One-site immunometric assays that utilize affinity microcolumns were developed and evaluated
Genomics Proteomics and Bioinformatics > Ankyrin Receptors > One-site immunometric assays that utilize affinity microcolumns were developed and evaluated
admin April 24, 2016 Ankyrin Receptors MK-0517 (Fosaprepitant), Vegfa
One-site immunometric assays that utilize affinity microcolumns were developed and evaluated for the analysis of protein biomarkers. (0.38-0.45 pM) were achieved when using fluorescein or a NIR fluorescent dye as the label with an assay precision of ± 0.1-4.2%. Several parameters were examined during the optimization of these assays and general guidelines and procedures were developed for the extension of MK-0517 (Fosaprepitant) this approach for use with other types of affinity microcolumns and protein biomarkers. is assumed to be negligible on the time scale that the non-retained peak spends within the column [7]. If the solution-phase reaction in Eqn. (1) is MK-0517 (Fosaprepitant) allowed to reach equilibrium and the concentration of is defined in this case as the ratio of the moles of applied analyte versus the moles of binding sites within the affinity column (= mol has become bound to the labeled binding agent and the total quantity of in the sample [7 20 27 Eqn. (4) predicts that a linear response with a positive slope MK-0517 (Fosaprepitant) will be obtained for a plot of the relative response vs. Load when and and in Eqn. (5) represents a combination of factors that include the association rate constant for the binding of = = 4 batches). The label content of this preparation ranged from 3-6 mol/mol antibodies with an average of 5 (± 1) mol/mol. These labeled antibodies were stable for up to two weeks when protected from light and when stored at 4 °C in pH 7.4 buffer. The second type of labeled antibody conjugate that was considered was one in which the antibodies were labeled with an NHS-ester of the NIR fluorescent tag IR-800CW. This type of label has previously been shown in work with other immunoassay formats to provide good limits of detection (i.e. pM to nM range) and low background signals for biological samples [16 17 35 The NIR fluorescent labeled antibodies that were prepared in this study had a final antibody concentration of 0.75-0.92 mg/mL (5.0-6.1 μM) and an average concentration of 0.86 (± 0.07) mg/mL (= 4 batches). These conjugates contained 0.5-1.5 mol label/mol antibody with an average label content of 1 1.0 (± 0.3) mol/mol. This type of labeled antibody was again found to be stable for up to two weeks when stored at 4 °C in pH 7.4 buffer and when protected from light. 4.3 Development of one-site immunometric assays using fluorescein as a label Once the affinity microcolumns and labeled antibodies had been evaluated these components were combined Vegfa and tested for use in one-site immunometric assays using HSA as a model target protein. Some typical chromatograms that were obtained by such a method are shown in Figure 2(a) when using on-line fluorescence detection and fluorescein as the label. As predicted by Eqn. (4) there was an increase in the signal due to the non-retained peak for the labeled antibodies as the amount of HSA was increased in the sample. The use of this non-retained peak for detection allowed results to be obtained within 2.5-2.8 min of sample injection at a flow rate of 0.10 mL/min. When the flow rate was increased to 0.5 mL/min the non-retained peak was observed within 1.3-1.5 min of sample injection and this peak appeared at 1.0 mL/min within 35-42 s of injection. Figure 2 (a) MK-0517 (Fosaprepitant) Representative chromatograms and (b) a typical calibration plot for a one-site immunometric assay as obtained by using 600 ng/mL fluorescein-labeled anti-HSA antibodies combined with samples that contained 0-100 ng/mL HSA in a 1:15 (= 3) as based upon the slope and standard deviation of the intercept for the best-fit line. The linear range extended up to approximately 25-40 ng/mL (0.38-0.60 nM) with the given preparation of labeled antibodies and the dynamic range went up to over 100 ng/mL (1.5 nM). As will be shown later in this section the limit of detection and usable range of this assay could be adjusted by varying the amount of labeled antibodies that was mixed with each sample. Based on Eqn. (4) linear behavior at low-to-intermediate target concentrations would be expected for a one-site immunometric assay when a 1:1 interaction can occur between the target and labeled binding agent [7]. This type of behavior has been noted in prior work with labeled Fab fragments [20]. For bivalent binding agents (e.g. intact antibodies or F(ab')2 fragments) some sigmoidal behavior has been observed in the calibration curves of one-site immunometric assays for small targets [20] although others possess discovered this curvature to be minimal and to approximate a linear response [27]. In this current study a linear response was.