Low molecular weight, high affinity targeting ligands for use in therapeutics and diagnostics systems

Antibodies are a rapidly developing class of molecules used in the treatment inflammatory diseases and cancers as well as in diagnostic systems. However, there are major limitations associated with the use of antibodies including specificity for only one type of disease, the lack of uptake in solid tumors, the immunogenicity of the molecule, non-specific uptake of antibodies into normal organs, reduced binding if derivatized and difficulty with production and purification. The most troublesome problem, however, is the loss of specificity through epitope mutation. As an alternative to antibody therapeutics, design of a high affinity low molecular weight molecule which reduces the aforementioned issues could revolutionize therapeutics and diagnostics by circumventing these problems while still providing selectivity. Surface epitopes are continually mutating without harming or altering functions in the cell and subsequently become undetectable by antibodies. Thus targeting surface receptors vital to cell survival with high affinity, low molecular weight ligands represent a unique method for use in therapeutics and diagnostics. There are several low molecular weight, high affinity ligands which could achieve these goals; however, few have been used therapeutically and fewer have been used in diagnostic assays. Here we present the use of several low molecular weight, high affinity ligands for use in cancer and inflammatory disease therapeutics and bacterial diagnostics systems. First, we designed a label free biosensor, employing a peptide sequence identified from a phage display library, capable of detecting as few as 34 anthrax causing Bacillus anthracis spores. Secondly, we have developed a flow cytometry based diagnostic system for the detection of Pseudomonas aeruginosa employing the siderophore, pyoverdine. Through simple conjugation to a latex microsphere, we were able to specifically detect as few as 10e4 bacteria/mL. Lastly, we prepared a folate conjugated verrucarin A prodrug designed to specifically target folate receptor expressing cancer cells and activated macrophages. The conjugate was found to be cytotoxic to human KB cells and murine RAW cells with IC50s of 70 nM and 4 nM respectively. Using this concept, we can develop a new class of therapeutic and diagnostic systems not limited to potentially mutable surface molecules

Selective Capture and Identification of Pathogenic Bacteria Using an Immobilized Siderophore

Rapid identification of infectious pathogens constitutes an important step toward limiting the spread of contagious diseases. Whereas antibody-based detection strategies are often selected because of their speed, mutation of the pathogen can render such tests obsolete. In an effort to develop a rapid yet mutation-proof method for pathogen identification, we have explored the use of immutable ligands to capture the desired microbe on a detection device. In this proof-of-principle study, we immobilize pyoverdine, a siderophore that Pseudomonas aeruginosa must bind to obtain iron, onto gold-plated glass chips and then examine the siderophore\u27s ability to capture P. aeruginosa for its subsequent identification. We demonstrate that exposure of pyoverdine-coated chips to increasing dilutions of P. aeruginosa allows detection of the bacterium down to concentrations as low as 10(2)/mL. We further demonstrate that printing of the siderophore in a periodic pattern on the detection chip enables a sensitive method of detecting the bound pathogen by a Fourier transform analysis of light scattered by the patterned chip. Because unrelated bacteria are not captured on the pyoverdine chip, we conclude that pyoverdine can be exploited for the specific binding and identification of P. aeruginosa. It follows that the utilization of other microbe-specific immutable ligands may allow the specific identification of their cognate pathogens

Detection of Bacillus subtilis spores using peptide-functionalized cantilever arrays

We move beyond anti body-antigen binding systems and demonstrate that short peptide ligands can be used to efficiently capture Bacillus subtilis (a simulant of Bacillus anthracis) spores in liquids. On an eight-cantilever array chip, four cantilevers were coated with binding peptide (NHFLPKV-GGGC) and the other four were coated with control peptide (LFNKHVP-GGGC) for reagentless detection of whole B. subtilis spores in liquids. The peptide-ligand-functionalized microcantilever chip was mounted onto a fluid cell filled with a B. subtilis spore suspension for similar to 40 min; a 40 nm net differential deflection was observed. Fifth-mode resonant frequency measurements were also performed before and after dipping microcantilever arrays into a static B. subtilis solution showing a substantial decrease in frequency for binding-peptide-coated microcantilevers as compared to that for control peptide cantilevers. Further confirmation was obtained by subsequent examination of the microcantilever arrays under a dark-field microscope. Applications of this technology will serve as a platform for the detection of pathogenic organisms including biowarfare agents

Detection of Folate Binding Protein with Enhanced Sensitivity Using a Functionalized Quartz Crystal Microbalance Sensor

In this report, we describe the development of a quartz crystal microbalance biosensor for detection of folate binding protein (FBP). Using a simple folate—BSA conjugate absorbed onto a Au-coated quartz sensor, a detection limit of 30 nM was achieved. Binding of FBP to the sensor surface could be blocked at concentrations as high as 1 uM with a 100-fold excess of folic acid, indicating the specificity of the folate—FBP interaction and the absence of nonspecific binding to the functionalized surface. Moreover, capture could be achieved in the presence of blood serum, making the assay amendable to the analysis of bodily fluids. Further signal enhancement based on an anti-FBP antibody and protein-A-coated gold nanosphere sandwich assay extended the detection limit to 50 pM (~3 orders-of-magnitude improvement). Given the overexpression of FBP in certain malignancies and inflammatory disorders, we expect the methodology described here to be useful to detect FBP as a possible biomarker for disease diagnosis