https://www.cell.com/cell/pdf/0092-8674(82)90400-7.pdf

The catalog of human cytokeratins: Patterns of expression in normal epithelia, tumors and cultured cells

Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes.

https://www.cell.com/cell/pdf/0092-8674(87)90579-4.pdf

Dystrophin: The protein product of the duchenne muscular dystrophy locus

A monoclonal antibody to the microtubule-associated protein tau (tau) labeled some neurofibrillary tangles and plaque neurites, the two major locations of paired-helical filaments (PHF), in Alzheimer disease brain. The antibody also labeled isolated PHF that had been repeatedly washed with NaDodSO4. Dephosphorylation of the tissue sections with alkaline phosphatase prior to immunolabeling dramatically increased the number of tangles and plaques recognized by the antibody. The plaque core amyloid was not stained in either dephosphorylated or nondephosphorylated tissue sections. On immunoblots PHF polypeptides were labeled readily only when dephosphorylated. In contrast, a commercially available monoclonal antibody to a phosphorylated epitope of neurofilaments that labeled the tangles and the plaque neurites in tissue did not label any PHF polypeptides on immunoblots. The PHF polypeptides, labeled with the monoclonal antibody to tau, electrophoresed with those polypeptides recognized by antibodies to isolated PHF. The antibody to tau-labeled microtubules from normal human brains assembled in vitro but identically treated Alzheimer brain preparations had to be dephosphorylated to be completely recognized by this antibody. These findings suggest that tau in Alzheimer brain is an abnormally phosphorylated protein component of PHF.

https://www.pnas.org/content/pnas/83/13/4913.full.pdf

Abnormal phosphorylation of the microtubule-associated protein tau (tau) in Alzheimer cytoskeletal pathology

NEW ways of making antibodies have recently been demonstrated using gene technology. Immunoglobulm variable (V) genes are amplified from hybridomas or B cells using the polymerase chain reaction, and cloned into expression vectors. Soluble antibody fragments secreted from bacteria are then screened for binding activities (see ref. 1 for review). Screening of V genes would, however, be revolutionized if they could be expressed on the surface of bacteriophage. Phage carrying V genes that encode binding activities could then be selected directly with antigen. Here we show that complete antibody V domains can be displayed on the surface of fd bacteriophage, that the phage bind specifically to antigen and that rare phage (one in a million) can be isolated after affinity chromatography.

Phage antibodies: filamentous phage displaying antibody variable domains

A method has been devised for the electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets. The method results in quantitative transfer of ribosomal proteins from gels containing urea. For sodium dodecyl sulfate gels, the original band pattern was obtained with no loss of resolution, but the transfer was not quantitative. The method allows detection of proteins by autoradiography and is simpler than conventional procedures. The immobilized proteins were detectable by immunological procedures. All additional binding capacity on the nitrocellulose was blocked with excess protein; then a specific antibody was bound and, finally, a second antibody directed against the first antibody. The second antibody was either radioactively labeled or conjugated to fluorescein or to peroxidase. The specific protein was then detected by either autoradiography, under UV light, or by the peroxidase reaction product, respectively. In the latter case, as little as 100 pg of protein was clearly detectable. It is anticipated that the procedure will be applicable to analysis of a wide variety of proteins with specific reactions or ligands.

https://www.pnas.org/content/pnas/76/9/4350.full.pdf

Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.

The most abundant inhibitory neurotransmitter in the central nervous system, γ-aminobutyric acid (GABA), exerts its main effects via a GABAA receptor that gates a chloride channel in the subsynaptic membrane1. These receptors can contain a modulatory unit, the benzodiazepine receptor, through which ligands of different chemical classes can increase or decrease GABAA receptor function2,3. We have now visualized a GABAA receptor in mammalian brain using monoclonal antibodies. The protein complex recognized by the antibodies contained high- and low-affinity binding sites for GABA as well as binding sites for benzodiazepines, indicative of a GABAA receptor functionally associated with benzodiazepine receptors. As the pattern of brain immunoreactivity corresponds to the autoradiographical distribution of benzodiazepine binding sites, most benzodiazepine receptors seem to be part of GABAA receptors. Two constituent proteins were identified immunologically. Because the monoclonal antibodies cross-react with human brain, they provide a means for elucidating those CNS disorders which may be linked to a dysfunction of a GABAA receptor.

Co-localization of GABA A receptors and benzodiazepine receptors in the brain shown by monoclonal antibodies

High frequencies of antigen-specific hybridomas: dependence on immunization parameters and prediction by spleen cell analysis

Purification and characterization of recombinant human leukocyte interferon (IFLrA) with monoclonal antibodies.

Monoclonal antibodies (mAb) against a gamma-aminobutyric acid/benzodiazepine receptor complex (GABAA/BZR) were produced by using spleen cells from a mouse immunized with GABAA/BZR purified from bovine cerebral cortex. The mAb, most of which were of the IgG1 isotype could be divided into four groups (I-IV) specifying different antigenic structures. On immunoblots, group I mAb recognized exclusively the Mr 55,000 beta-subunit, while groups II and IV mAb recognized the Mr 50,000 alpha-subunit of bovine GABAA/BZR. Three of the four groups of mAb (I, III, and IV) crossreacted with both human and rat GABAA/BZR with the same subunit specificity as in bovine brain; the fourth group (II) crossreacted with human but not with the rat receptor. The binding sites for benzodiazepines as well as the high and low affinity GABA sites reside on the same structural complex as shown by immunoprecipitation. Ligand binding to these sites was not inhibited by mAb. Since quantitative immunoprecipitation of GABAA/BZR was achieved with mAb selective for either the alpha- or beta-subunit, both subunits occur in each individual receptor complex. The pattern of immunoblot staining suggests that the smaller alpha-subunit is not a processing product of the larger beta-subunit. Both alpha- and beta-subunits were present in all brain areas and species tested (rat cerebral cortex, cerebellum, and hippocampus; bovine cerebral cortex and cerebellum; human cerebral cortex). This suggests a uniform subunit composition of the receptor throughout the brain in contrast to earlier evidence for a heterogeneous subunit composition based on photoaffinity labeling.

Theophil Staehelin

Papers

Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.

Co-localization of GABA A receptors and benzodiazepine receptors in the brain shown by monoclonal antibodies

High frequencies of antigen-specific hybridomas: dependence on immunization parameters and prediction by spleen cell analysis

Purification and characterization of recombinant human leukocyte interferon (IFLrA) with monoclonal antibodies.

Monoclonal antibodies reveal structural homogeneity of gamma-aminobutyric acid/benzodiazepine receptors in different brain areas