Improving an Immunoassay Response to Related Polychlorinated Biphenyl Analytes by Mixing Antibodies

Abstract

Immunoassays for detection of a class of closely related antigens, e.g., PCBs, have often been too specific (responding strongly to some members of the class and missing others) and no general method for adjusting the response has been described. In this paper, the difference in the response of a model immunoassay to different Kanechlors (Japanese commercial mixtures of PCBs, analogous to Aroclors in the United States) is reduced from 20- or 50-fold (depending on which antibody is used) to 3-fold when the antibodies are mixed at the proper ratio. A mathematical model based on competitive binding of two antibodies for up to four antigens has been developed and used to describe the assay performance and to predict optimum mix ratios for the antibodies used. The model (based on separate measurement of each antibody's effective Kd for each Kanechlor) provides an excellent fit to the measured mixed antibody assay response. The model is also successful in identifying cases where mixing monoclonal antibodies will not improve the response. It is thought the method described will have applicability in a variety of cases where the analytical goal is semiquantitative screening based on the total quantity of an unknown mixture of related compounds.

New Antibody and Immunoassay Pretreatment Strategy to Screen Polychlorinated Biphenyls in Korean Transformer Oil

“…The reactivity values of the sera to a mixture of Aroclor 1242, 1248, 1254, and 1260 were analyzed by a KinExA 3000 instrument from Sapidyne Instruments Inc. (ID) based on their ability to bind to a chlorinated phenyl BSA complex, described previously. [8][9][10] Briefly, it is based on kinetic exclusion immunoassay, and consists of a capillary flow cell in a filter fluorometer with the means to automatically replace immunoactive solid-phase particles and flow samples. A mouse whose serum gave the highest signal by KinExA was selected, and was given a final booster shot.…”

“…[8][9][10][11][12][13][14][15][16] All of our prior work has been focused on Kanechlors (Japanese technical PCB mixtures), which are analogous, but not identical, to U. S. technical PCB mixtures known as Aroclors. Previously described Aroclor immunoassays suffer from ambiguous results due to the antibody's differing reactivity to various Aroclors.…”

Development and Verification of a Model for Estimating the Screening Utility in the Detection of PCBs in Transformer Oil

“…We have previously reported a pretreatment method to extract PCBs from transformer oil 5 and the development of immunoassay systems to measure PCBs easily. [6][7][8] We also evaluated the performance (sensitivity and specificity) of the immunoassay systems when used to screen for PCB contamination in Japanese 9,10 and Korean 11 transformer oil.…”

A multiplexed immunoassay system based upon reciprocating centrifugal microfluidics

“…Generally, the rate of formation of antigen-antibody complexes, which is proportional to the concentration of the antibodies and antigens, dictates the required incubation time for generating a detectable result. This is why it is desirable to introduce other transport mechanisms such as migration and convection into the system to cause turbulence in the antibody solution and/or to generate flow and increase the rate of formation of antigen-antibody complexes 19,20 by employing different innovative methods. A promising methodology that has been examined in recent decades as a candidate for μTAS is the fabrication of microfluidic systems on rotating platforms, or compact discs (CD).…”

“…Beads of different sizes are often used to serve both as solid phases functionalized with capture antigens and to increase the interaction rate by intermittently mixing the reagents on the disk. [16][17][18][19][20][21][22][23] The rate of interaction will also increase by flowing the antibody solution over the array of immobilized antigens and hence increasing the rate of mass transport. [24][25][26] Rotating platforms are especially suitable for flow-through capture systems because the flow can be readily generated by the centrifugal acceleration.…”