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Studies of immobilized antibody supports and their use in chromatographic immunoassays
Abstract
A method of preparing dihydrazide-activated silica was developed for use in high-performance affinity chromatography (HPAC). This support was made by oxidizing diol-bonded silica and reacting it with oxalic or adipic dihydrazide. The steps involved in this synthesis were studied and confirmed by FTIR. Items considered in optimizing the preparation of the support included the amount of dihydrazide added and the reaction time or pH used. Control of dihydrazide bifunctional attachment was obtained by varying the extent of diol-bonded silica oxidation. This support was successfully used in the immobilization of oxidized antibodies, horse radish peroxidase and transfer RNA. The activity of the immobilized antibodies on dihydrazide activated silica were compared to those attached to silica by the Schiff base and CDI methods. The dihydrazide method produced antibodies with an activity that was equivalent to or greater than that produced by the amine-based coupling methods. This dihydrazide activated support was used in the immobilization of anti-HSA antibodies in the development of an automated immunoassay system for the simultaneous analysis of albumin and creatinine in urine. Albumin was determined by injecting samples onto a column containing immobilized anti-albumin antibodies. A flow injection analysis (FIA) system following this column was used to quantitate creatinine eluting in the non-retained peak. Items considered in the optimization of this system included the binding and elution properties of the immobilized antibody column and the reagent concentrations used in the FIA system. The total analysis time of the final method was five minutes per 20 $\mu$l injection. The limits of detection for albumin and creatinine were 0.005 and 0.008 mg/ml and the linear ranges covered a 200 to 1000-fold range in concentration. The within-run precision for both analytes was $\pm$2% or better and the results showed good agreement with those obtained by reference methods. The approach described is a general one that can be applied to other analytes in urine by using different immobilized antibody columns.
Subject Area
Analytical chemistry
Recommended Citation
Ruhn, Peggy Faye, "Studies of immobilized antibody supports and their use in chromatographic immunoassays" (1994). ETD collection for University of Nebraska-Lincoln. AAI9510979.
https://digitalcommons.unl.edu/dissertations/AAI9510979