Cross-reactivity between Thy-1 and a component of intermediate filaments demonstrated using a monoclonal antibody

Dulbecco, Renato; Unger, Michael; Bologna, Mauro; Battifora, Hector; Syka, Peter; Okada, Shunsuke

doi:10.1038/292772a0

Cited by 108 publications

(24 citation statements)

References 12 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Woodsworth et al (1983) prepared mcABs against iPA; certain of these antibodies were highly specific for iPA while others cross-reacted with selected structurally related molecules. The enhanced cross-reactivity of mcABs as compared to polyclonal antiserum is well documented for other antigens (Lane and Hoeffler 1980, PiUemar and Weissman 1981, Dulbecco et al 1981) and possible explanations have been offered (Lane and Koprowski 1982). The ability to prepare mixtures of mcABs may be very useful for specific removal of selected cytokinins from a plant extract for subsequent analysis on HPLC or similar instrumentation as suggested by MacDonald et al (1981).…”

Section: Discussionmentioning

confidence: 92%

The development of monoclonal antibodies against the cytokinin zeatin riboside

Trione

Krygier

Banowetz

et al. 1985

J Plant Growth Regul

View full text Add to dashboard Cite

Abstract. Seven monoclonal anti-zeatin riboside antibodies were characterized by radioimmunoassay (RIA) and found to measure femtomole (10-15 M) quantities (-20 pg) of this cytokinin. The antibodies had different measuring ranges defined by the linear portion of the logit/log plots; slopes and intercepts of the line varied considerably between the antibodies. Competitive binding trials against cis-zeatin riboside (cZR), dihydrozeatin riboside (diHZR), zeatin (Z), and isopentenyl adenosine (iPA) showed differences among the seven antibodies in their cross-reactivities towards these structurally related cytokinins. It was possible to combine selected antibodies to provide a mixture with a predictable measuring range and cross-reactivity; the ability to prepare a highly specific reagent in this manner with well-defined reactivity was noted and differences between monoclonal antibody and polyclonal antiserum probes for measurement of cytokinins were discussed.Cytokinins play important roles in many plant physiological processes including the regulation of growth and development. Plant hormone research has been hindered by the lack of specific and sensitive methods for qualitative and quantitative assays, in part because cytokinins and other growth regulators

show abstract

Section: Discussionmentioning

confidence: 92%

The development of monoclonal antibodies against the cytokinin zeatin riboside

Trione

Krygier

Banowetz

et al. 1985

J Plant Growth Regul

View full text Add to dashboard Cite

show abstract

“…2). More ,than 50% of the enzyme activity was lost within 60 min, and over 90% inhibition of enzyme activity was observed in about 3 is due to the binding of antibodies to an antigenic site that is present on the unrelated enzyme proteins dihydrofolate reductase, pigeon liver fatty acid synthetase, and yeast glucose-6-phosphate dehydrogenase. Because all these enzymes possess dehydrogenase activities, they would be expected to have a common structural domain that binds NADPH.…”

Section: Resultsmentioning

confidence: 99%

“…Recent experiments with monoclonal antibodies have shown the existence of crossreacting sites on protein molecules that do not have homology as detected by conventional serological methods (1)(2)(3). For example, some monoclonal antibodies against large tumor (T) antigen of simian virus (SV40) have been demonstrated to crossreact with host proteins (4).…”

mentioning

confidence: 99%

Antibodies specific for NADPH-binding region of enzymes possessing dehydrogenase activities.

Katiyar¹,

Porter²

1983

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

The results reported in this paper show the presence of a population of antibodies in rabbit polyclonal antiserum that recognize an antigenic site at the NADPH-binding region of enzymes possessing dehydrogenase activities. Antisera from rabbits immunized with glucose-6-phosphate dehydrogenase or fatty acid synthetase were found to inactivate the enzyme dihydrofolate reductase. The inhibitory effect of this site-specific antibody is a time-and concentration-dependent reaction. This immunoinactivation is prevented by preincubation of the enzyme with NADPH. Immunization Protocol and Production of Antibodies. New Zealand White rabbits (5 to 6 months old) were immunized with purified preparations of fatty acid synthetase and glucose-6-phosphate dehydrogenase. Initially, 1 mg of enzyme emulsified with 2 ml of complete Freund's adjuvant was injected into each rabbit subcutaneously at 10 different sites. The animals were given booster injections at 2 and 4 weeks after the first injection at intramuscular and subcutaneous sites, and blood was withdrawn 10 days thereafter. In all cases blood was allowed to clot at 370C for 1 hr and then stored at 00C overnight. After removal of the clot, the rabbit serum IgG fraction was purified by precipitation with 50% saturated ammonium sulfate and chromatography on DEAE-cellulose (14).Enzyme Assays. Glucose-6-phosphate dehydrogenase activity was assayed spectrophotometrically by measuring the-rate of reduction of NADP at 340 nm in the presence of glucose 6-phosphate (15). The activity of pigeon liver fatty acid synthetase was determined by following the oxidation of NADPH in the presence of the substrates acetyl-and malonyl-CoA (16). Dihydrofolate reductase activity was measured by following the oxidation of NADPH spectrophotometrically in the presence of dihydrofolate (17).Immunotitration of Enzyme Activities. The antigen-antibody reaction was followed by an immunotitration procedure that we reported earlier (14,18,19). Immunotitrations were performed by incubating enzyme with various amounts of DEAEcellulose-purified (or ammonium sulfate-purified) antibody to glucose-6-phosphate dehydrogenase or pigeon liver fatty acid synthetase. Incubations were carried out in a buffer, pH 7.0, in the presence of 1 mM EDTA and bovine serum albumin at 1 mg/ml in a final volume of 100 A.l for various time intervals at 300C. In a typical titration increasing amounts of anti-pigeon liver fatty acid synthetase IgG (0-500 ,ug) were added to 3.44 ,ug of dihydrofolate reductase in the presence of 100 ,ug of bovine serum albumin in 0.1 M potassium phosphate buffer, pH 7.0/1 mM EDTA in a final volume of 100 A.1 After a fixed incubation time, aliquots (10 p.l) were assayed for the remaining dihydrofolate reductase activity. A similar protocol was used for immunotitrations involving other enzymes. RESULTS AND DISCUSSIONThe presence of an antibody population that is specific for binding at a NADPH-binding region of an enzyme suggested that t Permanent address:

show abstract

“…Our results may therefore be relevant to the interpretation of crossreactivities as they are occasionally observed with monoclonal antibodies (i.e., see refs. [18][19][20][21][22][23]. In the case of monoclonal antibodies the chemical nature of the immunogenic site is generally unknown, but in the case of anti-peptide reagents this site is very well defined as a short amino acid sequence, and the interpretation of crossreactivities is considerably facilitated.…”

Section: Discussionmentioning

confidence: 99%

On the nature of crossreactions observed with antibodies directed to defined epitopes.

Nigg¹,

Walter²,

Singer³

1982

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Antibodies directed against a synthetic peptide (src-c) containing the six carboxyl-terminal amino acids of p60rc, the transforming protein of Rous sarcoma virus, recognize p6Oarc. However, when used at sufficiently high concentrations they also react with a number ofconstituents ofuntransformed cells. These reactions can be completely inhibited by src-c peptide. Crossreactivities are to different components in cells from different species and cannot be attributed to p60C §, the ubiquitous cellular homologue ofp6j.C* By indirect immunofluorescence microscopy and immunochemical techniques we have identified three cytoskeletal proteins, myosin, tubulin, and vimentin, as well as an unknown intranuclear antigen, as major targets of anti-src-c antibodies in different untransformed cells. These crossreactivities probably reflect identities or similarities in the amino acid sequence ofthe immunogenic peptide and segments ofthe otherwise unrelated crossreactive proteins. These findings are discussed with respect to the interpretation of crossreactivities that are occasionally observed with anti-peptide sera and with monoclonal antibodies.An attractive possibility for producing antisera against particular proteins is to immunize animals with hapten conjugates made with synthetic oligopeptides corresponding in sequence to selected portions of these proteins (1-8). Because rapid progress in nucleotide sequence analysis allows the prediction of amino acid sequences of an increasing number of proteins, it may be expected that this synthetic approach to the production of monospecific antibodies will find widespread applications in the near future.Recently antibodies have been raised to a synthetic peptide (src-c) corresponding to the carboxyl-terminal hexapeptide sequence of p6src, the transforming protein kinase coded for by the src gene of Rous sarcoma virus (RSV). The amino acid sequence was deduced from the nucleotide sequence (9). These antibodies (referred to as anti-src-c) were then used to investigate the intracellular distribution of p6Osrc (10). By using low concentrations ofaffinity-purified anti-src-c antibodies, specific immunofluorescent labeling ofp6OsfC could be achieved. However, with increasing concentrations of antibody we observed labeling ofconstituents present in untransformed cells or in cells transformed by agents other than RSV. In the present study, these crossreactivities were investigated in detail in order to determine their significance, and in particular whether they might reveal functional or structural relationships between p60srC and the crossreactive normal cell constituents. The results of this study, however, have suggested to us that these crossreactions are fortuitous, but they may occur with some frequency with such anti-peptide antisera. These results also bear on the interpretation of unexpected crossreactivities observed occasionally with monoclonal antibodies. MATERIALS AND METHODSCell Culture and Immunochemical Reagents. Untransformed and RSV strain B77-transformed normal ...

show abstract

Cross-reactivity between Thy-1 and a component of intermediate filaments demonstrated using a monoclonal antibody

Cited by 108 publications

References 12 publications

The development of monoclonal antibodies against the cytokinin zeatin riboside

The development of monoclonal antibodies against the cytokinin zeatin riboside

Antibodies specific for NADPH-binding region of enzymes possessing dehydrogenase activities.

On the nature of crossreactions observed with antibodies directed to defined epitopes.

Contact Info

Product

Resources

About