Stability of specific immunoglobulin secretion by EBV-transformed lymphoblastoid cells and human-murine heterohybridomas

The purpose of this project was to determine if stability of specific antibody secretion improved after fusion of Epstein-Barr virus (EBV)-transformed lymphoblastoid cells with P3X63Ag8.653 murine myeloma cells. Production of human monoclonal antibodies by Epstein-Barr virus transformation and somatic cell fusion has been used by many laboratories, however the steps involved have not been fully optimized. B lymphocytes isolated from the peripheral blood of normal donors were enriched for Thomsen-Friedenreich (T) antigen-reactive cells by panning on asialoglycophorin. The EBV-transformed lymphoblastoid cell lines generated from asialoglycophorin-adherent B lymphocytes were treated in three different manners: (1) cloned and maintained in culture as monoclonal lymphoblastoid cell lines, (2) cloned and fused with murine myeloma cells or (3) fused shortly after transfomation without prior cloning. Cloned lymphoblastoid cell lines maintained in culture without fusion either died or lost specific antibody secretion within five months. Uncloned lymphoblastoid cells remained viable for up to three months but lost specific antibody secretion within two months probably due to overgrowth by nonspecific clones. In an attempt to increase longevity and to stabilize specific antibody secretion by these cells, the cloned lymphoblastoid cells were fused with murine myeloma cells. In nine of ten fusions no hybrids were recovered. As an alternate approach, uncloned lymphoblastoid cells secreting T antigen-specific antibody were hybridized with murine myeloma cells, hybrids secreting T antigen-specific antibody were recovered in six of seven fusions. Furthermore, T antigen-specific antibodies of high titer were secreted by the heterohybridoma clones for more than five months of continuous culture. These heterohybridoma cells secreted more immunoglobulin, produced greater titers of antibody and maintained specific antibody secretion longer than either monoclonal or polyclonal EBV-transformed lymphoblastoid cells. These studies have conclusively demonstrated that fusion of polyclonal lymphoblastoid cells secreting T antigen-specific antibody with murine myeloma cells results in prolongation of human monoclonal antibody production compared with unfused monoclonal or polyclonal lymphoblastoid cell lines. This procedure should be generally applicable for the production of stable human monoclonal antibody-secreting cells lines from peripheral blood lymphocytes. ^

Immunochemical interactions of peptides with site-directed polyclonal immunoglobulin G

The objective of this study was to investigate the immunochemical nature of the polyclonal immune response to the 14mer peptide TINKEDDESPGLYG and to identify interactions among antibodies to more than one epitope. Two groups of rabbits were immunized with the 14mer peptide and a Keyhole Limpet hemocyanin (KLH) carrier, but with KLH attached either to the 14mer's N- or C-terminus. Two approximate epitopes were mapped by an antibody-capture enzyme-linked immunosorbent assay method using antiserum obtained when KLH was oriented on the C-terminus of the 14mer. A precise mapping of the epitopes performed with inhibition enzyme immunoassays (iEIAs) resulted in an N-terminal 6mer epitope TINKED and a C-terminal 10mer epitope EDDESPGLYG. The epitopes overlapped by two amino acids. IEIAs and iEIAs incorporating antibody-blocking peptides indicated that the two anti-epitope antibody fractions did not interfere with one anothers' epitope binding. It was postulated that the anti-TINKED and anti-EDDESPGLYG antibody fractions individually bind their respective hydrophobic epitope "core" region at the N- or C-terminal of peptide TINKEDDESPGLYG, while sharing the two hydrophilic overlap amino acids. This antibody "lap joint" binding interaction can be accomplished by each of the anti-epitope antibodies binding an opposite side of the epitope overlap region in the shallow periphery of its binding site. ^

RELATIONSHIP OF MOUSE IMMUNOGLOBULIN GENES TO INVERTED REPETITIVE DNA SEQUENCES

The purpose of this study was to examine the relationship of immunoglobulin genes, more specifically the C regions, to the inverted repetitive sequences found in the mouse genome. Total mRNA as well as mRNA for light chain kappa was purified from mouse plasmacytoma MOPC 321 cells. Complementary DNA molecules were synthesized from the mRNA templates and hybridized to DNA fractionated on hydroxyapatite columns. This fractionation separates DNA according to the presence of inverted repetitive sequences which will be retained by hydroxyapatite while the remaining fraction will be unbound.^ The results obtained during the course of this investigation suggested the following conclusions. Firstly, it was shown that inverted sequences were not found within the transcribed DNA region. Secondly, inverted sequences are not found within the kappa gene. And finally, it was shown that the inverted sequences may not be representative of all the sequences found in MOPC 321 DNA. ^

Stratification and compartmentalisation of immunoglobulin responses to commensal intestinal microbes

The gastrointestinal tract is heavily colonized with commensal microbes with the concentration of bacteria increasing longitudinally down the length of the intestine. Bacteria are also spatially distributed transversely from the epithelial surface to the intestinal lumen with the inner mucus layer normally void of bacteria. Maintenance of this equilibrium is extremely important for human health and, as the dominant immunoglobulin at mucosal sites, IgA influences mutualism between the host and its normal microbiota. In this review we focus on the links between immune and microbial geography of the mammalian intestinal tract.

Protein-glycan interactions as targets of intravenous/subcutaneous immunoglobulin (IVIg/SCIg) preparations

Post Presentation

The dog as a genetic model for immunoglobulin A (IgA) deficiency: identification of several breeds with low serum IgA concentrations

Immunoglobulin A (IgA) serves as the basis of the secretory immune system by protecting the lining of mucosal sites from pathogens. In both humans and dogs, IgA deficiency (IgAD) is associated with recurrent infections of mucosal sites and immune-mediated diseases. Low concentrations of serum IgA have previously been reported to occur in a number of dog breeds but no generally accepted cut-off value has been established for canine IgAD. The current study represents the largest screening to date of IgA in dogs in terms of both number of dogs (n = 1267) and number of breeds studied (n = 22). Serum IgA concentrations were quantified by using capture ELISA and were found to vary widely between breeds. We also found IgA to be positively correlated with age (p < 0.0001). Apart from the two breeds previously reported as predisposed to low IgA (Shar-Pei and German shepherd), we identified six additional breeds in which ≥10% of all tested dogs had very low (<0.07 g/l) IgA concentrations (Hovawart, Norwegian elkhound, Nova Scotia duck tolling retriever, Bullterrier, Golden retriever and Labrador retriever). In addition, we discovered low IgA concentrations to be significantly associated with canine atopic dermatitis (CAD, p < 0.0001) and pancreatic acinar atrophy (PAA, p = 0.04) in German shepherds.

Short communication: Immunoglobulin variation in quarter-milked colostrum

Whereas whole first-milked colostrum IgG1 variation is documented, the IgG1 difference between the quarter mammary glands of dairy animals is unknown. First colostrum was quarter-collected from healthy udders of 8 multiparous dairy cows, all within 3h of parturition. Weight of colostrum produced by individual quarters was determined and a sample of each was frozen for subsequent analysis. Immunoglobulin G1 concentration (mg/mL) was measured by ELISA and total mass (g) was calculated. Standard addition method was used to overcome colostrum matrix effects and validate the standard ELISA measures. Analysis of the data showed that cow and quarter (cow) were significantly different in both concentration and total mass per quarter. Analysis of the mean IgG1 concentration of the front and rear quarters showed that this was not different, but the large variation in individual quarters confounds the analysis. This quarter difference finding indicates that each mammary gland develops a different capacity to accumulate precolostrum IgG1, whereas the circulating hormone concentrations that induce colostrogenesis reach the 4 glands similarly. This finding also shows that the variation in quarter colostrum production is a contributor to the vast variation in first milking colostrum IgG1 content. Finally, the data suggests other factors, such as locally acting autocrine or paracrine, epigenetic, or stochasticity, in gene regulation mechanisms may impinge on colostrogenesis capacity.