Craig M. Story

Human placental Fc receptors and the transmission of antibodies from mother to fetus

During human pregnancy, maternal IgG is transported across the placenta to the fetus. On the way, some maternal antibodies against fetal antigens are removed as immune complexes. The placenta contains several known Fc receptors and also other proteins that bind immunoglobulins. A consideration of the binding properties and distribution of these proteins suggests that the neonated Fc receptor (FcRn) transports IgG across the syncytiotrophoblast, and possibly the fetal blood vessel endothelium. Fc gamma RI, Fc gamma RII and Fc gamma RIII on Hofbauer cells in the stroma probably clear immune complexes, together with Fc gamma RII on endothelial cells.

Screening individual hybridomas by microengraving to discover monoclonal antibodies

The demand for monoclonal antibodies (mAbs) in biomedical research is significant, but the current methodologies used to discover them are both lengthy and costly. Consequently, the diversity of antibodies available for any particular antigen remains limited. Microengraving is a soft lithographic technique that provides a rapid and efficient alternative for discovering new mAbs. This protocol describes how to use microengraving to screen mouse hybridomas to establish new cell lines producing unique mAbs. Single cells from a polyclonal population are isolated into an array of microscale wells (∼105 cells per screen). The array is then used to print a protein microarray, where each element contains the antibodies captured from individual wells. The antibodies on the microarray are screened with antigens of interest, and mapped to the corresponding cells, which are then recovered from their microwells by micromanipulation. Screening and retrieval require approximately 1–3 d (9–12 d including the steps for preparing arrays of microwells).

Visualization of the ER-to-cytosol dislocation reaction of a type I membrane protein

The human cytomegalovirus gene products US2 and US11 induce proteasomal degradation of MHC class I heavy chains. We have generated an enhanced green fluorescent protein–class I heavy chain (EGFP–HC) chimeric molecule to study its dislocation and degradation in US2‐ and US11‐expressing cells. The EGFP–HC fusion is stable in control cells, but is degraded rapidly in US2‐ or US11‐expressing cells. Proteasome inhibitors induce in a time‐dependent manner the accumulation of EGFP–HC molecules in US2‐ and US11‐expressing cells, as assessed biochemically and by cytofluorimetry of intact cells. Pulse–chase analysis and subcellular fractionation show that EGFP–HC proteins are dislocated from the endoplasmic reticulum and can be recovered as deglycosylated fluorescent intermediates in the cytosol. These results raise the possibility that dislocation of glycoproteins from the ER may not require their full unfolding.

Profiling antibody responses by multiparametric analysis of primary B cells

Determining the efficacy of a vaccine generally relies on measuring neutralizing antibodies in sera. This measure cannot elucidate the mechanisms responsible for the development of immunological memory at the cellular level, however. Quantitative profiles that detail the cellular origin, extent, and diversity of the humoral (antibody-based) immune response would improve both the assessment and development of vaccines. Here, we describe a novel approach to collect multiparametric datasets that describe the specificity, isotype, and apparent affinity of the antibodies secreted from large numbers of individual primary B cells (≈103-104). The antibody/antigen binding curves obtained by this approach can be used to classify closely related populations of cells using algorithms for data clustering, and the relationships among populations can be visualized graphically using affinity heatmaps. The technique described was used to evaluate the diversity of antigen-specific antibody-secreting cells generated during an in vivo humoral response to a series of immunizations designed to mimic a multipart vaccination. Profiles correlating primary antibody-producing cells with the molecular characteristics of their secreted antibodies should facilitate both the evaluation of candidate vaccines and, broadly, studies on the repertoires of antibodies generated in response to infectious or autoimmune diseases.

Optimization of the surfaces used to capture antibodies from single hybridomas reduces the time required for microengraving.

The most common method for the generation of monoclonal antibodies involves the identification and isolation of hybridomas from polyclonal populations. The discovery of new antibodies for biochemical and immunohistochemical assays in a rapid and efficient manner, however, remains a challenge. Here, a series of experiments are described that realize significant improvements to an approach for screening large numbers of single cells to identify antigen-specific monoclonal antibodies in a high-throughput manner (10(5)-10(6) cells in less than 12 h). The soft lithographic process called microengraving yields microarrays of monoclonal antibodies that can be correlated to individual hybridomas; the cells can then be retrieved and expanded to establish new cell lines. The factors examined here included the glass slide used for the microarray, the buffer used to deposit capture antibodies onto the glass, the type of polyclonal antibodies used to capture the secreted antibodies, and the time required for microengraving. Compared to earlier reports of this method, these studies resulted in increased signal-to-noise ratios for individual elements in the microarrays produced, and a considerable decrease in the time required to produce one microarray from a set of cells (from 2-4 h to 3-10 min). These technical advances will improve the throughput and reduce the costs for this alternative to traditional screening by limiting serial dilution.

A monoclonal anti-glycoconjugate antibody defines a stage and position-dependent gradient in the developing sympathoadrenal system

SummaryThe expression of complex carbohydrate antigens was analysed in developing sympathoadrenal cells of the rat using monoclonal antibodies that react with unique carbohydrate structures. CC1 and CC4 are monoclonal antibodies that react specifically with β-N-acetylgalactosamine and α-galactose/α-fucose moieties, respectively. CC1-reactive glycoconjugates are expressed in embryonic superior cervical ganglion (SCG) cells as early as embryonic day 15 (E15). CC4 is expressed in the SCG only for a brief period starting at E18 and then disappearing at P5. During their transient period of expression, CC1 antigens are expressed uniformly throughout the SCG at E15–17, but are then restricted to the rostral portion of the SCG from E18 to P4. CC4 is also concentrated in the rostral portion of the SCG between E21 and P4. In the adrenal medulla, CC1 and CC4 antigens display a post-natal onset of expression commencing approximately at P14 and continue to be expressed on a subset of cells which contain tyrosine hydroxylase (TH). The expression of CC1, however, is restricted to phenylethanolamine-N-methyltransferase-(PNMT)-negative chromaffin cells, whereas CC4 is not. CC1 and CC4-expressing cells appear to be scattered throughout the adrenal medulla without any particular topographic orientation. These findings suggest that the CC1 monoclonal antibody defines a stage-specific differentiation antigen in the sympathoadrenal lineage. Additionally, the CC1 antigen may confer important positional information in the embryonic SCG by distinguishing rostral from caudal neuronal cell bodies.