Production of a recombinant anti-morphine-3-glucuronide single-chain variable fragment (scFv) antibody for the development of a "real-time" biosensor-based immunoassay.

A recombinant single-chain variable fragment (scFv) antibody to morphine-3-glucuronide (M3G) was produced using genetic material obtained from the spleen cells of mice immunised with a morphine-3-glucuronide-bovine serum albumin (M3G-BSA) conjugate. Immunoglobulin light (V(L)) and heavy (V(H)) chain genes were amplified and cloned into pAK vectors for generation of recombinant antibody fragments in Escherichia coli. A competition ELISA assay was developed in PBS to characterise the ability of the antibody fragments to recognise free drug and the detection limits were found to be as low as 3 ng ml(-1). Surface plasmon resonance-based inhibition immunoassays were developed. The recombinant antibody was pre-incubated with various concentrations of free drug followed by injection over a morphine-3-glucuronide-thyroglobulin (M3G-THY) immobilised surface. The response of antibody binding to the surface of the chip was inversely proportional to the amount of free drug in solution. Regeneration conditions for antibody binding to the surface were optimised resulting in a binding-regeneration capacity of at least 30 cycles. The inhibition assay for M3G was tested with assay ranges between 3 and 195 ng ml(-1) and 3 and 97 ng ml(-1) in PBS and urine, respectively.

Immunoassay for the determination of morphine-3-glucuronide using a surface plasmon resonance-based biosensor.

Polyclonal antibodies were produced for the development of competitive ELISA's and surface plasmon resonance (SPR)-based BIAcore inhibition assays for the detection of morphine-3-glucuronide (M3G, the main metabolite of heroin and morphine). A conjugate consisting of M3G and ovalbumin was produced and used for the generation of antibodies, for the coating of immunoplates and for immobilisation onto BIAcore chips. Competition ELISA's were developed in PBS and urine to characterise the antibodies ability to recognise free M3G. SPR-based inhibition immunoassays on BIAcore were developed. The regeneration of the surface of a chip immobilised with conjugate following antibody binding, essential for the development of inhibition assays was investigated. Regeneration of the conjugate-coated surface was optimised for both polyclonal antibodies resulting in binding-regeneration capacities of approximately 60 cycles for one antibody and 50 cycles for the second antibody. The inhibition assays developed in urine had ranges of detection of 762-24,400 (antibody 1) and 976-62,500 pg ml(-1) (antibody 2). The inter-day coefficients of variation for the assays ranged from 1.48 to 11.24%.

Nuclear factor kappaB inhibitors induce adhesion-dependent colon cancer apoptosis: implications for metastasis.

The transcription factor nuclear factor kappaB (NFkappaB) is constitutively active in many types of cancercells and regulates the expression of several antiapoptotic genes. Previous studies demonstrated a role for the inhibition of NFkappaB in cancer therapyusing a transgenic approach in mice. We found that NFkappaB was transiently activated much greater than background constitutive levels during colon cancer cell readhesion, which rendered the readhering colon cancer cells exquisitely susceptible to apoptosis in the presence of soluble NFkappaB inhibitors. These compounds greatly reduced colon cancer cell implantation in an in vivo seeding model of metastasis. The ability of soluble NFkappaB inhibitors to significantly induce apoptosis of readherent colon cancer cells makes them prospective candidates for preventing colon cancer metastasis.

A role for the peroxisome proliferator-activated receptor alpha in T-cell physiology and ageing immunobiology.

Peroxisome proliferator-activated receptor (PPAR) represents an important member of the nuclear hormone receptor superfamily that can be activated by a variety of natural fatty acids, some of their metabolites and by commonly-used anti-lipidaemic drugs. We recently demonstrated PPAR expression in T lymphocytes, where it controls the initiation of transcription of T-box expressed in T-cells (T-bet) independent of added agonist. T-bet is an activation-inducible transcription factor regulator of interleukin 2 (suppression) and interferon (stimulation) synthesis. A suppressed ability to produce interleukin 2 and an enhanced production of interferon occurs in activated T-cells from PPAR-/- mice, as well as in T-cells from wild-type aged animals whose lymphocytes express lowered basal levels of PPAR. The dysregulated expression and/or function of cytokines, glucocorticoids or leptin that occurs with advanced age could all be responsible for the reduced expression of PPAR. Dietary supplementation of aged mice with vitamin E, or supplementation with known agonists of PPAR, was associated with elevation of lymphocyte expression of this nuclear hormone receptor, restoration of control over T-bet expression and elimination of the dysregulated production of interleukin 2 and interferon following lymphocyte activation. Interleukin 2 and interferon play very important roles in the initiation and/or regulation of immune, inflammatory and autoimmune disease states. Thus, the mechanisms that control the timing, magnitude and duration of specific cytokine production by activated T lymphocytes need clarification before appropriate nutritional or therapeutic strategies can be devised to treat disease conditions where cytokine expression and/or activities are deemed to be dysregulated and responsible.