Antibodies in diagnostic applications.

Immunoassays, or assays that are using antibodies as the specific binding reagents, have become one of the most common methodologies in diagnostic laboratories. In this paper we review different configurations of immunoassays as applied to a variety of analytes and sensitivity limits, along with common detection techniques and strategies. Progress in developing of ultra high affinity antibodies as a direction to improved immunoassays is also reviewed. Finally, we specifically concentrate on determination of antibody binding constants and performing immunoassays at the single molecule level using Fluorescence Correlation Spectroscopy (FCS). This technique has become a powerful tool in molecular binding characterizations and assay development, and possibly will grow into a quantitative analytical method suitable for diagnostic tests.

Relating biomarkers to efficacy and the efficacy curve: a review of EGFR therapies in oncology.

Personalized medicine attempts to provide the right drug to the right patient. In oncology, the mechanisms driving an individual's tumor need to be identified for appropriate therapy selection. Progressing from an individual's biomarker characterization to a population-based characterization is necessary for clinical trial design and success. This article will review recent EGF receptor therapy trials in non-small-cell lung cancer and colorectal cancer, and in defined subgroups will demonstrate a relationship between biomarkers and efficacy (response rate) that can be visualized with an 'efficacy curve'. A method for predicting therapeutic response in subgroups of patients defined by biomarker tests is provided.

Immiscible phase nucleic acid purification eliminates PCR inhibitors with a single pass of paramagnetic particles through a hydrophobic liquid.

Extraction and purification of nucleic acids from complex biological samples for PCR are critical steps because inhibitors must be removed that can affect reaction efficiency and the accuracy of results. This preanalytical processing generally involves capturing nucleic acids on microparticles that are then washed with a series of buffers to desorb and dilute out interfering substances. We have developed a novel purification method that replaces multiple wash steps with a single pass of paramagnetic particles (PMPs) though an immiscible hydrophobic liquid. Only two aqueous solutions are required: a lysis buffer, in which nucleic acids are captured on PMPs, and an elution buffer, in which they are released for amplification. The PMPs containing the nucleic acids are magnetically transported through a channel containing liquid wax that connects the lysis chamber to the elution chamber in a specially designed cartridge. Transporting PMPs through the immiscible phase yielded DNA and RNA as pure as that obtained after extensive wash steps required by comparable purification methods. Our immiscible-phase process has been applied to targets in whole blood, plasma, and urine and will enable the development of faster and simpler purification systems.

Solid phase assembly of defined protein conjugates.

We have developed a solid-phase procedure for protein-protein conjugation that gives greater control over product size and composition than previous methods. Conjugates are assembled by sequential addition of activated proteins to the support under conditions suitable for maintaining the activity of the proteins. The total number of conjugate units to be prepared is fixed in the first step by the quantity of the first protein absorbed by the support. In each following step, the added protein links only to previously bound protein. The final conjugate is released to solution by cleaving the linker holding the first protein to the support. This stepwise assembly provides uniformly sized conjugates of the desired size and composition with placement of components at the desired positions within the structure. Using this approach, we have prepared a series of conjugates containing R-phycoerythrin as the central protein, with varying quantities of alkaline phosphatase and IgG with expected molecular masses ranging from 1.6 to 11.5 MDa. Size-exclusion chromatography and atomic force microscopy demonstrate homogeneity and control of the conjugate size. In an immunoassay for human thyroid stimulating hormone, the conjugates show signals consistent with their compositions.