Immunoaffinity Column Validation Steps

1. Calculation of binding capacity: if IgG is the antibody used for immobilization the molar ratio between the IgG and the antigen (analyte) is 1:2 because IgG is bivalent. Thus, the theoretical binding capacity of the column is as follows:

Binding capacity (mass) = (mass IgG x 2 x mol. wt. analyte)/mol wt. IgG.

It should be noted that the mass of IgG used in the numerator refers to the mass of IgG immobilized per column (50 mg in our example), the multiplier of "2" denotes the bivalent nature of IgG, and the molecular wt. of the analyte should be provided on a millimole basis (i.e. mg/mmol). The nominal molecular weight of 150,000 mg/mmol for IgG is used in the denominator.

For small molecule analytes (see Note 6) for which a radiotracer form is available, sequentially load a known quantity of tracer dissolved in buffer and determine the amount of analyte in the eluant. When the radioactivity not retained by the immunoaffinity column plateaus, the column binding sites are saturated. Wash the column, and elute the retained radioactivity. The mass of analyte in the eluted volume is the apparent column capacity. In many instances a radiolabeled analyte may not be available. In such cases, high-performance liquid chromatography, UV spectroscopy, or any other analytical tool capable of selectively quantifying the analyte may be used to determine column capacity.

2. Determination of column stability: test the column stability by applying a known amount of analyte; perform sample loading, column wash, analyte elution, column regeneration, and storage cycle steps for multiple analyses. Initially we test the columns' reusability daily for a week, then weekly for a month, then monthly for up to 3 mo.

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