Michael J. Lacy

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 Freunds 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.

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.

Mechanisms that generate junctional diversity in α and δ chains that use the Tcrd-V3 gene product

The signals that dictate whether a thymocyte will express the αß or γδ 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 α and δ chains. By examining the products of rearrangements in αß or γδ 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 Vδ3 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 α and δ chain junctional diversity is generated primarily by variability in the position of joining two gene segments (i.e., Tcrd-V3 to Tcra-J in α chains; Tcrd-V3 to Tcrd-D2 and Tcrd-D2 to Tcrd-J1 in δ chains). The pattern of base pair deletion from the end of the Tcrd-V3 gene was identical in α and δ chains and deletions occurred in fetal as well as adult T cells. In later development T cells use not only this mechanism for α and δ 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 α and δ chains shows that a notable difference is the variability in length of the CDR 3 region which can be significantly longer in δ-chains than in α-chains.

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).