Fluoresceinated Antibodies Research Articles

Detection of Antinuclear Antibodies in Canine Serum by Immunoperoxidase in MDCK and Indirect Immunofluorescence in HEp2 Cells

Antinuclear antibodies are found in animals suffering from Systemic Lupus Erythematosus (SLE) and some other diseases, their presence in the blood is determined by antinuclear antibody (ANA) test using indirect immunofluorescence (IF) with HEp2 cells as a substrate. In this work, an immunoperoxidase (IP) assay was developed to evaluate the ANAs in canine sera, using canine kidney cell lines (MDCK) and compared with a commercial immunofluorescence test on Hep2 cells for this system, a fluoresceinated anti-canine Ig antibody was standardized. The study was performed on 50 sera from dogs submitted to the laboratory with different clinical diagnoses of autoimmune-associated diseases. The procedures on both cells were unified to perform comparisons of the reactions, direct sera or at different dilutions were added to a monolayer of permeabilized MDCK cells, followed by a peroxidized anti-canine IgG conjugate, and a substrate for the IP reaction. The same sera were tested on the commercial IF assay on Hep2 cell system. In 22/50 cases, the presence of LE cells in peripheral blood was determined. A high correlation was found in the detection of antinuclear antibodies between both cell lines and techniques, however there were differences in the reaction patterns in the nucleus and cytoplasm between cell lines. The diffuse nuclear pattern observed in MDCK cells was more related to the presence of high percentages of LE cells in peripheral blood. The differences found in the results were possibly associated with the presence of homologous antigens between the MDCK cells and the dog. In addition, the methodology and standardization for the use and interpretation of a reference serum was developed to unify the interpretation criteria in the laboratory.

Rapid and simple quantitative identification of Listeria monocytogenes in cheese by isothermal sequence exchange amplification based on surface-enhanced Raman spectroscopy.

Foodborne pathogens detection is important to ensure food safety and human health. In this study, we designed a comet structure to rapidly and sensitively detect foodborne Listeria monocytogenes. This method combined isothermal sequence exchange amplification (SEA) and surface-enhanced Raman spectroscopy. Listeria monocytogenes DNA could be rapidly amplified at a constant temperature via SEA with a pair of modified primers, which rendered the precise thermal control instrumentation unnecessary. Efficient SEA amplification generated a large number of DNA duplexes that could be easily captured by streptavidin-modified magnetic bead and AuMB@Ag-isothiocyanate fluorescein antibody (anti-FITC). AuMB@Ag-anti-FITC was used as a signal probe, which generated a significant excitation signal at 1,616 cm-1 for quantitative detection and analysis. The results displayed sensitive detection of L. monocytogenes in cheese from 2.0 × 101 cfu/mL to 2.0 × 106 cfu/mL within 1.0 h with a detection limit of 7.8 cfu/mL. Furthermore, this comet structure displayed the desirable specificity as its specific primers and amplified DNA ends were attached to streptavidin-modified magnetic beads and AuMB@Ag-anti-FITC, respectively. We expected that the method devised would provide a promising new approach to screening for L. monocytogenes and guarantee the microbiological safety of dairy products.

Increased frequency of regulatory T cells in pediatric inflammatory bowel disease at diagnosis: a compensative role?

Regulatory T cells (Tregs) play a critical role in maintaining immune homeostasis. We investigated two main types of Tregs, the CD4+FOXP3+ and IL-10+ Tr1, in pediatric subjects with inflammatory bowel disease (IBD) both at diagnosis and after the clinical remission. Peripheral blood Tregs were analyzed in 16 children with Crohn's disease (CD), 19 with ulcerative colitis (UC), and 14 healthy controls (HC). Two cocktails of fluoresceinated antibodies were used to discriminate between CD4+FOXP3+ and Tr1. We observed in both CD and UC groups a higher frequency of Tr1 at diagnosis compared to controls, which decreased at follow-up compared to diagnosis, in particular in UC. Similarly, in UC patients the percentage of CD4+FOXP3+ Tregs markedly decreased at follow-up compared to the same patients at diagnosis and compared to HC. The expression of CTLA-4 in CD4+FOXP3+ Tregs increased in both groups at clinical remission. This study shows that IBD children present at diagnosis an increased frequency of circulating Tregs, probably as a compensative reaction to tissue inflammation. During the clinical remission, the Treg frequency diminishes, and concomitantly, their activation status increases. Notwithstanding, the high Treg density at diagnosis is not sufficient to counteract the inflammation in the childhood IBD.

Investigation of Coxiella burnetii Prevalence in Women Who Had Miscarriage and Their Spouses by Serological and Molecular Methods

Coxiella burnetii is the causative agent of the common zoonotic disease known as Q fever. Human infection is mostly maintained by inhalation of contaminated aerosols that originate from infected birth products, milk and urine. Sexual transmission has also been reported. In pregnant women the disease causes abortion during the first trimester, while at later stages it tends to become chronic causing low birth weight babies and premature birth. The aim of this study was to investigate the prevalence of C.burnetii in women who had miscarriages, their spouses and in a control group composed of women with normal delivery by using serological and molecular methods. A total of 89 cases (58 female, 31 male; age range: 21-64 years, mean age: 33.1 ± 7.6 years) were included in the study. Women who had abortion (n= 36) were recruited along with their husbands (n= 31), and 22 women who had normal pregnancy were accepted as controls. Blood and placental tissue samples (after abortion or normal delivery) were collected from all of the female subjects, while blood samples were collected from the males. C.burnetii IgG and IgM antibodies in the sera of patients and controls were analysed by ELISA and indirect fluorescein antibody (IFA) methods, and the presence of C.burnetii DNA was searched in whole blood and placenta samples by using polymerase chain reaction (PCR). In our study, C.burnetii Phase II IgG antibody positivity rates in women who had miscarriages, their spouses and in women with normal delivery were found as 27.8% (10/36), 38.7% (12/31) and 4.5% (1/22), respectively by ELISA, while those rates were detected as 27.8% (10/36), 41.9% (13/31) and 9.1% (2/22), respectively by IFA which was accepted as the reference method. However C.burnetii Phase I IgM, Phase I IgG and Phase II IgM antibodies were not detected in none of the subjects by both methods. The relatively high seropositivity rate in our study group (25/89; 28.1%) was thought to be associated with high rates of livestock breeding in our region. Although C.burnetii IgG seropositivity rate in in women who had miscarriages was higher than women with normal delivery, the difference was not found to be statistically significant (x2= 2.906, p= 0.088). When the results of the women with miscarriages and their spouses were evaluated together, it was detected that C.burnetii IgG antibodies were not determined in the spouses of four seropositive women (two positive with 1/64, two with 1/128 titer); titer was 1/64 in four women and their spouses and two women with 1/128 titer had spouses with 1/64 titer. The determination of high titer phase II IgG positivity in 13% (4/31) of the spouses of women who had miscarriages was of notice. All of the blood (n= 89) and placenta samples (n= 51, 29 were from aborted and 22 from normal delivered women) were negative for C.burnetii DNA by PCR. In conclusion, since livestock breeding is common in our region, in cases with recurrent abortion and premature births, women and their husbands should be screened for C.burnetii.

Babesia ovis infections: Detailed clinical and laboratory observations in the pre- and post-treatment periods of 97 field cases

Ovine babesiosis, caused by Babesia ovis, is of major economic importance in Turkey. The changes in the blood profile of infected animals are informative about the course of infection. The aim of the present study was to evaluate the hematological and biochemical changes in the pre- and post-treatment periods of the natural B. ovis infections. The presence of the parasites was confirmed by microscopy and polymerase chain reaction (PCR) analysis. On the basis of the clinical and laboratory findings, the infections were categorized into different groups according to the degree of anemia and the level of parasitemia. All infected sheep were treated with imidocarb dipropionate (IMDP). The blood pictures in the pre- and post-treatment periods were compared.Pancytopenia occurred in animals with severe anemia and very high parasitemia, and bicytopenia in the other groups. The platelet count (PLT), plateletcrit (PCT) and mean platelet volume (MPV) returned to the normal ranges after treatment, except those in the group with severe anemia. In the biochemical profile, B. ovis infection caused an increase in blood urea nitrogen and total bilirubin, and these parameters returned to normal levels after treatment.The indirect fluorescein antibody test (IFAT) results showed that 38.1% of the cases raised specific antibodies during the period of infection, with titers ranging from 1/160 to 1/640. All of 45 animals re-examined after treatment were seropositive, with high titers that rose up to 1/5120.

FTY720 Markedly Increases Alloengraftment but Does Not Eliminate Host Anti-Donor T Cells that Cause Graft Rejection on Its Withdrawal

The immunomodulator FTY720 (FTY) is beneficial in models of graft-versus-host disease, solid organ transplantation, and autoimmunity and has been approved for use in patients with multiple sclerosis. FTY modifies the homing and migration of many cell types. We report that FTY has profound positive and negative effects on allogeneic bone marrow (BM) engraftment in sublethally irradiated recipients. FTY increased donor hematopoietic progenitors in the BM, resulting in high donor engraftment in the B cell, myeloid cell, and natural killer cell, but not T cell, lineages. Donor T cell progenitors within the thymus of FTY-treated recipients were dramatically reduced, resulting in a lack of donor T cell reconstitution. In addition to preventing the ingress of donor (and host) T cell progenitors, FTY prevented the egress of fully functional host CD4+CD8- and CD4-CD8+ thymocytes that on cessation of FTY administration were able to exit from the thymus and contribute to a rapid and complete rejection of a well-established donor BM graft. When used in combination with anti-CD40L mAbs to block the CD40L:CD40 costimulatory pathway, FTY markedly enhanced anti-CD40L mAb-mediated alloengraftment promotion. In contrast to FTY alone, the combination of anti-CD40L mAb and FTY resulted in a surprisingly stable, multilineage, long-term donor chimerism. These data illustrate FTY's profound migration modulating effects and suggest a use in combinatorial therapy in achieving stable alloengraftment under nonmyeloablative conditions.

Adhesion through Single Peptide Aptamers

Aptamer and antibody mediated adhesion is central to biological function and is valuable in the engineering of "lab on a chip" devices. Single molecule force spectroscopy using optical tweezers enables direct nonequilibrium measurement of these noncovalent interactions for three peptide aptamers selected for glass, polystyrene, and carbon nanotubes. A comprehensive examination of the strong attachment between antifluorescein 4-4-20 and fluorescein was also carried out using the same assay. Bond lifetime, barrier width, and free energy of activation are extracted from unbinding histogram data using three single molecule pulling models. The evaluated aptamers appear to adhere stronger than the fluorescein antibody under no- and low-load conditions, yet weaker than antibodies at loads above ∼25 pN. Comparison to force spectroscopy data of other biological linkages shows the diversity of load dependent binding and provides insight into linkages used in biological processes and those designed for engineered systems.

Immobilized antibody orientation analysis using secondary ion mass spectrometry and fluorescence imaging of affinity-generated patterns.

This study assesses the capability of high-resolution surface analytical tools to distinguish immobilized antibody orientations on patterned surfaces designed for antibody affinity capture. High-fidelity, side-by-side copatterning of protein A (antibody Fc domain affinity reagent) and fluorescein (antibody Fab domain hapten) was achieved photolithographically on commercial amine-reactive hydrogel polymer surfaces. This was verified from fluorescence imaging using fluorescently labeled protein A and intrinsic fluorescence from fluorescein. Subsequently, dye-labeled murine antifluorescein antibody (4-4-20) and antibody Fab and Fc fragments were immobilized from solution onto respective protein A- and fluorescein- copatterned or control surfaces using antibody-ligand affinity interactions. Fluorescence assays support specific immobilization to fluorescein hapten- and protein A-patterned regions through antigen-antibody recognition and natural protein A-Fc domain interactions, respectively. Affinity-based antibody immobilization on the two different copatterned surfaces generated side-by-side full antibody "heads-up" and "tails-up" oriented surface patterns. Time-of-flight secondary ion mass spectrometry (TOF-SIMS) analysis, sensitive to chemical information from the top 2 to 3 nm of the surface, provided ion-specific images of these antibody patterned regions, imaging and distinguishing characteristic ions from amino acids enriched in Fab domains for antibodies oriented in "heads-up" regions, and ions from amino acids enriched in Fc domains for antibodies oriented in "tails-up" regions. Principal component analysis (PCA) improved the distinct TOF-SIMS amino acid compositional and ion-specific surface mapping sensitivity for each "heads-up" versus "tails-up" patterned region. Characteristic Fab and Fc fragment immobilized patterns served as controls. This provides first demonstration of pattern-specific, antibody orientation-dependent surface maps based on antibody domain- and structure-specific compositional differences by TOF-SIMS analysis. Since antibody immobilization and orientation are critical to many technologies, orientation characterization using TOF-SIMS could be very useful and convenient for immobilization quality control and understanding methods for improving the performance of antibody-based surface capture assays.

Leakage of serum proteins in brain tissues in experimentally induced renal hypertension.

The immunohistological method using the fluorescein antibody technique (direct method) was demonstrated to be proper for detecting the edema fluid in the brain. Serum leakage was observed mainly in the cerebral white matter in the renal hypertensive rat. Although the pathogenesis of the edema formation could not be clarified, the nature of edema fluid and the extent and location of fluid deposition in the brain can be detected. Advanced study can be expected with this method.

Laser-based transfection with conjugated gold nanoparticles

The irradiation of cells combined with the immunoconjugate of gold nanoparticles by the short pulse laser can make the plasma membrane be transiently permeabilized, which can be used to transfer exogenous molecules into the cells. We explore this technique as a novel gene transfection method for floating cells. Three different floating cells exposed to the laser are selectively transfected with fluorescein isothiocyanatedextran, antibody, and green fluorescent protein (GFP) coding plasmids, and the viability of cells are determined by propidium iodide. For fluorescein isothiocyanate-dextran, the best transfection efficiency of 65% is obtained; for the antibody, it is 74%; whereas for the green fluorescent protein coding plasmids, a very small transfection efficiency is gained. If the transfection efficiency is improved, gold nanoparticles will be very useful as mediator for gene transfection in living cells.

Screening for Chlamydia trachomatis Infection Using Direct Fluorescein Antibody Method in Female Sex Workers Registered in Ankara, Turkey: Pros and Cons

Aim: This study aimed to screen for Chlamydia trachomatis infection by direct fluorescein antibody (DFA) method in a high-risk population, registered female sex workers (FSWs) in Ankara, Turkey, to investigate the advantages and disadvantages of using this method in a routine screening program of the FSW population. Materials and Methods: All registered FSWs (n = 152) in Ankara were enrolled in the study. None of them were showing any genital signs or symptoms of sexually transmitted infections (STIs). Endocervical swabs were collected and examined for C. trachomatis by DFA method (Fluorotect Chlamydia, Omega Diagnostics, UK). Cellular adequacy of the specimens was examined for the presence of at least five columnar epithelial cells per X400 field. A positive diagnosis was made when fixed and stained specimens showed at least 10 chlamydial bodies under the fluorescein microscope. A positive control slide was also stained and examined on each study day. Results: Of the 152 specimens, only 112 (73.7%) were adequate; it was not possible to obtain adequate specimen from the remainder of the women because the cervical os was too tight to insert the swab. Of the 112 adequate specimens, 3.6% (4/112) were positive for C. trachomatis serotype-specific major outer membrane proteins (MOMP), based on DFA method. Conclusions: C. trachomatis infection rate of 3.6% was relatively low in this group of women compared to a priori expectations, yet still remarkable. A significant level of ´´inadequacy´´ in obtaining endocervical specimens (in 26.3% of FSWs) could have limited the external validity of the prevalence rate estimation in this population and the practicality of DFA use for C. trachomatis screening. C. trachomatis screening should be integrated in the STI control program for sex workers in Turkey. However, further research is warranted to optimize the method to be used in screening and/or diagnosis in such high-risk groups.

Development of immunofiltration assay by light addressable potentiometric sensor with genetically biotinylated recombinant antibody for rapid identification of Venezuelan equine encephalitis virus

A genetically biotinylated single chain fragment variable antibody (scFv) against Venezuelan equine encephalitis virus (VEE) was applied in a system consisting of an immunofiltration enzyme assay (IFA) with a light addressable potentiometric sensor (LAPS) for the rapid identification of VEE. The IFA involved formation of an immunocomplex sandwich consisting of VEE, biotinylated antibody, fluoresceinated antibody and streptavidin, capture of the sandwich by filtration on biotinylated membrane, and labeling of the sandwich by anti-fluorescein urease conjugate. The concentration ratio of biotinylated to fluoresceinated antibodies was investigated and optimized. By the IFA/LAPS assay, the limit of detection (LOD) of VEE was approximately 30 ng/ml, similar to that achieved when chemically biotinylated monoclonal antibody (mAb) was applied. Total assay variance of the IFA/LAPS assay for both intra- and inter-assay precision was less than 20%. Assay accuracy was measured by comparing VEE concentrations estimated by IFA/LAPS standard curve to those obtained by conventional protein assay. VEE concentrations were found to differ by no more than 10%. The IFA/LAPS assay sensitivity was approximately equal to that of a conventional enzyme-linked immunosorbent assay (ELISA) utilizing polystyrene plates and a chromogenic substrate; however, less time and effort were required for performance of the IFA/LAPS assay. More importantly, use of genetically biotinylated scFv in the IFA/LAPS assay obviates the need for chemical biotinylation of antibody with resultant possible impairment of the antigen-binding site. Furthermore, the potential for batch-to-batch variability resulting from inequality in the number of biotin molecules labeled per antibody molecule is eliminated.

Fine-needle aspiration biopsy and flow cytometry immunophenotyping of lymphoid and myeloproliferative disorders of the spleen.

Flow cytometry (FC) is a useful adjunct to fine-needle aspiration biopsy (FNAB) in the evaluation of lymphoproliferative disorders. The application of FC to FNAB of the spleen (sFNAB) is reported. Flow cytometry was performed on 18 sFNAB collected over 3 years. The series comprised 10 cases of non- Hodgkin lymphomas (NHL), 2 cases insufficient for diagnosis, 2 cases of reactive hyperplasia (RH), and 4 cases of myeloid metaplasia (MM). FNAB was performed under ultrasound guidance using a 22-gauge needle. One or two passes were sufficient to prepare a conventional smear that was immediately evaluated to select the cases studied and to prepare a cell suspension for FC. The following fluoresceinated antibodies were used: CD3, CD19/kappa/lambda, FMC7/CD23/CD19, Bcl-2, and CD13/HLA-DR. In six cases, cytospins were also prepared for immunocytochemistry and were tested for CD20 (L26), CD45Ro, and kappa and lambda light chain expression. Flow cytometry contributed to the diagnosis of all cases of NHL by assessing light chain restriction. The specific subtype was also diagnosed by CD19/CD5 and CD 19/CD10 coexpression in two cases. Flow cytometry quantified the percentage of myeloid cells in MM cases and contributed to the cytologic diagnosis showing a polyclonal light chain expression in RH cases. Immunocytochemistry was effective and concordant in four cases. Patients tolerated the sFNAB well and no complications were reported. Cytologic and FC diagnoses were confirmed by follow-up and by histologic evaluation in cases in which splenectomy was performed for therapeutic purposes. Flow cytometry applied to sFNAB corroborates the cytologic diagnosis in lymphoid and myeloproliferative disorders of the spleen and allows therapeutic decisions avoiding splenectomy.

Clinical and epidemiological features of acute follicular conjunctivitis with special reference to that caused by herpes simplex virus type 1

BACKGROUND/AIMSIt is reported by the national surveillance of ocular infectious diseases in Japan that 4.3% of cases of epidemic keratoconjunctivitis (EKC) diagnosed clinically were caused by herpes simplex virus (HSV)....

Three-Day Course of Oral Azithromycin vs Topical Oxytetracycline/ Polymyxin in Treatment of Active Endemic Trachoma

Purpose: The aim of this study on endemic trachoma was to carry out a comparison of azithromycin (3-day course, oral dose of 10 mg/kg per day) with conventional treatment (topical oxytetracycline/polymyxin ointment; twice a day for 2 months) in a rural area near Sanliurfa, Turkey. Methods: Ninety-six subjects with active trachoma were randomly assigned conventional or azithromycin treatment. Subjects were examined 1, 2, 3, and 6 months after the start of treatment. Clinical findings were recorded for each eye. Swabs were taken from upper eyelids 3 and 6 months after the start of treatment for direct fluorescein antibody test. Results: By six-month follow-up, trachoma had resolved clinically in 43 (89.58%) of the 48 subjects who received azithromycin, compared with 33 (68.75%) of the 48 who were treated conventionally. Microbiological success rates (direct fluorescein antibody test negativity) were 83.33% in the azithromycin group and 62.50% in the conventional therapy group. Compliance with both treatments was good. By 6 months, 14.58% of the subjects in azithromycin group and 33.33% of the subjects in the topical treatment group were reinfected. There were significant differences in the efficacy of the treatment effects and the re-emergence of disease between the two treatment groups. Azithromycin was well-tolerated. Conclusions: These results indicate that azithromycin may be an effective alternative for patients with active trachoma. As a systemic treatment, a 3-day course oral dose has important potential for trachoma control.

Autoimmune-related pancreatitis is associated with autoantibodies and a Th1/Th2-type cellular immune response

Background & Aims: Although autoimmunity may be involved in some cases of pancreatitis, the mechanism is still unknown. To clarify this, we studied serum autoantibodies, subsets of lymphocytes, and the Th1/Th2 balance of cellular immune responses in patients with autoimmune-related pancreatitis (AIP). Methods: Seventeen patients with AIP (8 men and 9 women; age, 53.2 ± 13.0 years) were studied. Autoantibodies including antilactoferrin (ALF) or carbonic anhydrase II antibody (ACA-II) were examined using the enzyme-linked immunosorbent assay (ELISA) or the indirect fluorescein antibody method. Intracellular cytokines (interferon gamma and interleukin 4) and subtypes of peripheral blood lymphocytes were examined by flow cytometry and ELISA. Results: More than one autoantibody was observed in all 17 patients. Serum antinuclear antibody was detected in 13 of 17 patients, ALF antibody in 13, ACA-II antibody in 10, rheumatoid factor in 5, and anti–smooth muscle antibody in 3, but antimitochondrial antibody in none. The serum levels of ACA-II and LF antibody were not correlated. HLA-DR +CD8 + and HLA-DR +CD4 + cells were significantly increased in peripheral blood ( P < 0.05). CD4 + cells producing interferon gamma and the secreted levels were significantly increased compared with those in controls ( P < 0.05), but interleukin 4 was not increased. Conclusions: An autoimmune mechanism against CA-II or LF, and Th1-type immune response, may be involved in AIP. GASTROENTEROLOGY 2000;118:573-581

Angiography of fluoresceinated anti-vascular endothelial growth factor antibody and dextrans in experimental choroidal neovascularization.

To determine if anti-vascular endothelial growth factor antibody and a range of dextrans with varying diffusion radii and molecular weights are permeable through experimental choroidal neovascularization (CNV). Choroidal neovascularization was induced in 10 cynomolgus monkey retinas by means of argon laser injury. Digital fundus fluorescein angiograms were performed with fluorescein sodium, fluoresceinated IgG antibodies (anti-vascular endothelial growth factor and a control antibody), and fluoresceinated dextrans with molecular weights of 4, 20, 40, 70 and 150 kd. The 40- and 70-kd dextrans straddle the effective diffusion radius of IgG. For each reagent, early and late angiograms were performed in a standardized fashion, with follow-up images obtained to monitor residual fluorescence. Perfusion of retinal vessels and choroidal vasculature was seen with all reagents. Fluorescein and 4- and 20-kd dextran leaked rapidly from the CNV within the first minute. Angiography with the use of 40-kd dextran and fluoresceinated antibody, either anti-vascular endothelial growth factor or control IgG, showed fluorescence within the CNV that increased during the first 1 to 5 hours, with mild leakage from the CNV. By 24 hours, fluorescence in the CNV was minimal, although in some cases persistent fluorescence in the surrounding tissue was evident up to 2 weeks. The 70-kd dextran showed fluorescence within the CNV and leakage in 1 of 3 eyes. The 150-kd dextran showed fluorescence within the CNV but did not demonstrate leakage. Fluoresceinated antibodies and dextran with smaller effective diffusion radii showed CNV perfusion and leakage. Dextrans with larger effective diffusion radii (70 kd and 150 kd) perfused into CNV but did not show leakage consistently. Determining the permeablity of antibodies and molecules of similar size through CNV can help ascertain the feasibility of using intravenously administered antibodies against angiogenic growth factors as a future treatment for choroidal neovascularization.

APOPTOSIS EXPRESSION IN CONGENITAL HEPATIC FIBROSIS

93 Abstract. Congenital hepatic fibrosis is a rare disease characterized by portal fibrosis and biliary ductular ectasia. The stellate cell is the major source of fibrosis in the liver. Stellate cell action is mediated mainly by serine threonine kinase receptors types I and II. The receptors mediate signal transducer by incompletely defined pathways and result in the programmed cell death or apoptosis. This signal is instrumental in organ development and in the inhibition of epithelial proliferation by TGF β1. The increased expression of TGF β1 and the abnormal distribution of the type I and II receptors in CHF led us to speculate about the extent of apoptosis on hepatocytes and biliary ductular epithelial cells in this classic condition of Ductal Plate Malformation. Methods. Three normal livers, two CHF livers, and two CHF wedge biopsies were used. The Oncor Apoptag Plus In situ detection kit was used (Oncor, Gaithesburg, MD). The method identifies 3′ OH DNA fragments produced by the action of endonuclease in the process of apoptosis. The TdT enzyme (terminal deoxynucleotidyl transferase) that attaches the digoxigenin to the 3′ OH DNA fragments was prepared within 6 hours. The antidigoxigenin fluorescein antibody was then applied and the reaction proceeded for 30 minutes at room temperature in a humidified chamber. Counter staining was then performed with Propidium iodide and the slides were read with a florescent microscope at 494 nm wavelength. Two positive and one negative control were supplies by the manufacturer. Results. Normal liver showed minimal apoptosis as expected. There was an increase in the degree of apoptosis in CHF livers. This may reflect the increased expression of TGF β1 previously described. The ectatic biliary epithelium appears to lack any evidence of apoptosis. Conclusion. The effect of an increased expression of TGF β1 in CHF results in hepatocyte apoptosis in CHF. The increase in apoptosis may eventually lead to a decrease in parenchymal cell mass. The lack of apoptosis in the ectatic biliary epithelium may reflect the lack of TGF β1 receptor II expression and may explain the hyperplasia in this condition.

Sensitive assay for measurement of antibodies to Clostridium botulinum neurotoxins A, B, and E: use of hapten-labeled-antibody elution to isolate specific complexes.

The measurement of chicken and human antibodies to Clostridium botulinum neurotoxins A, B, and E was accomplished by affinity isolation of complexes containing these antibodies. By this approach, a mixture of toxin with the test antibody, fluoresceinated antibody, and enzyme (Russell's viper venom factor X activator)-labeled antibody is allowed to form a complex in solution phase. This complex is then bound to a matrix containing antifluorescein antibody. All components not bound to the matrix are washed off, and the complex is isolated intact by elution with fluorescein, which competes with the complex for binding to the antifluorescein matrix. The eluted complex is then bound to a matrix which specifically binds the test antibody (anti-chicken immunoglobulin Y [IgY] or anti-human IgG), and the bound complex is measured by using the enzyme label. Using this approach, we were able to measure as little as 1 ng of specific antibody per ml from affinity-isolated, monospecific chicken antibody preparations and to measure antibody specifically from IgY fractions of monospecific chicken antibody preparations. Human antibodies from subjects immunized with pentavalent toxoid preparations were detectable at dilutions as great as 24,300-fold, and undiluted serum from most control subjects showed no measurable antibody. Antibody was also measured in 65 subjects who were receiving preparations of neurotoxin A (BOTOX) for the treatment of spastic disorders. Eighteen of them had toxin-specific antibody reactive with toxin B, and two of them had toxin-specific antibody reactive with toxin A. The two patients having antibody to toxin A were refractory to treatment with this toxin. This approach of isolation of hapten-labeled immune complexes under nondenaturing conditions with hapten is broadly applicable to the specific measurement of antibodies present at very low concentrations in serum.

Photochemically-activated electrodes: application in design of reversible immunosensors and antibody patterned interfaces

Antigen monolayers assembled onto Au electrodes associated with a quartz crystal act as electrochemical or microgravimetric quartz-crystal-microbalance (QCM) sensing interfaces for the complementary antibody. Electrochemical analysis of the antibody (Ab) is based on the insulation of the antigen monolayer electrode by the associated Ab towards a redox probe in the electrolyte solution. Ferrocene-modified glucose oxidase (Fc-GOx) and glucose are employed as redox probes for the amperometric transduction of the Ab association to the electrode. Bioelectrocatalyzed oxidation of glucose provides an electrochemical route to amplify the antigen-Ab complex formation. Electrochemical analysis of the dinitrophenyl antibody, DNP-Ab, by a dinitrophenyl-lysine monolayer electrode is presented. QCM analysis of the Ab is based on the frequency changes of the quartz crystal resulting from the association of the Ab to the crystal assembly. This method is discussed with the analysis of the fluorescein antibody, Flc-Ab, using a fluorescein monolayer-modified quartz crystal. A novel method to tailor reversible immunosensor devices by the application of photoisomerizable antigen monolayers on electrodes is presented. The antigen is modified by photoactive units exhibiting reversible photoisomerizable properties. In one photoisomer state, the antigen exhibits affinity for the Ab and enables its electrochemical or QCM analysis. Photoisomerization to the complementary state perturbs the antigen structure and the monolayer lacks affinity for the Ab. This enables the washing-off of the Ab and the regeneration of the actively sensing interface by a second illumination process that restores the antigen monolayer-modified surface. This method is exemplified by the development of a reversible DNP-Ab sensing electrode. N-Mercaptobutyl dinitrospopyran was assembled as a photoisomerizable monolayer on a Au electrode. The dinitrospiropyran monolayer, SP-state, exhibits affinity for the DNP-Ab and enables the amperometric detection of the Ab using Fc-GOx and glucose as redox probe. The complementary photoisomerized protonated dinitromerocyanine monolayer, MRH +-state, lacks affinity for the DNP-Ab. By photoisomerization of the DNP-Ab associated with the SP-monolayer electrode to the MRH +-monolayer state, the DNP-Ab is washed-off, and by a second illumination process, the MRH +-monolayer is re-isomerized to the SP-monolayer assembly, which is the active interface for further analysis of the DNP-Ab. Cyclic amperometric detection of the DNP-Ab by the photoisomerizable dinitrospiropyran monolayer is demonstrated. The association of the DNP-Ab to the SP-monolayer electrode and the dissociation of the Ab from the MRH +-monolayer electrode are confirmed by QCM experiments using a dinitrospiropyran monolayer-modified quartz crystal. The insulating features of an antigen-Ab complex on a conductive surface and the photochemically controlled association of an antibody to a photoisomerizable monolayer assembled onto the surface were used to develop means for micropatterning of surfaces by the antibody. A dinitrospiropyran antigen monolayer was assembled onto conductive ITO glass. A DNP-Ab solution was used as ‘ink solution’ to pattern the surface. The Ab-pattern was imaged by electrochemical copper deposition onto the Ab-lacking surface domains. The dinitrospiropyran monolayer assembled onto ITO or Pyrex glass surfaces was employed as an active interface for the photolithographic patterning of the surface with the DNP-Ab. Irradiation of the dinitrospiropyran monolayer, SP-state, through a grid-mask generated surface domains of protonated dinitromerocyanine, MRH +-state, which lacks affinity for the DNP-Ab. The DNP-Ab covalently-linked to agarose beads (50 μm) was selectively associated to the SP-monolayer sites and the micro-pattern of the resulting antibody was imaged. Methods for employing this photolithographic patterning of DNP-Ab to generate microstructures of any biomaterial, and, specifically, neural networks, are discussed.