Kinetics and proposed mechanism of the reaction of an immunoinhibition, particle-enhanced immunoassay

Abstract

We report kinetic studies on the reaction of a latex agglutination immunoassay used to quantify phenytoin in serum. In this assay, polystyrene particles with a covalently attached analog of phenytoin react with an antiphenytoin monoclonal antibody to form light-scattering aggregates, with the rate of this reaction being decreased by addition of phenytoin from sample. In the absence of free (sample) phenytoin, this reaction did not exhibit a maximum rate of agglutination in the presence of excess antibody, i.e., an equivalence point. Furthermore, agglutination was inhibitable by free phenytoin even when the latter was added after agglutination of particles with antibody had begun. Most significantly, the immunoagglutination proceeded in an identical fashion with monovalent F(ab) fragment. These data are consistent with low-affinity immunospecific particle–antibody complexation, which then induces colloidal aggregation, without requiring immunospecific bridging by antibody molecules. The described mechanism is not generalizable to all latex agglutination immunoassays, although disturbance of colloidal stability may be a component in most assays.

Effects of the Ionic Environment, Charge, and Particle Surface Chemistry for Enhancing a Latex Homogeneous Immunoassay of C-Reactive Protein

“…It is, however, interesting to note that some studies indicate an alternative mechanism of an initial immunorecognition event followed by colloidal instability, resulting in aggregation. This was witnessed by immunoagglutination for the PETINIA format with latex particles labeled with hapten in the presence of monovalent F AB fragments in solution that, therefore, could not allow any cross-linking to occur …”

Mechanism of Nanoparticle-Enhanced Turbidimetric Assays Applying Nanoparticles of Different Size and Immunoreactivity

“…1 Furthermore, proteins can show highly specific interactions such as antigen-antibody interactions 2 or enzymecatalyzed reactions. 2 Hence, specific protein interactions can be exploited to gain control over the process of particle aggregation of protein-coated particles 3 and used to design analytical techniques based on analyte-triggered particle aggregation. For diagnostic purposes, latices coated with antibodies that react with a specific analyte can be applied to relate the triggered particle aggregation to an analyte concentration by means of a fast and easy measurement like turbidimetry.…”

A modified quick PETIA for detecting anti-CCP antibodies in human serum

“…Due to the connection of antigen with antibody, the absorbance of the reaction solution is changed in a short duration time, and the change of absorbance is correlated with the change of concentration of the test antigen. 11 This detection mode has been used for some macromolecular protein antigens, such as C-reactive protein, rheumatoid factor etc. 12,13 PETIAs are characterized as methods that are simple, rapid, accurate, low cost, and easily automated and standardized.…”