A highly sensitive affinity-column-mediated immunometric assay, as exemplified by digoxin.

Abstract

We describe a highly sensitive heterogeneous enzyme-linked immunoassay in which digoxin is used as the model analyte. An excess of enzyme-labeled monovalent antibody is incubated with sample containing the analyte such that all analyte is rapidly and quantitatively bound. Excess antibody that does not acquire a antigen in its binding site is rapidly removed from the mixture by passage through a porous affinity column containing immobilized analyte (or analog), present in vast excess. Only the labeled monovalent antibody that possesses an antigen in its binding site elutes from the column in the unbound fraction. The label present in this fraction is then quantified. Such an assay is extremely sensitive and obviates the limitations imposed by antibody affinity constants on homogeneous and competitive heterogeneous immunoassays. This assay can be performed rapidly and is readily amenable to automation.

Development of a microcolumn one-site immunometric assay for a protein biomarker: Analysis of alpha1-acid glycoprotein

“…The 0.25 mg L -1 AGP was added to all samples and standards to avoid the small non-linear response seen at low AGP concentrations in Figure 5. As mentioned in the previous section, this non-linearity was probably due to binding by some antibodies to both soluble and immobilized AGP through their two available binding sites to this target [9]. The overall mixture was allowed to incubate for at least 5 min…”

“…Under these conditions, the binding of Ab* with A in the reaction given earlier in Eq. (1) can be approximated by an irreversible second-order reaction [9] with the following rate expression.…”

“…A one-site immunometric assay is one format that can be used in a chromatographic immunoassay (see Figure 1) [9][10][11][12][13][14][15][16][17]. In this method, a sample or standard that contains the target analyte is combined and incubated with an excess amount of a labeled binding agent for the target (e.g., an intact antibody or F ab fragment).…”

“…The amount of non-bound labeled agent that remains after the incubation step is removed by passing this mixture through a column or support that contains an immobilized analog of the target. A signal that is related to the amount of analyte in the original sample is then obtained by measuring the amount of labeled binding agent that elutes non-retained from the column or that is captured by this column [9][10][11][12][13][14][15][16][17].…”

“…One-site immunometric assays have been previously used with traditional-sized columns and a number of targets with small-to-intermediate sizes (i.e., molar mass < 18 kDa) [9][10][11][12][13][14][15][16][18][19][20][21]. Examples have included assays for targets such as digoxin [9,10], thyroxine [11,15], β-estradiol [12], α-(difluoromethyl)ornithine [13], 2,4-dichlorophenoxyacetic acid [14], D-phenylalanine [16], interleukin-10, and human methionyl granulocyte column stimulating factor [9][10][11][12][13][14][15][16][17][18]. It has only recently been shown that the same method can be used for proteins, as has been demonstrated for human serum albumin (HSA, the major protein in serum; normal concentration, 39-53 g L -1 ; molar mass, 66.5 kDa) [19,20].…”