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D W Metzger

Publications and source records attributed to D W Metzger.

At least 55 records · Page 3Linked to original sources
PubMed1986

Heating of immunoglobulins for immunoblot analysis destroys variable region antigenicity.

The antigenicity of rabbit immunoglobulin heavy chain variable region allotypes was examined by immunoblotting after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The standard method of sample preparation resulted in a total loss of immunoreactivity with anti-allotype antibody due to heating of the samples prior to their electrophoresis. Thus, caution is warranted in interpreting results obtained from analysis of Ig when such proteins are heated before or during separation. In addition, we describe a method that permits not only separation of Ig heavy and light chains in SDS-PAGE but also preserves V region antigenicity.

Animals↗
PubMed1986

Multiple epitopes in a dodecapeptide of myelin basic protein determined by monoclonal antibodies.

Three custom synthesized myelin basic protein (MBP) peptides, bovine peptide 79-88, human peptide 80-89, and human peptide 82-91, were used to produce four murine monoclonal antibodies (MAb) that were selected on the basis of reaction in a solid phase radioimmunoassay (SRIA) with human MBP. The MAb were compared with respect to antigen specificity against intact MBP and 10 overlapping MBP peptides. One MAb recognized an epitope near the amino-terminus of bovine MBP peptide 79-88. A second MAb was directed towards an epitope that is more reactive in human MBP peptide 45-89 than in intact MBP, but is not recognized in any of the small MBP peptides examined. The third MAb detected an epitope near the middle of human MBP peptide 80-89, whereas the fourth MAb reacted with the carboxyl-terminal portion of human MBP peptide 82-91. Epitopes recognized in SRIA were sometimes not detected by the same MAb in a fluid phase double antibody radioimmunoassay. These results demonstrate the multiplicity of potential epitopes in a dodecapeptide of MBP and do not support the concept of a single, dominant epitope in the region of MBP peptide 80-89.

Amino Acid Sequence↗
PubMed1986

Mouse monoclonal antibodies induced by anti-allotype antibody display internal images of the rabbit VHa1 allotype: direct visualization by immunoelectron microscopy.

We have previously shown that injection of adult rabbits with anti-immunoglobulin (Ig) antibody (Ab) induces the expression of genetically unexpected Ig markers, i.e., a1 allotypic determinants. We now show that these rabbit Ig markers can also be induced in mice by a similar treatment; in the latter case the a1 determinants are located in the antigen-combining site and thus represent "internal images". Two monoclonal antibodies (mAb) were developed from mice treated with anti-allotype Ab. These mAb were reactive with all homologous and heterologous anti-a1 Ab but not normal Ig; efficiently inhibited the binding of rabbit a1 Ig to anti-a1 Ab; and elicited the production of anti-allotype Ab when injected into normal mice. To determine whether the a1-like determinants on these mAb were located in the antigen-combining site, immunoelectron microscopy was utilized to directly visualize Ab complexes. Complexes composed of intact Ab and anti-a1 Fab fragments yielded uniform binding patterns which were identical to those produced by anti-idiotypic reactions. In each case, an identical tip-to-tip binding configuration was observed with a single Fab fragment attached at an approximate 180 degree angle to the V region terminus of each Ab arm. In contrast, rabbit a1 Ig bound as many as two anti-a1 fragments per Ig arm; these fragments were attached laterally and at right angles to the intact molecule. These mAb thus provide the first direct evidence that Ab2 beta determinants are located in, or near, the antigen-combining site.

Animals↗
PubMed1985

The nature of antiidiotype molecules induced by antiallotype. Presence of both latent allotype and allotypic internal images.

Previously (9), I found that immunization of rabbits with antibody directed against variable region heavy chain VH polypeptides of a1 allotype induced the production of antiidiotype (anti-Id) molecules that appeared to bear images of the original a1 allotype. I now show that these anti-Id molecules can be fractionated into two populations: one population (a2a3- anti-Id) that lacks the nominal VH a2 or a3 allotype of the rabbit from which it was derived, and another population (a2a3+ anti-Id) that expresses these allotypes. Both anti-Id populations display epitopes that resemble a1 since: (a) they were capable of inhibiting 125I-a1 Ig binding to rabbit anti-a1, goat anti-a1, and mouse anti-a1 mAb; and (b) immunization of normal a2a3 rabbits with either anti-Id fraction led to the formation of specific anti-a1 antibody. Reductive cleavage of the anti-Id molecules showed that the a1 determinants in the a2a3- population were fully displayed on isolated H chains, consistent with the presence of latent a1 Ig. On the other hand, as expected for internal images encoded by the antigen-combining site, the a2a3+ anti-Id population required intact H and L chains for maximal a1 expression. The a1-like images within the a2a3+ anti-Id population do not appear to be identical to nominal or latent a1, however, since a2a3- anti-Id was invariably a more efficient inhibitor of a 1 Ig-anti-a1 binding than a a2a3+ anti-Id. These results indicate that immunization with antiallotype can result in the simultaneous production of both latent allotypes and allotypic internal images.

Animals↗
PubMed1985

Monoclonal antibodies to glycoproteins of Vinca alkaloid-resistant human leukemic cells.

Drug resistance is a major problem in the successful treatment of cancer. Resistance to one drug is often associated with cross-resistance to other anticancer agents. This is commonly seen with the "natural product" anticancer drugs such as the Vinca alkaloids and anthracyclines. In experimental systems, specific changes in plasma membranes characterize this "multiple drug resistance." The most prominent of these is the enhanced expression in several systems of high-molecular-weight glycoproteins ranging from Mr approximately equal to 150,000 to approximately equal to 180,000, the amount of which has been shown to be related to the degree of drug resistance. We report here the production of three monoclonal antibodies that bind preferentially to the surfaces of cultured human leukemic lymphoblasts resistant to the Vinca alkaloid vinblastine. Each antibody recognizes a surface membrane glycoprotein with molecular weights of 180,000 to 210,000. Additionally, two of the antibodies also recognize a second surface glycoprotein with molecular weights of either approximately equal to 155,000 or approximately equal to 130,000. All of these glycoproteins are overexpressed in the alkaloid-resistant cells. While a Mr approximately equal to 180,000 protein has been shown to be associated with multiple drug resistance, the other two glycoproteins have not been described previously in these cells. These antibodies may be useful in studies of the mechanisms of drug resistance, as well as in screening cells from drug-resistant patients for these resistance-associated glycoproteins.

Antibodies, Monoclonal↗
PubMed1984

The expressed lysozyme-specific B cell repertoire. I. Heterogeneity in the monoclonal anti-hen egg white lysozyme specificity repertoire, and its difference from the in situ repertoire.

A panel of closely and distantly related lysozymes and lysozyme-peptide fragments were utilized in assessing the specificity repertoire of murine anti-hen egg white lysozyme hybridomas. The 44 monoclonal antibodies could be divided into a minimum of 18 fine specificity groups in tests using the lysozyme panel. Two hybridoma products were specific for epitopes containing amino acids 68 and 121, respectively; and another was specific for an epitopes containing amino acids 113-114. Several hybridomas demonstrated unique heteroclitic binding, for example, to bob-white lysozyme (BEL), but not other closely related lysozymes, suggesting lysine at position 68 in BEL as an important residue of recognition. Radioimmunoassay using lysozyme peptides bound to plastic plates specified the regional specificity of 6 additional antibodies of the 44. A comparison of the specificities of monoclonal antibodies with antibody produced in vivo showed some major differences suggesting that those cells proceeding on to antibody formation in the regulatory milieu of the whole animal are a selected subpopulation.

Animals↗
PubMed1984

A mouse monoclonal antibody against rabbit VH allotype shares the predominant idiotype with a rabbit antibody of the same specificity.

A monoclonal antibody, termed 3-2F1, is specific for the rabbit VH al allotypic marker. This antibody appears to recognize a common al determinant since it reacts with all of the a1 immunoglobulin (Ig) that is bound by polyclonal rabbit anti-a1, and completely inhibits the binding of polyclonal anti-a1 to a1 Ig. When tested for the predominant IdX-a1 idiotype previously shown to be expressed on virtually all rabbit anti-a1 antibody, it was found that 3-2F1 also bears this idiotype. In fact, sequential absorption and inhibition assays indicate that 3-2F1 displays the entire set of rabbit IdX-a1 determinants. The results are consistent with the concept that the anti-IdX reagent bears a latent internal image of the common VH a1 epitope.

Absorption↗
PubMed1984

Induced latent allotypes within rabbit anti-cross-reactive idiotype reagents. Direct immunoelectron microscopic evidence.

Immunization of rabbits with anti-VHa allotype antibody leads to the production of an "anti-cross-reactive idiotype (IdX)" reagent which appears to recognize a highly conserved IdX on anti-allotype antibodies. In many rabbits, a large percentage of this "anti-IdX" reagent does not express the nominal VHa framework allotype of the rabbit from which it was derived and is otherwise serologically indistinguishable from the original antigen, i.e. a1 or a2 allotype. To determine whether these anti-IdX molecules were in fact anti-IdX (or internal images) or were induced immunoglobulins bearing latent VHa allotypes, immunoelectron microscopic analysis of the molecules in complex with Fab anti-allotype antibody was conducted. The results show that the complexes do not resemble idiotype-anti-idiotype interactions but are essentially indistinguishable from allotype-anti-allotype complexes. We conclude that a major component of the "anti-idiotype" response of these anti-VHa-immunized rabbits is the production of Ig molecules bearing latent VHa allotypes.

Animals↗
PubMed1984

Characterization of a monoclonal antibody reactive with rabbit T lymphocytes and neutrophils.

An IgG1 monoclonal antibody, termed ACM-1, has been shown to react with rabbit T cells, but not Ig+ cells or macrophages. This antibody appears to recognize the same epitope as the previously described 9AE10 antibody and, together with 9AE10, has been used to obtain highly pure and fully functional T- and B-cell populations. However, the relevant epitope does not appear to be homologous to rodent Thy-1 since quantitative absorptions failed to show reactivity with rabbit brain. Furthermore, attempts to obtain in vivo T-cell depletion resulted in larger decreases in white blood cells than would be expected for simple T-cell removal. In vitro assays on enriched neutrophil preparations revealed that 80-95% of these cells were reactive with ACM-1 and 9AE10. Thus, it appears that in the rabbit, T cells and neutrophils share a major epitope.

Animals↗
PubMed1984

Localization of frog virus 3 proteins using monoclonal antibodies.

Thirty-seven monoclonal antibodies to seven frog virus 3 (FV3) structural proteins were isolated and used to examine the distribution of viral proteins within virions and infected cells. Three monoclonal antibodies, one to the major capsid protein, VP55, and two to VP38 had detectable neutralizing activity suggesting that these proteins are located on the surface of virions. Immunofluorescent studies showed that VP108, VP57, VP55, and VP16 were localized mainly within virus assembly sites, while VP17 was detected in both assembly sites and the surrounding cytoplasm. The abundance of viral structural proteins within assembly sites is consistent with the idea that virion maturation occurs exclusively within assembly sites.

Animals↗
PubMed1984

Cross-reactive idiotypes on heterologous anti-allotype antibody.

Specific anti-a1 Ab was isolated from rabbits, guinea pigs, mice, chickens, and a goat. Each of these preparations was able to inhibit the reaction between a1 IgG and rabbit anti-a1 Ab as well as the reaction between rabbit IdX (anti-a1 Ab) and rabbit anti-IdX (anti-anti-a1 Ab). Maximal levels of inhibition ranged from 75 to 100% in the latter assay. Although the relative binding efficiencies of each preparation varied widely, there was generally a positive correlation between the ability of an anti-allotype reagent to bind to a1 IgG and to anti-IdX Ab. Each of the heterologous anti-a1 Ab samples was able to form precipitin bands with rabbit anti-Id Ab. These bands fused with each other and with rabbit anti-a1 Ab. These results weaken the interpretation that the ubiquitous expression of IdX previously observed in the rabbit reflects shared conserved genes. We suggest that either 1) the anti-Id Ab represents high fidelity internal images of the a1 epitopes, or 2) latent a1 allotype Ig is present in the anti-IdX preparation. In either case, any anti-a1 Ab would be potentially reactive with the anti-IdX preparation.

Animals↗
PubMed1983

A monoclonal antibody (SJ-9A4) to P24 present on common alls, neuroblastomas and platelets - I. Characterization and development of a unique radioimmunometric assay.

We report the development and characterization of SJ-9A4, a monoclonal antibody (MoAb) produced against common acute lymphoblastic leukemia (C-ALL) cell lines. SJ-9A4 reacted with C-ALL, B-cell chronic lymphocytic leukemia (B-CLL), platelets and C-ALL neuroblastoma (NB) and the K562 cell lines. It had no significant reactivity with erythrocytes, granulocytes, circulating T or B lymphocytes, monocytes, granulocytic cell lines or a Ewing's sarcoma cell line. SJ-9A4 was shown to recognize the same region as two other MoAb to the p24 antigen, BA-2 and DU-ALL-1, as demonstrated by their ability to inhibit the binding of labeled SJ-9A4 to NALM-1 and NB cells. Other MoAb: J5, PI 153/3 and monoclonal anti-HLA-DR antibodies gave no inhibition. A solid phase indirect radioimmunometric assay (IRA) was developed which enabled the detection of P24 from C-ALL cells, utilizing its ability to bind the Ricinus communis agglutinin (RCA1) or wheat germ agglutinin (WGA) and SJ-9A4 simultaneously. When BA-2 and DU-ALL-1 were used in place of SJ-9A4, similar IRA results were obtained. Using the RCA1/SJ-9A4-IRA, P24 from as few as 1.6 X 10(4) cells of a C-ALL cell line could be detected; however, similar extracts of NB cell lines were negative despite high levels of SJ-9A4 binding to intact cells. The presence of P24 in NB extracts was demonstrated by (1) preincubation of NB extracts with SJ-9A4 which blocked MoAb binding to P24 and (2) immunoadsorption of P24 from solubilized membranes of 35S-methionine (met) labeled NB cells. Treatment of NB cells with neuraminidase did not result in IRA binding when either RCA1 or WGA were used as the solid phase lectin indicating that the differences in lectin affinity are not due to over sialation of NB membrane glycoproteins. These findings demonstrate a difference in the glycosylation of P24 from C-ALL and NB cells.

Antibodies, Monoclonal↗
PubMed1982

A predominant idiotype on rabbit anti-VH a1 allotype antibodies: sharing by both major and minor VH subgroups.

In every rabbit examined, greater than or equal to 80% of antibodies directed against the VH allotypic marker, a1, bears a predominant idiotype (IdX-a1). The IdX-a1 marker is site-associated and expressed on H chains, but not L chains, of anti-a1 antibody. Experiments using rabbits suppressed for the VH a subgroup demonstrated that IdX-a1 can be associated with both major (a+) and minor (a-) VH subgroup gene products and that a1 rabbits contain IdX-a1 within their genetic repertoire. The genetic and regulatory implications of our results are discussed.

Animals↗
PubMed1982

Immunoelectron microscopic localization of idiotypes and allotypes on immunoglobulin molecules.

We have developed a novel approach to the analysis of antigenic (allotypic and idiotypic) determinants on intact immunoglobulin molecules. Immune complexes composed of IgG in combination with anti-idiotype or anti-allotype antibody were "visualized" by transmission electron microscopy. Individual Fab fragments of anti-idiotype or anti-allotype antibody, when bound to the IgG, altered the "Y" configuration in a reproducible and interpretable manner. Anti-idiotype antibody (either as Fab or IgG) bound to the terminus of the presumed V region of the IgG molecule, thus extending the apparent length of the Fab arms. Analysis of a rabbit VH framework allotype (a1) revealed that the determinant(s) is (are) located on the lateral portion of the V region of IgG. Binding of the anti-a1 Fab fragments was always at approximately right angles to the axis of the Fab arms of IgG. Fab antibody to the rabbit kappa light chain (b4) allotype bound to the lateral portion of the terminal half of the IgG Fab arms. This technique should be of value in localizing less well defined immunoglobulin determinants.

Animals↗
PubMed1981

Idiotypic repertoire of anti-hen eggwhite lysozyme antibodies probed with hybridomas. Selection after immunization of an IdX marker common to antibodies of distinct epitope specificity.

A panel of hybridoma antibodies obtained from lymphoid cells that were fused during a primary response ("early") or a secondary response ("late") gave results concordant with analysis of conventional, in vivo-produced anti-lysozyme idiotypes: early antibodies did not display the predominant anti-hen eggwhite lysozyme idiotype (IdX-HEL), whereas late antibodies all displayed IdX-HEL. Furthermore, individual late hybridomas could each remove the entire anti-IdX-HEL activity by absorption, whereas early hybridomas could not. The epitope specificities of the hybridomas in both the early and late populations were heterogenous. We conclude that epitypic specificity in the response to HEL is determined independently from idiotypic specificity and that the predominant idiotype is selected for during the maturation of the anti-lysozyme response.

Animals↗