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Biomedical subjects

F S Ligler

Publications and source records attributed to F S Ligler.

At least 55 records · Page 3Linked to original sources
PubMed1992

Detection of Clostridium botulinum toxin A using a fiber optic-based biosensor.

A rapid, sensitive, analytical method for the detection of Clostridium botulinum toxin has been developed. The fiber optic-based biosensor utilizes the evanescent wave of a tapered optical fiber for signal discrimination. A 50 mW argon-ion laser, which generates laser light at 514 nm, is used in conjunction with an optical fiber probe that is tapered at the distal end. Antibodies specific for C. botulinum are covalently attached to the surface of the tapered fiber. The principle of the system is a sandwich immunoassay using rhodamine-labeled polyclonal anti-toxin A immunoglobin G (IgG) antibodies for generation of the specific fluorescent signal. Various anti-toxin antibodies were immobilized to the fibers. Affinity-purified polyclonal horse anti-toxin A antibodies performed better than the IgG fraction from the same horse serum or than the monoclonal anti-toxin A antibody BA11-3. Botulinum toxin could be detected within a minute, at concentrations as low as 5 ng/ml. The reaction was highly specific and no response was observed against tetanus toxin.

Animals↗
PubMed1992

Biological responses to exchange transfusion with liposome-encapsulated hemoglobin.

The present study evaluates the biological responses to 10% blood volume infusion ("top-loading," n = 7) or 50% blood volume exchange transfusion (n = 8) with a new preparation of liposome-encapsulated hemoglobin (LEH) in the conscious rat. Top-loading did not affect systemic hemodynamic variables but induced transient thrombocytopenia (-36% +/- 7%; P less than 0.05) and elevation of plasma thromboxane B2 (12.4 +/- 3.8 pg/100 microliters vs. undetectable levels at the control; P less than 0.05). Rats exchanged with LEH maintained mean arterial pressure (MAP) during the 3 hr observation period, whereas control (0.9% NaCl or lactated Ringer's solution) animals developed hypotension (-23 +/- 5 mm Hg, P less than 0.05). The maintenance of MAP in the LEH group resulted from the opposing effects of LEH-induced increase in total peripheral resistance index (TPRI; +0.06 +/- 0.01 mm Hg/ml/min/kg; P less than 0.05), and decreased cardiac index, (Cl, -25 +/- 6 ml/min/kg, P less than 0.05). In contrast, the decline in MAP in the control groups could be attributed to the lower Cl (-65 +/- 8 ml/min/kg; P less than 0.05). All rats developed metabolic acidosis (base excess of -9.7 +/- 0.6 mmol/liter; P less than 0.05), which was transient in the LEH group but progressive in the control groups. These data support the possible use of LEH as a blood substitute when massive transfusions are needed.

6-Ketoprostaglandin F1 alpha↗
PubMed1991

Novel trifunctional carrier molecule for the fluorescent labeling of haptens.

We developed a novel trifunctional carrier molecule for the synthesis of hapten-fluorophore conjugates as reporter molecules in immunoassays. This carrier eliminates some of the disadvantages associated with currently used fluorophore-labeling procedures including high nonspecific binding. The backbone of the carrier consists of the 21 amino acid residues of the insulin A-chain molecule. This polypeptide provides a single site (terminal amino group) for covalent coupling of the hapten, three carboxyl groups for the attachment of fluorophores, and four sulfhydryl groups for derivatization with hydrophilic residues to compensate for the hydrophobic effect of the attached fluorophores. The sites for fluorophore attachment are 4, 17, and 21 amino acids away from the hapten attachment site. This spatial separation minimizes quenching of the fluorescence signal due to interaction of the fluorophores with each other and with the attached hapten. In this study, 2,4-dinitrophenol (DNP) was selected as model hapten, fluorescein as label, and S-sulfonate groups as hydrophilic residues. The properties of the DNP-insulin A-chain-fluorescein conjugate (DNP-Ins-Fl) were compared to those of a DNP derivative labeled with a single fluorescein moiety via a small lysine spacer (DNP-Lys-Fl). The DNP-Ins-Fl conjugate exhibited a 3-fold lower nonspecific adsorption to immobilized non-immune IgG contributing to an approximately 3-fold more efficient displacement from the binding sites of an immobilized monoclonal anti-DNP antibody by the antigen DNP-lysine. Furthermore, at equimolar concentrations the DNP-Ins-Fl generated a 2.6-fold higher fluorescent signal than DNP-Lys-Fl.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1990

A continuous flow immunoassay for rapid and sensitive detection of small molecules.

An immunosensor operating in continuous flow and capable of detecting low molecular weight antigens is described. The approach differs from previously described continuous flow assays by not requiring incubation steps or the introduction of additional reagents following the loading of the sample into the system. Detection of the antigen is rapid, occurring within 3 min in the system described. The assay is based on the binding of labeled antigen to an immobilized antibody, with subsequent displacement of the labeled antigen when antigen is present in the buffer flow. Signal detection occurs downstream of the antigen recognition event. In this study, the hapten 2,4-dinitrophenol (DNP) as DNP-lysine was used as model antigen. To generate a labeled antigen, DNP was coupled to the terminal amino group of insulin A chain (tetra S-sulfonate form) which provides two tyrosine residues for the introduction of an 125I-label (DNP-Ins-125I) or three carboxyl groups for the attachment of three fluorescein residues (DNP-Ins-Fl). The radiolabeled antigen was used to establish assay conditions. Subsequently, fluorescein was substituted for the radioisotope label in order to develop an assay independent of the restrictions associated with isotopes. Using this flow immunoassay, we were able to detect DNP-lysine down to a detection limit of 143 nM (29 pmol/200 microliters) using DNP-Ins-125I or DNP-Ins-Fl as labeled antigen. The density of immobilized antibody and the flow rate were identified to be critical parameters for the sensitivity of the assay.

Animals↗
PubMed1990

Liposome-encapsulated hemoglobin: an oxygen-carrying fluid.

From the original concept of encapsulating hemoglobin in an inert shell, LEH has evolved into a fluid proven to carry oxygen, capable of surviving for reasonable periods in the circulation, and amenable to large-scale production. The formula for the outer shell evolved from synthetic, nonlipid materials, to egg-lecithin-based lipid mixtures, to distearoyl-phosphatidylcholine-based blends. The fabrication technology started with the production of milliliter quantities and methods detrimental to the hemoglobin and developed into high-pressure extrusion systems producing multi-liter quantities without damaging the hemoglobin. The development of methods for analysis and quality control of LEH has been difficult: even techniques for measuring basic characteristics of size and methemoglobin are still being standardized. In vivo studies have established that LEH has a circulation half-life of 16-20 hr and can carry oxygen sufficient to sustain life, but safety has yet to be proven. In each of the general areas mentioned above, there are opportunities for further improvement and characterization. The source of the hemoglobin and the coencapsulation of hemoglobin modifiers needs to be reassessed now that human hemoglobin has been cloned and functional hemoglobin can be produced by using fermentation techniques. The development of routine methods for quality control and assurance must accompany the production of large quantities of LEH for preclinical studies. Whether or not the LEH can and should be manufactured as a lyophilized product must be assessed. Animal studies must done to prove safety as well as efficacy in a variety of clinical models, including hemorrhagic and septic shock as well as various levels of isovolemic exchange. One approach toward the improvement of the LEH is to alter the liposome surface to increase its biocompatibility. The evolution of biocompatible liposome surfaces has included carbohydrate moieties, as carbohydrates are expressed on the majority of biological membrane surfaces including the red cell. It has been demonstrated that inclusion of carbohydrate components such as gangliosides into the liposomal bilayer results in increased circulation times. As a result, these ganglioside-containing liposomes may exhibit a reduced impact on the RES system. Goins et al. have examined methods of introducing the ganglioside GM1 into LEH preparations. The long-term preservation of LEH is essential for its use by paramedics both in civilian and military trauma settings.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1989

Use of thiol-terminal silanes and heterobifunctional crosslinkers for immobilization of antibodies on silica surfaces.

A procedure for covalent immobilization of functional proteins on silica substrates was developed using thiol-terminal silanes and heterobifunctional cross-linkers. Using this procedure, a high density of functional antibodies was bound to glass cover slips and silica fibers. The amount of anti-IgG antibody immobilized was determined to be in the range of 0.66 to 0.96 ng/mm2 using radiolabeled antibody. The relative amount of IgG antigen bound by the immobilized antibody (0.37 to 0.55 mol antigen/mol antibody) was three to five times greater than other investigators have reported. In addition, the amount of protein nonspecifically adsorbed to the antibody-coated surface was further reduced by the addition of blocking agents so that nonspecific adsorption of protein antigens represented only 2-6% of the total antigen binding. With this low background, IgG antigen binding could be measured at levels as low as 150 fmol when an antigen concentration of 3 pmol/ml was applied. The process for antibody immobilization is straightforward, easy to perform, and adaptable for modifying mass quantities of biosensor components.

Antibodies↗
PubMed1989

Liposome encapsulated hemoglobin: stabilization, encapsulation, and storage.

Liposome encapsulated hemoglobin has numerous advantages as a red cell substitute. LEH has no blood type and can be made virus-free and sterile in large quantities. The technology is compatible with the use of either human or bovine hemoglobin and does not require chemical modification of the hemoglobin. Cofactors can be included in the liposome to modify the P50 and to prevent methemoglobin formation. The inclusion of dissacharides stabilizes the LEH during freezing and dehydration and provides a method for long-term storage. Most importantly, LEH can sustain life in animals after removal of red cells to lethal levels for periods commensurate with a 16-20 hour circulation half-life.

Animals↗
PubMed1988

Fabrication of key components of a receptor-based biosensor.

A three-pronged approach was taken to the development of receptor-based bisensors. First, asymmetric bilayer membranes were developed with one monolayer adaptable to the particular receptor of interest and the other monolayer polymerized to enhance membrane stability. Second, alamethicin and calcium channel complexes were introduced into the stabilized membrane and tested for ion-channel function. Third, a porous support for the receptor-containing membrane was fabricated, which is compatible with silicon technology. Preliminary devices incorporating these components were constructed.

Alamethicin↗
PubMed1987

A homogeneous immunoassay for the mycotoxin T-2 utilizing liposomes, monoclonal antibodies, and complement.

The trichothecene mycotoxin T-2 is a fungal metabolite known to contaminate agricultural products and cause intoxication of humans and animals. We have developed a homogeneous competition inhibition assay for T-2 mycotoxin based on complement-mediated lysis of liposomes. The T-2 mycotoxin was converted to an acid chloride derivative, subsequently coupled to the amino group of phosphatidylethanolamine, and incorporated with the phospholipid into unilamellar liposomes. Carboxyfluorescein, which is self-quenched at high concentrations, was entrapped in the liposomes as a release marker. We used a monoclonal IgG1 antibody specific for T-2 mycotoxin and a polyclonal anti-mouse Ig as a secondary antibody since the anti-T-2 IgG1 does not activate complement. In the absence of free T-2, the liposomes were lysed within 30 min after the addition of complement, releasing carboxyfluorescein into the surrounding buffer. In the presence of free T-2 toxin, the binding of antibodies to the liposomes was reduced, causing a corresponding decrease in lysis. This assay proved to be sensitive to T-2 toxin levels as low as 2 ng, which is 10-fold more sensitive than the present enzyme immunoassay using the same antibodies.

Antibodies, Monoclonal↗
PubMed1987

Cytogenetics and cell surface marker analysis in chronic myelocytic leukemia. II. Implications for patient management.

Chronic myelocytic leukemia (CML) is a model system for the study of many aspects of malignant disease. One aspect that correlates with decreasing therapeutic response is tumor progression. This progression is often accompanied by clonal evolution. In those cases where aggressive therapy does not prevent this evolution, the clinical response to therapy usually proves to be poor and of short duration. Investigators are concentrating their efforts in three primary, but not mutually exclusive, areas with respect to the clinical management of CML. These include: an attempt to distinguish patients at risk for early transformation from those who will have a prolonged chronic phase; the cryopreservation of autologous bone marrow or buffy coat early in chronic phase for subsequent use in the accelerated phase and; endeavors to identify early markers for disease progression allowing intervention before an irreversible blast crisis occurs. This report deals with two types of potential prognostic markers of transformation: chromosomal and cell surface characteristics. The appearance of nonrandom abnormal chromosomal patterns has been correlated with myeloblastic transformation by many investigators. However, there has always been a subset of CML patients who do not undergo clonal evolution. Additionally, the type(s) of transformation in CML may vary depending on the cell lineages involved. Unlike myeloblastic transformants, many of our patients who do not exhibit clonal evolution as a concomitance of disease progression develop a lymphoblastic transformation. Cytofluorometric analysis can distinguish small populations of abnormal cells with lymphoblastic characteristics (HLA DR+). Initial data suggests that patients expressing the HLA DR+ in their "normal" peripheral blood cells are at risk of undergoing lymphoblastic transformation. The combined use of clinical, cytogenetic, and cytofluorometric data to predict an impending transformation and to discriminate between myeloblastic and lymphoblastic populations allows clinicians to manage their patients more effectively.

Adolescent↗
PubMed1986

Effect of covalent attachment of immunoglobulin fragments on liposomal integrity.

Liposome stability during and after covalent coupling of Fab' antibody fragments was investigated. Large unilamellar vesicles containing entrapped 5(6)-carboxyfluorescein (CF) as a marker for liposomal integrity were prepared by extrusion through polycarbonate membranes. N-[4-(p-Maleimidophenyl)-butyryl]phosphatidylethanolamine (MPB-PE) was employed as a liposomal anchor for the covalent coupling of Fab' fragments. We observed that coupling of Fab' fragments to liposomes containing 5 mol % MPB-PE caused a concentration-dependent increase in size and polydispersity of the liposomes. Dependent on the concentration of the MPB-PE anchor in the membrane and the concentration of Fab' added, coupling was associated with the release of up to 95% of the entrapped CF. Rupture of the liposomes was identified as the primary mechanism of CF release during Fab' coupling. Reduction of the MPB-PE concentration to 1 mol % resulted in liposomes that were stable during and after Fab' coupling. The increased stability of these liposomes was due to the lower MPB-PE concentration and not to the lower number of attached Fab' fragments. By proper adjustment of the experimental conditions for coupling, the number of Fab' fragments attached to the 1 mol % MPB-PE liposomes could be increased without affecting the stability of the resulting liposomes. These stable liposomes, made by an extrusion method that avoids the use of organic solvents, detergents, or sonication, are therefore suitable for entrapment of labile compounds and can be used for immunotargeting or immunoassays.

Animals↗
PubMed1985

Cytogenetics and cell surface marker analysis in CML--1. Prediction of phenotype of acute phase transformation.

Thirty-nine patients with Philadelphia chromosome-positive (Ph1) chronic myelocytic leukemia (CML) were monitored using cytofluorometry and cytogenetics. During chronic phase, abnormal populations could be detected using commercially available monoclonal antibodies. Most of these abnormal populations had either an HLA.DR+ or an OKM1+ HLA.DR- phenotype. Thirteen patients progressed to acute phase disease. Five patients with HLA-DR+ abnormal cells during chronic phase showed no clonal evolution detectable using standard cytogenetic techniques and underwent lymphoid transformation. Seven patients with OKM1+-HLA.DR- abnormal cells exhibited chromosomal abnormalities in addition to the Ph1 and progressed to a myeloblastic acute phase. One patient with HLA.DR+ OKM1+ 63D3+ abnormal cells in chronic phase showed clonal evolution and progressed to a myelomonocytic acute phase. These results suggest that cell surface markers on chronic phase cells can be used in conjunction with cytogenetic monitoring to predict the phenotype of cells involved in the acute transformation of CML. B cells (6, 13), but not T cells (18), bearing the Ph1 have been isolated from chronic phase CML patients. Thus cells other than those of myeloid lineage may be involved. Periodic cytogenetic monitoring has been shown to detect clonal evolution (i.e. genetic changes in addition to the PH1) in as many as 75% of patients prior to the acute phase (14, 37). However, neither cytogenetic changes nor morphologic analysis appear to predict the lymphoblastic type of transformation (14). Since fluorescence analysis of cell surface markers has shown reduced numbers of normal cells (28), the identity of the 'abnormal' cells was probed with antibodies recognizing a variety of markers on immature and mature lymphoid and myeloid cells. Not only were significant numbers of abnormal cells detected in the peripheral blood of chronic phase patients, but the phenotype of the abnormal population, in combination with cytogenetic analyses, predicted the type of blasts involved in acute phase disease.

Adult↗
PubMed1985

Cytogenetics and flow cytometry may predict phenotype of CML blast crisis.

Twenty-eight patients with chronic myelocytic leukemia (CML) and the Philadelphia chromosome (Ph1) were monitored using cytofluorometry and cytogenetics. During chronic phase, abnormal populations could be detected using commercially available monoclonal antibodies. These abnormal populations fell into two categories based on the presence or absence of HLA.DR. Patients with HLA.DR+ abnormal cells showed no clonal evolution and progressed to lymphoid blast crisis. Patients with HLA.DR- abnormal cells exhibited chromosomal abnormalities in addition to the Ph1 and progressed to a myeloblastic acute phase.

Adult↗
PubMed1983

Monocyte markers and the common acute lymphoblastic leukemia antigen on chronic lymphocytic leukemia cells.

The mononuclear cells from 42 patients with chronic lymphocytic leukemia (CLL) were analyzed for surface markers using monoclonal antibodies and flow cytofluorimetry. All 42 CLLs were Ig+HLA.DR+OKT3-OKT4-OKT6-OKT8- and monoclonal with respect to light chain type. Thirty of the 42 tumors expressed the common acute lymphocytic leukemia antigen, and cells from 18 leukemias stained with the antibodies OKM1 and 63D3, which are specific for different antigens characteristic of myeloid cells. Eight of the leukemias failed to stain with PI153, which has been reported to recognize all B-cell CLLs.

Antibodies, Monoclonal↗
PubMed1983

Immunoregulatory cell subsets in Goodpasture's syndrome: evidence for selective T suppressor-cell depletion during active autoimmune disease.

In a patient with documented untreated Goodpasture's syndrome, serial determination of T-cell subsets using monoclonal antibodies and flow cytometry revealed a persistent but variable deficiency of T-suppressor cells during the period of active disease. As the percentage of OKT8+ cells (suppressor/cytotoxic T cells) returned to normal levels, anti-basement membrane autoantibody production decreased and finally disappeared. Possible pathogenetic implications of immunoregulatory cell imbalance specifically in reference to Goodpasture's syndrome are discussed.

Adult↗