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Activated, N-substituted acrylamide polymers for antibody coupling: application to a novel membrane-based immunoassay.

A room-temperature-precipitable, activated terpolymer consisting of N-isopropylacrylamide (NIPAAm)/N-n-butylacrylamide(nBAAm)/N-acryloxysuccinimide (NASI) (LCST = 7-13 degrees C) at a monomer feed ratio of 60:40:2.5, respectively, was prepared and conjugated to an antibody. The conjugate was evaluated in a novel cellulose acetate (CA) membrane-based immunoassay which utilizes the especially strong physical attachment of the polymer to CA to bind and concentrate the polymer attached protein onto the membrane. When compared in the CA membrane immunoassay to the antibody-poly(NIPAAm) conjugate prepared via anhydrous copolymerization of NIPAAm and NASI at the monomer feed ratio of 40:1, respectively, the performance of the NIPAAm/nBAAm/NASI terpolymer was superior to that of the NIPAAm/NASI copolymer (LCST = 32 degrees C) when the studies were carried out at room temperature. However, the terpolymer and copolymer gave equivalent performance when the assay mixture was heated to 45 degrees C. These results indicate the importance of the LCST of the polymer component of the Ab-polymer conjugate to its adsorption and binding on the CA membrane.

Acrylamides↗

Inhibition of leukotriene production by N-[4-[4-(diphenylmethyl)-1- piperazinyl]butyl]-3-(6-methyl-3-pyridyl) acrylamide (AL-3264), a new antiallergic agent.

The effects of AL-3264, which exhibits a 5-lipoxygenase (5-LO) inhibiting property by blocking histamine H1-receptors and inhibition of histamine release, were examined on leukotriene (LT) production and LT-mediated responses. AL-3264 (1-30 microM) inhibited the A23,187-induced LT production from human leukocytes with almost the same potency as that of nordihydroguaiaretic acid. AL-3264 (30-100 mg/kg, p.o.) inhibited the antigen-induced LT production in the abdominal cavity of passively sensitized rats; its effect was as potent as that of AA-861, a 5-LO inhibitor. AL-3264 (30 microM) suppressed both the initial and sustained phases of the antigen-induced contractions in isolated trachea from actively sensitized guinea pig. Phenidone (3 microM), a dual inhibitor of 5-LO and cyclooxygenase (CO), suppressed the sustained phase, while indomethacin was without effect on either phase. AL-3264 (40-160 mg/kg, p.o.) suppressed the arachidonic acid-induced ear edema in mice, for which 5-LO inhibitors were effective but antihistamines were not. The anti-edematous effect of AL-3264 (160 mg/kg) was reduced by intradermal administration of LTC4 (0.1 microgram). These results suggest that AL-3264 suppresses LT production in vivo and in vitro by inhibiting 5-LO activity, and this property may contribute to the antiallergic effect of AL-3264.

Acrylamides↗

Transfer in SDS of biotinylated proteins from acrylamide gels to an avidin-coated membrane filter.

Avidin was covalently linked to aldehyde-derivatized polyethersulfone membrane filters. These filters were used in Western blot analysis of proteins reacted with biotinylation reagents and electrophoresed in sodium dodecyl sulfate (SDS) on polyacrylamide gels. Electrophoretic transfer from the gels to these filters was in 0.1% SDS, in which the covalently bound avidin retained its biotin-binding capacity. We compared Western blots on avidin-coated membrane filters of biotinylated and nonbiotinylated forms of mouse immunoglobulin G (IgG), mouse IgG heavy chain, muscle-type acetylcholine receptor alpha subunit, and fused alpha and beta subunits of receptor. Biotinylated proteins were captured with high specificity compared to their nonbiotinylated counterparts and sensitively detected on the avidin-coated membranes.

Acrylamides↗