Description of a non-competitive ELISA based on time course analysis of ligand binding at saturation, and a direct method for calculating the affinity of monoclonal antibodies.

We present a time-course saturation ELISA for measuring the equilibrium constant of the monoclonal antibody (mAb) SIM 28 against horse radish peroxidase (HRP). The curves of HRP binding to a series of fixed mAb dilutions were plotted to completion, and the K (= K) value (time to occupy 50 % of the mAb paratopes) was determined for each mAb dilution and HRP concentration. Analysis of the kinetic mechanism of the reaction by Lineweaver-Burk and Hanes plots showed that the slope and y-intercept were affected, indicating that mAb ligand saturation follows non-competitive inhibition kinetics in this assay format.

Monoclonal antibody on-rate constant determined from time-course data of ligand binding by capture ELISA: Evaluation of eight data analysis methods.

We describe an ELISA method with which to determine monoclonal antibody (mAb) on-rate constants (k) based on time-course data of ligand (L) binding to plate-bound mAb. The assay was performed in pseudo-first order kinetic conditions ([L] > > [mAb]) and at various starting ligand concentrations. Time-course initial velocity was analyzed by several methods to derive the pseudo-first order (k) and second order (k) association rate constants of the antibody; the methods included i) an exponential first order rate equation, ii) reaction half-time from the Michaelis-Menten relationship, iii) the V/K tangent of the time-course curve, iv) Boeker's extrapolated-v method, v-vi) modified Hanes-Woolf and Lineweaver-Burk linear plots, vii) a LOS plot, and viii) initial velocity gradient.

Variant analyses of candidate genes in orofacial clefts in multi-ethnic populations.

Objectives: Cleft lip with/without cleft palate and cleft palate only is congenital birth defects where the upper lip and/or palate fail to fuse properly during embryonic facial development. Affecting ~1.2/1000 live births worldwide, these orofacial clefts impose significant social and financial burdens on affected individuals and their families.

Development of a competitive inhibition kinetic ELISA to determine the inhibition constant (K) of monoclonal antibodies.

Antibody-antigen interactions are mediated by the same molecular recognition mechanisms as those of an enzyme and its substrate. On this basis, we developed a competitive inhibition kinetic ELISA to measure monoclonal antibody (mAb) inhibition constants. Serially diluted samples of ligand (mAb) and inhibitor (soluble antigen) were incubated to equilibrium in ELISA plates coated with a fixed concentration of antigen (receptor).

Non-random distribution of deleterious mutations in the DNA and protein-binding domains of IRF6 are associated with Van Der Woude syndrome.

Background: The development of the face occurs during the early days of intrauterine life by the formation of facial processes from the first Pharyngeal arch. Derangement in these well-organized fusion events results in Orofacial clefts (OFC). Van der Woude syndrome (VWS) is one of the most common causes of syndromic cleft lip and/or palate accounting for 2% of all cases.

A kinetic ELISA to determine the immunoreactive fraction of monoclonal antibodies.

We developed a two-step ELISA to determine the immunoreactive fraction of monoclonal antibodies in conditions of antigen excess. An antibody aliquot at limiting dilution was incubated in wells coated with increasing amounts of antigen up to concentrations that bind 100% of antibody. At equilibrium, a supernatant aliquot was transferred to a second plate coated with excess of antiglobulin, and the captured antibody was incubated with peroxidase-conjugated anti-IgG.

Quantitation of monoclonal antibody by capture ELISA based on initial enzyme activity rate.

We developed a noncompetitive two-site sandwich ELISA to quantitate monoclonal antibodies in culture supernatant. This assay measures the initial enzyme activity rate during the first minute of the reaction, which ensures linear velocity relative to time and a progress curve slope proportional to analyte concentration. During this period, the enzyme substrate is in large excess relative to the analyte/antibody-enzyme complex, and enzyme catalysis proceeds in steady-state conditions.

Development and characterization of mouse monoclonal antibodies to eight human complement components: Analysis of reactivity with orthologs of nine mammalian genera.

To study complement function in mammalian leishmanioses, we developed mouse monoclonal antibodies to the human complement system components C1q, C4, factor D, factor H, factor B, properdin, C5 and C9. Antibody specificity was determined by indirect and capture ELISA and by Western blot. In flow cytometry analysis, seven antibodies recognized the cognate component on human serum-opsonized Leishmania promastigotes.

An Insight into the proteome of Crithidia fasciculata choanomastigotes as a comparative approach to axenic growth, peanut lectin agglutination and differentiation of Leishmania spp. promastigotes.

The life cycle of the trypanosomatid Crithidia fasciculata is monogenetic, as the unique hosts of these parasites are different species of culicids. The comparison of these non-pathogenic microorganisms evolutionary close to other species of trypanosomatids that develop digenetic life cycles and cause chronic severe sickness to millions of people worldwide is of outstanding interest. A ground-breaking analysis of differential protein abundance in Crithidia fasciculata is reported herein.

Evidence of Leishmania infantum infection in rabbits (Oryctolagus cuniculus) in a natural area in Madrid, Spain.

Leishmaniasis is one of the most important neglected zoonosis and remains endemic in at least 88 developing countries in the world. In addition, anthropogenic environmental changes in urban areas are leading to its emergency world wide. Zoonotic leishmaniasis control might only be achieved by an integrated approach targeting both the human host and the animal reservoirs, which in certain sylvatic cycles are yet to be identified.

Detection of anti-Leishmania infantum antibodies in sylvatic lagomorphs from an epidemic area of Madrid using the indirect immunofluorescence antibody test.

An outbreak of human leishmaniasis was confirmed in the southwest of the province of Madrid, Spain, between July 2009 and December 2012. Incidence of Leishmania infection in dogs was unchanged in this period, prompting a search for alternative sylvatic infection reservoirs. We evaluated exposure to Leishmania in serum samples from animals in the area with an indirect immunofluorescence test (IFAT).

Effect of allicin on promastigotes and intracellular amastigotes of Leishmania donovani and L. infantum.

Anti-leishmanial activity of allicin (=diallyl thiosulphinate) has been tested in vitro against promastigotes and intracellular amastigotes of Leishmania donovani and Leishmania infantum. Macrophage infections have been carried out in vitro in the murine cell line J774 and ex vivo with peritoneal macrophages from BALB/c mice with a modified method to isolate metacyclic promastigotes. The compound has shown a significant in vitro effect on the multiplication of promastigotes of L.

Study of peripheral blood cell populations involved in the immune response of goats naturally infected with Mycobacterium caprae.

Tuberculosis in goats caused by Mycobacterium bovis and Mycobacterium caprae has noteworthy sanitary and economic implications. Current diagnostic assays are based on cellular immunity and although they have demonstrated a high sensitivity, some animals remain undetected. In the present study, flow cytometry has been used to determine changes in CD4+, CD8+ and CD25+ T cell populations in peripheral blood from naturally infected goats.

Kinetic analysis of ex vivo human blood infection by Leishmania.

The leishmanioses, vector-borne diseases caused by the trypanosomatid protozoan Leishmania, are transmitted to susceptible mammals by infected phlebotomine sand flies that inoculate promastigotes into hemorrhagic pools created in host skin. We assumed that promastigotes are delivered to a blood pool, and analyzed early promastigote interactions (0-5 min) with host components, which lead to parasite endocytosis by blood leukocytes, and to host infection. Promastigotes were incubated with NHS or with heparinized blood in near-physiological conditions, and we used cell radioimmunoassay and flow cytometry to measure the on-rate constants (k(+1)) of promastigote interactions with natural opsonins and erythrocytes.

Temperature increase prevails over acidification in gene expression modulation of amastigote differentiation in Leishmania infantum.

Background: The extracellular promastigote and the intracellular amastigote stages alternate in the digenetic life cycle of the trypanosomatid parasite Leishmania. Amastigotes develop inside parasitophorous vacuoles of mammalian phagocytes, where they tolerate extreme environmental conditions. Temperature increase and pH decrease are crucial factors in the multifactorial differentiation process of promastigotes to amastigotes.

Comparative real-time kinetic analysis of human complement killing of Leishmania infantum promastigotes derived from axenic culture or from Phlebotomus perniciosus.

Although Leishmania metacyclic promastigotes are generally considered resistant to human complement, studies of in vitro-cultured axenic stationary promastigotes using serum concentrations that approximate physiological plasma conditions indicate complement sensitivity. Natural Leishmania infection is caused by sand fly-inoculated promastigotes, whose complement resistance has not been analyzed systematically. We compared Leishmania susceptibility to human complement in L.

Early mechanisms of Leishmania infection in human blood.

In human blood, promastigotes bind natural antibodies and activate the classical complement pathway. C3-opsonized promastigotes immune-adhere within seconds to erythrocytes. Promastigote lysis by complement parallels C3 deposition kinetics, and ~90% of promastigotes are killed after 2.

Complement interaction with trypanosomatid promastigotes in normal human serum.

In normal human serum (NHS), axenic promastigotes of Crithidia, Phytomonas, and Leishmania trigger complement activation, and from 1.2 to 1.8 x 10(5) C3 molecules are deposited per promastigote within 2.

Leishmania immune adherence reaction in vertebrates.

In normal human blood, C3-opsonized Leishmania promastigotes immune adhere to erythrocytes, a mechanism believed to enhance their clearance from blood and phagocytosis. Given the potential importance of this reaction in host defence against infection, the promastigote-erythrocyte interaction was studied in blood of individuals from one avian and 12 mammalian genera; [111In]-labelled promastigotes were found to bind only to primate erythrocytes. Nevertheless, previous experiments coincubating platelets isolated from nonprimate mammals with C3-opsonized promastigotes led to promastigote-platelet adherence.

Bile acid secretion during rat liver carcinogenesis.

Retro-differentiation of liver parenchyma during neoplastic processes is characterized by the expression of tumor antigens, such as alpha-fetoprotein and the placental isoenzyme of glutathione-S-transferase (GST-P). To investigate whether this may also affect a typical liver function such as bile acid secretion was the aim of this work. Rat hepatocarcinogenesis was induced by diethylnitrosamine (i.

Immune adherence-mediated opsonophagocytosis: the mechanism of Leishmania infection.

To mimic the sandfly pool feeding process and characterize the cellular and biochemical events that occur during the early stages of promastigote-host interaction, we developed an ex vivo model of human blood infection with Leishmania promastigotes. Within 30 s of blood contact, Leishmania promastigotes bind natural anti-Leishmania antibodies, which then activate the classical complement pathway and opsonization by the third component of complement. The opsonized promastigotes undergo an immune adherence reaction and bind quantitatively to erythrocyte CR1 receptors; opsonized Leishmania amastigotes also bind to erythrocytes.

Periodate oxidation of R36A pneumococci greatly enhances production of hybridomas secreting anti-protein antibodies.

Most hybridomas derived from mice immunized with non-capsulated Streptococcus pneumoniae strains secrete antibodies to C-polysaccharide epitopes and very rarely against cell wall proteins. Mild periodate oxidation of the non-capsulated R36A pneumococcal strain destroys C-polysaccharide antigenicity without a noticeable loss in the immunizing capacity for proteins. Following immunization of BALB/c mice with periodate-treated R36A cells, most hybridomas obtained (87.

Correlation of mid-sternum and mid-spine positions in 82 patients irradiated for breast cancer.

The validity of designing anterior internal mammary node (IMN) radiation therapy fields based on vertebral body landmarks was tested by reviewing computer tomography (CT) images in 82 patients treated for breast cancer. The horizontal distance between the geometric center of the sternum and the vertebral body was 0-5, 6-10 and > 10 mm in approximately 30%, 25%, and 45% of patients, respectively. This suggests that using the projection of the vertebral bodies to design an anterior internal mammary node field is frequently inaccurate and may lead to inadequate coverage of these lymph nodes.

Split tolerance induced by chick embryo thymic epithelium allografted to embryonic recipients.

To test the capacity of the epithelial component of the chick embryo thymus to induce tolerance to major histocompatibility complex (MHC) antigens, pre-colonized thymic rudiments were grafted into chick embryonic recipients. Semi-allogeneic or allogeneic transplantations were done between two lines of chickens histocompatible at the MHC locus. Approximately 10% of these thymic chimeras hatched and were studied 3 mo after hatching.

Post-natal ontogenesis of graft-versus-host reactivity of peanut agglutinin lectin-negative thymocytes in the chicken.

Chicken thymocyte fractionation by peanut agglutinin lectin yields two cell subpopulations which differ in their GvH competence when injected into major histocompatibility complex (MHC)-(B locus) incompatible embryos. The nonagglutinated PNA- fraction displayed a degree of alloreactivity similar to that of peripheral blood lymphocytes while the PNA+ cells were nonreactive. Analysis of chickens at the age of 4 to 24 weeks showed that the development of GvH-reactive PBL preceded by 12 weeks the maturation of GvH-reactive PNA- thymocytes.