Isolation of monoclonal antibodies from anti-synthetase syndrome patients and affinity maturation by recombination of independent somatic variants.

The autoimmune disease known as Jo-1 positive anti-synthetase syndrome (ASS) is characterized by circulating antibody titers to histidyl-tRNA synthetase (HARS), which may play a role in modulating the non-canonical functions of HARS. Monoclonal antibodies to HARS were isolated by single-cell screening and sequencing from three Jo-1 positive ASS patients and shown to be of high affinity, covering diverse epitope space. The immune response was further characterized by repertoire sequencing from the most productive of the donor samples. In line with previous studies of autoimmune repertoires, these antibodies tended to have long complementarity-determining region H3 sequences with more positive-charged residues than average. Clones of interest were clustered into groups with related sequences, allowing us to observe different somatic mutations in related clones. We postulated that these had found alternate structural solutions for high affinity binding, but that mutations might be transferable between clones to further enhance binding affinity. Transfer of somatic mutations between antibodies within the same clonal group was able to enhance binding affinity in a number of cases, including beneficial transfer of a mutation from a lower affinity clone into one of higher affinity. Affinity enhancement was seen with mutation transfer both between related single-cell clones, and directly from related repertoire sequences. To our knowledge, this is the first demonstration of somatic hypermutation transfer from repertoire sequences to further mature in vivo derived antibodies, and represents an additional tool to aid in affinity maturation for the development of antibodies.

Methods for multiplex template sampling in digital PCR assays.

The efficient use of digital PCR (dPCR) for precision copy number analysis requires high concentrations of target molecules that may be difficult or impossible to obtain from clinical samples. To solve this problem we present a strategy, called Multiplex Template Sampling (MTS), that effectively increases template concentrations by detecting multiple regions of fragmented target molecules. Three alternative assay approaches are presented for implementing MTS analysis of chromosome 21, providing a 10-fold concentration enhancement while preserving assay precision.

Parylene C coating for high-performance replica molding.

This paper presents an improvement to the soft lithography fabrication process that uses chemical vapor deposition of poly(chloro-p-xylylene) (parylene C) to protect microfabricated masters and to improve the release of polymer devices following replica molding. Chemical vapor deposition creates nanometre thick conformal coatings of parylene C on silicon wafers having arrays of 30 µm high SU8 pillars with densities ranging from 278 to 10,040 features per mm(2) and aspect ratios (height : width) from 1 : 1 to 6 : 1. A single coating of parylene C was sufficient to permanently promote poly(dimethyl)siloxane (PDMS) mold release and to protect masters for an indefinite number of molding cycles. We also show that the improved release properties of parylene treated masters allow for fabrication with hard polymers, such as poly(urethane), that would otherwise not be compatible with SU8 on silicon masters. Parylene C provides a robust and high performance mold release coating for soft lithography microfabrication that extends the life of microfabricated masters and improves the achievable density and aspect ratio of replicated features.

Megapixel digital PCR.

We present a microfluidic 'megapixel' digital PCR device that uses surface tension-based sample partitioning and dehydration control to enable high-fidelity single DNA molecule amplification in 1,000,000 reactors of picoliter volume with densities up to 440,000 reactors cm(-2). This device achieves a dynamic range of 10(7), single-nucleotide-variant detection below one copy per 100,000 wild-type sequences and the discrimination of a 1% difference in chromosome copy number.

Polyshrink™ based microfluidic chips and protein microarrays.

A new approach for the rapid production of microfluidic chips integrating protein spots is described. The technology, called "Print-n-Shrink", is based on the screen-printing of a microfluidic design (using a dielectric ink) onto Polyshrink™ polystyrene sheets. The initial printing which have a minimum size of 15 µm (height)×230 µm (width) was thermally treated (30s, 163°C) to shrink and generate features of 85 µm (height)×100 µm (width). Protein spots were also demonstrated to be shrinkable and arrays of 50 µm-size spots with density up to 6400 spots/cm(2) were achieved. Proteins such as monoclonal antibodies or cellular adhesion proteins were thus spotted onto the Polyshrink™ sheets and shrunk together with the microfluidic design, creating complete biochips integrating both complex microfluidic designs and protein spots for bioanalytical applications. These shrunk spots were shown to host enough active proteins to enable the achievement of both sensitive sandwich immunoassays (Brain Natriuretic Peptide, C-Reactive Protein and c-Troponin I) and localized cell culture.

"Print-n-Shrink" technology for the rapid production of microfluidic chips and protein microarrays.

An innovative method for the production of microfluidic chips integrating protein spots is described. The technology, called "Print-n-Shrink", is based on the screen-printing of a microfluidic design (using a dielectric ink) onto Polyshrink polystyrene sheets. The initial print which has a minimum size of 15 microm (height) x 230 microm (width) is thermally treated (30 seconds, 163 degrees C) to shrink and generate features of 85 microm (height) x 100 microm (width). Concomitantly, proteins such as monoclonal antibodies or cellular adhesion proteins are spotted onto the Polyshrink sheets and shrunk together with the microfluidic design, creating a complete biochip integrating both complex microfluidic designs and protein spots for bioanalytical applications.

Straightforward protein immobilization using redox-initiated poly(methyl methacrylate) polymerization.

Rigid poly(methyl methacrylate) (PMMA) biochips directly modified with active protein spots were obtained, using a redox-initiated PMMA polymerization process. The protein immobilization mechanism is believed to be a combination of both a covalent binding through transient amino acid radical generation and a direct entrapment of the biomolecules in the PMMA polymer. Three different immunoassays (binding, capture, and sandwich) were performed using the developed system, and really promising limits of detection (160-200pg/mL) were obtained, demonstrating a novel straightforward route to fabricate plastic biochips.

"Macromolecules to PDMS transfer" as a general route for PDMS biochips.

"Macromolecules to PDMS transfer" technique relying on the direct entrapment of macromolecules spots during PDMS polymerisation is proposed as an alternative for the easy and simple PDMS surface modification. In the present work, the development of three different applications based on this procedure is presented as proof of the method potentialities. First, C-reactive protein (CRP) sandwich immunoassay using immobilised monoclonal anti-CRP antibodies was developed for sepsis diagnosis. The preserved integrity of the immobilised monoclonal immunoglobulin permitted the sensitive detection of free CRP in human sera (LOD=12.5 microg/L, detection ranging over two decades). Then, rheumatoid arthritis diagnosis through the rheumatoid factor (RF) detection based on rabbit immunoglobulins immobilisation allowed the detection of specific antibodies in human sera samples down to low RF levels (detection range 5.3-485 IU/mL). Finally, the "Macromolecules to PDMS transfer" procedure was used to easily and rapidly produce fibronectin-based cell culture arrays. The successful attachment of HeLa and BALB/3T3 cells was demonstrated with optical microscopy and specific staining of actin and vinculin.

Microfluidic biochip for chemiluminescent detection of allergen-specific antibodies.

Protein microarrays for allergen-specific antibodies detection were integrated in microfluidic chips, with imaging chemiluminescence as the analytical technique. This paper demonstrates the feasibility of miniaturized chemiluminescent ELISA by presenting rapid, reproducible and sensitive detection of protein antibodies using microfluidics. Three different proteins, beta-lactoglobulin, peanut lectin and human IgG were immobilized via a "macromolecules to polydimethylsiloxane elastomer (PDMS) transfer" protocol and used as capturing agent for the detection of specific antibodies. A convenient and reversible procedure was used to bond the PDMS microarray substrate to complimentary SU-8/glass microfluidic reaction chambers. The hydrodynamic behaviours of the three proteins interactions within the micro-chambers were investigated to select the most efficient flowing parameters (come to terms with the assay time and performances). The use of optimized conditions led to the concomitant detection of three specific antibodies at pM level in 300 microL and using 6 min sample incubation time. Finally, sera from allergic patients were assayed using the microfluidic device modified with apple hazelnut and pollen allergen. The results obtained compared favourably with those obtained with the classical Pharmacia CAP system.

Biochips: non-conventional strategies for biosensing elements immobilization.

The present article draws a general picture of non-conventional methods for biomolecules immobilization. The technologies presented are based either on original solid supports or on innovative immobilization processes. Polydimethylsiloxane elastomer will be presented as a popular immobilization support within the biochip developer community. Electro-addressing of biomolecules at the surface of conducting biochips will appear to be an interesting alternative to immobilization processes based on surface functionalization. Finally, bead-assisted biomolecules immobilization will be presented as an open field of research for biochip developments.

Straightforward protein immobilization on Sylgard 184 PDMS microarray surface.

In this work, a straightforward technique for protein immobilization on Sylgard 184 is described. The method consists of a direct transfer of dried protein/salt solutions to the PDMS interface during the polymer curing. Such non-conventional treatment of proteins was found to have no major negative consequence on their integrity. The mechanisms of this direct immobilization were investigated using a lysine modified dextran molecule as a model. Clear experimental results suggested that both chemical bounding and molding effect were implicated. As a proof of concept study, three different proteins were immobilized on a single microarray (Arachis hypogaea lectin, rabbit IgG, and human IgG) and used as antigens for capture of chemiluminescent immunoassays. The proteins were shown to be easily recognized by their specific antibodies, giving antibody detection limits in the fmol range.