Production of monoclonal antibodies and ELISA for thebaine and codeine

The ratio of hapten and bovine serum albumin in antigen conjugate was exactly determined by matrix-assisted laser desorption/ionization mass spectrometry. Monoclonal antibodies against thebaine and codeine were produced by hybridoma fused with the sprenocytes immunized with thebaine- and codeine-bovine serum albumin conjugate and HAT-sensitive mouse myeloma cell line, P3-X63-Ag8-653. No cross-reaction of anti-thebaine antibody against morphine was observed. Very small cross-reaction appeared in codeine (0.004%). The cross-reaction of anti-codeine antibody against morphine and thebaine was 2.97 and 5.98%, respectively. The full measuring range of the assay extends from 60 pg mL to 1 ng mL for thebaine and 1 ng mL to 100 ng mL for codeine.     

Physicochemical and immunological characterization of the type E botulinum neurotoxin binding protein purified fromClostridium botulinum

Type E botulinum neurotoxin is produced byClostridium botulinum along with a neurotoxin binding protein which helps protect the neurotoxin from adversepH, temperature, and proteolytic conditions. The neurotoxin binding protein has been purified as a 118-kDa protein. Secondary structure content of the neurotoxin binding protein as revealed by far-UV circular dichroism spectroscopy was 19% -helix, 50%-sheets, 28% random coils, and 3%-turns. This compared to 22% -helix, 44%-sheets, 34% random coils, and no-turns of the type E botulinum neurotoxin. The complex of the two proteins revealed 25%-helix, 45%-sheets, 27% random coils, and 3%-turns, suggesting a significant alteration at least in the-helical folding of the two proteins upon their interaction. Tyrosine topography is altered considerably (28%) when the neurotoxin and its binding protein are separated, indicating strong interaction between the two proteins. Gel filtration results suggested that type E neurotoxin binding protein clearly complexes with type E neurotoxin. The interaction is favored at lowpH as indicated by an initial binding rate of 8.4 min^–1 atpH 5.7 compared to 4.0 min^–1 atpH 7.5 as determined using a fiber optic-based biosensor. The neurotoxin and its binding protein apparently are of equivalent antigenicity, as both reacted equally on enzyme-linked immunosorbent assay to polyclonal antibodies raised against the toxoid of their complex.     

Antibody reaction of human anti-Toxoplasma gondii positive and negative sera with Neospora caninum antigens

Anti-Neospora caninum antibody was detected in anti-Toxoplasma gondii positive and negative human sera by ELISA, western blot and immunofluorescence assay (IFA). Twelve cases out of 172 (6.7%) Toxoplasma-positive sera cross-reacted with both T. gondii and N. caninum antigens, and one out of 110 Toxoplasma-negative sera reacted with N. caninum antigen by ELISA. By western blot, all 12 sera reacted with T. gondii antigens with various banding patterns but specifically at 30 kDa (SAG1) and 22 kDa (SAG2) bands. With N. caninum antigen, the number of reactive bands was reduced, however a 43 kDa band reacted in three cases in Toxoplasma-positive sera in addition to one in Toxoplasma-negative control sera. All sera of the Toxoplasma-positive group labeled surface membrane of T. gondii, but reacted differently with N. caninum. Fluorescence was detected in surface membrane, subcellular organelles, or both in N. caninum. And one case in the Toxoplasma-negative group also reacted with N. caninum strongly in subcellular organelles. This suggested that the antibody against N. caninum may be present in human sera although the positive rate was very low in this study. The possibility of human infection with N. caninum remains to be evaluated further.     

Postharvest changes in endogenous cytokinins and cytokinin efficacy in potato tubers in relation to bud endodormancy

Using an indirect enzyme‐linked immunosorbent assay (ELISA), the effects of postharvest storage duration and temperature on endogenous cytokinins in potato ( Solanum tuberosum L. cv. Russet Burbank) tuber apical bud tissues in relation to endodormancy status were determined. Following fractionation by HPLC, a total of eight cytokinins were detected and these were: zeatin riboside‐5'‐monophosphate (ZRMP), zeatin‐ O ‐glucoside (ZOG), zeatin (Z), zeatin riboside (ZR), isopentenyl adenosine‐5'‐monophosphate (IPMP), isopentenyl adenine‐9‐glucoside (IP‐9‐G), isopentenyl adenine (IP) and isopentenyl adenosine (IPA). Regardless of postharvest storage temperature or endodormancy status, IP‐9‐G was the most abundant cytokinin detected while ZRMP and ZOG were the least abundant ones. In tubers preincubated at a growth‐permissive temperature (20°C) prior to extraction, the loss of endodormancy was preceded by significant increases in the endogenous levels of Z, ZR, IPMP and IP‐9‐G. When stored continuously at a growth‐inhibiting temperature (3°C), significant increases in ZR, IP‐9‐G and IP + IPA were observed. The total content of cytokinins increased by over 7‐fold during postharvest storage and this increase was a result of de novo biosynthesis. Dose‐response studies using IPA and ZR demonstrated a time‐dependent increase in apparent cytokinin sensitivity during postharvest storage. With the exception of IP‐9‐G, injection of any of these endogenous cytokinins resulted in the rapid and complete termination of tuber endodormancy. The significance of these results with respect to endodormancy regulation and the possible mechanisms controlling cytokinin levels in potato tubers are discussed.     

Evidence that cytokinin controls bud size and branch form in Norway spruce

Shoot elongation in many coniferous species is predetermined during bud formation the year before the shoot extends. This implies that formation of the primordial shoot within the bud is the primary event in annual shoot growth. Hormonal factors regulating bud formation are consequently of utmost importance. We followed the levels of the endogenous cytokinins zeatin riboside (ZR) and isopentenyladenosine (iPA) in terminal buds, whorl buds and lower lateral buds of the uppermost current-year whorl shoots of 15- to 20-year-old trees of Norway spruce [ Picea abies (L.) Karst.] from June to September. Cytokinins were isolated with affinity chromatography columns, purified by high performance liquid chromatography, and quantified by ELISA. The level of ZR was low in June but increased gradually in all buds until September. Throughout the measurement period, the ZR level was highest in terminal buds and lowest in the scattered lateral, buds, with the whorl buds intermediate. The level of iPA peaked in July and decreased later without any consistent differences among the three classes of buds. The development of different kinds of buds was followed by scanning electron microscopy. We found that bud growth was greatest during August and September. The final size of primordial shoots within the buds varied considerably and the weight of the terminal bud was three times that of the whorl buds and more than five times that of the other lateral buds.
We conclude that the increase in ZR level during the period of active bud development is indicative of the importance of cytokinin for this process. Furthermore, the positive correlation between the level of ZR and bud growth during the period of predetermination of next year's branch growth suggests that this hormone indirectly controls the form of single branches in the spruce tree.     

Schistosoma japonicum: immunological characterization and detection of circulating polysaccharide antigens from adult worms

The antigenic constituents of a trichloroacetic acid (TCA)-soluble fraction of adult Schistosoma japonicum were studied with immunoelectrophoresis, and compared with those of Schistosoma mansoni. Eight TCA-soluble antigens of S. japonicum were demonstrated, five of which showed immunological identity with S. mansoni antigens. Of the eight antigens, five antigens with anodic motility were found as circulating antigens in S. japonicum-infected hamster and rabbit sera; the major circulating antigen was the circulating anodic antigen (CAA). Two other antigens, with cathodic motility, including the circulating cathodic antigen (CCA), were demonstrable as circulating antigens in S. mansoni infections, but not in S. japonicum infections. Most of the circulating antigens were shown to be gut-associated. Only one antigen, line 2, which was not demonstrable as circulating antigen and which was present in the parenchyma of the worms, was found to be specific for S. japonicum. Using an ELISA for the detection of CAA in the sera of S. japonicum-infected rabbits, a lower detection level of 100 ng CAA/ml serum was achieved. Moreover, at 7-8 weeks after infection, a direct relationship between worm burden and CAA level was demonstrated.     

Schistosoma japonicum: Immunological response to circulating polysaccharide antigens in rabbits with a light infection

To study the detectability of circulating polysaccharide antigens and the immunological response to such antigens in rabbits with a light Schistosoma japonicum infection, sera of five rabbits infected with 50 cercariae were studied up to 29 weeks post infection (p.i.). While one rabbit developed no worm burden, the other rabbits developed low worm burdens (4 to 16 worms). In the sera of these rabbits, the only polysaccharide antigen demonstrable with immunoelectrophoresis (IEF), was the circulating anodic antigen (CAA). With the enzyme-linked immunosorbent assay (ELISA), CAA was detectable from 5 to 6 weeks p.i. in the sera of the two rabbits with the highest number of worm couples. The lowest CAA level which was detectable in unconcentrated sera from which serum proteins had been removed was 125 ng CAA/ml, corresponding with a worm burden of 4.5 worm/kg body wt. During the entire infection, CAA-specific immune complexes were only demonstrable in very low concentrations. Antibodies against polysaccharide antigens were assessed with immunofluorescent antibody (IFA) on Rossman's fixed sections of adult worms, with the ELISA, and with IEF. Specific IgA, IgG, and IgM antibodies were detectable from 2 to 3 weeks p.i. with IFA and ELISA. These early antibodies were shown to be directed against gut-associated antigens, while antibodies against parenchyma-associated antigens were found later in the infection. With IEF, antibodies against two trichloroacetic acid (TCA)-soluble antigens were detectable, including the major, S. japonicum-specific antigen 2.     

Use of monoclonal and polyclonal antibodies as structural and topographical probes for hepatic epoxide hydrolase

Monoclonal antibodies have been prepared against rat liver epoxide hydrolase (EH), some of which gave precipitation lines on immunodiffusion against pure EH suggesting the presence of repetitive structural domains on the enzyme. Using ELISA, with polyclonal antibodies to rat and rabbit liver EH, reactivity and therefore structural similarities between EH of all species tested, including human, were observed. This was in contrast to immunodiffusion results demonstrating the limitations of the latter technique. Using monoclonal antibodies in ELISA, greatest structural similarity was between rat, mouse, and Syrian hamster EH and relatively little between rat and human. Two of the antibodies reacted with nearly all species tested and may be directed towards critical sites on the enzyme. This and most of the EH molecule would appear to be localised on the cytoplasmic surface of the endoplasmic reticulum.     

Affinity of pertussis toxin produced by Bordetella pertussis for human haptoglobin: application to the in vitro assay of the toxin

Pertussis toxin formed a stable complex with human (Hp). Hemagglutinating activity of the toxin was inhibited in the presence of Hp, but leukocytosis activity was not.An enzyme linked immunosorbent assay for the toxin, Hp-ELIS, was developed on the basis of its specific affinity for Hp. A polystyrene microplate coated with Hp was incubated with samples containing the toxin. The bound was measured by sequential reaction with antipertussis toxin goat IgG, alkaline-labelled anti-goat IgG and p-nitrophenylphosphate.The Hp-ELISA method showed high specificity, high sensitivity and good correlation with leukocytes promoting activity in vivo. One ng of pertussis toxin could be detected within 3 h.     

Recognition and differentiation of seven whitefly-transmitted geminiviruses from India, and their relationships to African cassava mosaic and Thailand mung bean yellow mosaic viruses

Particles resembling those of geminiviruses were found by immunosorbent electron microscopy in extracts of plants infected in India with bhendi yellow vein mosaic, croton yellow vein mosaic, dolichos yellow mosaic, horsegram yellow mosaic, Indian cassava mosaic and tomato leaf curl viruses. All these viruses were transmitted by Bemisia tabaci whiteflies, all reacted with at least one out of ten monoclonal antibodies to African cassava mosaic virus (ACMV), and all reacted with a probe for ACMV DNA-1, but scarcely or not at all with a full-length probe for ACMV DNA-2. Most of the viruses were distinguished by their host ranges when transmitted by whiteflies, and the rest could be distinguished by their pattern of reactions with the panel of monoclonal antibodies. Horsegram yellow mosaic virus was distinguished from Thailand mung bean yellow mosaic virus by its lack of sap transmissibility, ability to infect Arachis hypogaea, failure to react strongly with the probe for ACMV DNA-2 and its pattern of reactions with the monoclonal antibodies. Structures resembling a ‘string of pearls’, but not geminate particles, were found in leaf extracts containing malvastrum yellow vein mosaic virus. Such extracts reacted with two of the monoclonal antibodies, suggesting that this whitefly-transmitted virus too is a geminivirus. All seven viruses from India can therefore be considered whitefly-transmitted geminiviruses.