Specific Protein Analysis by Light-Scatter Measurement with a Miniature Centrifugal Fast Analyzer

Abstract

A miniature Centrifugal Fast Analyzer has been modified for fluorescence and light-scatter measurements by using several rotors developed for this purpose. The modified system has been used to evaluate the feasibility of adapting specific protein analyses, such as IgG, IgA, IgM, C'3 complement component, and α-1-antitrypsin, to the Centrifugal Fast Analyzer. A study of reaction conditions has revealed that the addition of polyethylene glycol 6000 (Carbowax) to the dilution medium increases the rate of reaction and allows apparent equilibrium to be achieved in less than 60 s. Furthermore, scatter intensity is enhanced. This system can be used to make rapid immunoglobulin measurements with only microliter volumes of antibody (2-7 µl), without the need of sample blanks. Determination of antigen excess by a method that involves dynamic injection of antibody is also discussed.

Automated Instrumentation and the Laboratory Diagnosis of Bleeding and Thrombotic Disorders

“…Several other investigators have also reported on the adaptation of centrifugal analyzers in light scatter technology. 22 ' 41,125 It is expected that many automated methods based on nephelometry will become available for coagulation proteins.…”

Multipoint kinetic method for the immunochemical quantitation of isoenzymes developed and evaluated with creatine kinase inhibition as a model system

“…For a zero-order enzyme-catalyzed reaction producing product, P, the velocity can be written as V7 = dPt/df = feo-Et (1) in which Vy is the velocity, P is product concentration, k0 is a zero-order rate constant, and Et is the instantaneous enzyme concentration. For the uninhibited subunit, the velocity will remain constant, and the time-dependent product concentration will be given by , = V\0t (2) in which Pt[0 is the time-dependent product concentration for the zero-order (uninhibited) component of the reaction and V'i|0 is the initial velocity of the zero-order component.…”

“…Although these equilibrium-based procedures have proven effective, they do not represent the only option and it is probable that kinetic-based procedures could offer complementary capabilities as has been the case with more traditional types of reactions. A few studies that involve measurements during the kinetic phases of antigen/antibody reactions have been reported (e.g., ref [1][2][3], but these studies only begin to exploit the capabilities of kinetic-based immunoassays. To date no one has reported the use of multipoint kinetic data to resolve two or more components simultaneously.…”

Standardization of light scattering measurement in conjunction with immunochemical analysis

“…The centrifugal analyzer has been applied to the automated kinetic measurement of antigen antibody reactions. Centrifugal analyzers have been used to measure scatter from antigen antibody complexes at 90° [ 7 ] and at angles between 15 and 30 degrees [ 8 ], with the smaller angles providing greater sensitivity due to the large complex size.…”