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A simple method for covalent coupling of proteins to filter paper modified with quinone groups is described. This paper, termed ‘Key paper’ is flexible, stable on storage and does not require any activation before use. Proteins bound to Key paper can be detected by enzyme immunoassay, radioimmunoassay or Coomassie blue staining. Bound enzymes retain their enzymatic activity. Nucleic acids do not bind and do not interfere with the activity of the bound proteins. Because of its mechanical and chemical properties Key paper is a good matrix for electroblotting and for direct in situ analysis of proteins.

Conventional cleavage of linked polypeptide chains by heating in SDS can so alter molecular structure as to interfere with antibody binding, on which both immunoprecipitation and 'western blotting' depend. As an alternative, gentle treatment with acid at room temperature or at 0 degrees C was effective in separating the alpha and beta chains of human MHC Class II glycoprotein dimers and proved superior in terms of preservation of at least one labile epitope on the beta chain.

The reductive methylation procedure for proteins in solution has been adapted for tritium radiolabelling of surface components of macrophages, including the Fcγ receptor. High specific radioactivity of the receptor was obtained with no loss of IgG binding activity, tested by the solid-phase radioassay. To prepare the support for the assay, cellulose filter paper was CNBr-activated and a diamine hydrocarbon reagent was attached as the spacer chain. Next, TNP-hapten residues were covalently coupled to free amine groups. Antibody-coated TNP-paper discs were used as the solid phase for the competitive binding radioassay for the determination of IgG-binding activity of purified Fcγ receptor. The developed radioassay permits the detection of low affinity interactions and/or low quantities of Fc receptors.

Separation of mixtures of proteins by polyacrylamide gel electrophoresis followed by transfer of the proteins to support media such as nitrocellulose and detection by immunologic procedures provides a powerful analytic tool for assaying the antibody specificity of antisera or for following the purification of antigens. This technique requires fewer assumptions about antigenic solubility or antibody reactivity than immunoprecipitation methods. We present a glucose oxidase immunoenzyme staining procedure for protein blots and illustrate its use for detecting antibody to several viruses. The glucose oxidase immunoenzyme stain has a lower background than some peroxidase stains. We have detected as little as 1 μg/ml of antiviral antibody using this stain.

An immunoblotting procedure using a peroxidase-anti-peroxidase (PAP) complex was developed for the detection of autoantigens in crude mixtures by human autoimmune sera. Thymus proteins were transferred to a nitrocellulose sheet after electrophoresis in polyacrrylamide gels and probed with a 1:100 dilution of serum. The location and extent of immunoglobulin G (IgG) binding was determined by sequential reaction with: rabbit anti-human IgG, goat anti-rabbit IgG and rabbit peroxidase-anti-peroxidase complex. The peroxidase was allowed to react with chloronaphthol and low levels of autoantigen/autoantibody complex were detectable with virtual absence of background colour. The inclusion of human IgG and its pepsin-generated fragment provided a means of controlling and calibrating the blotting procedure.