Biomedical subjects
Publications and source records attributed to R Thorpe.
Two monoclonal antibodies were found to give enhanced affinity for beta-lipotropin when mixed, as evidenced by competitive radioimmunoassay. Both monoclonals were found to react with a pentapeptide Ala-Glu-Leu-Glu-Tyr, which is a sequence of high local hydrophilicity within the N-terminal section of beta-lipotropin.
A monoclonal antibody to porcine beta-lipotropin has been produced which binds to the N-terminal (gamma-lipotropin) portion of the molecule. The antibody can be used to detect beta-lipotropin as well as other beta-endorphin precursors (predominantly a Mr 38 000 polypeptide) using radiobinding assay or the immunoblotting technique. Purification of the peptides can be readily achieved by affinity chromatography using the monoclonal antibody covalently bound to Sepharose 4B. As the antibody recognises the N-terminal part of beta-lipotropin, it can be used to detect and purify beta-lipotropin and other beta-endorphin precursors in the presence of beta-endorphin.
Investigation of the immunological reactions with individual poliovirus capsid polypeptides of antisera and monoclonal antibodies raised against poliovirus type 3 antigens are described. Virus polypeptides were separated by electrophoresis, transferred electrophoretically to nitrocellulose sheets and treated with antibody preparations. Antibody binding specifically to the virus polypeptides was then detected by application of 125I-labelled anti-immunoglobulin followed by autoradiography. The technique readily enabled the identification of the polypeptides recognized by the antibody. Antibodies present in polyclonal, type-specific neutralizing sera to poliovirus type 3 bound to the two largest capsid polypeptides (VP1 and VP2) of the homotypic poliovirus, and also to the VP1 of poliovirus type 1 and type 2. There was no obvious difference between the antibody binding patterns obtained with neutralizing and non-neutralizing antisera or between C-specific and D-specific antisera. VP1 appeared to be the immunodominant virus polypeptide. Among monoclonal antibodies specific for the C antigen of poliovirus type 3, a proportion reacted homotypically with the VP1 of poliovirus type 3. Other monoclonal antibodies of C antigen or D antigen specificity, or which reacted both with D and C antigens, some of which had potent virus-neutralizing activity, failed to give demonstrable binding reactions. The non-correlation of neutralization and immunoblot reactivity suggests that sequence determinants alone do not mediate virus neutralization which may depend on antigenic determinants specified by complex conformational arrangements of the virus capsid proteins.
We have produced a monoclonal antibody that reacts with all classes of intermediate filaments in immunofluorescence assays, including glial filaments in astrocytes, neurofilaments in axons, tonofilaments in epithelial PtK2 cells and intermediate filaments in fibroblasts. It also binds to Z lines in skeletal muscle. In SDS-polyacrylamide gels, the antibody binds to most and perhaps all of the major intermediate filament proteins that have been previously defined, including glial fibrillary acidic protein, the three vertebrate neurofilament proteins (the "neurofilament triplet"), vimentin, desmin, several cytokeratins and the neurofilament proteins of squid and the marine worm Myxicola. In addition, the antibody binds to a protein with an approximate molecular weight of 66,000 that may be a component of all intermediate filaments. These findings suggest that all vertebrate and invertebrate intermediate filament proteins share a common antigenic determinant and raise the possibility that all intermediate filaments contain a 66,000 molecular weight protein.
Rabbit antisera have been produced to bovine brain 10 nm filament preparations and to purified polypeptide constituents of brain 10 nm filaments. Antisera to the former preparation stain both neurons and astrocytes, whereas antisera to two polypeptides of mol. wt 155 000 and 210 000 are neuron specific. It is therefore concluded that these latter polypeptides are components of neurofilaments and the mixed staining pattern obtained with the antisera to the whole brain 10 nm filament preparation is due to the presence in this material of a mixture of neurofilaments and glial filaments which are thus biochemically distinct forms of 10 nm filament.
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Brain 10 nm filaments were isolated from bovine, rabbit and rat brains by a modification of an existing procedure. The overall polypeptide composition of these preparations was similar to that previously reported for brain neurofilaments. In addition to the major polypeptide component, which has mol. wt. approx. 50 000, three other polypeptides with chain mol. wts. approx. 210 000, 155 000 and 70 000, which correspond to peripheral-nerve neurofilament polypeptides, were consistently found to be present. The mol. wt.-50 000 species was found to be heterogeneous and may contain a component derived from the mol. wt. 70 000 polypeptide. The three higher-molecular-weight polypeptides did not appear to be obviously homologous or to be homologous with myosin or Myxicola neurofilament polypeptides. These same three higher-molecular-weight components were shown to be identical with the polypeptides probably responsible for the 10 nm filaments formed during the early cycles of the tubulin-purification protocol.
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An antiserum has been raised to purified alpha-L-fucosidase. Levels of cross-reaction with serum of two unrelated fucosidosis patients and normal individuals with low activity are consistent with the presence of very low amounts of normal enzyme. Similarly no cross-reacting material could be found in cultured fibroblasts from fucosidosis patients. It is deduced that in these cases there is no production of mutant enzyme in quantities comparable to normal levels. Some observations on the interrelations of fucosidases I and II are reported.
The levels of three lysosomal glycosidase, alpha-D-mannosidase, a-L-fucosidase and beta-D-hexosaminidase have been determined in normal hair roots and in hair roots obtained from a patient with mannosidosis. The most active glycosidase in normal hair roots was beta-D-hexosaminidase, followed by alpha-L-fucosidase and alpha-D-mannosidase. There was no alpha-D-mannosidase activity in the hair roots of the patient with mannosidosis. The significance of these results is discussed in relation to the detection of lysosomal storage diseases.
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