Monoclonal antibodies to a higher-plant nitrate reductase: Differential inhibition of enzyme activities

A set of monoclonal antibodies has been raised against NADH-nitrate reductase (NR; EC 1.6.6.1) from spinach (Spinacea oleracea L.) leaves. Antibodies were screened by enzyme-linked immunosorbent assay and by their ability to inhibit various activities of the enzyme. The six monoclonals selected (AFRC MAC 74 to 79) are all gamma globulins; four (MAC 74 to 77) inhibit all terminal donating activities (NADH-NR; flavin mononucleotide, reduced form (FMNH₂)-NR; and methyl viologen, reduced form (MV)-NR) and two (MAC 78 and 79) inhibit the acceptor activities (NADH-NR, and NADH-cytochrome c reductase). MAC 74 to 77 inhibit the NADH-NR activity of crude extracts of a variety of species (mono- and dicotyledoneae) while MAC 78 and 79 are effective against spinach and marrow, but not oil-seed rape, cucumber, oats, wheat and barley.Abbreviations Cyt c Rase cytochrome c reductase - ELISA enzyme-linked immunosorbent assay - FAD(H₂) flavin adenine dinucleotide (reduced form) - FMN(H₂) flavin mononucleotide (reduced form) - McAb monoclonal antibody - MV methyl viologen reduced form - NR nitrate reductase     

Comparison of different enzyme immunoassays for measuring indole-3-acetic acid in vegetative citrus tissues

An enzyme-linked immunosorbent assay (ELISA) using polyclonal antibodies, which were raised against indole-3-acetic acid (IAA) conjugated to bovine serum albumin (BSA) via the indolic nitrogen (IAA-N₁-BSA), has been developed. The sensitivity and specificity of these antibodies were compared to those of polyclonal and monoclonal antibodies raised against IAA conjugated to BSA via C₁ of the carboxyl group (IAA-C₁-BSA). The sensitivity of the assays improved in the following order: monoclonal antibodies > antibodies to IAA-C₁-BSA > antibodies to IAA-C₁-BSA. Antibodies against IAA-C₁-BSA had less cross-reactivity to indoles structurally related to IAA, excluding indole-3-pyruvic acid. A rapid and effective method for purification of IAA in citrus tissues before analysis by ELISA is described. Values of IAA in citrus ( Citrus sinensis [L.] Osbeck cv. Shamouti orange) shoot tips obtained with all three antibodies were similar. However, in leaf tissues which contain lower amounts of IAA compared to shoot tips, monoclonal antibodies gave higher values of IAA than polyclonal antibodies. Estimation of free IAA levels in purified extracts of citrus shoot tips, very young leaves, and mature leaves was ca 380, 248, and 74 ng (g fresh weight)⁻¹ respectively.     

Comparison of ELISA and immunoblotting techniques for the detection of cherry mottle leaf virus

Formaldehyde treated cherry mottle leaf virus (ChMLV) and the isolated coat protein were used successfully for the production of polyclonal and monoclonal antibodies. The monoclonal antibodies had a titre of 1:51 200 and consisted of IgG1 and IgG2. The antibodies reacted with all 11 isolates of ChMLV, from five locations in Canada and the USA, included in this study. Several serological procedures were assessed to compare their sensitivity for detecting ChMLV. Plate-trapped antigen ELISA (PTA-ELISA) and dot-blot immunobinding assay (DBIA), using virus specific MAbs, were the most sensitive tests in this study. Triple antibody sandwich ELISA (TAS-ELISA) and Western blot were found to be less sensitive. Dilution of the samples appeared to increase the sensitivity of both PTA-ELISA and Western blot detection. Young leaves and flowers of Prunus avium were the best tissue for detecting the virus which could also be detected in the fruit and leaves of P. tomentosa. April and May were optimal for detection of the virus in the field, whereas both April to May and August to September were optimal for screenhouse-grown plants.     

Preparation of mycoplasma immunogens from plants and a comparison of polyclonal and monoclonal antibodies made against primula yellows MLO-associated antigens

A method is described for obtaining from plants partially purified preparations of mycoplasma-like organisms (MLO) which are suitable for use as immunogens for polyclonal or monoclonal antibody production, and as antigens for directly coating ELISA plates. Using this method a mouse monoclonal antibody to primula yellows MLO was prepared, and its characteristics compared with those of primula yellows polyclonal antibodies from rabbits and also against polyclonal antibodies made to similar preparations of European aster yellows MLO. No serological distinction was obtained between any of the homologous or heterologous combinations of antibody and MLO preparation using ELISA, fluorescence microscopy with FITC-labelled antibodies, or immunoprobes of western blots of partially purified MLO preparations. By contrast, there were no cross-reactions between the primula or aster yellows antibodies or MLO preparations and preparations of clover phyllody or tomato big bud MLOs or their respective polyclonal antibodies. The primula yellows MLO monoclonal and polyclonal antibodies, and also the European aster yellows MLO polyclonal antibodies, all appeared to recognize only a single major antigen of approximate M, = 22 400 daltons. Some possible explanations for the apparent specificity of the polyclinic antisera for a single antigen, and the relevance to MLO preparation procedures are discussed.     

In situ localization of antigens of Histoplasma capsulatum using colloidal gold immune electron microscopy

Histoplasma capsulatum contains multiple antigens, among them the H antigen and M antigen, which are useful in serologic testing for histoplasmosis. We prepared 7 mouse monoclonal antibodies (5 IgG, 2 IgM) to histoplasmin, and compared these with polyclonal histoplasmin antibodies raised in rabbits and mice. Both monoclonal and polyclonal antibodies were high titered by ELISA. Colloidal gold immune electron microscopy (CGIEM) showed that polyclonal antibodies to histoplasmin or H antigen bound at multiple sites in the cell wall, cytoplasm, and nucleus of Histoplasma yeast cells. In contrast, antibodies to M antigen selectively label the cell membrane and antibodies to alkali soluble cell wall antigen label only the cell wall. Polyclonal antibodies cross reacted extensively with other fungi, both by ELISA and CGIEM. Monoclonal antibodies stained only cytoplasmic epitopes, but also cross reacted with other fungi by electron microscopy. Only periodate treated H antigen elicited polyclonal antibodies which were more specific than those of untreated H antigen or histoplasmin.     

Preparation of Monoclonal Antibodies against NADH: Nitrate Reductase from the Red Alga Porphyra yezoensis

Twenty-two monoclonal antibodies were raised against the nativeform of nitrate reductase (NR) from Porphyra yezoensis, a marinered alga. All the antibodies were able to bind to NR from P.yezoensis, with resultant inhibition of full (NADH:NR) and/orpartial (NADH:FR, NADH:CR, FMNH₂:NR, and MV:NR) enzymatic activity.Fifteen of the antibodies recognized the denatured form of theenzyme. Size-exclusion gel nitration and immunoblotting of theproducts of the limited proteolysis of NR from P. yezoensisby trypsin or Staphylococcus aureus V8 protease revealed that2 out of the 15 subunit-recognizing antibodies bound to theFAD domain, 6 bound to the heme domain, and 7 bound to the Mo-pterindomain. The products of limited proteolysis of NR from P. yezoensisalso revealed that the sites of proteolytic cleavage that encompassedthe heme domain were inverted as compared to the analogous sitesin NRs of higher plants. Some of the monoclonal antibodies cross-reactedwith NRs from plants belonged to different phyla. From threeto five of the antibodies bound subunits of NR from multicellularred algae, but failed to bind NRs from unicellular red algae.Three or four of the antibodies crossreacted with NRs from higherplants, such as tobacco and spinach. One of the antibodies boundNRs from several types of plant, namely, members of Cryptophyta,Chromophyta, and Chlorophyta. All of the monoclonal antibodiesthat cross-reacted with NRs from plants other than the red algaewere specific for the Mo-pterin domain of NR from P. yezoensis. (Received May 10, 1994; Accepted September 7, 1994)     

单克隆抗体与多克隆抗体配对ELISA方法比较

以人绒毛膜促性腺激素(HCG)为抗原,制备出对HCG的多克隆抗体和特异性单克隆抗体,并进行抗体纯化和特性分析,利用辣根过氧化物酶(HRP)分别对其进行了标记.采用双抗夹心ELISA试验,探讨了多克隆抗体与单克隆抗体配对的若干事项.结果表明,利用单克隆抗体和酶标多克隆抗体配对,并用含动物血清的稀释液稀释酶标抗体,可实现对检测原的高特异性和高灵敏度检测.     

Discrimination between the red- and far-red-absorbing forms of phytochrome from Avena sativa L. by monoclonal antibodies

A set of rat monoclonal antibodies (ARC MAC 48 to 52 and 54 to 56), raised to phytochrome from dark-grown seedlings of Avena sativa L. was tested for the ability to discriminate between the red-absorbing (Pr) and far-red-absorbing (Pfr) forms of phytochrome by indirect enzyme-linked immunosorbent assay. MAC 50 bound more strongly to Pfr and MAC 49 and 52 showed preferential binding to Pr from extracts of dark-grown Avena seedlings; MAC 50 also bound more strongly to Pfr from brushite-purified phytochrome. The remainder of the monoclonal antibodies and a rabbit polyclonal antiphytochrome preparation did not discriminate between Pr and Pfr. The results provide evidence for conformational changes in defined regions of the phytochrome apoprotein upon photoconversion.Abbreviations ELISA enzyme-linked immunosorbent assay - FR far-red light - McAb monoclonal antibody(ies) - PBS phosphate-buffered saline - Pfr far-red-absorbing form of phytochrome - Pr red-absorbing form of phytochrome - R red light - PMSF phenylmethylsulphonylfluoride     

Regulation of sucrose phosphate synthase by gibberellins in soybean and spinach plants

Exogenous applications of gibberellins (GAs) increased the extractable activity of leaf sucrose phosphate synthase (SPS) in soybean (Glycine max [L.]) and spinach (Spinacia oleracea [L.]). The response to GA applications was detectable within 2 h postapplication and was still observed 6 h, 24 h, and 7 d after treatment. When paclobutrazol, a GA biosynthesis inhibitor, was applied to intact soybean and spinach plants, decreased extractable SPS activity resulted within 24 h following the treatment. Different methods of GA application (spray, injection, capillary wick, and excised leaf systems) produced similar effects on SPS activity of soybean leaves. Protein synthesis in soybean leaves appeared to be necessary for GA-promoted SPS activity because gibberellic acid only partially reversed the inhibitory effect of pretreatment with cycloheximide. Levels of SPS protein from crude extracts of spinach plants were measured by a dot blot technique using monoclonal antibodies against SPS. Application of gibberellic acid to spinach leaves increased levels of SPS protein 2 h, 24 h, and 7 d after treatment. The results suggest that, in both soybean and spinach, GA is one of the endogenous hormonal factors that regulate the steady-state level of SPS protein and, hence, its activity.     

Rapid modulation of nitrate reductase in pea roots

The regulatory properties of nitrate reductase (NR; EC 1.6.6.1) in root extracts from hydroponically grown pea (Pisum sativum L. cv. Kleine Rheinländerin) plants were examined and compared with known properties of NR from spinach and pea leaves. Nitrate-reductase activity (NRA) extracted from pea roots decreased slowly when plants were kept in the dark, or when illuminated plants were detopped, with a half-time of about 4 h (= slow modulation in vivo). In contrast, the half-time for the dark-inactivation of NR from pea leaves was only 10 min. However, when root tip segments were transferred from aerobic to anaerobic conditions or vice versa, changes in NRA were as rapid as in leaves (= rapid modulation in vivo). Nitrate-reductase activity was low when extracted from roots kept in solutions flushed with air or pure oxygen, and high in nitrogen. Okadaic acid, a specific inhibitor of type-1 and type-2A protein phosphatases, totally prevented the in vivo activation by anaerobiosis of NR, indicating that rapid activation of root NR involved protein dephosphorylation. Under aerobic conditions, the low NRA in roots was also rapidly increased by incubating the roots with either uncouplers or mannose. Under these conditions, and also under anaerobiosis, ATP levels in roots were much lower than in aerated control roots. Thus, whenever ATP levels in roots were artificially decreased, NRA increased rapidly. The highly active NR extracted from anaerobic roots could be partially inactivated in vitro by preincubation of desalted root extracts with MgATP (2 mM), with a half-time of about 20 min. It was reactivated by subsequently incubating the extracts with excess AMP (2 mM). Thus, pea root NR shares many of the previously described properties of NR from spinach leaves, suggesting that the root enzyme, like the leaf enzyme, can be rapidly modulated, probably by reversible protein phosphorylation/ dephosphorylation.     

Assay sensitivity and differentiation of monoclonal antibody specificity in ELISA with different coating buffers

Buffers of different pH and ionic strength were employed as coating buffers for antigen adsorption to microtitre plates. Their efficiency for coating plates with rinderpest virus (RPV) and foot-and-mouth disease virus (FMDV) antigens was studied by ELISA with polyclonal and monoclonal antibody preparations. While the adsorption and detection of RPV antigen with polyclonal antiserum was highly dependent on the ionic strength and pH of coating buffer, adsorption of antigenically active FMDV antigen was relatively unaffected by the buffering conditions. Both antigens were adsorbed optimally in 0.01 M phosphate buffer, pH 8.0. When monoclonal antibodies were used to detect antigen, there was a greater degree of dependence on the coating buffer than that found with polyclonal antisera. Moreover, when they were used to detect antigen adsorbed under several buffering conditions, monoclonal antibodies showed a variety of preferred buffers. The usefulness of this differential reactivity in distinguishing epitope specificity is demonstrated.     

检测乙肝表面抗原夹心法中使用不同抗体的对比配伍研究

在提高乙型肝炎表面抗原（ＨＢｓＡｇ）酶联免疫诊断盒灵敏度的研究中对包被抗体、酶标记用抗体作了大量的筛选、对比实验。结果表明,包被单克隆抗体配伍酶标记山羊或豚鼠多克隆抗体可使乙肝表面抗原检测灵敏度达到０．２ｎｇ,超过中国药品生物制品检定所要求的１ｎｇ批批检合格标准,同时,特异性及变异系数均合乎要求,从而提高了试剂盒的质量。这一研究结果对提高其它以夹心法为原理的检测灵敏度有重要意义。     

Chicken egg yolk antibodies (IgY) for detecting circulating antigens of Schistosoma japonicum

BackgroundIgY isolated from egg yolk has been widely used in immunodiagnostic tests, including tests to detect circulating antigen (soluble egg antigen or SEA) of Schistosoma japonicum.ResultsA sandwich ELISA was established using a combination of anti-S. japonicum SEA-IgY polyclonal antibodies and IgM monoclonal antibodies. To explore sensitivity and specificity of the sandwich ELISA, serum samples from 43 patients infected with S. japonicum were tested. All acute cases and 91.3% of the chronic cases showed a positive reaction. Only 5% of the control sera from healthy persons gave a positive response. Cross-reactions with antibodies to nine other parasites were rare.ConclusionThe developed immunoassay is reasonably sensitive and specific. It could be used for field research and treatment efficacy assessments.     

Nitrate reductase activity of two leafy vegetables as affected by nickel and different nitrogen forms

The author studied the effect of different nickel concentrations (0, 0.4, 40 and 80 μM Ni) on the nitrate reductase (NR) activity of New Zealand spinach (Tetragonia expansa Murr.) and lettuce (Lactuca sativa L. cv. Justyna) plants supplied with different nitrogen forms (NO₃ ⁻–N, NH₄ ⁺–N, NH₄NO₃). A low concentration of Ni (0.4 μM) did not cause statistically significant changes of the nitrate reductase activity in lettuce plants supplied with nitrate nitrogen (NO₃ ⁻–N) or mixed (NH₄NO₃) nitrogen form, but in New Zealand spinach leaves the enzyme activity decreased and increased, respectively. The introduction of 0.4 μM Ni in the medium containing ammonium ions as a sole source of nitrogen resulted in significantly increased NR activity in lettuce roots, and did not cause statistically significant changes of the enzyme activity in New Zealand spinach plants. At a high nickel level (Ni 40 or 80 μM), a significant decrease in the NR activity was observed in New Zealand spinach plants treated with nitrate or mixed nitrogen form, but it was much more marked in leaves than in roots. An exception was lack of significant changes of the enzyme activity in spinach leaves when plants were treated with 40 μM Ni and supplied with mixed nitrogen form, which resulted in the stronger reduction of the enzyme activity in roots than in leaves. The statistically significant drop in the NR activity was recorded in the aboveground parts of nickel-stressed lettuce plants supplied with NO₃ ⁻–N or NH₄NO₃. At the same time, there were no statistically significant changes recorded in lettuce roots, except for the drop of the enzyme activity in the roots of NO₃ ⁻-fed plants grown in the nutrient solution containing 80 μM Ni. An addition of high nickel doses to the nutrient solution contained ammonium nitrogen (NH₄ ⁺–N) did not affect the NR activity in New Zealand spinach plants and caused a high increase of this enzyme in lettuce organs, especially in roots. It should be stressed that, independently of nickel dose in New Zealand spinach plants supplied with ammonium form, NR activity in roots was dramatically higher than that in leaves. Moreover, in New Zealand spinach plants treated with NH₄ ⁺–N the enzyme activity in roots was even higher than in those supplied with NO₃ ⁻–N.     

The detection of Rickettsia prowazekii in the vectors of louse-born typhus Pediculus humanus by using monoclonal antibody-based preparations]

The results of this investigation indicate that preparations obtained on the basis of monoclonal antibodies have proved to be suitable for the detection of R. prowazekii antigens in the natural carrier of typhus when used in all types of the enzyme immunoassay; of these, the assay made by the capture method has been found to possess the highest sensitivity. The testing of the sensitivity limits has shown that this method known as ELISA-mu-capture, i.e. ELISA carried out with the use of antibodies to the mu chain of human immunoglobulin, is capable of detecting the antigen in a dose of 0.5-1 ng. Preparations based on monoclonal antibodies to species-specific R. prowazekii antigen permit the identification of the causative agent of typhus in its natural carrier within 24 hours.     

Organ-specific expression of hepatitis B surface antigen in potato

Summary The gene encoding the hepatitis B surface antigen (HBsAg) under the control of cauliflower mosaic virus 35S-promoter was constructed and expressed in transgenic potato plants. The HBsAg expression were measured by ELISA kit containing monoclonal antibodies. The amount of HBsAg in roots was found 5–10 fold higher than in leaf tissues     

NADH-Ferricyanide Reductase of Leaf Plasma Membranes : Partial Purification and Immunological Relation to Potato Tuber Microsomal NADH-Ferricyanide Reductase and Spinach Leaf NADH-Nitrate Reductase

Plasma membranes obtained by two-phase partitioning of microsomal fractions from spinach (Spinacea oleracea L. cv Medania) and sugar beet leaves (Beta vulgaris L.) contained relatively high NADH-ferricyanide reductase and NADH-nitrate reductase (NR; EC 1.6.6.1) activities. Both of these activities were latent. To investigate whether these activities were due to the same enzyme, plasma membrane polypeptides were separated with SDS-PAGE and analyzed with immunoblotting methods. Antibodies raised against microsomal NADH-ferricyanide reductase (tentatively identified as NADH-cytochrome b₅ reductase, EC 1.6.2.2), purified from potato (Solanum tuberosum L. cv Bintje) tuber microsomes, displayed one single band at 43 kilodaltons when reacted with spinach plasma membranes, whereas lgG produced against NR from spinach leaves gave a major band at 110 kilodaltons together with a few fainter bands of lower molecular mass. Immunoblotting analysis using inside-out and right-side-out plasma membrane vesicles strongly indicated that NR was not an integral protein but probably trapped inside the plasma membrane vesicles during homogenization. Proteins from spinach plasma membranes were solubilized with the zwitterionic detergent 3-[(3-cholamidopropyl) dimethylammonio] 1-propane-sulfonate and separated on a Mono Q anion exchange column at pH 5.6 with fast protein liquid chromatography. One major peak of NADH-ferricyanide reductase activity was found after separation. The peak fraction was enriched about 70-fold in this activity compared to the plasma membrane. When the peak fractions were analyzed with SDS-PAGE the NADH-ferricyanide reductase activity strongly correlated with a 43 kilodalton polypeptide which reacted with the antibodies against potato microsomal NADH-ferricyanide reductase. Thus, our data indicate that most, if not all, of the truly membrane-bound NADH-ferricyanide reductase activity of leaf plasma membranes is due to an enzyme very similar to potato tuber microsomal NADH-ferricyanide reductase (NADH-cytochrome b₅ reductase).     

The protein kinase, protein phosphatase and inhibitor protein of nitrate reductase are ubiquitous in higher plants and independent of nitrate reductase expression and turnover

Nitrate reductase activity and NR protein levels in various leaf tissues were drastically decreased (<3.5% of normal activity) either by keeping detached leaves in continuous darkness for up to 6 d (spinach), or by growing plants (pea, squash) hydroponically on ammonium as the sole N-source, or by germinating and growing etiolated seedlings in complete darkness (squash). The presence of nitrate reductase protein kinase (NRPK), nitrate reductase protein phosphatase (NRPP) and inhibitor protein (IP) was examined by measuring the ability of NR-free desalted extracts to inactivate (ATP-dependent) and reactivate (5-AMP/EDTA-dependent) added purified spinach NR in vitro. Extracts from low-NR plants (ammonium-grown pea and squash) were also prepared from leaves harvested at the end of a normal light or dark phase, or after treating leaves with anaerobiosis, uncouplers or mannose, conditions which usually activate NR in nitrategrown normal plants. Without exception, extracts from NR-deficient plant tissues were able to inactivate and reactivate purified spinach NR with normal velocity, irrespective of pretreatment or time of harvest. Considerable NRPK, NRPP and IP activities were also found in extracts from almost NR-free ripe fruits (cucumber and tomato). Activities were totally absent, however, in extracts from isolated spinach chloroplasts. The NRPK and IP fractions were partially purified with normal yields from NR-deficient squash or spinach leaves, following the purification protocol worked out for nitrate-grown spinach. The Ca²⁺/Mg²⁺-dependent kinase fraction from NR-deficient squash or spinach phosphorylated added purified spinach NR with -[³²P]ATP and inactivated the enzyme after addition of IP. It is suggested (i) that the auxiliary proteins (NRPK, IP, NRPP) which modulate NR are rather species- or organ-unspecific, (ii) that they do not turn over as rapidly as does NR, (iii) that they are probably expressed independently of NR, and (iiii) that they are not covalently modulated, but under control of metabolic and/or physical signals which are removed by desalting.Abbreviations IP inhibitor protein - NR NADH-nitrate reductase - NRA nitrate reductase activity - NRPK nitrate reductase protein kinase - NRPP nitrate reductase protein phosphatase - PK protein kinase This work was supported by the Deutsche Forschungsgemeinschaft (SFB 251).     

Oxygen Diffusion in Lupin Nodules: II. MECHANISMS OF DIFFUSION BARRIER OPERATION

The oxygen diffusion resistance of Lupinus albus (L.) cv. Multoluparoot nodules was increased by subjection to short-term stresses;lowering rhizosphere temperature from 25 to 16 °C (2 h),detopping plants (3 h), darkening plants (21 h) or exposingroots to 20 mol m^–3 KN03 for 2, 4 or 6 d. Microscopicobservations and measurements showed that this resulted in thearea of open intercellular spaces within the inner cortex beingreduced due to both cell expansion and increased productionof an occluding glycoprotein. Electrophoretic and Western Blotanalysis using the monoclonal antibodies MAC236 and MAC265 showedtwo distinct glycoprotein antigens with molecular weights of240 and 135 kDa, respectively. Both antigens are localized withinintercellular spaces of the inner cortex. The amount of glycoproteinwas determined using either ELISA, with MAC265, or quantificationof immunolabelling with MAC236. This immunolabelling also localizedthe glycoprotein within globules adhering to the inside of theinner cortical cell walls. Key words: Oxygen diffusion resistance, glycoprotein, nodules, nitrogen fixation, Lupinus albus