are fast enough to reach equilibria in less... 

Fig. 3 Electropherograms used for the determination of the dissociation constant between FITC-insulin and the antibody. All samples contained 0.1 nM FITC-proline (internal standard) and 2 nM antibody. Total concentrations of FITC-insulin were (A) 1 nM, (B) 2 nM, (C) 3 nM, (D) 4 nM, (E) 6 nM. (Reprinted with permission from Ref. 9. Copyright 1997 Wiley-VCH.)...

Tao and Kennedy (9) also used a competitive approach to determine the dissociation constants of unlabeled insulins from several species to an antibody by fitting bound over free FITC-insulin as a function of unlabeled insulin concentration. The Kd values for the different insulins were between... 

Fluorescein isothiocyanate (FITC)-labeled insulin was used to visualize the pathway insulin took when traversing the nasal epithelial layer in vivo following the administration of TDM. FITC-insulin was visualized in the cytoplasm of nasal epithelial cells, but only when added in the presence of TDM. FITC-insulin was not found exclusively in the paracellular spaces, either in the presence or absence of TDM. These results were consistent with animal studies in which transmission electron microscopy of nasal septal tissue exposed to formulations containing 0.125% TDM showed increased endocytotic internalizations compared to septal tissue from rats treated with saline. Taken together, these results suggest that the AGs increase both the transcytotic and paracellular pathways. 

Figure 2 Affinity CE for the determination of competitive binding constant between unlabeled bovine insulin and the antibody. All samples contained fixed amount of FITC-insulin and antibody and increasing amount of bovine insulin. (Reprinted from Ref. 12.)...

Figure 6 Representative electropherograms showing the principle of noncompetitive CE-IA. The top trace shows the blank sample of FITC-labeled insulin (note the presence of multiple labeled products) the bottom trace shows CE separation of a mixture of FITC-insulin and Fab. (Reprinted from Ref. 24.)...

Figure 5 (Top) Electropherogram of 100 nM FITC-insulin under HPCE conditions that employed uncoated capillaries, 25 im i.d. and 150 im o.d., total lengths of 25-30 cm, length to detector 12-15 cm, buffer of 0.05 M sodium phosphate with 0.025 M K2S04 at pH 7.5, applied voltage 1000 V/cm, hydrostatic injection. (Bottom) Electropherogram of 100 nM FITC-insulin and 50 nM Fab. Peaks 2, 3, and 5 are FITC-insulin, while peaks 1 and 4 are due to the formation of the complex of Fab with FITC-insulin in peaks 2 and 5, respectively. An He-Cd laser was used as the excitation source (66). (Reproduced with permission from the copyright holder, American Chemical Society and Analytical Chemistry.)...

Figure 7 Confocal microscopy images of FITC-insulin loaded microspheres. (Left) Msp A (right) Msp B. (From Ref 49 with permission.)...

The observed release pattern from both types of microspheres lies in the distribution of the protein inside a microsphere, which is associated with the preparation method. In order to see this, FITC (fluorescein isothiocyanate)-insulin incorporated microspheres were observed under a confocal microscope, as shown in Fig. 7. For Msp A, a homogeneous distribution of fluorescence was observed while Msp B exhibited a rather heterogeneous distribution of FITC-insulin. In addition, Msp B shows significant surface fluorescence. These observations are, hence, consistent with the observed initial burst from Msp B and from the constant insulin release from Msp A over a prolonged period of time. It is reported that the constant release of insulin from triblock copolymer hydrogel may be attributed to the hydrophilic/hydrophobic domain structure of the gel. The incorporation of a significant fraction of insulin in the hydrophobic domain may have made possible... 

FIGURE 10.3 Repeated on-off release of FITC-insulin from the glucose-responsive hydrogel at 28 °C, pH 9.0, in response to external glucose concentration. Reproduced from Ref. [32] with permission. Copyright 1998 American Chemical Society. 

Morimoto et al. [33] demonstrated that the ocular absorption of hydrophilic compounds over a wide range of molecular weights could be increased by 2 and 10 mM sodium taurocholate and sodium taurodeoxycholate in a dose-dependent manner. The compounds were glutathione (307 Da), 6-carboxyfluorescein (376 Da), FTTC-dextran (4 kDa), and insulin (5.7 kDa). Of the two bile salts, sodium taurodeoxycholate was more effective. At 10 mM, this bile salt increased the permeability of 6-carboxyfluorescein from 0.02% to 11%, glutathione from 0.08% to 6%, FITC-dextran from 0% to 0.07%, and insulin from 0.06% to 3.8%. Sodium taurocholate, on the other hand, increased the permeability to 0.13%, 0.38%, 0.0011%, and 0.14%, respectively. Taurodeoxycholate was more effective than taurocholate in the nasal epithelium as well [202], This difference in activities can possibly be attributed to their micelle-forming capability, which is higher for taurodeoxycholate, a dihydroxy bile salt [190],... 

Applications. The general feasibility of CE to separate immunocomplex from unbound reagent was first demonstrated by Grossman et al. [4] and Nielsen et al. [23], whose reports set the stage for further development of quantification techniques in CE-IA. The first quantitative noncompetitive CE assay of insulin was reported by Schultz and Kennedy [24], To increase the sensitivity of analysis, the authors used FITC-labeled insulin and LIF detection. Due to the separation power of CE, multiple products of fluorescence labeling of insulin did not hamper analysis (see Figure 6). Two types of calibration curves were con-... 

The use of antibodies labeled with fluorescein isothiocyanate (FITC) in immunochromatographic assays in sandwich format increases the assay sensitivity compared with the on/oif detection method [55,116]. The effect of Ab-Ag binding constants on sensitivity was shown by the higher peak size obtained for insulin B chain than for insulin in their sandwich immunochromatographic analysis using FITC-labeled antipeptides antibodies with a higher affinity constant for the first protein [55]. No differences in sensitivity were observed between sequential and simultaneous procedures. 

The utility of FITC-labeled insulin has been proved in noncompetitive and competitive CZE assays with laser-induced fluorescence (LIF) detection (scheme shown in Fig. 8). The DL for Fab anti-insulin in the noncompetitive format was 2nM (280zmol) [86]. A similar DL (3nM) was achieved in competitive assays... 

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