Inhibition of cell division in mouse B-cell hybridomas: an overlooked property of 2-mercaptoethanol and its impact on in vitro antibody production.

Thiol 2-mercaptoethanol (2-ME) has been reported to enhance growth in lymphocytes by various investigators. Some have used 50 µM for growing hybridomas in vitro. Concentrations of 50 and 5 µM in 5% FBS supplemented D-MEM were tested to determine their effects on the growth of 5 monoclonal antibody secreting mouse B cell hybridomas and the myeloma Sp2/O-Ag14. Viability after 24 and 48 h exposure was determined by Trypan blue exclusion. Analysis by one-way ANOVA confirmed that 50 µM 2-ME has a significant negative impact (p<0.05) on hybridoma as well as on myeloma growth, whereas no significant difference (p>0.05) between the control and the 5 µM treatment group was observed after 48 h. Also, no significant difference (p>0.05) in the mortality rates between the control and the treatment groups was found. When combined with the observed protracted doubling time in the 50 µM treatment group, these results indicate that the impact of 2-ME is due to inhibition of cell division. The degree of inhibition was observed to vary between the different hybridomas as well as the myeloma. Although the impact of 2-ME on mitosis has been demonstrated in organisms such as the ciliated protozoan Tetrahymena pyriformis, the yeast Saccharomycess cerevisiae, and the egg of the echinoid the sand dollar Dendraster excentricus, this work demonstrates for the first time that 2-ME impedes the growth of mouse B cell hybridomas. We conclude that adding 2-ME to mouse B cell hybridoma growth media may not be beneficial.

Detection of recombinant Alt a1 in a two-site, IgM based, sandwich ELISA opens up possibilities of developing alternative assays for the allergen.

Alternaria alternata is well known to induce IgE-mediated asthma in humans. Alt a1, a 29 kD glycoprotein doublet composed of 14.5 and 16 kD subunits, is the major allergen of this mould. Detection of Alt a1 relies on a two-site sandwich ELISA using the same IgG subclass immunoglobulin as primary and secondary antibody. In this study, we have compared two IgM monoclonal antibodies against recombinant and native Alt a1 in detecting the allergen in a two-site sandwich ELISA. Although both IgM clones detected the native and the recombinant allergen by SDS-PAGE immunoblotting and by the antibody-capture ELISA, only the IgM against recombinant Alt a1 was able to detect the corresponding, and not the native allergen, in a two-site sandwich ELISA. The IgM against native Alt a1 was unable to detect either allergen by this method. A combination of the two IgM clones and with a commercially available IgG failed to detect both allergens. However, atopic human IgE detected both forms of the allergen with the two IgM clones as primary antibody. This is the first time to demonstrate detection of Alt a1 in a two-site, IgM based, sandwich ELISA opening up possibilities for exploring novel detection methods, based on this approach.

A novel isoform of sarcolemmal membrane-associated protein (SLMAP) is a component of the microtubule organizing centre.

The microtubule organizing centre (MTOC) or the centrosome serves a crucial role in the establishment of cellular polarity, organization of interphase microtubules and the formation of the bipolar mitotic spindle. We have elucidated the genomic structure of a gene encoding the sarcolemmal membrane-associated protein (SLMAP), which encodes a 91 kDa polypeptide with a previously uncharacterized N-terminal sequence encompassing a forkhead-associated (FHA) domain that resides at the centrosome. Anti-peptide antibodies directed against SLMAP N-terminal sequences showed colocalization with gamma-tubulin at the centrosomes at all phases of the cell cycle. Agents that specifically disrupt microtubules did not affect SLMAP association with centrosomes. Furthermore, SLMAP sequences directed a reporter green fluorescent protein (GFP) to the centrosome, and deletions of the newly identified N-terminal sequence from SLMAP prevented the centrosomal targeting. Deletion-mutant analysis concluded that overall, structural determinants in SLMAP were responsible for centrosomal targeting. Elevated levels of centrosomal SLMAP were found to be lethal, whereas mutants that lacked centrosomal targeting inhibited cell growth accompanied by an accumulation of cells at the G2/M phase of the cell cycle.