Mimetic-enzyme fluorescence immunoassay using a thermal phase separating polymer

Abstract

Poly-N-isopropylacrylamide (PNIP), a water-soluble, thermally precipitated synthetic polymer, has been conjugated together with a monoclonal antibody and utilized as a novel separation method for an immunoassay. PNIP precipitates out of water above a critical temperature of 31 degrees C, enabling a polymer-bound immune complex to be separated from the solution. These characteristics were used to develop a novel polymer-mimetic enzyme immunoassay method for determination of alpha-1-fetoprotein (AFP) with hemin as a labeling reagent to catalyze the reaction of p-hydroxyphenyl acetic acid (HPA) and hydrogen peroxide in alkaline medium. After a one-step competitive immunoreaction, the polymer-antibody-antigen-hemin conjugate moiety was determined by coupling the fluorogenic reaction of HPA and hydrogen peroxide, The calibration graph for AFP was linear over the range of 0-380 ng cm(-3) with a detection limit of 1.0 ng cm(-3). This method combines some advantages of both homogeneous and heterogeneous immunoassays, and has been applied to determine AFP in human blood serum with satisfactory results

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

“…We have known that the appropriate immune reaction temperature was 25 ℃ when PNIP was used as a carrier. 5 However, in the case of using P(NIP-AA) as a carrier, the reaction temperature can be raised to 33 ℃, which is closer to the physiological environment temperature of an organism.…”

“…Afterwards PNIP was widely utilized as the separation carrier in immunoassays. [5][6][7][8] However, such assays can only be conducted under the LCST of the polymers, and not at the physiological environment temperature of an organism. This, therefore, would inevitably affect the immune reaction efficiency.…”

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].…”

“…The influence of the amount used for A-Ag or B-Ag conjugate was studied. Different volumes (10,20,30, 40 L) of immobilized Ag were chosen to get the different calibration curves for the determination of Ag. The linear range was 500-2000 ng mL −1 for 30 and 40 L of immobilized Ag and the fluorescence intensity hardly changed with concentrations below 500 ng mL −1 .…”

“…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]. However, such assays can only be conducted under the LCST of the polymers, and therefore, this would inevitably affect the immune reaction efficiency.…”