CHEMICAL ACTIVATION OF NITROCELLULOSE MEMBRANES FOR PEPTIDE ANTIGEN-ANTIBODY BINDING-STUDIES - DIRECT SUBSTITUTION OF THE NITRATE GROUP WITH DIAMINOALKANE

A method to covalently link peptide and proteins, through a diaminoalk ane spacer to nitrocellulose membrane was developed for immunochemical applications. Initially the nitrocellulose membrane was modified by c ovalent incorporation of diaminoalkane spacers without any prior activ ation. The incorporation was shown primarily to involve alpha-eliminat ion of the nitrate groups, and an imine was formed between the carbony l group on the membrane and the diaminoalkane. The rate of incorporati on increased exponentially with the length of the diaminoalkane as det ermined 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 ord er to retain the strength of the membrane. The primary amino groups of the modified membrane was glutaraldehyde activated and the octapeptid e, angiotensin II, was covalently bound. A dot immunoassay was perform ed where specific anti-angiotensin II antibodies reacted with the pept ide and was visualized by peroxidase coupled secondary antibodies. The results were quantified by video densitometry above 0.005 mug AII per cm2. The immunoassay showed improved detection of the peptide on the activated as compared to unactivated membrane as well as increased ret ention of radiolabeled [I-125]angiotensin II.