Preparation of antibodies to roridin A and their applications

Polyclonal rabbit antibodies against a conjugate synthesized through condensing BSA and disubstituted roridin A hemisuccinate allowed roridin A to be determined in solutions at a sensitivity of 0.2 ng/ml. The cross-reactivity of structural analogues--roridin A, verrucarin, and verrucarol--amounted to 100, 2.5, and 0.03%, respectively. The data showed that these antibodies determine roridin A in an indirect heterogeneous enzyme immunoassay in cereal straw samples at a sensitivity of 20 micrograms/kg.     

Enzyme immunoassay for the macrocyclic trichothecene roridin A: production, properties, and use of rabbit antibodies

Antisera against roridin A were prepared by using a roridin A-hemisuccinate derivative coupled to human serum albumin as the immunogen. Antibodies could be detected in the sera of the immunized rabbits as early as 4 weeks after the initial exposure. After one booster injection at week 14, high antibody titers were measured over a period of 21 weeks. The specificity and sensitivity of the antibodies were tested by using roridin A-hemisuccinate coupled to horseradish peroxidase as an enzyme-linked toxin in a competitive assay with a double-antibody solid phase. The assay was most specific for the tested macrocyclic trichothecenes, and the relative cross-reactivities with roridin A, roridin J, verrucarin A, satratoxin H, and satratoxin G were 1, 0.41, 0.15, 0.15, and 0.07, respectively. When 16 nonmacrocyclic trichothecenes were tested, only diacetylverrucarol (0.0015) and verrucarol (0.0005) showed minor cross-reactivity. The sensitivity of the enzyme immunoassay for the detection of roridin A was in the range of 5 to 50 ng/ml (0.16 to 1.6 ng per assay).     

Enzyme immunoassay for the macrocyclic trichothecene roridin A: production, properties, and use of rabbit antibodies.

Antisera against roridin A were prepared by using a roridin A-hemisuccinate derivative coupled to human serum albumin as the immunogen. Antibodies could be detected in the sera of the immunized rabbits as early as 4 weeks after the initial exposure. After one booster injection at week 14, high antibody titers were measured over a period of 21 weeks. The specificity and sensitivity of the antibodies were tested by using roridin A-hemisuccinate coupled to horseradish peroxidase as an enzyme-linked toxin in a competitive assay with a double-antibody solid phase. The assay was most specific for the tested macrocyclic trichothecenes, and the relative cross-reactivities with roridin A, roridin J, verrucarin A, satratoxin H, and satratoxin G were 1, 0.41, 0.15, 0.15, and 0.07, respectively. When 16 nonmacrocyclic trichothecenes were tested, only diacetylverrucarol (0.0015) and verrucarol (0.0005) showed minor cross-reactivity. The sensitivity of the enzyme immunoassay for the detection of roridin A was in the range of 5 to 50 ng/ml (0.16 to 1.6 ng per assay).     

Production and characterization of monoclonal antibodies to the macrocyclic trichothecene roridin A.

Two murine monoclonal antibodies to the macrocyclic trichothecene roridin A are described. Screening for antibody production was performed on absorbed anti-mouse immunoglobulin serum as double-antibody solid phase, and further characterization was done on affinity-purified anti-mouse IgG serum. The antibodies, designated 5G11 and 4H10, had affinity constants for roridin A of 9.25 X 10(7) and 1.7 X 10(7) liters/mol, respectively. In monoclonal antibody-based direct enzyme immunoassays, these IgG1 antibodies had detection limits for roridin A of 0.4 ng/ml (0.02 ng per assay) and 1.8 ng/ml (0.09 ng per assay), respectively. Both antibodies were most specific for the tested macrocyclic trichothecenes. The relative cross-reactivities of antibody 5G11 with roridin A, roridin J, verrucarin A, satratoxin G, and satratoxin H were 100.0, 43.8, 16.7, 3.7, and 18.9%, respectively; for antibody 4H10 they were 100.0, 6.3, 64.0, 4.4, and 4.9%, respectively.     

Production and characterization of monoclonal antibodies to the macrocyclic trichothecene roridin A

Two murine monoclonal antibodies to the macrocyclic trichothecene roridin A are described. Screening for antibody production was performed on absorbed anti-mouse immunoglobulin serum as double-antibody solid phase, and further characterization was done on affinity-purified anti-mouse IgG serum. The antibodies, designated 5G11 and 4H10, had affinity constants for roridin A of 9.25 X 10(7) and 1.7 X 10(7) liters/mol, respectively. In monoclonal antibody-based direct enzyme immunoassays, these IgG1 antibodies had detection limits for roridin A of 0.4 ng/ml (0.02 ng per assay) and 1.8 ng/ml (0.09 ng per assay), respectively. Both antibodies were most specific for the tested macrocyclic trichothecenes. The relative cross-reactivities of antibody 5G11 with roridin A, roridin J, verrucarin A, satratoxin G, and satratoxin H were 100.0, 43.8, 16.7, 3.7, and 18.9%, respectively; for antibody 4H10 they were 100.0, 6.3, 64.0, 4.4, and 4.9%, respectively.     

Design of a test system based on magnetic particles with immobilized monoclonal antibodies for selective <Emphasis Type="Italic">Bacillus anthracis</Emphasis> spore concentration

Magnetic immunosorbents (MIS) have been designed using antianthrax monoclonal antibodies immobilized on aluminum silicate—iron oxide matrix activated by sodium perchlorate. MIS allows us to concentrate the analyzed microorganism and to decontaminate culture from concomitant microflora. Diagnostic test systems constructed on the basis of MIS were tested on pure Bacillus anthracis cultures and in the model experiments. The results testify to the high specificity and sensitivity (10²–10³ spore/ml) of the designed test systems.     

Combination of the peroxidase anti-peroxidase (PAP)-and avidin-biotin-peroxidase complex (ABC)-techniques: an amplification alternative in immunocytochemical staining

Summary A combination of the PAP- and ABC-techniques was developed to enhance the intensity of the immunocytochemical staining with monoclonal antibodies at light and electron microscopical levels. This amplification technique could be performed in 4 (single PAP + ABC) or 6 (double PAP + ABC) sequential steps depending on the quality of the primary antibodies used and the processing of the tissue before the immunocytochemical reaction: First step — Incubation of the tissue sections with the monoclonal primary antibodies; Second step — biotinylated anti-rat or anti-mouse IgG; Third step — monoclonal PAP complex; Fourth step — ABC complex which binds to the biotinylated secondary antibody. If stronger enhancement of the immunostaining has required the steps 2 and 3 could be repeated followed by the 6th step — the ABC complex. Choline acetyltransferase-like immunoreactivity of the rat hypoglossal nucleus and desmin- and vimentin-like immunoreactivity of human testis were studied. After the 4-and more pronounced the 6-step reaction a significant increase of the staining intensity was observed for all the reactions under study. ChAT-like immunoreactivity was observed to longer distances of the nerve cell dendrites after their emerging from the perikarya and within a greater number of structures in the neuropil as compared to the standard techniques. At electron microscopical level the technique permits longer fixation of the tissue which is important for the better preservation of the ultrastructure as well as for the easier recognition of the reaction product even in the smallest dendrite branches and the axons of the nerve cells. Stronger staining intensity was obtained for desmin-like immunoreactivity (LI) (within myofibroblasts of the lamina propria and muscle cells of the blood vessels)-and vimentin-LI (within Sertoli cells, myofibroblasts of the lamina propria and fibroblasts of the interstitium) of the human testis. The full combination (6 step-reaction) permits the detection of smallest quantities of an antigen in sections of different tissues using highly diluted primary antibodies. The technique represents a further alternative among the immunocytochemical amplification methods.     

Effect of organic and inorganic toxic compounds on luminescence of luminous fungi

The possibility of the development of the solid phase bioluminescent biotest using aerial mycelium of luminous fungi was investigated. Effect of organic and inorganic toxic compounds (TC) at concentrations from 10⁻⁶ to 1 mg/ml on luminescence of aerial mycelia of four species of luminous fungi—Armillaria borealis (Culture Collection of the Institute of Forest, Siberian Branch, Russian Academy of Sciences), A. mellea, A. gallica, and Lampteromyces japonicus (Fungi Collection of the Botanical Institute, Russian Academy of Sciences)—has been studied. Culture of A. mellea was shown to be most sensitive to solutions of the model TC. It was demonstrated that the sensitivity of the luminous fungi is comparable with the sensitivity of the bacteria that are used for environmental monitoring. Use of the aerial mycelium of luminous fungi on the solid support as a test object is a promising approach in biotesting for the development of continuous biosensors for air monitoring.     

Trichothecenes Production by the Hypocrealean Epibiont of Baccharis coridifolia

Abstract: The Hypocrealean epibiont of Baccharis coridifolia has been shown to synthesize de novo a series of macrocyclic trichothecene antibiotics, such as roridin A, roridin E, verrucarin A and verrucarin J. The structures of the compounds were determined by spectroscopic methods (¹H- and ¹³C-NMR and FABMS).     

A colorimetric technique for detecting trichothecenes and assessing relative potencies

We tested a novel colorimetric toxicity test, based on inhibition of beta-galactosidase activity in the yeast Kluyveromyces marxianus, for sensitivity to a range of mycotoxins. A variety of trichothecene mycotoxins could be detected. The order of toxicity established with this bioassay was verrucarin A > roridin A > T-2 toxin > diacetoxyscirpenol > HT-2 toxin > acetyl T-2 toxin > neosolaniol > fusarenon X > T-2 triol > scirpentriol > nivalenol > deoxynivalenol > T-2 tetraol. The sensitivity of detection was high, with the most potent trichothecene tested, verrucarin A, having a 50% effective concentration (concentration of toxin causing 50% inhibition) of 2 ng/ml. Other mycotoxins (cyclopiazonic acid, fumonisin B1, ochratoxin A, patulin, sterigmatocystin, tenuazonic acid, and zearalenone) could not be detected at up to 10 micrograms/ml, nor could aflatoxins B1 and M1 be detected at concentrations up to 25 micrograms/ml. This test should be useful for trichothecene detection and for studies of relevant interactions-both between trichothecenes themselves and between trichothecenes and other food constituents.     

PCR Analysis of cry7 Genes in Bacillus thuringiensis by the Five Conserved Blocks of Toxins

As the prevalence of antibiotic-resistant strains of bacteria increases, novel ways of treating infections need to be developed. This is particularly pertinent with respect to the periodontal diseases—the most common chronic bacterial infections of man. The use of a photosensitizer in combination with red light has been demonstrated to be effective in killing several human pathogens, including the oral bacterium, Porphyromonas gingivalis, a major pathogen in periodontitis. Killing was associated with alterations in the molecular masses of several outer membrane and plasma membrane proteins and these may be therapeutic targets for photodynamic therapy and other antimicrobial approaches. To identify these photolabile proteins, we have used a panel of monoclonal antibodies raised to whole P. gingivalis. A number of the antibodies recognized various photolabile proteins. Using a combination of Western blotting and protein sequencing the predominant photolabile proteins in P. gingivalis have been identified as the major secreted/cell surface proteases—Lys and Arg gingipain. Received: 30 August 2000 / Accepted: 1 January 2001     

Growth hormone-like activities of macrocyclic trichothecenes in in vitro callus induction and growth of four Baccharis species

The ability of two plant-produced macrocyclic trichothecenes (baccharinoid B4 and roridin E) to induce callus growth of two trichothecene-producing Baccharis species (B. coridifolia and B. megapotamica) and two nontrichothecene-producing species (B. halimifolia and B. neglecta) was investigated. Roridin E had no effect in the induction of callus of B. coridifolia, a roridin-producing plant, but induced callus of nonroridin-producing plants (B. megapotamica, B. halimifolia, and B. neglecta). Baccharinoid B4 stimulated callus growth of B. megapotamica, a baccharinoid-producing plant, and inhibited growth of B. coridifolia, B. halimifolia, and B. neglecta callus tissues. The ability of roridin E to induce callus was most effective at concentrations of 10^–8 and 10^–6 M and when synergistically coupled with auxin, 2,4-dichlorophenoxyacetic acid (2,4-D). The ability of baccharinoid B4 to stimulate callus growth appeared to increase with increased concentration in the culture medium. Analysis of callus cultures grown in medium amended with roridin E showed that B4, roridin E, and 8-hydroxyroridin E and verrucarols were formed in the tissues but not in the medium. The results of this study indicated that while the callus-inducing ability of roridin E seemed to be nonspecies-specific in nature, the ability of B4 to stimulate callus was a highly species-specific phenomena. Callus-inducing activity of roridin E may depend on the capacity of plant species to transform exogenous roridin E into baccharinoids or other macrocyclic trichothecene derivatives.     

The serologic screening for celiac disease in the general population (blood donors) and in some high-risk groups of adults (patients with autoimmune diseases,osteoporosis and infertility) in the Czech republic

The prevalence of celiac disease (CD) was determined in healthy blood donors and in high-risk groups of adults (a total of 1835 adults—randomly selected 1312 healthy blood donors, 102 patients with primary osteoporosis, 58 patients with autoimmune diseases and 365 infertile women). It was calculated on the basis of a two-step serologic screening method—in the first step IgA and IgG antigliadin antibodies (AGA) and IgA anti-γ-glutamyltransferase (‘transglutaminase’) antibodies (ATG) were estimated, in the second step sera positive for IgA AGA and/or IgA ATG were examined for antiendomysial IgA (AEA) antibodies. Immunoenzymic assay (ELISA) was used for determining of AGA and ATG antibodies; immunoflurescence method, performed on human umbilical cord tissue, was used for assaying of AEA antibodies. Total serum IgA level in only IgG AGA positive subjects was measured by routine turbidimetric method. 0.45% of healthy blood donors, 0.98% of osteoporotic patients, 2.7% of patients suffering from autoimmune disease and 1.13% of women with infertility considered as immunologically mediated were found to be positive in both steps of serologic screening (AGA and/or ATG and antiendomysium positive). The presumed high prevalence of seropositivity for CD in apparently healthy Czech adult population was confirmed. In the high-risk groups, the prevalence of seropositivity for CD was approximately 2–4 times higher than in healthy blood donors. The real prevalence of CD in the tested groups, however, can be estimated after performing small intestinal biopsy in the seropositive patients.     

Differences in the immune response of rabbits to p-aminobenzoic acid and sulphanilic acid,conjugated with BGG

1) After immunization of rabbits with two structurally similar haptenes—p-aminobenzoic acid and sulphanilic acid—conjugated with the same BGG molecule, three groups of animals can be differentiated, according to the type of antibody response; a) animals forming antibodies well against both haptenes, b) animals forming no, or low titres of antibodies against both haptenes and c) animals forming antibodies well against one of the given haptenes, but not (or only in very low titres) against the other. 2) Hybridization experiments showed that the capability or incapability to synthesize antibodies against a given haptene depends on the genotypical constitution of the individual concerned.     

A genetic variant of immunoglobulin γ2 is strongly associated with immunity to mucin 1 in patients with breast cancer

High levels of antibodies to mucin 1 (MUC1), a membrane-bound glycoprotein that is overexpressed in adenocarcinomas, are associated with good prognosis in patients with breast cancer. The aim of the present investigation was to determine whether GM and KM allotypes—genetic markers of IgG heavy chains and κ-type light chains, respectively—contribute to the magnitude of natural antibody responsiveness to MUC1 in patients with breast cancer. A total of 153 Caucasian subjects with breast cancer were allotyped for several GM and KM markers. These subjects were also characterized for IgG and IgM antibodies to MUC1. Anti-MUC1 IgG antibody levels in subjects who were carriers of the immunoglobulin γ2 allele GM 23 were significantly higher than in those who were noncarriers (P = 0.003). These results could potentially divide the population into high or low responders to MUC1, which has important implications for MUC1-based immunotherapeutic interventions in breast cancer.     

Mannose-binding lectin haplotypes influence Brucella abortus infection in the water buffalo (Bubalus bubalis)

A case-control study established that the haplotype pair HYA/HYA at the MBL (mannose binding lectin) locus of water buffalo is associated with resistance to Brucella abortus infection (P < 10⁻⁷) and the haplotype pairs LYD/LYD with susceptibility to the same pathogen (P < 10⁻⁷). The subjects included in the present study were tested twice—at a 1-month interval—for the presence of anti-B. abortus antibodies in the serum by agglutination, complement fixation and flow cytometry. Cases (335 subjects) included animals consistently positive to all these tests; controls (335 subjects) comprised animals exposed yet negative by the same tests. The serum from genetically resistant subjects displayed in vitro significantly higher antibacterial activity compared to the serum from genetically susceptible subjects, lending biological significance to the results from the association study. Inhibition of the antibacterial activity following heat treatment of the serum, addition of specific MBL inhibitors (EDTA, mannose, N-acetyl-d-glucosamine) or anti-human MBL antiserum provide convincing evidence that the antibacterial activity present in the serum results from the interaction between MBL and B. abortus. A replication study (comprising 100 cases and 100 controls) confirmed the results from the original study.     

Trends in trace element determinations in blood,serum, and urine of the dutch population,and the role of neutron activation analysis

A critical review on the quality of literature data on trace elements in human blood, serum, and urine of inhabitants in the Netherlands has shown that many of the currently available data have been established 15–20 years ago. Only in a few publications are quality indicators mentioned, which should be considered typical—and minimal —for studies resulting at reference values. The use of neutron activation analysis for determination of trace elements in human body fluids was restricted to a few studies in the 1970s. However, although it is frequently assumed that the sensitivity of NAA might be insufficient, it is demonstrated that modern, large, well-type Ge detectors may serve well for the determination of trace elements in human body fluids via radiochemical NAA, for example.     

Modeling immunotherapy for allergy

Type I hypersensitivity, which functions to protect the organism from parasites, is caused by binding of antigen to IgE antibodies pre-attached to the cell surface of tissue mast cells and their circulating counterparts, the basophils. In “allergy,” type I hypersensitivity is inappropriately induced by protein-based foreign substances (such as pollen) or protein components of insect stings, which in the normal course of events would be cleared from the organism without causing any damage. Paradoxically, a successful clinical treatment of allergy involves repeated immunization of allergic persons with low doses of the allergen—immunotherapy. Investigation of the available experimental evidence leads to the conclusion that the phenomena of immunotherapy are best addressed in terms of the interplay among the mechanism(s) of immune memory—Th1/Th2 cross-regulation—and the physical compart-mentalization of the immune system. These conclusions are illustrated with a numerical simulation.     

A fluorometric assay for the measurement of endothelial cell density in vitro

Summary A fluorometric assay for determining endothelial cell numbers based on the endogenous enzyme acid phosphatase is described. In preliminary studies, three substrates—p-nitrophenyl phosphate, 4-methylumbelliferyl phosphate, and 2′-[2-benzthiazoyl]-6′-hydroxy-benthiazole phosphate (AttoPhos™)—were compared with respect to their kinetic, optimum assay conditions, sensitivity, and detection limits. Only AttoPhos™ was found to have a high degree of sensitivity, reliability, and reproducibility for measuring both high and low cell numbers in the same plate. In subsequent experiments, assay conditions were validated for measuring endothelial cell density in response to basic fibroblast growth factor and fumagillin. Furthermore, the AttoPhos™ assay revealed a linear correlation between acid phosphatase activity and cell number in many cell types, including BALB/3T3, CHO-K1, A431, MCF7, 2008, SK-OV-3, T47-D, and OVCAR-3. This assay is potentially valuable for use in many in vitro systems in which the quantitation of cell density and proliferation is necessary. The practical advantages of AttoPhos™ assay for measuring endothelial cell numbers include (1) nonradioactivity, (2) simplicity, (3) economy, (4) speed of assessment of proliferation of large number of samples, and (5) amenability to high-throughput drug screening.     

Expression in promoter variant of the ERα gene in bos taurus liver

Due to the variant functions that estrogens play in the regulation of reproduction, development of the mammary gland, growth and differentiation of cells, estrogen receptors and their genes are considered as a candidates for the markers of production and functional traits in farm animals, including cattle. In the earliest study, a 2853-bp bovine ER gene 5′-region was PCR amplified and sequenced. Moreover, for the first time, a polymorphism was described within 5′ region of the bovine ERα gene—A/G transition lying upstream at position 2591 from acceptor splice site +85, possibly within its promoter—which could be recognized with RFLP-BglI. In other study we are found second polymorphism—A/G transition at position 1213 from acceptor splice site +85, located in promoter for exon B. We have examined the specific mRNA expression of ERα in various genotypes using real-time RT-PCR. We used four animals from each genotype group—AG, GG for BglI and AA, AG for SnaBI—to analyse liver ERα expression at the level of Real-time PCR. Liver samples were taken from the 16 young Friesian bulls of the different ERα genotypes, slaughtered at the local abattoir. As shown by Real-Time PCR, on the livers of animals with different genotype ERα mRNA for BglI polymorphism we didn’t found variability, but for SnaBI we have found variability between AG and AA genotypes.