Inhibitory monoclonal antibodies against rat liver alcohol dehydrogenase

Eleven hybridoma clones which secrete monoclonal antibodies against purified rat liver alcohol dehydrogenase (EC 1.1.1.1) were isolated. Antibodies (R-1-R-11) were identified by their ability to bind to immobilized pure alcohol dehydrogenase in an enzyme-linked immunoadsorbent assay, in which antibody R-9 showed the highest binding capacity. Except for R-1 and R-7, all antibodies inhibited catalytic activity of the enzyme isolated from inbred (Fischer-344) or outbred (Sprague-Dawley) strains (R-11 greater than R-9 greater than R-4 greater than R-6 greater than R-10 greater than R-8 greater than R-2 = R-3 = R-5). The inhibition of enzyme activity by antibodies was noncompetitive for ethanol and NAD+, and was dependent on antibody concentration and incubation time. Antibodies R-4, R-9, and R-11 were most effective when enzyme activity was assayed below pH 7.7-7.8, a condition thought to protonate the enzyme's active center. These three antibodies did not inhibit horse liver alcohol dehydrogenase activity, indicating their species specificity. Such antibodies will be useful to delineate structural and functional roles of rat liver alcohol dehydrogenase.     

Developmental profile and tissue distribution of Drosophila alcohol dehydrogenase: an immunochemical analysis with monoclonal antibodies.

To analyze Drosophila alcohol dehydrogenase gene (Adh) expression and tissue distribution at various developmental stages, we devised several immunochemical techniques making use of monoclonal antibodies against Drosophila alcohol dehydrogenase (ADH), which had been obtained previously. We here report their application to analyze the expression of Adh in a wild-type strain of D. melanogaster. s-ELISA tests were performed to evaluate fluctuations in ADH content and specific activity during development in individual organs as well as in whole individuals. In all cases, ADH specific activity appeared to be quite constant, which implies that variations in enzyme activity reflect differences in protein content. Immunoblottings of crude homogenates revealed immunoreactive low relative molecular mass peptides in addition to the 27 KD monomeric band, showing a conserved banding pattern in different organs and developmental stages. Immunohistochemical assays on whole organs were used to analyze the general pattern of ADH distribution. Immunoperoxidase staining of cryosections proved to be of crucial relevance, as it yielded full details of the tissue localization of ADH within the ADH-positive organs. We have shown not only that ADH displays a specific distribution in some organs but also that the enzyme is restricted to certain cell types.     

Monoclonal antibodies recognizing cell surface antigens in Drosophila melanogaster

Monoclonal antibodies have been made against cell surface antigens from Drosophila melanogaster, as probes for “differentiation antigens.” The immunogens used were 0–16 hr embryonic cells and mass isolated imaginal discs. The tissue distributions of the antigens recognized by three antiembryonic antibodies and two antiimaginal disc antibodies have been defined by indirect immunofluorescent (IIF) assays on differentiated embryonic cell cultures and on dissected third instar larval organs. These antigens fall into two major categories being either ubiquitous or tissue-limited in distribution. In indirect radioimmunoassays against 12 Drosophila cell lines the antigens showing ubiquitous tissue distributions were detectable on all cell lines whereas the tissue-limited antigens were absent from some cell lines. Such a screen against cell lines therefore provides a straightforward means of identifying antibodies against nonubiquitous antigens. One antibody recognizing a tissue-limited antigen was detected as muscle-specific by IIF assays on differentiated embryonic cell cultures. The second tissue-limited antigen was found to label basement membranes, by IIF assays against third instar larval organs. The temporal distribution of the antigens during embryogenesis (3–21 hr) has been studied by indirect radioimmunoassay. In this respect the antigens fall into three classes: (1) abundant throughout embryogenesis (a ubiquitous antigen), (2) present throughout embryogenesis but increasing markedly in abundance between 12 and 15 hr (two ubiquitous antigens and the muscle-specific antigen), and (3) absent early in development but appearing at about 12 hr postfertilization (the tissue-limited, basement membrane antigen).     

Allozyme variation in the fruit flies Dacus tryoni and Dacus neohumeralis (Tephritidae)

Electrophoretic variation in four specific proteins, an alcohol dehydrogenase, and an octanol dehydrogenase from adult body homogenates, an esterase from adult heads, and a tyrosinase from larval hemolymph, is described for laboratory populations of two sibling species, Dacus tryoni and Dacus neohumeralis. For each enzyme, a number of test crosses between different electrophoretic forms provided evidence that the observed variation was due to segregation of alleles at one structural gene locus.     

Inter-relatedness of some isoenzymes of cytochrome P-450 from rat, rabbit and human, determined with monoclonal antibodies.

Monoclonal antibodies have been raised to rat liver cytochromes P-450 b and c, and rabbit liver cytochrome P-450 form 4. A total of six antibodies have been studied. Each antibody reacted strongly both with its homologous antigen and with microsomal fractions selectively enriched with that antigen by treatment of animals with inducing compounds. However, several of the antibodies showed cross-reactivity, either within or between species. A combination of enzyme-linked immunosorbent assay, immunoadsorption, Western blotting and competitive radioimmunoassay revealed that each of the antibodies reacted with a different epitope. Proteolytic digestion of antigen followed by Western blotting of the peptide fragments enabled antibodies, otherwise identical in their reactivity, to be distinguished. It is concluded that complex structural relationships exist amongst the different isoenzymes of cytochrome P-450 and that epitope mapping will help in characterizing both animal and human cytochromes P-450.     

The primary structure of alcohol dehydrogenase from Drosophila lebanonensis. Extensive variation within insect 'short-chain' alcohol dehydrogenase lacking zinc

Insect alcohol dehydrogenase is highly different from the well-known yeast and mammalian alcohol dehydrogenases. The enzyme from Drosophila lebanonensis has now been characterized by protein analysis and was found to have a 254-residue protein chain with an acetyl-blocked N-terminal Met. Comparisons with the structures of the enzyme from other species allows judgement of the extent of variability within the insect alcohol dehydrogenases. They have diverged to a considerable extent; two forms analyzed at the protein level differ at 18% of all residues, and all the known Drosophila alcohol dehydrogenase structures reveal differences at 72 positions. Some deviations, against a background similarity, in the extent of changes are noted among the parts corresponding to different exons. The structural variation within Drosophila is about as large as the one for the mammalian zinc-containing alcohol dehydrogenase. Consequently, the results illustrate Drosophila relationships and establish great variations also for group of alcohol dehydrogenases lacking zinc.     

Chicken antichromatin antibodies: specificity to different chromatin fractions.

Specific complement-fixing and precipitin antibodies were induced in chicken to chromatin from WI-38 human diploid fibroblast. Chicken antibodies were chosen because they present two major advantages with respect to rabbit antibodies: (a) the optimal NaCl concentration for chicken precipitin is 1.5 M, which is also an optimal solvent for chromatin proteins that are insoluble at the lower salt concentration required by rabbit precipitin; (b) chicken antichromatin antibodies require an antigen concentration much lower with respect to rabbit antibodies for saturation at the complement-fixation reaction. These antibodies are species specific and they react at the complement-fixation and precipitin assay not only with the whole chromatin but also with dissociated proteins, albeit at a higher concentration of both antigen and antibodies. The specificity of these antibodies to different fractions of chromosomal proteins and of the chromatin after washing with a solution at a different sodium chloride concentration has been investigated.     

Genetic variation in the expression of ADH in Drosophila melanogaster

Several chromosomes derived from natural populations have been identified that affect the expression of alcohol dehydrogenase (ADH). Second chromosomes, which also carry the structural gene Adh, show a great deal of polymorphism of genetic elements that determine how much enzyme protein accumulates. The level of enzyme was measured in third instar larvae, 6-to-8-day-old males and in larval fat bodies and alimentary canals. In general, activities in the different organs and stages are highly correlated with one another. One line was found, however, in which the ADH level in the fat body is more than twice the level one would expect on the basis of the activity in alimentary canal. We have also found evidence of third-chromosome elements that affect the level of ADH.     

Metabolic response to ethanol and isopropanol in D. funebris and D. immigrans

Ethanol at low concentration increases larval viability in D. funebris and D. melanogaster and decreases it in D. immigrans. Isopropanol decreases viability in all three species. Isopropanol depresses alcohol dehydrogenase activity. This is accompanied by a change in the relative proportion of electrophoretic forms of the enzyme. Acetone appears in the medium when larvae of D. funebris and D. immigrans develop in the presence of isopropanol. It can be transformed again into isopropanol on reaching a threshold value. It seems that this transformation cannot be carried out by alcohol dehydrogenase alone.     

Production, characterization, and applications of monoclonal antibodies reactive with soybean nodule xanthine dehydrogenase

Seven monoclonal antibodies were produced against soybean nodule xanthine dehydrogenase, an enzyme involved in ureide synthesis. Specificity of the seven monoclonal antibodies for xanthine dehydrogenase was demonstrated by immunopurifying the enzyme to homogeneity from a crude nodule extract using antibodies immobilized to Sepharose 4B beads. Each monoclonal antibody was covalently bound to Sepharose 4B beads for the preparation of immunoaffinity columns for each antibody. All seven antibodies were found to be of the IgG1,K subclass. A competitive, indirect enzyme-linked immunosorbent assay demonstrated that two of the seven antibodies shared a common epitope while the remaining five antibodies defined unique determinants on the protein. Rapid, large scale purification of active xanthine dehydrogenase to homogeneity was performed by immunoaffinity chromatography. The presence of xanthine dehydrogenase activity and protein in every organ of the soybean plant was determined. Crude extracts of nodules, roots, stems, and leaves cross-reacted with all seven monoclonal antibodies in an indirect enzyme-linked immunosorbent assay. A positive correlation was observed between the degree of cross-reactivity of a given organ and the level of enzyme activity in that organ. These data demonstrate that xanthine dehydrogenase is not nodule specific. Antigenic variability of xanthine dehydrogenase present in crude extracts from nodules of soybean, wild soybean, cowpea, lima bean, pea, and lupin were detected in the indirect enzyme-linked immunosorbent assay which corresponded to six binding patterns for xanthine dehydrogenase from these plant species. These results correspond well with the epitope determination data which showed that the seven antibodies bind to six different binding determinants on the enzyme.     

La glutamate déshydrogénase au cours de la gamétogénèse mâle chez Medicago sativa

Glutamate dehydrogenase during male gametogenesis in Medicago sativa.
The multiple forms of the alfalfa ( Medicago sativa L.) glutamate dehydrogenase (GDH; EC 1.4.1.2–4) are much more numerous in the pollen than in other organs, and in particular in the floral parts. The appearance of the pollen-specific GDH pattern during male gametogenesis has been followed using electrophoretic, cytological and immunochemical techniques. After the tetrad stage the uninucleate microspore has no glutamate dehydrogenase activity. This activity appears only in the mitochondria of the binucleate young pollen. At this stage the electrophoretic pattern displays a faint intensity, but is already identical to that of mature pollen. On the other hand, the use of monospecific antibodies has revealed the presence of antigen in the microspore, and in the young pollen there is as much antigen as in the mature pollen. Therefore the synthesis of GDH precedes its activation, which follows the first pollen mitosis.     

Serologic cross-reactivity of human IgM and IgG antibodies to five species of Ebola virus

Five species of Ebola virus (EBOV) have been identified, with nucleotide differences of 30-45% between species. Four of these species have been shown to cause Ebola hemorrhagic fever (EHF) in humans and a fifth species (Reston ebolavirus) is capable of causing a similar disease in non-human primates. While examining potential serologic cross-reactivity between EBOV species is important for diagnostic assays as well as putative vaccines, the nature of cross-reactive antibodies following EBOV infection has not been thoroughly characterized. In order to examine cross-reactivity of human serologic responses to EBOV, we developed antigen preparations for all five EBOV species, and compared serologic responses by IgM capture and IgG enzyme-linked immunosorbent assay (ELISA) in groups of convalescent diagnostic sera from outbreaks in Kikwit, Democratic Republic of Congo (n=24), Gulu, Uganda (n=20), Bundibugyo, Uganda (n=33), and the Philippines (n=18), which represent outbreaks due to four different EBOV species. For groups of samples from Kikwit, Gulu, and Bundibugyo, some limited IgM cross-reactivity was noted between heterologous sera-antigen pairs, however, IgM responses were largely stronger against autologous antigen. In some instances IgG responses were higher to autologous antigen than heterologous antigen, however, in contrast to IgM responses, we observed strong cross-reactive IgG antibody responses to heterologous antigens among all sets of samples. Finally, we examined autologous IgM and IgG antibody levels, relative to time following EHF onset, and observed early peaking and declining IgM antibody levels (by 80 days) and early development and persistence of IgG antibodies among all samples, implying a consistent pattern of antibody kinetics, regardless of EBOV species. Our findings demonstrate limited cross-reactivity of IgM antibodies to EBOV, however, the stronger tendency for cross-reactive IgG antibody responses can largely circumvent limitations in the utility of heterologous antigen for diagnostic assays and may assist in the development of antibody-mediated vaccines to EBOV.     

Population genetics and geographic variation of alcohol dehydrogenase (Adh) paralogs and glucose-6-phosphate dehydrogenase (G6pd) in Drosophila mojavensis

Populations of Drosophila mojavensis from the deserts of the Baja California peninsula and mainland Mexico utilize different cactus hosts with different alcohol contents. The enzyme alcohol dehydrogenase (ADH) has been proposed to play an important role in the adaptation of Drosophila species to their environment. This study investigates the role of ADH in the adaptation of the cactophilic D. mojavensis to its cactus host. In D. mojavensis and its sibling species, D. arizonae, the Adh gene has duplicated, giving rise to a larval/ovarian form (Adh-1) and an adult form (Adh-2). Studies of sequence variation presented here indicate that the Adh paralogs have followed different evolutionary trajectories. Adh-1 exhibits an excess of fixed amino acid replacements, suggesting adaptive evolution, which could have been a result of several host shifts that occurred during the divergence of D. mojavensis. A 17-bp intron haplotype polymorphism segregates in Adh-2 and has markedly different frequencies in the Baja and mainland populations. The presence of the intron polymorphism suggests possible selection for the maintenance of pre-mRNA structure. Finally, this study supports the proposed Baja California origination of D. mojavensis and subsequent colonization of the mainland accompanied by a host shift.     

Three surface antigens dominate the mucosal antibody response to gastrointestinal L3-stage strongylid nematodes in field immune sheep

Although gastrointestinal nematode parasites are a major human and veterinary health problem, little is known about how the host is sometimes able to mount an effective immune rejection response. In previous work, we identified a carbohydrate larval surface antigen (CarLA) as the target of mucosal antibodies that can elicit rejection of Trichostrongylus colubriformis L3s in sheep. Here we characterise the natural mucosal antibody responses to L3s from three major strongylid gastrointestinal parasites of sheep, Trichostrongylus colubriformis, Haemonchus contortus and Teladorsagia circumcincta. The mucosal antibody repertoire of naturally field-immune sheep was displayed on bacteriophage as single-chain antibodies (scFvs) and phage were selected for the ability to bind to the surface of living L3s of the three nematode species. All nematode-binding scFvs were found to recognize one of three different antigen classes that are each found in the three strongylid species. These three antigen classes appear to represent all of the major antigens recognized on Western blots by pooled mucosal antibodies from field-immune sheep. One of the antigen classes is a heterogeneous, high molecular weight molecule that is protease-sensitive. The scFvs recognizing this surface antigen also recognize a similar antigen in all strongylids tested. A second antigen class is a protease-insensitive, low molecular weight antigen found only in sheaths and scFvs recognizing this antigen cross-react with a similar molecule found in all strongylids tested. The third surface antigen class is CarLA and all of the anti-CarLA scFvs obtained from the field-immune sheep repertoire were specific to L3s of only one species and often recognized only a subset of the worms. Thus three different L3-stage surface antigens, two that lack a protein component, dominate the natural mucosal antibody response to L3-stage gastrointestinal strongylid nematodes in sheep.     

Hybridoma antibody immunoassays for the detection of parasitic infection: development of a model system using a larval cestode infection in mice

A prototype immunodiagnostic assay has been developed using chronic infection with the larval cestode, Mesocestoides corti, as a model system in mice. The assay is highly sensitive, it appears to be absolutely specific for M. corti infection, and is based on the inhibition of binding (by sera from infected mice) of a radiolabelled anti-M. corti hybridoma antibody to a crude M. corti antigen extract. The hybridoma antibody binds to living M. corti larvae and is an IgG1 protein. In large scale experiments no false positives were detected and the only M. corti-infected mice not detected by the assay were hypothymic nude (nu/nu) mice. Only limited success has been achieved in attempts to convert the assay to one not requiring parasite antigen and based on the inhibition of binding of radiolabelled anti-parasite hybridoma antibody and a large pool of anti-idiotype antiserum. Monoclonal antibodies derived from anti-parasite antibody-secreting hybridoma cell lines will be of particular use in the development of new, highly specific, immunodiagnostic reagents for the detection of parasite infection, exposure and disease.     

The metabolism of ethanol-derived acetaldehyde by alcohol dehydrogenase (EC 1.1.1.1) and aldehyde dehydrogenase (EC 1.2.1.3) in Drosophila melanogaster larvae.

Both aldehyde dehydrogenase (ALDH, EC 1.2.1.3) and the aldehyde dehydrogenase activity of alcohol dehydrogenase (ADH, EC 1.1.1.1) were found to coexist in Drosophila melanogaster larvae. The enzymes, however, showed different inhibition patterns with respect to pyrazole, cyanamide and disulphiram. ALDH-1 and ALDH-2 isoenzymes were detected in larvae by electrophoretic methods. Nonetheless, in tracer studies in vivo, more than 75% of the acetaldehyde converted to acetate by the ADH ethanol-degrading pathway appeared to be also catalysed by the ADH enzyme. The larval fat body probably was the major site of this pathway.     

Quality control test for immunoreactivity of radiolabeled antibody

A rapid method for the measurement of the immunoreactive fraction of a radiolabeled monoclonal antibody or antibody fragment has been developed. This may be used as a quality control test prior to patient administration of the radiolabeled antibody preparation. The test employs solid phase antigens and the assay is conducted under conditions of antigen excess. Assay parameters have been evaluated and a standardized procedure has been developed. The assay has been compared to a standard extrapolation method and found to give approximately the same result. The test has been used on four different radiolabeled antibodies currently in clinical trials in patients with colorectal cancer. Mean immunoreactive fractions for these radiolabeled antibodies ranged from 35 to 65% and the variability of the immunoreactive fraction ranged from 140 to 240% for different antibodies. We conclude that the quality, defined as the immunoreactive fraction, of radiolabeled antibodies is both low and highly variable, indicating the need for a quality control test of these radiopharmaceuticals in the clinic prior to patient administration.     

Alcohol dehydrogenase activities and ethanol tolerance in Anastrepha (Diptera, Tephritidae) fruit-fly species and their hybrids

The ADH (alcohol dehydrogenase) system is one of the earliest known models of molecular evolution, and is still the most studied in Drosophila. Herein, we studied this model in the genus Anastrepha (Diptera, Tephritidae). Due to the remarkable advantages it presents, it is possible to cross species with different Adh genotypes and with different phenotype traits related to ethanol tolerance. The two species studied here each have a different number of Adh gene copies, whereby crosses generate polymorphisms in gene number and in composition of the genetic background. We measured certain traits related to ethanol metabolism and tolerance. ADH specific enzyme activity presented gene by environment interactions, and the larval protein content showed an additive pattern of inheritance, whilst ADH enzyme activity per larva presented a complex behavior that may be explained by epistatic effects. Regression models suggest that there are heritable factors acting on ethanol tolerance, which may be related to enzymatic activity of the ADHs and to larval mass, although a pronounced environmental effect on ethanol tolerance was also observed. By using these data, we speculated on the mechanisms of ethanol tolerance and its inheritance as well as of associated traits.     

Isolation of a cDNA encoding functional Drosophila alcohol dehydrogenase in Escherichia coli and purification of the bacterially produced enzyme

Because of the severe limitations on growing large quantities of Drosophila affinidisjuncta in the laboratory, direct purification of alcohol dehydrogenase (ADH) from this species has proven impossible. As an alternative source of this enzyme, a cDNA encoding functional ADH was isolated from a newly constructed cDNA library made from larval poly(A)-containing RNA. The cDNA was recovered by virtue of its hybridization to a previously isolated genomic ADH gene. Nucleotide sequence analysis confirmed the identity of the newly isolated cDNA. When the cDNA was inserted in the proper orientation downstream of the lac promoter on the vector pUC8, the cDNA directed the synthesis of functional ADH by the bacterial host. The bacterially produced enzyme was purified to homogeneity and used to elicit polyclonal antibodies in rabbits. The purified ADH has identical apparent subunit molecular weight to that of authentic ADH in larval fly extracts as determined by immunoblotting. Further, comparisons of the kinetic parameters of the bacterially produced enzyme and ADH activity in larval fly extracts indicate similar substrate preferences, pH dependencies, and Km values for 2-propanol and NAD. These results show that expression of a cDNA in Escherichia coli is a valid strategy for isolation of an ADH that would otherwise be difficult or impossible to purify.     

Genetic sexing in the mediterranean fruit fly,Ceratitis capitata,using the alcohol dehydrogenase locus

Summary Using the alcohol dehydrogenase (ADH) locus a genetic sexing system is being developed in the Mediterranean fruit fly Ceratitis capitata based on the sensitivity of ADH null mutations to environmental ethanol. A series of null mutants have been induced at this locus, however, none proved viable as homozygotes. One of these null mutants was translocated to the male determining chromosome and this line can be used for genetic sexing. When larvae from this line were reared on larval medium containing various concentrations of allyl alcohol, 97% of the emerging adults were males; in the absence of the allyl alcohol the sex ratio in the line is distorted in favour of the females. It is proposed that the higher ADH activity of the females (homozygous positive) in comparison with the males (heterozygous null) is responsible for their lower survival in larval medium containing allyl alcohol. ADH converts the allyl alcohol to the lethal ketone. The possible use of this line to sex large populations of medflies for use in sterile insect release programmes is discussed.