Phase separation immunoassay

Phase-separation immunoassays have been reported, in which the solid phase particles are formed after the immunoreaction is completed.(42) Phase-separation immunoassays are advantageous since the unstirred layer of solution near a solid surface alters diffusion and binding kinetics at the surface in comparison with the properties of the bulk solution. In phase-separation assays for IgG and IgM, capture antibodies are bound with monomers suitable for styrene or acrylamide polymerization.(42) Monomer-labeled capture antibodies are reacted with analyte and with fluorescein- and/or phycoerythrin-labeled antibodies in a sandwich assay, followed by polymerization of the monomers. Fluorescence of the resulting particles is quantitated in a FACS IV flow microfluorometer, and is directly proportional to analyte concentration. 

This thermally induced phase separation immunoassay has been extended by grafting PNIPAM to porous nylon membranes on microfiuidic cards. At temperatures above the LCST of PNIPAM, the polymer-antibody conjugates are aggregated and retained on the modified filters, while other proteins can pass through. Below that temperature, the captured and labelled antigens are released as a concentrated pulse (Golden et al, 2010 Nash era/., 2010). 

Fig. 1. Schematic representation of double-antibody solid phase enzyme immunoassay using cross-linked second antibody particles for separation. Encircled E, enzyme label.

Fraction-separation methods used in immunoassay can be divided into two major groups liquid-phase and immunoreagent modified solid-phase separations. 

Two-site immunometric or sandwich assays that made use of two or more antibodies directed at different parts of the PRL molecule were next to be developed. As with other two-site IRMA assays, the capture antibody is attached to a solid phase separation system and the second or signal antibody is labeled with a detection molecule (e.g., radio-isotope, enzyme,fluorophor, or chemiluminescence tag ). In some assays, the capture antibody is attached to the wall of test tubes, plastic beads, microtiter plates, ferromagnetic particles, or glass-fiber paper. Other assays have used the strep-avidin approach that couples biotin to the signal antibody with avidin linked to a solid phase. Most of the current immunometric assays for PRL have been adapted to fully automated immunoassay systems. Compared with the older traditional RIA methods, these automated immunometric assays for PRL generally achieve lower detection limits (0.2 to 1.0 ig/L) and improved precision (interlaboratory coefficients of variation of <8% at all concentrations), and have superior specificity (<0.05% crossreactivity with GH). 

Enzyme immunoassay and RIA kits for the measurement of sahvary DHEA and DHEA-S are commercially available for research purposes. " Analysis of DHEA by immunoassay usually requires pretreatment of serum samples, because the serum concentration of DHEA is 1000-fold lower than that of DHEA-S. Several extraction and chromatographic procedures have been suggested for this purpose. Celite is the preferred adsorbent, and dichloromethane and ethyl acetate are common choices for extraction solvents. Commercial RIA kits using solid-phase separation techniques and I-... 

A number of nonisotopic immunoassays for estradiol have been developed and adapted for use on fuHy automated immunoassay systems. All are heterogeneous assays (separation step needed), but most are direct assays and do not require prehminary extraction. Most procedures offer the convenience of solid-phase separation methods. For routine clinical applications, the greatest experience is with enzyme immunoassays. Most commercial enzyme immunoassays use horseradish peroxidase or alkaline phosphatase to label estradiol antigens enzyme activity is determined using a variety of photometric,fluorescent,or chemiluminescent substrates. ... 

Monji N, Hoffman AS. A novel immunoassay system and bioseparation process based on thermal phase separating polymers. Appl Biochem Biotechnol 1987 14 107-120. 

In the case of an immunoassay (Fig. 8.13) only the free fraction of the cationic tracers is extracted from the mixture and accumulates at the surface of the electrode where it is detected electrochemically. The antibody-cationic tracer complexes are too large to penetrate the polymer and are thus not detected. This is an elegant approach to the separation of free and bound fractions of tracers, permitting a homogeneous-phase like immunoassay. 

Reversed-phase HPLC uses a nonpolar stationary phase and a polar mobile phase. The characteristics are operational simplicity, high efficiency, column stability, and ability to analyze simultaneously a broad spectrum of both closely related and widely different compounds. Separation is based on hydrophobicity.33 Findlay et al. provide a comparison of chromatography methods with immunoassays (Table ll.l).27... 

CAE employing antibodies or antibody-related substances is currently referred to as immunoaf-hnity capillary electrophoresis (lACE), and is emerging as a powerful tool for the identification and characterization of biomolecules found in low abundance in complex matrices that can be used as biomarkers, which are essential for pharmaceutical and clinical research [166]. Besides the heterogeneous mode utilizing immobilized antibodies as described above, lACE can be performed in homogeneous format where both the analyte and the antibody are in a liquid phase. Two different approaches are available competitive and noncompetitive immunoassay. The noncompetitive immunoassay is performed by incubating the sample with a known excess of a labeled antibody prior to the separation by CE. The labeled antibodies that are bound to the analyte (the immuno-complex) are then separated from the nonbound labeled antibody on the basis of their different electrophoretic mobility. The quantification of the analyte is then performed on the basis of the peak area of the nonbonded antibody. 

These requirements are difficult to satisfy with, e.g., immunoassays. Instrumental techniques like MS/MS require sample clean-up and sometimes even an online coupled separation method. Thus separation techniques, first of all SPE, GC, and HPLC, remain indispensable tools of analysts. An expert in analytical separation can nearly always today solve the complex tasks described above. The large variety of commercial equipment and stationary phases provide him with the necessary tools in most cases. The question may arise, then, if he really needs MIP stationary phases. At this time there are many possible answers to this question and only the future will give the final answer. 

Cross-reactivity (e.g. due to metabolites) can only be eliminated by collecting appropriate fractions from HPLC separations or applying solid phase extractions (SPE) preceeding the immunoassay procedure. 

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