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    • The conjugated gold is an essential

    2021-10-12

    The conjugated gold is an essential part of rapid test kits. For this PIs-IC strip the gold nanoparticles were conjugated with an optimal concentration of antibody to produce a stable nanoparticle solution (Fig. 3). From TEM images it became obvious that the colloid gold contained particles of different sizes (Fig. 4a and b) with most gold particles approximately 10-30 nm. The absorbance spectrum of gold colloid depends on the particle size and in general the smaller size of gold nanoparticles has a red color. Following conjugation there was a shift in the peak absorbance wavelength (from 523 nm to 530 nm) due to a change in the native refractive index surrounding the gold [33], indicating that anti-MA HB-8975 mAb was bound to the gold surface (Fig. 4c). To test the efficacy of conjugated gold and antibody binding activity after conjugation, an anti-MA HB-8975 mAb was conjugated with colloidal gold and subsequently use to develop a direct and sandwich dot blot immunoassay for the detection of HIV-PR activity. To conclude, anti-MA HB-8975 mAb conjugate specifically bound to MA after HIV-PR cleavage and anti-MA G53 mAb bound to another epitope of the intact or cleaved form of MA (Fig. 5a and b). These results verified that this system could be applied to develop an immunochromatographic (IC) strip test. The PIs-IC strip developed has a unique “two-step” detection process starting with the proteolysis of HIV-PRH6 followed by an IC reaction. The PIs-IC strip composes of the conjugated pad which absorbs the anti-MA HB-8975 mAb (binds to the free C-terminus of the cleaved HIV-MA) conjugated with gold particles (anti-MA HB-8975-CGC). Anti-mouse Igs are immobilized on the control line. Anti-MA G53 mAb, which binds against both intact and cleaved MA, are immobilized on the test line. Before dipping the IC strip in the plasma sample, the Aurora Kinase Inhibitor III sale reaction performed by incubating the HIV-PRH6 substrate with H6MA-CA. The cleaved products bind to the anti-MA HB-8975-CGC dissolved on the conjugate pad and then migrate up the surface of the nitrocellulose membrane. The complexes are finally captured by the immobilized anti-MA G53 mAb and immobilized anti-mouse Igs generating a red-purple color at the test and control lines, respectively. A schematic describing the components of the PIs-IC strip and the immunochromatographic reaction are shown in Fig. 6. Regarding the specific binding of anti-MA HB-8975 mAb to free C-terminus of MA after cleaving by HIV- PRH6, the PIs-IC strip could be used to measure the inhibitory effects of all FDA-approved HIV PIs. In the present study, we assessed the PIs-IC strip to semi-quantify the plasma concentrations of LPV in samples from HIV-infected patient receiving LPV/r-based ART. Nevertheless, as LPV and other commonly prescribed PIs are coadministered with RTV [29,30], it was essential to determine the impact of low dose RTV on the strip detection limit. It was revealed that LPV/r concentrations below 1,000/150 ng mL-1 did not inhibit HIV-PRH6 activity while LPV/r over 1,000 ng mL-1 (e.g. 4,000/150, 8,000/500 and 15,000/1,500 ng mL-1) fully inhibited HIV-PRH6 activity. These results showed that low-dose RTV (150-1,500 ng mL-1) used in boosting the pharmacokinetic properties did not interfere in the semi-quantification of LPV using the PIs-IC strip. In addition, cobicistat is a pharmacoenhancer similar RTV [8, 34,35] so the influence of low-dose cobicistat on the PIs-IC strip must also be determined. In the present study, we applied the PIs-IC strip to determine the protease inhibitor activity in clinical samples beside the spiked plasma. Heat-inactivation of serum specimens before testing for HIV-1 drug levels is required in this step as it is an effective means of abolishing HIV-1 [36,37]. To confirm that heating samples have no effect on the enzymatic reaction, the samples were heated at 56 °C for 30 min prior test with the PIs-IC strip. The results showed that heat inactivation of spiked plasma had no interference on detectable levels of LPV when compared to non-heated samples. This result is supported by F. Akeb et al. 2002 who also reported that the inactivation process did not change the PI drug level in spiked plasma samples [36].