Illustration of microengraving.

<p>Arrays of nanowells with dimensions of 50 µm×50 µm×50 µm were used for microengraving. Spleen or cervical lymph nodes cells were loaded in the nanowells. Cells in the nanowells were imaged using an automated epifluorescence microscope. Micrograving is performed by hybridizing nanowells with capture slides containing anti-mouse Ig for 2 hrs at 37°C with 5% CO₂. After incubation, nanowells containing intact live cells and capture slides were separated. A mixture of antibodies containing IgG1-Alexa Fluor 488, B6 SG lysate-Alexa Fluor 594 and <i>Aec1Aec2</i> SG lysate-Alexa Fluor 555 were added to the capture slides. Micrographs of microarrays were generating by scanning using a Genepix 4200AL microarray scanner.</p

Frequency of IgG1.

<p>Enumeration of IgG1-secretion cells from arrays of nanowells occupied by single cells from the spleens and cervical lymph nodes of C57BL/6 (n = 4) and C57BL/6.NOD-<i>Aec1Aec2</i> mice (n = 4). Data extracted from the image processing using Genepix software were used to identify the appropriate signals. The data were correlated with the nanowell image data in which nanowells contained a single cell positive for both Calcein (live cells) and CD19. The frequency was determined by using the ratio of positive IgG1 signal from wells with single cells and the total number of wells with single cells. *p<0.05 by unpaired t test. NS: not significant.</p

Characterization of cells from the cervical lymph nodes of C57BL/6 and C57BL/6.NOD-<i>Aec1Aec2</i> mice using microengraving.

<p><b>A.</b> Representative micrographs of live cells from C57BL/6 cervical lymph nodes (n = 4) in nanowells labeled with Calcein (live cells), CD19-FITC and CD4-Cy7. Micrographs of matching microarray showing detection signals for IgG1-Alexa Fluor 488, C57BL/6 (B6) salivary glands proteins labeled with Alexa Fluor 594 and C57BL/6.NOD-<i>Aec1Aec2</i> (SjS) salivary glands proteins labeled with Alexa Fluor 555. The last vertical panel illustrates the close-up features (arrows) (Live cell: CD19FITC, IgG1: IgG1-488 signal, B6 gland: signal of antibody binding to salivary gland proteins isolated from B6 mice. SjS gland: signal of antibody binding to salivary proteins isolated from SjS mice. <b>B.</b> Representative micrographs of live cells from C57BL/6.NOD-<i>Aec1Aec2</i> cervical lymph nodes (n = 4) in nanowells labeled with Calcein (live cells), CD19-FITC and CD4-Cy7. Micrographs of matching microarray showing detection signals for IgG1-Alexa Fluor 488, B6 salivary glands proteins labeled with Alexa Fluor 594 and SjS salivary glands proteins labeled with Alexa Fluor 555. The last vertical panel illustrates the close-up features pointed by the arrows (Live cell: CD19FITC, IgG1: IgG1-488 signal, B6 gland: signal of antibody binding to salivary gland proteins isolated from B6 mice. SjS gland: signal of antibody binding to salivary proteins isolated from SjS mice. All experiments were repeated at least twice for consistency.</p

The distribution of polysaccharide-specific antibody-secreting and memory B cells against the four polysaccharides.

<p>The number of PS-specific ASCs (A) and MBCs (B) specific for each antigen at days 0, 7 and 28 post-vaccination are depicted. The radius of each circle in the center panel is proportional to the number of PS-specific ASCs or MBCs detected, and the antigen specificity is indicated by the color of each circle. The antigen distribution for all ASCs and MBCs within each vaccine-group are depicted in pie charts. Numbers in the center of the pie charts indicate the total numbers of ASCs or MBCs detected. The distribution of antigen specificity was compared using a 2XN Fisher’s test.</p

The heavy-chain gene-family usage and CDR3 length of polysaccharide-specific memory B cells.

<p>Sequences recovered from PS-specific MBCs were analyzed by IMGT/HighV-QUEST using the rhesus macaque immunoglobulin database. The heavy chain alleles of each B cell were generated by RT-PCR. (A) Heavy-chain gene-family usage at day 0 and 7 after immunization with 23vPS or 13vPnC. The number in the pie chart is the average number of variable sequences recovered from PS-specific MBCs. Pairwise statistical analysis was performed by Fisher’s Exact Test. (B) Distribution of heavy-chain gene-family usage for each PS serotype from PS-specific MBCs at 7 days following immunization with 13vPnC. The number in the pie chart is the average number of PS-specific MBCs against each antigen. Statistical analysis was performed by Fisher’s Exact Test. (C) The average CDR3 amino acid length of PS-specific MBCs at 0 and 7 days after immunization.</p

The distribution of polysaccharide-specific antibody-secreting and memory B cells against the four polysaccharides.

<p>The number of PS-specific ASCs (A) and MBCs (B) specific for each antigen at days 0, 7 and 28 post-vaccination are depicted. The radius of each circle in the center panel is proportional to the number of PS-specific ASCs or MBCs detected, and the antigen specificity is indicated by the color of each circle. The antigen distribution for all ASCs and MBCs within each vaccine-group are depicted in pie charts. Numbers in the center of the pie charts indicate the total numbers of ASCs or MBCs detected. The distribution of antigen specificity was compared using a 2XN Fisher’s test.</p

Emergent Properties of Nanosensor Arrays: Applications for Monitoring IgG Affinity Distributions, Weakly Affined Hypermannosylation, and Colony Selection for Biomanufacturing

It is widely recognized that an array of addressable sensors can be multiplexed for the label-free detection of a library of analytes. However, such arrays have useful properties that emerge from the ensemble, even when monofunctionalized. As examples, we show that an array of nanosensors can estimate the mean and variance of the observed dissociation constant (<i>K</i>_D), using three different examples of binding IgG with Protein A as the recognition site, including polyclonal human IgG (<i>K</i>_D μ = 19 μM, σ² = 1000 mM²), murine IgG (<i>K</i>_D μ = 4.3 nM, σ² = 3 μM²), and human IgG from CHO cells (<i>K</i>_D μ = 2.5 nM, σ² = 0.01 μM²). Second, we show that an array of nanosensors can uniquely monitor weakly affined analyte interactions <i>via</i> the increased number of observed interactions. One application involves monitoring the metabolically induced hypermannosylation of human IgG from CHO using PSA-lectin conjugated sensor arrays where temporal glycosylation patterns are measured and compared. Finally, the array of sensors can also spatially map the local production of an analyte from cellular biosynthesis. As an example, we rank productivity of IgG-producing HEK colonies cultured directly on the array of nanosensors itself