Design of rapid, selective, and sensitive DNA and ribonucleic acid (RNA) biosensors capable of minimizing false positives from nuclease degradation is crucial for translational research and clinical diagnostics. We present proof-of-principle studies of an innovative micro-ribonucleic acid (miRNA) reporter-probe biosensor that displaces a self-complementary reporter, while target miRNA binds to the probe. The freed reporter folds into a hairpin structure to induce a decrease in the fluorescent signal. The self-complementarity of the reporter facilitates the reduction of false positives from nuclease degradation. Nanomolar limits of detection and quantitation were capable with this proof-of-principle design. Detection of miRNA occurs within 10 min and does not require any additional hybridization, labeling, or rinsing steps. The potential for medical applications of the reporter-probe biosensor is demonstrated by selective detection of a cancer regulating microRNA, Lethal-7 (Let-7a). Mechanisms for transporting the biosensor across the cell membrane will be the focus of future work.

Human peripheral blood lymphocytes (PBL) were cultured for various time periods (up to 8 d) in the presence of pokeweed mitogen (PWM), lipopolysaccharide, or Epstein-Barr virus. Cell-free supernates were fractionated on a standardized ultrogel AcA 22 column and the proportion of polymeric and monomeric IgA was determined by radioimmunoassay. The results demonstrate that PBL stimulated with these mitogens produce IgM and IgG with molecular characteristics identical to those found in serum, but that the IgA produced is predominantly of the polymeric type. To prove that this IgA represented disulfide bond-linked polymers rather than aggregated monomers, we have demonstrated that the high molecular weight IgA (a) maintains its polymeric form upon treatment with acidic buffers, (b) contains J chain, a glycoprotein associated only with polymeric immunoglobulins, and (c) dissociates to the monomeric form upon reduction of disulfide bonds. After 1 wk in culture, approximately 60% of the PWM-stimulated cells that contained IgA were positive for IgA2, whereas 40% were IgA1 positive as determined by immunofluorescence. Therefore, peripheral blood contains a population of lymphocytes with the potential to display, after appropriate stimulation and differentiation, characteristics similar to IgA cells found in external secretory tissues. The demonstration of the presence of such cells in the peripheral circulation suggests that these cells are precursors of IgA-producing plasma cells with the potential to populate mucosal tissues.

It is widely believed that antibodies to the group B streptococcal (GBS) typespecific polysaccharides are of major importance in human host defenses against GBS infection. All four of the type-specific polysaccharides elicit antibodies against at least two forms of the antigens: a complete antigen that contains Nacetylneuraminic acid (sialic acid) at the nonreducing ends of the side chains and an incomplete or nonsialated antigen (1-4). This was first demonstrated for the type II polysaccharide by Lancefield and Freimer (1) when they showed that rabbit antisera directed against either the complete or incomplete antigen would protect mice from a lethal challenge of live type II organisms. They were unable to carry out studies with type III GBS using similar passive protection tests because these organisms were not by themselves pathogenic for mice.The question of antibody specificity in protection against type III GBS infections remains controversial. There are conflicting reports regarding the protective properties of human and rabbit antibodies directed against the type III antigens and against the capsular polysaccharide of the type 14 pneumococcus, which cross-reacts with the incomplete GBS type III antigen (5). Although it is possible to select for certain specificities in anti-GBS serum by specific absorptions with purified antigens, as was done by Lancefield and Freimer (1), the polyclonal nature of antisera makes it difficult to clearly determine the immunoprotective effects of various specificities and different isotypes of the antibodies in question.Harris et al. (6) recently reported that mice could be protected against GBS types Ia, Ib, Ic, or II by antibody-producing tumors formed 2 wk after subscapular injection of hybridoma cells. The protection was serotype specific. Similar experiments could not be carried out using type III GBS because this serotype was not virulent in their mouse model. This report describes the production of mouse hybridoma antibodies of different isotypes directed against both sialated and nonsialated forms of the GBS type III polysaccharide. The protective properties of antibodies were examined under conditions where type III GBS were lethal for mice.

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