A novel immunoassay system and bioseparation process based on thermal phase separating polymers

Abstract

Poly-N-isopropylacrylamide (polyNIPAAm), a water-soluble, thermally precipitating synthetic polymer, has been conjugated together with a monoclonal antibody (MAb) and utilized in a novel separation method for an immunoassay. The PolyNIPAAm precipitates out of water above a critical temperature of 31 degres C, enabling a polymer-bound immune complex to be separated from the solution. The principal advantages of this method are that it utilizes a homogeneous incubation for the antigen-antibody reaction, plus, it has the ability to assay large-molecular-weight antigens with sensitivities equivalent to other nonisotopic heterogeneous immunoassays. In addition, since the polymer-immune complex may be reversibly redissolved by cooling, the method may be used both to concentrate the signal and isolate the analyte. This general technique may also be used for a wide variety of separation processes in addition to immunoassays, in which a specific component in a biological fluid, industrial process stream, or body of water is to be isolated for analysis, recovery, or disposal. Thus, product recovery and/or toxin or pollutant removal processes are possible with this methodology.

Preparation of pH-sensitive polymer by thermal initiation polymerization and its application in fluorescence immunoassay

“…The results showed that the immune reaction would reach equilibrium within 4 min, which is much shorter than that of solid immunoreaction [26], and immuoreactions modulated by temperature-sensitive polymers [6][7][8][9][10].…”

“…Among the temperature-sensitive polymers, poly-N-isopropylacrylamide (PNIP) is known to have a LCST of about 31 • C throughout a wide concentration range. The special thermo-sensitive characteristic of PNIP was successfully exploited in immunoassays by Hoffman et al decades ago [6]. Then PNIP has been widely utilized as the separation carrier in immunoassays [7][8][9][10].…”

Determination of doping methyltestosterone by capillary electrophoresis immunological analysis with thermally reversible hydrogel and laser-induced fluorescence

“…A stock solution of 5.3 mg/mL FITC-antigen was made by mixing 0.15 mL of 0.36 mg/mL FITC solution with 1.5 mL of 5.3 mg/mL antigen (dissolved in 0.5 M sodium carbonate buffer at pH 9.5) in a 10 mL beaker [3]. The mixture was vortex-mixed and left in the dark at room temperature for 4±6 h before use.…”

Competitive Enzymatic Fluorescence Immunoassay for Human IgG by Using a Temperature‐Sensitive Phase Separating Polymer with Regulated Phase Transition Temperature

“…PNIP precipitates out of water when the temperature is raised above the lower critical solution temperature (LCST, about 31 ), and ℃ re-dissolve when the solution temperature is lowered below the LCST. The special temperature-sensitive characteristic of PNIP was successfully exploited in immunoassays by Hoffman et al 4 decades ago. Afterwards PNIP was widely utilized as the separation carrier in immunoassays.…”