Akira Kosaka

Improved reaction buffers for solid-phase enzyme immunoassay without interference by serum factors

A gelatin moderately digested by a protease, was as effective as original gelatin to remove the interference by serum factors with a sandwich immunoassay for insulin. Digestion of gelatin resulted in decrease in the viscosity, and the buffer containing the digested gelatin did not show gelation at 4 degrees C. Therefore the buffer involving the digested gelatin might be widely useful for the immunoassay without interference by serum factors.

Enzymatic determination of bilirubin fractions in serum

Novel enzymatic methods using bilirubin oxidase from Myrothecium verrucaria are described for the determination of bilirubin fractions in serum. These fractions include an unconjugated form (Bu), a conjugated form (Bc), and a delta fraction of bilirubin that reacts with direct diazo reagent and is irreversibly bound to serum albumin (B delta). The determination is based on the different reactivities of the enzyme to bilirubin fractions at different pH in the presence or absence of anionic detergent such as sodium dodecyl sulfate (SDS) or sodium cholate. Thus, in the absence of detergents, Bc and B delta are oxidized at acidic pH, while Bc is oxidized at alkaline pH; Bu is not oxidized at either acidic or alkaline pH. The first approach is based on measuring the decreased absorbance of bilirubin colour at 450 nm caused by the enzymatic oxidation. Total bilirubin is measured at pH 8.0 in the presence of SDS. Direct bilirubin is measured at pH 3.7 and Bc is measured at pH 10.0 in the absence of SDS, respectively. The second approach is made by coupling the diazo reaction with the enzyme reaction. Total and direct bilirubin are determined by a conventional diazo method without prior treatment by the enzyme. B delta is determined with a direct diazo method after the serum sample is treated at pH 10.0 by the enzyme to oxidize the Bc fraction. The precision and the accuracy of these methods were compared with a diazo method, an HPLC method and a slide method, and good results were obtained.

Use of antibody Fab′ fragments to remove interference by rheumatoid factors with the enzyme-linked sandwich immunoassay

Recently, we reported a method to remove the nonspecific interference by serum factors with the enzyme-linked sandwich immunoassay [ 11. Inclusion of gelatin with a relatively high concentration of salt in the immunoassay mixture was effective to remove the nonspecific interference by serum with the assay. However a specific interference with the assay system was still observed when sera containing rheumatoid factors were subjected to the assay. Interference by rheumatoid factors with sandwich immunoassay systems have been reported in [2]. Here, we describe a method to remove the interference by rheumatoid factors with the enzymelinked sandwich immunoassay of insulin. Use of Fab’ fragments of antibody IgG fractions for preparing the antibody-immobilized solid phase and the antibodyenzyme complex is effective to remove interference by rheumatoid factors with the enzyme-linked sandwich immunoassay system.

Use of gelatin to remove interference by serum with the solid phase enzyme-linked sandwich immunoassay of insulin

An enzyme-linked sandwich immunoassay system using the antibody Fab’-~-D~galactosidase complex and ~tibud~~ound glass or silicone rubber pieces as solid phases is highly sensitive 11-41. Even attomole amounts of macromolecular antigens are measurable in specified conditions [3]. However, the assay system is interfered with non-specifically by serum factors, when serum is present in the sample being analysed f4f* Interference by serum factors has been reported with both ~~dio~mmunoass~y [S,6] and enzyme immunoassay [7], especially when solid phases are used as a separation technique. Here, we describe a method to remove interference by serum with the enzyme-linked sandwich ~mmunoassay of insulin. fnclusion of gelatin, a h~dropl~o~~c protein, with a relatively high concentration of salt in the assay mixture is found to remove effectively interference by serum with the sandwich immunoassay of insulin.