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M J Lacy

Publications and source records attributed to M J Lacy.

12 recordsLinked to original sources
PubMed2001

A panel immunoblot using co-incubated monoclonal antibodies for identification of melanoma cells.

Antigen expression in melanoma is heterogeneous. Immunophenotyping using a panel of monoclonal antibodies may facilitate immunotherapy. An immunoblot procedure was developed to detect antigens in melanoma cells. Numerous monoclonal antibodies were tested to determine if (1) antigens were detected after transfer to membranes, (2) single bands or discrete multiple bands were obtained, (3) co-incubation of multiple monoclonal antibodies had no interference, and (4) banding patterns were non-overlapping. Antigens were selected based upon their association with melanoma and the availability of respective monoclonal antibodies. Antigens were melanoma antigen recognized by T-cells (MART-1), tyrosinase, tyrosinase-related protein 1 (TRP-1), S100, vimentin, glycoprotein 130 (gp130), a carcinoembryonic antigen (CEA)-like marker, KBA-62 and NKI-C3. Actin positive controls could be assessed simultaneously. Test samples were separated by polyacrylamide gel electrophoresis in a 4-15% polyacrylamide gradient, transferred to polyvinylidine fluoride membrane, blotted using a Fast-Blot apparatus (Pierce), and developed using diaminobenzidine/metal. Melanoma cell lines were immunophenotyped using this panel immunoblot, and were compared to a standard control and to non-melanoma cells. Up to four antigens could be detected simultaneously in a single lane of the immunoblot, using a single test sample of greater than 100000 cells.

Antibodies, Monoclonal↗
PubMed1992

Characterization of a single-chain T-cell receptor expressed in Escherichia coli.

Despite progress in defining the nature of major histocompatibility complex products that are recognized by the T-cell antigen receptor, the binding properties and structure of the receptor have not been solved. The primary problem has been the difficulty in obtaining sufficient quantities of active receptor. In this report we show that a single-chain T-cell receptor gene can be expressed in Escherichia coli. The protein consists of the variable (V) regions of the alpha and beta chains (V alpha and V beta) encoded by the cytotoxic T-lymphocyte clone 2C (a H-2b anti-H-2d alloreactive cell line) linked by a 25-amino acid flexible peptide. Solubilized extracts that contain the 27-kDa V alpha 3V beta 8 protein are positive in solid-phase immunoassays with the anti-V beta 8 antibody KJ16 and the anti-clonotypic antibody 1B2. Approximately 1% of the protein can be specifically purified on a 1B2-conjugated column. These results indicate that a fraction of the protein is able to fold into a native conformation and that single-chain proteins should be useful not only as immunogens for eliciting anti-T-cell receptor antibodies but in the study of T-cell receptor structure and function.

Amino Acid Sequence↗
PubMed1992

Mechanisms that generate junctional diversity in alpha and delta chains that use the Tcrd-V3 gene product.

The signals that dictate whether a thymocyte will express the alpha beta or gamma delta T-cell receptors are unknown. Although it is also not known if these two different cell types use identical recombinational machinery during rearrangement, the same variable (V) region genes can be used by both alpha and delta chains. By examining the products of rearrangements in alpha beta or gamma delta thymocytes that express identical V genes, we hoped to determine whether these cell types might differ in particular aspects of their recombinational activity. The polymerase chain reaction was used to show that the Tcrd-V2, Tcrd-V3, and Tcra-V3 genes are expressed as both Tcra and Tcrd transcripts in fetal and adult BALB/c mice. Sequencing of V delta 3 isolates was performed in order to compare the contribution of various mechanisms to the generation of junctional diversity. Extensive junctional diversity was present at all stages of development examined (fetal, newborn, and adult). During early development both alpha and delta chain junctional diversity is generated primarily by variability in the position of joining two gene segments (i.e., Tcrd-V3 to Tcra-J in alpha chains; Tcrd-V3 to Tcrd-D2 and Tcrd-D2 to Tcrd-J1 in delta chains). The pattern of base pair deletion from the end of the Tcrd-V3 gene was identical in alpha and delta chains and deletions occurred in fetal as well as adult T cells. In later development T cells use not only this mechanism for alpha and delta chains but also the addition of bases at gene segment junctions, presumably through the action of terminal deoxynucleotidyl transferase (TdT). Finally, a comparison of the variable domains of these alpha and delta chains shows that a notable difference is the variability in length of the CDR 3 region which can be significantly longer in delta-chains than in alpha-chains.

Amino Acid Sequence↗
PubMed1989

Quantitative analyses of immune network components.

Polyclonal immune network antibodies were quantitated and characterized in a syngeneic BALB/c murine system. Immunizations of BALB/c antifluorescein mAb 9-40 conjugated to keyhole limpet hemocyanin, produced anti-Id (anti-9-40, 39 to 190 micrograms/ml) as well as anti-fluorescein (anti-Fl, 12 to 109 micrograms/ml). Separately, immunizations of polyclonal anti-9-40, developed significant anti-Fl serum levels in the secondary (2 degrees) response (50 to 270 micrograms/ml), which decreased in the 3 degrees response (50 to 180 micrograms/ml) and thereafter, although levels of 9-40 idiotypically related antibodies increased. Polyclonal 2 degrees anti-anti-9-40 exhibited variant anti-Fl active sites, was antigenically more cross-reactive than polyclonal 2 degrees anti-Fl, but did not exhibit affinity maturation for fluorescein relative to 1 degrees anti-anti-9-40. In addition, the 9-40 idiotype constituted a small (less than 1.0%) percent of the 2 degrees and 3 degrees anti-Fl (ab1) immune response. When viewed within the context of an antigenic system that possesses widely diverse idiotypy, continued introduction of polyclonal anti-Id appears eventually to: 1) induce polyclonal ab3 with quantitative expression of idiotypically related antibodies in preference to ab3 of ancestral (9-40) antigenic specificity, 2) relative to ab1, induce a 100-fold increase in the level of ab3 antibodies that have both ancestral idiotype and ancestral antigen reactivity, and 3) induce polyclonal ab3 antibodies with a measurably wider range of antigenic reactivities than those of polyclonal ab1. These quantitative data may reflect the natural state of an immune network in a diverse antigenic response.

Animals↗
PubMed1989

Direct adsorption of ssDNA to polystyrene for characterization of the DNA/anti-DNA interaction, and immunoassay for anti-DNA autoantibody in New Zealand White mice.

DNA was adsorbed directly to polystyrene in concentrated NaCl (1.2 M) during simultaneous denaturation to single stranded form by high pH (12.0). Adsorption of single stranded DNA to polystyrene under these conditions (HsDNA) was complete within 15 min. DNA was not dissociated from polystyrene by NaCl concentrations as high as 5.0 M, nor by Tween 20 concentrations up to 50%. The method was ineffective for dT15 adsorption to polystyrene. HsDNA adsorption to polystyrene was compared to an indirect method in which methylated bovine serum albumin (mBSA) was adsorbed to polystyrene after which DNA was electrostatically bound to mBSA. DNA/mBSA interaction was affected by as low as 0.3 M NaCl. Using the DNA/mBSA assay, sera obtained from New Zealand White (NZW) mice showed IgG anti-DNA activity in approximately 50% of mice tested. HsDNA assay found greater than 62% of NZW mice have 10 micrograms anti-ssDNA or more per ml serum. Complex formation involving C1q complement and ssDNA in HsDNA assay were shown to be negligible. Anti-DNA autoantibody production by NZW implicated the NZW parental strain in autoimmunity of F1 progeny obtained from the New Zealand Black X NZW cross.

Adsorption↗
PubMed1989

Structural properties of an anti-fluorescein monoclonal IgM cryoglobulin.

Prior studies with murine monoclonal anti-fluorescein IgM 18-2-3 indicated both a high affinity for Fl (Ka = 2.9 x 10(10)/M), a relatively lower affinity for phenyloxazolone and an active site mediated cryoprecipitability in the absence of bound ligand. Active site related electrostatic interactions appeared to correlate with the low temp insolubility of 18-2-3, since auto-aggregation was sensitive to pH, ionic strength, temp and protein concn. Results of solid phase binding assays indicated that 18-2-3 complexed with murine and human IgM molecules but not with murine anti-Fl Mab 4-4-20 (IgG2a, kappa). Hemagglutination studies showed that 18-2-3 was not a cold agglutinin. Pentameric monoclonal antibody 18-2-3 exhibited a slower association rate with fluorescyl ligand relative to the rate observed with non-cryoglobulin anti-Fl monoclonal antibodies. However, Fab fragments of 18-2-3 displayed relatively faster kinetics, normally observed with anti-Fl monoclonal antibodies. The slower association rate exhibited by pentameric 18-2-3 was attributed to competitive binding between the fluorescein ligand and 18-2-3 determinants. Derivation and characterization of 18-2-3 (Fc)5 fragments indicated co-purification of Fv fragments possessing functional antigen binding sites, providing further evidence for binding of 18-2-3 Fab fragments with isologous Fc. Since 18-2-3 bound other IgM molecules, the mechanism of cryoprecipitation appeared to be an interaction of the fluorescein antigen binding site with specific Fc epitope(s).

Animals↗
PubMed1989

T-cell receptor delta-chain diversity in peripheral lymphocytes.

A small percentage (approximately 5%) of the cells in the adult thymus expresses a heterodimeric receptor, gamma delta, that exhibits extensive clonal diversity. The specificity and function of these cells are unclear. Furthermore, it is not known if their role in the immune system is primarily one that operates within the thymus during the selection of the T-cell repertoire or if they function primarily in an antigen-recognition capacity in the peripheral lymphoid system. To examine if gamma delta+ T cells in the periphery are as diverse as those in the thymus, we used the polymerase chain reaction to amplify delta-chain transcripts from polyclonal populations of thymic and splenic lymphocytes (the latter were derived from allogeneic mixed lymphocyte cultures). The nucleotide sequences of delta chains from the spleen, like those from the thymus, were all different. Most of the diversity was present in the region between the variable (V) and joining (J) gene segments and was generated through the use of the two known diversity (D) elements, D delta 1 and D delta 2, and by the addition or deletion of bases at the V delta D delta 1, D delta 1D delta 2, and D delta 2J delta junctions. The extensive gamma delta repertoire among peripheral cells suggests that they have the potential to recognize an array of ligands that could be as diverse as those recognized by alpha beta+ cells. The amplification strategy described here can be used to analyze rapidly the diversity exhibited by any of the members of the immunoglobulin-like gene families that undergo rearrangement.

Animals↗
PubMed1988

Selection of variable-joining region combinations in the alpha chain of the T cell receptor.

Most T lymphocytes express an antigen-specific receptor composed of two subunits, alpha and beta, each of which can exhibit structural variability. A complex selection process operates on T cells during development in the thymus such that cells expressing only particular alpha beta-receptors migrate to the periphery. The alpha-chain repertoire was dissected at different stages of the selection process by using the polymerase chain reaction (PCR) technique to amplify only those transcripts of a particular variable region gene (V58). Sequences from these V58 cDNAs reveal the predominant expression of four joining (J) segments by T cells in the adult thymus, suggesting that molecular or cellular processes select particular V alpha J alpha combinations during development. T cells expressing one of these V58J alpha chains appear to have been negatively selected at a later stage, since these transcripts were present in the spleen at approximately one-tenth the level in the thymus. Results also indicate that residues present at the V alpha J alpha junction may be important in an early selection process.

Animals↗
PubMed1986

A modified method to induce immune polyclonal ascites fluid in BALB/c mice using Sp2/0-Ag14 cells.

Immunized BALB/c mice were injected with 10(6) viable Sp2/0-Ag14 cells to induce ascites fluid concurrently with maximum serum antibody activity. Ascites fluids developed in a systematic manner, yielded large amounts of specific polyclonal antibody even when the mice were injected with relatively small amounts of immunogen. A combination of an initial pristane injection prior to 3 intraperitoneal immunizations of immunogen emulsified in Freund's complete adjuvant (total volume of 0.1-0.2 ml per injection), followed 4 days later by injection of 10(6) viable Sp2/0-Ag14 cells, resulted in a consistent production of ascites fluid with substantial specific antibody activity.

Animals↗
PubMedDate not supplied

Analysis of T cell receptor transcripts using the polymerase chain reaction.

The immune system is composed of two major types of lymphocytes, called B and T cells, that recognize foreign antigens. Recognition of antigens is accomplished through the generation of a large repertoire of different cell surface receptors, called immunoglobulins (Igs) on B cells and T cell receptors (TCRs) on T cells. The elucidation of Ig structure and molecular genetics preceded that of the TCR because of the greater abundance of Ig protein and mRNA. Although studies of TCRs have recently shed light on many of the issues of T cell recognition, the process of examining TCR gene structure has been tedious. Such analyses are also difficult because of the time required for the production, maintenance, and culturing of T cell clones. This report describes several strategies that use the polymerase chain reaction (PCR) to analyze very rapidly the structure of TCRs. Specific manipulations of the amplified material are discussed, as are the advantages of using the PCR to study TCR diversity.

Animals↗