Chemical activation of nitrocellulose membranes for peptide antigen‐antibody binding studies: Direct substitution of the nitrate group with diaminoalkane

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Abstract
A method to covalently link peptide and proteins, through a diaminoalkane spacer to nitrocellulose membrane was developed for immunochemical applications. Initially the nitrocellulose membrane was modified by covalent incorporation of diaminoalkane spacers without any prior activation. The incorporation was shown primarily to involve α-elimination of the nitrate groups, and an imine was formed between the carbonyl group on the membrane and the diaminoalkane. The rate of incorporation increased exponentially with the length of the diaminoalkane as determined by a ninhydrin colorometric reaction, which was developed for the study. More than 200 nmole diamine per mg nitrocellulose could be incorporated, but less than 11 nmol/mg (63 nmol/cm2) was chosen in order to retain the strength of the membrane. The primary amino groups of the modified membrane was glutaraldehyde activated and the octapeptide, angiotensin II, was covalently bound. A dot immunoassay was performed where specific anti-angiotensin II antibodies reacted with the peptide and was visualized by peroxidase coupled secondary antibodies. The results were quantified by video densitometry above 0.005 μg All per cm2. The immunoassay showed improved detection of the peptide on the activated as compared to unactivated membrane as well as increased retention of radiolabeled [125I]angiotensin II.