Disposable microliter immunoabsorbent columns: construction and operation

Abstract

Disposable microliter immunoabsorbent columns were constructed from pasteur pipets. The bed support was a cube of gelatin surgical sponge, which was tamped into the pipet tip. Column dead space, represented by the compressed volume of the sponge, was 5 microliters. The columns were used with protein A-Sepharose; settled bed volumes were 50 microliters. It was possible to pour columns that functioned as immunoabsorbents with bed volumes as small as 10 microliters. There was no gravity flow through these columns. Flow was achieved by touching column tips to absorbent paper if liquid was to be discarded or to 50 microliters capillary tubes for fluid collection. A simple capillary collection tube assembly was designed for operation of a row of 10 columns at a time. In a test system of [3H]methotrexate and IgG anti-methotrexate, 90% of applied antigen was bound to antibody columns, whereas 90% was recovered in the eluates from control columns. The columns were used in the initial step of screening uncloned hybridoma culture fluids for anti-MSP.