Antibody microarrays for native toxin detection.

We have developed antibody-based microarray techniques for the multiplexed detection of cholera toxin beta-subunit, diphtheria toxin, anthrax lethal factor and protective antigen, Staphylococcus aureus enterotoxin B, and tetanus toxin C fragment in spiked samples. Two detection schemes were investigated: (i) a direct assay in which fluorescently labeled toxins were captured directly by the antibody array and (ii) a competition assay that employed unlabeled toxins as reporters for the quantification of native toxin in solution. In the direct assay, fluorescence measured at each array element is correlated with labeled toxin concentration to yield baseline binding information (Langmuir isotherms and affinity constants). Extending from the direct assay, the competition assay yields information on the presence, identity, and concentration of toxins. A significant advantage of the competition assay over reported profiling assays is the minimal sample preparation required prior to analysis because the competition assay obviates the need to fluorescently label native proteins in the sample of interest. Sigmoidal calibration curves and detection limits were established for both assay formats. Although the sensitivity of the direct assay is superior to that of the competition assay, detection limits for unmodified toxins in the competition assay are comparable to values reported previously for sandwich-format immunoassays of antibodies arrayed on planar substrates. As a demonstration of the potential of the competition assay for unlabeled toxin detection, we conclude with a straightforward multiplexed assay for the differentiation and identification of both native S. aureus enterotoxin B and tetanus toxin C fragment in spiked dilute serum samples.

Functional antibody immobilization on 3-dimensional polymeric surfaces generated by reactive ion etching.

Reactive ion etching (RIE) was used to pattern antibodies onto the surfaces of polymer substrates. A low pressure, inductively coupled oxygen plasma was used to anisotropically etch 25-30 mum deep features into poly(methyl methacrylate) (PMMA), Zeonex, and polycarbonate (PC). Scanning electron microscopy and contact angle measurements show that the resulting surfaces exhibit significant microroughness and enhanced hydrophilicity. Fourier transform infrared spectroscopy suggests that, in addition to enhanced surface area, chemical modifications may contribute to antibody immobilization. Polyclonal antibodies preferentially bind to the etched areas in RIE-patterned PMMA and Zeonex substrates but localize in unetched regions of RIE-patterned PC surfaces. Simple immunoassays were performed to demonstrate a potential application for RIE-modified polymer surfaces. Antibodies specific for the capture of fluorescently labeled cholera toxin, S. aureus enterotoxin B, and B. anthracis protective antigen were immobilized onto etched PMMA surfaces and shown to specifically capture their labeled antigen from solution. This work demonstrates a potentially useful fabrication methodology for constructing antibody microarrays on plastic substrates.

Sequence specific fluorescence detection of double strand DNA.

Methods for the fluorescent detection of specific sequences of double strand DNA in homogeneous solution may be useful in the field of human genetics. A series of hairpin polyamides with tetramethyl rhodamine (TMR) attached to an internal pyrrole ring were synthesized, and the fluorescence properties of the polyamide-fluorophore conjugates in the presence and absence of duplex DNA were examined. We observe weak TMR fluorescence in the absence of DNA. Addition of >/=1:1 match DNA affords a significant fluorescence increase over equimolar mismatch DNA for each polyamide-TMR conjugate. Polyamide-fluorophore conjugates offer a new class of sensors for the detection of specific DNA sequences without the need for denaturation. The polyamide-dye fluorescence-based method can be used to screen in parallel the interactions between aromatic ring pairs and the minor groove of DNA even when the binding site contains a non-Watson-Crick DNA base pair. A ranking of the specificity of three polyamide ring pairs-Py/Py, Im/Py, and Im/Im-was established for all 16 possible base pairs of A, T, G, and C in the minor groove. We find that Im/Im is an energetically favorable ring pair for minor groove recognition of the T.G base pair.

Targeted derepression of the human immunodeficiency virus type 1 long terminal repeat by pyrrole-imidazole polyamides.

The host factor LSF represses the human immunodeficiency virus type 1 long terminal repeat (LTR) by mediating recruitment of histone deacetylase. We show that pyrrole-imidazole polyamides targeted to the LTR can specifically block LSF binding both in vitro and within cells via direct access to chromatin, resulting in increased LTR expression.

Polyamides reveal a role for repression in latency within resting T cells of HIV-infected donors.

BACKGROUND: The persistence of human immunodeficiency virus (HIV) type 1 within resting CD4+ T cells poses a daunting therapeutic challenge. Histone deacetylase (HDAC)-1, a chromatin-remodeling enzyme that can mediate gene silencing, is recruited to the HIV-1 long terminal repeat by the host transcription factor LSF. Pyrrole-imidazole polyamides, small molecules that target specific DNA sequences, can access the nucleus of cells and specifically block transcription-factor binding. METHODS: We used polyamides to directly test the role of chromatin remodeling in HIV quiescence in primary resting CD4+ T cells obtained from HIV-infected patients. RESULTS: After exposure to any of 4 different polyamides that specifically block HDAC-1 recruitment by LSF to the HIV promoter, replication-competent HIV was recovered from cultures of resting CD4+ T cells in 6 of 8 HIV-infected patients whose viremia had been suppressed by therapy. In comparison, HIV was not recovered after exposure to control, mismatched polyamides but was recovered from 7 of 8 of these patients' samples after the activation of T cells. CONCLUSIONS: We identify histone deacetylation as a mechanism that can dampen viral expression in infected, activated CD4+ T cells and establish a persistent, quiescent reservoir of HIV infection.