Robert S. Matson

Printing Low Density Protein Arrays in Microplates

Here, we provide methods for the creation of protein microarrays in microplates. The microplate consists of 96 wells with each well capable of holding a protein microarray at a spot density of up to 400 (20 x 20) individual elements. Arrays of capture monoclonal antibodies, corresponding to specific interleukins, were printed onto the bottom of the wells which had been surface activated for covalent attachment. A Biomek 2000 laboratory automation workstation (Beckman Coulter, Inc., Fullerton, CA) equipped with a high-density replicating tool was used for printing low density 3 x 3 to 5 x 5 arrays. For higher density arrays, a microarrayer system (Cartesian PS7200, Genomic Solutions, Inc., Ann Arbor, MI) was employed. Multiple antigens were simultaneously analyzed without detectable cross-reactivity associated with capture antibody or secondary antibody interactions. Detection of interleukin antigens, spiked into cell culture media containing 10% fetal calf serum, was specific and sensitive.

Overprint Immunoassay Using Protein A Microarrays

The ability to perform microarray-based immunoassays without the need for wells or other fluid barriers were demonstrated. Both contact and noncontact microarray printing technology is used to prepare spotted arrays of analyte binding sites, as well as, to deliver samples, secondary antibodies and other signal development reagents directly to these sites in a parallel fashion are called as overprint immunoassays. A micro-ELISA is demonstrated based upon the use of Protein A as a universal microarray. All components of the assay (capture antibody, antigen, and signal development reagents) were site-specifically dispensed in parallel fashion to the surface in nanoliter volumes. This represents a 1000-fold reduction in reagent consumption from that used in a conventional 96-well microtiter plate assay. Overprinting nanoliter volumes directly onto 200-300 microm spots yields similar levels of sensitivity achieved with the bulk dispensing of microliter volumes into wells.

Multiplex ELISA Using Oligonucleotide Tethered Antibodies

Multiplex assays represent a new paradigm for diagnostics. The simultaneous measure of multiple analytes from a single sample is advantageous in creating disease-associated panels that enable more accurate prognosis or diagnosis of the disease state. Furthermore, multiplexing may reduce reagent consumption, sample requirements and labor thereby lowering the cost per test. Here we describe a novel multiplex immunoassay technology based upon creating microarrays in microtiter plates that are formed upon the self-assembly of oligonucleotide-antibody conjugates.

Hybridization Analysis Using Oligonucleotide Probe Arrays

This chapter describes methodology for the labeling, hybridization, and detection of amplicon target DNA to arrays of oligonucleotide probes attached to plastic substrates. A systematic approach to target discrimination based on both hybridization and wash stringency is provided.

Construction of In Situ Oligonucleotide Arrays on Plastic

The concept of DNA arrays was first introduced in the early 1980s, by Sir Edwin Southern. Since then, many research institutions and biotechnology companies have investigated the potential use of arrays in fields ranging from genetic diagnostics to forensics investigations. A 64-channel automated chemical delivery system, known as the Southern Array Maker, which synthesizes oligonucleotides directly onto an aminated polypropylene substrate has been constructed. Many different arrays have been synthesized for the purpose of detecting single point mutations, which might be either indicators of, or directly responsible for, many different types of genetic diseases and cancers. These include cystic fibrosis, H-ras, K-ras, and other mutations. In addition to the synthesized arrays, we are also looking into various alternative methods of producing both high-and low-density DNA arrays. This chapter is intended to demonstrate the synthesis of oligoarrays by in situ method using standard phosphoramidite chemistry. Phosphoramidate linkage to the aminated polypropylene is quite stable under oligo cleavage and deprotection conditions. Oligonucleotide density is approx 3 pmole or 10(1)2 molecules/mm(2).

Oligonucleotide Arrays for the Detection of ras Mutations

Although a variety of genetic instabilities have been suggested to be involved in the etiology of human tumors, much of our current understanding of this cellular transformation process strongly implicates the role of oncogenes and anti-oncogenes (tumor suppressor genes). Oncogenes are the activated form of normal genes (proto-oncogenes) which are generally involved in cell growth and proliferation. We know that proto-oncogene activation to oncogenes may involve alteration in the regulation of gene expression resulting in over amplification of the gene product; or by direct genetic mutation which leads ultimately to abnormal biological function, such as uncontrolled cell proliferation. The function of a tumor suppressor gene is to control cell proliferation, thus inactivation of this gene concomitant with activation of the Oncogene is thought to be a key factor in the initiation and the progression of tumorgenesis (Coleman and Tsongalis, 1997).