Immunomagnetic (IM) separation and concentration of specific target ligands or particles, such as bacteria or leukocytes, from complex mixtures, such as bone marrow, blood and other body fluids, is now a widely accepted technique. IM methodologies require high affinity antibodies or other receptors, but are potentially as effective as density gradient separations. Thus, a computer-controlled first-generation immunomagnetic assay system (IMAS) biodetector is being developed for clinical diagnostics. This system is fully automated and affords the advantage of rapid flow-through capture of all types of magnetic beads (MBs) and obviates operator contact with body fluid samples during the collection and analysis phases. In the present work, biotinylated capture antibodies were bound to streptavidin-coated MBs for capture of E. coli O157:H7, T cells and T cell subsets. Samples were automatically vortex mixed with antibody-coated MBs, stained with an acridine dye or fluorescent antibody and collected in a specially designed flow cell containing multiple steel pins, which concentrate external magnetic field lines. IM complexes were rapidly (within minutes), separated from their media in the magnetic field. Magnetically captured particles were automatically rinsed in the flow cell to remove unwanted materials and detection was achieved via a flow-through fluorimeter. Samples can be subsequently captured on a microbiological filter for microscopic visualization and image analysis. Preliminary results demonstrate that rapid detection of target bacteria and leukocytes at low concentrations in body fluids is possible with a total assay time under 1 h. This IM technology has many other potential clinical, industrial and environmental monitoring applications.

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A comparative study of PCR product detection and quantitation by electro-chemiluminescence and fluorescence.

Amplification and detection of target DNA sequences are made possible in a polymerase chain reaction (PCR) by using a mixture of biotinylated and ruthenium(II) trisbipyridal (Ru(bpy)3(2+))-end-labelled primers. In this way, biotin for capture and Ru(bpy)3(2+) for detection are directly incorporated into the PCR product obviating subsequent probe hybridization. PCR of a bacterial DNA template from Alteromonas species strain JD6.5 using a cocktail of biotin- and Ru(bpy)3(2+)-labelled primers amplified a 1 kilobase region. Serial dilution of PCR product followed by magnetic separation with Streptavidin (SA)-coated magnetic beads and an electrochemiluminescence (ECL) assay using the semi-automated QPCR System 5000 demonstrated sensitive (pg range) DNA detection. ECL assay of probe hybridization to a human immunodeficiency virus (HIV) sequence also produced pg level sensitivity. Quantitative DNA determination by ECL assay correlated well with visual detection of DNA in electrophoretic gels. However, DNA detection by ECL assay was 10 to 100 times more sensitive than conventional ethidium bromide staining. The combination of DNA-based magnetic separation with ECL assay provides a very sensitive and rapid method of quantitating DNA which, owing to its rapid and facile nature, may have many applications in the research, environmental monitoring, industrial and clinical fields.

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Cyclosporine and tacrolimus alter renin-angiotesin system in mouse medullary-thick ascending limb cultured cells.

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Binding properties of a monoclonal antibody directed toward lead-chelate complexes.

A monoclonal antibody (2C12) that recognizes a Pb(II)-cyclohexyldiethylenetriamine pentaacetic acid complex was produced by the injection of BALB/c mice with a Pb(II)-chelate complex covalently coupled to a carrier protein. The ability of purified antibody to interact with a variety of metal-free chelators and metal-chelate complexes was assessed by measuring equilibrium dissociation constants. The antibody bound to metal-free trans-cyclohexyldiethylenetriamine pentaacetic acid (CHXDTPA) with an equilibrium dissociation constant of 2.3 x 10(-)(7) M. Addition of Pb(II) increased the affinity of the antibody for the complex by 25-fold; Pb(II) was the only metal cation (of 15 different di-, tri-, and hexavalent metals tested) that increased the affinity of the antibody for CHXDTPA. The increased affinity was due primarily to an increase in the association rate constant. The antibody also had the ability to interact with ethylenediamine tetraacetic acid (EDTA), diethylenetriamine pentaacetic acid (DTPA), and structurally related derivatives, but with affinities from 50- to 10000-fold less than that determined for CHXDTPA. Addition of metals to EDTA-based chelators reduced the affinity of the antibody for these ligands. However, when DTPA was used as the chelator, addition of Pb(II) increased the affinity of the antibody for the complex by 200-fold. The sensitivity of prototype immunoassays for Pb(II) could be modulated by changing the structure of the immobilized metal-chelate complex and/or the soluble chelator used to complex Pb(II) in the test solution.

Amino Acid Sequence↗

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Enhanced adjuvant effect of granulocyte-macrophage colony-stimulating factor plus interleukin-12 compared with either alone in vaccine-induced tumor immunity.

Using the poorly immunogenic D5 murine melanoma, we examined the adjuvant effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-12 (IL-12) secretion by gene-modified tumor cells inoculated as a vaccine to prime tumor-draining lymph nodes (TDLNs). D5 transfectants that secreted IL-12 or GM-CSF alone were compared with a double transfectant that secreted equivalent amounts of both cytokines. TDLN cells harvested 9-10 days after subcutaneous tumor inoculation were cultured sequentially in anti-CD3 and IL-2 and assessed for antitumor reactivity against wild-type D5 tumor. The double transfectant-induced TDLN effector cells had greater cytotoxicity in a long-term assay than TDLN cells primed by single transfectants. In adoptive immunotherapy, the TDLN cells primed by the double transfectant were significantly better at mediating the regression of established tumors compared with the TDLN cells elicited by the single transfectants. Both IL-12 and GM-CSF had adjuvant effects in promoting tumor-reactive TDLN cells, but the combination was better than either alone. These observations suggest that the immunomodulation roles of IL-12 and GM-CSF are different and complementary.

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Interleukin-12 cDNA skin transfection potentiates human papillomavirus E6 DNA vaccine-induced antitumor immune response.

Human papillomaviruses are associated with >90% of all cases of uterine cervical tumors. The E6 and E7 oncoproteins of human papillomavirus are potentially ideal targets of immune therapy for cervical cancer, because their expression is necessary for cellular transformation. Although both E6 and E7 proteins contain numerous predicted cytotoxic T lymphocyte (CTL) epitopes that are capable of binding to human leukocyte antigens, the majority of earlier in vivo tumor rejection studies have focused on E7. We show here that gene gun-mediated skin transfection of plasmid vector encoding the nontransforming, amino-terminal half of E6 resulted in the induction of E6-specific CTL activity and tumor rejection in a murine model. The use of recombinant murine interleukin-12 (rmIL-12) as a vaccine adjuvant has been shown to result in both an enhancement and suppression of immune responses, depending upon the doses of rmIL-12 and the experimental systems used. We demonstrate here that local expression of transgenic mIL-12 at the E6 DNA vaccination site potentiated E6-specific CTL responses and increased vaccine-induced antitumor therapeutic efficacy. Our results indicate that transfection of the mIL-12 gene at the vaccination site may represent an attractive adjuvant for cancer gene immunotherapy.

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Vulnerable caregivers of patients with Alzheimer's disease have a deficit in circulating CD62L- T lymphocytes.

OBJECTIVE: The cell adhesion molecule, L-selectin (CD62L), serves a crucial role in the migration of naive T lymphocytes and is typically shed on cell activation. The objective of this study was to determine the effects of chronic stress on L-selectin expression on peripheral lymphocytes in elderly spousal caregivers of patients with Alzheimer's disease. METHODS: Twenty caregivers (mean age, 73.5 years) had their lymphocytes and catecholamine levels sampled at rest and in response to an acute psychological stressor. Ten of the caregivers were classified as susceptible or "vulnerable" based on the large amount of care required by the patient relative to the amount of respite the caregiver received during the previous 6 months. RESULTS: At rest, vulnerable caregivers had 60% fewer L-selectin negative CD8+ T cells (CD8+CD62L-) (p=.01) but no difference in CD8+CD62L+ cells. Vulnerable caregivers also showed significantly fewer CD4+CD62L- T lymphocytes (p=.04) but no difference in CD4+CD62L+ lymphocytes. Resting plasma epinephrine levels were 44% higher in vulnerable caregivers as compared with nonvulnerable caregivers (p=.01). The acute stressor increased circulating levels of CD8+CD62L- and CD8+CD62L+ lymphocytes and catecholamines similarly in both groups. CONCLUSIONS: The findings suggest that caregivers who are more vulnerable to the chronic stress of caregiving show a decrement in circulating CD62L- T lymphocytes, possibly by adrenomedullary activation. The data also suggest the identity of lymphocyte subsets that may underlie prior observations of immunologic decrements associated with the chronic stress of caregiving.

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