李痘病毒单克隆抗体及TAS-ELISA试剂盒的研制

将纯化的李痘病毒(Plum pox virus,PPV)制剂免疫BALB/c小鼠,用SP2/0骨髓瘤细胞与经李痘病毒免疫的BALB/c小鼠的脾细胞融合,有限稀释法克隆和间接ELISA法筛选出2株稳定分泌李痘病毒单克隆抗体的杂交瘤细胞株3F1,7A8。用间接ELISA方法对所获得的2个杂交瘤细胞株进行亚型鉴定分别为IgG1、IgG3。间接ELISA方法测定腹水效价分别为3F1:1.0×106,7A8:1.0×105。以多克隆抗体为包被抗体、单克隆抗体为检测抗体的TAS-ELISA试剂盒与李痘病毒的D株系、M株系的病毒分离物均有反应,与同属的马铃薯A病毒、莴苣花叶病毒、西瓜花叶病毒2号、马铃薯Y病毒坏死株系不发生交叉反应。     

Detection of plum pox potyvirus using monoclonal antibodies to structural and non-structural proteins1

Eleven monoclonal antibodies specific to plum pox potyvirus (PPV) coat protein were obtained by hybridoma technology from Spanish PPV isolates. In addition, two monoclonal antibodies specific for PPV cylindrical inclusions (CIP non-structural proteins) were obtained. The monoclonal antibodies specific for PPV coat protein were assayed by DASI ELISA against 81 PPV isolates. At least nine different epitopes were found and 21 distinct serological patterns of reaction (serogroups) were established using nine selected monoclonal antibodies against the collection of PPV isolates, indicating the high variability of coat protein among PPV isolates. Changes in epitope composition were observed after aphid and mechanical transmission, indicating the occurrence of mixtures of isolates in field trees. Monoclonal antibody 5B reacted with all PPV isolates assayed, with very high affinity, using DASI ELISA. This method was compared with immunocapture-PCR on field samples in spring, and showed very good coincidence of results. The efficiency of PPV detection can be slightly increased using monoclonal antibodies specific to cylindrical inclusions mixed with monoclonal antibodies against structural proteins, and using mixtures of monoclonal antibodies against different epitopes of coat protein. ELISA-I and immunoprinting-ELISA were able to detect CIP and PPV in extracts and tissue section, respectively, of woody plants. Two monoclonal antibodies offer the possibility of distinguishing between Marcus and Dideron PPV types (M or D). These D-specific monoclonal antibodies can be used in routine tests with high affinity.     

Polyclonal and monoclonal antibodies specific to the chrysanthemic acid moiety of pyrethroid insecticides

Two polyclonal and two monoclonal antibodies raised against chrysanthemic acid (CAA) were prepared for ELISA of pyrethroid insecticides with the common CAA moiety. The monoclonal antibody KCA226 showed the highest reactivity towards allethrin among the antibodies in C-ELISA, although KCA226 was more sensitive to higher concentrations of methanol than the polyclonal antibodies in ELISA. KCA226 was reacted linearly with allethrin at concentrations ranging from 1 to 10 μg litre^-1 in 0·5% methanol and from 300 to 10000 μg litre^-1 in 30% methanol. KCA226 reacted specifically with the pyrethroid insecticides with the CAA moiety but was much less reactive with CAA itself. These results suggest that C-ELISA based on KCA226 is useful for the assay of pyrethroid residues with the common CAA moiety. © 1998 Society of Chemical Industry     

Diversity of Plum pox virus isolates in Bosnia and Herzegovina

Sixteen Plum pox virus (PPV) isolates from several stone fruit cultivars, host species, orchards and geographical areas of Bosnia and Herzegovina were selected for typing, using serotype-specific monoclonal antibodies (MAbs) and PCR–RFLP, targeting the 3' terminal region of the coat protein (CP) and P3-6K₁ with restriction enzymes Rsa I and Dde I. Four PPV isolates were identified as PPV-M by serology and PCR; eight isolates were identified as PPV-D based on PCR–RFLP on both genomic regions, but were not recognized by the D-specific MAb4DG5. Four isolates from plum were identified as natural D/M recombinants (PPV-Rec), based on conflicting results of CP and P3-6K₁ typing. To investigate the genetic diversity of Bosnian PPV isolates in more detail, five isolates (three PPV-Rec, one PPV-M and one PPV-D) were partially sequenced in the region spanning the 3' terminal part of the NIb gene and the 5'-terminal part of the CP gene, corresponding to nucleotides 8056–8884. Nucleotide sequence alignment of recombinant isolates showed that they were closely related at the molecular level to previously characterized recombinants from other European countries, and shared the same recombination break point in the 3' terminal part of the NIb gene. This is the first report of naturally infected Prunus trees with PPV-M, PPV-D and PPV-Rec in Bosnia and Herzegovina. The high variability of the Bosnian PPV isolates fits with the presence of this virus in the country over a long period.     

Susceptibility of some North American and Yugoslav plum cultivars to plum pox potyvirus in Poland1

All North American plum cultivars investigated in Poland for their reaction to plum pox potyvirus appeared to be susceptible, showing severe symptoms on the leaves. Fruits of cvs Valor and Empress were also heavily damaged. Of the Yugoslav cultivars evaluated, Cacanska Najbolja was most tolerant whereas Cacanska Rodna appeared to be most sensitive to PPV.     

Detection and recovery of Rhizoctonia solani in naturally infested glasshouse soils using a combined baiting, double monoclonal antibody ELISA

A combined baiting, double monoclonal antibody immunoassay was developed that allows specific and sensitive detection of the economically important soil-borne plant pathogen Rhizoctonia solani in naturally infested soils. The assay is quick, taking only three days to complete from receipt of soil samples and the immunoassay format allows recovery of Rhizoctonia isolates from colonized baits for determination of anastomosis group (AG) affiliation and pathogenicity. The assay was tested on naturally infested soils from commercial glasshouses used to grow lettuce. Using the immunoassay, conventional anastomosis tests against known AG isolates, and pathogenicity tests, it was shown that R. solani isolates recovered from soil samples were pathogenic towards lettuce and belonged to AG4. Furthermore, those isolates that exhibited strong pathogenicity towards lettuce were recovered from sites that had experienced severe Rhizoctonia damage in previous lettuce crops. The possibility of developing a preplanting test to predict damage to specific crop plants due to the presence of particular AGs in the soil is discussed.     

南芥菜花叶病毒单克隆抗体的研制及检测应用

将纯化的南芥菜花叶病毒(Arabis mosaic virus,ArMV)制剂免疫Balb/c小鼠,末次免疫后第3天取其脾细胞与SP2/0细胞融合,采用选择性培养基、有限稀释法克隆和间接ELISA方法进行筛选,成功获得了2株稳定分泌ArMV单克隆抗体的杂交瘤细胞株并分别命名为3F7,4G10。用间接ELISA方法对所获得的2个杂交瘤细胞株进行亚型鉴定分别为IgA(3F7)、IgG1(4G10)。间接ELISA效价测定结果:3F7为1:106,4G10为1:108。以单克隆抗体为包被抗体、多克隆抗体为检测抗体的TAS-ELISA检测试剂盒能检测感染ArMV的昆诺藜病汁液的灵敏度为1:1600。     

Genetic analysis suggests a long and largely isolated evolutionary history of plum pox virus strain D in Turkey

Plum pox virus (PPV) strain D is globally distributed and causes serious losses in stone fruits in over 40 countries. Here, full-length genomic sequences were analysed for 44 PPV-D isolates from all regions of Turkey, together with partial sequences for a larger number of isolates. PPV-D isolates from Turkey are similar to other PPV-D isolates in all major genomic features. However, the majority of Turkish PPV-D isolates form separate phylogenetic clusters from all other isolates and show a geographical clustering tendency, suggestive of limited movement between regions. In particular, PPV-D isolates from Thrace and Central Anatolia formed a monophyletic sister cluster to the cluster that includes all previously known PPV-D isolates. Two isolates with strong evidence of recombination with the PPV-T strain were identified, together with two isolates with weaker evidence for intra-D strain recombination. The genetic diversity of PPV-D was found to be particularly high in Turkey (0.017 ± 0.001%), close to that observed for PPV-D world diversity once the over-represented isolates from Japan, the USA and Canada have been excluded (0.020 ± 0.001%). Taken together, these results suggest a long and largely isolated evolutionary history of PPV-D in Turkey and further extend knowledge of the diversity of this highly successful strain. The high diversity of PPV-D in Turkey, together with the basal phylogenetic position of Turkish isolates, are compatible with a hypothesis making Turkey the centre of origin of the D strain.     

甘薯潜隐病毒单克隆抗体的制备及初步鉴定

以原核表达的甘薯潜隐病毒(SPLV)的外壳蛋白(CP)为抗原免疫小鼠,经过细胞融合和亚克隆,筛选出2株稳定分泌抗SPLV CP的单克隆抗体杂交瘤细胞株(5B11-2和5G8-2),并分别制备了单克隆抗体腹水。间接ELISA结果表明,用SPLV CP包被酶联板,5B11-2和5G8-2单克隆抗体的效价均为1∶512 000;用感染SPLV的甘薯叶片汁液包被酶联板,2株单克隆抗体的效价均为1∶6 400。抗体类型及亚类鉴定结果表明,2株单克隆抗体均为IgG1、κ轻链。Western blot分析表明,2株单抗均能与SPLV CP和感染SPLV的甘薯叶片汁液有特异性反应。利用单克隆抗体建立的间接抗原包被ELISA(ACP-ELISA)检测SPLV方法,病叶1∶3 840倍稀释仍能检测到病毒。血清学和RT-PCR检测结果表明,制备的单克隆抗体可用于田间甘薯样品的检测。     

Evaluation of peach and nectarine cultivars in Bulgaria for their resistance to plum pox potyvirus

In spring and summer 1989, it was established that plum pox potyvirus (PPV) was present in certain peach cultivars in Bulgaria. At the same time, we started to investigate the distribution of PPV in naturally infected 4–5 year-old peach and nectarine cultivars and hybrids in order to optimize PPV detection. Over 160 peach and nectarine cultivars and hybrids were evaluated. In about 40% of the genotypes, typical plum pox symptoms were observed. The latter were estimated and divided into groups depending on their susceptibility to PPV. Observations were made on the population density of seven aphid species established in the peach orchards. Five proved to be vectors of the virus. Myzus persicae was the vector that played the main role in spreading the virus on peach.     

Recent results on resistance to plum pox potyvirus in plum in Romania,and on the epidemiology of the disease1

Of 50 plum cultivars investigated for infection by plum pox potyvirus (PPV), three had the virus in the latent state (Mansan, Twilight and Frontier), 19 showed very slight or slight symptoms on leaves, 16 showed moderate symptoms and 12 severe symptoms. No symptoms were seen on fruits of cvs Diana, Ialomita, Abundance, Silvia, Mont Royal, Red Glow, Locale de Turt, Krikon and Blue Free. Slight symptoms present only on the fruit skin were noted on cvs Gras ameliorat, Seneca, Rosior de Zalau, Prun protuberat, Anna Späth, Early Rivers, LU, Pitestean, Pamiat Timiriazeva, Dunarea Albastra (Blue Danube), Gras romanesc and others. Few plum cultivars had sensitive or very sensitive fruit. Most hybrids obtained in Romania are tolerant in terms of symptoms on fruits. Of the rootstocks, Red Dwarf Myrobalan, Buburuz, Marianna and P. besseyi showed no or very slight symptoms. Plum plantations located close to a PPV infection source for 6–9 years were 13.2–45.5% infected. Trees planted in an infected orchard were up to 74% infected in 6 years.     

番茄溃疡病菌单克隆抗体的制备与鉴定

为制备并鉴定番茄溃疡病菌(Clavibacter michiganensis subsp.michiganensis,Cmm)的单克隆抗体(McAbs),用全菌皮下免疫BALB/C小鼠,采用B细胞杂交瘤技术,经免疫、融合、间接ELISA筛选和克隆等,获得稳定分泌抗体的阳性杂交瘤细胞株,得到了抗番茄溃疡病菌的单克隆抗体。经免疫后获得3株单抗分别为1A4、1C3和1B7,经亚类鉴定分别是IgM、IgG1、IgG1;纯化腹水间接ELISA效价分别为1:3.2×106、1:8.1×105、1:3.2×106;与其他同属不同亚种无交叉反应。结果表明:3株单克隆抗体均具有较高特异性和敏感性,可作为番茄溃疡病菌的检测抗体,其中,1A4的效果最好。番茄溃疡病菌单克隆抗体的获得为进一步研发番茄溃疡病检测试剂盒奠定了基础。     

Comparison of monoclonal antibodies and polymerase chain reaction assays for the typing of isolates belonging to the d and m serotypes of plum pox potyvirus

ABSTRACT Plum pox potyvirus (PPV) isolates may be divided into four groups separated by serological, molecular, and epidemiological differences. Monoclonal antibodies specific for the two major groups of isolates, represented by the D and M serotypes of the virus, have been obtained. Polymerase chain reaction (PCR)-based assays allowing the direct detection and differentiation of PPV isolates have also been developed. We now report on a large-scale comparison of these two typing approaches. The results obtained show an overall excellent correlation between the results obtained in indirect double-antibody sandwich enzyme-linked immunosorbent assay using PPV-D- and PPV-M-specific monoclonal antibodies and those derived from either specific PCR assays or restriction fragment length polymorphism analysis of PCR fragments. Without exception, all isolates reacting positively with the PPV-M-specific monoclonal antibody were found to belong to the M serotype using the PCR-based assays, while 51 out of 53 isolates recognized by the D-specific monoclonal antibodies belonged to the D serotype according to the PCR typing results. However, failure to react with a specific monoclonal antibody did not prove as effective a predictor of the serotype of the isolate analyzed. In a few cases, the results obtained with the various techniques diverged, indicating low level variability of the epitopes recognized by the serotype-specific monoclonal antibodies. Isolates belonging to the two minor groups of PPV (El Amar and Cherry) also gave divergent results, indicating that the current typing assays are not suited for the analysis of such isolates.     

Detection and characterization of plum pox potyvirus isolates from southern Germany by polymerase chain reaction (PCR)1

Plum pox potyvirus (PPV) isolates show quantitative and qualitative differences in host plants, symptomatology, aphid transmissibility and molecular biology. In order to gain an overview of the different PPV strains that exist in Germany, several PPV isolates, mainly deriving from southern Germany, were analysed by polymerase chain reaction (PCR), the reaction yielding a 243 bp product. All PPV isolates tested were amplified and the amplified fragments were analysed by restriction endonuclease digestion. An Rsa I restriction site polymorphism in the amplified fragments allowed the discrimination of two groups of isolates.     

Susceptibility of apricot cultivars to artificial inoculation with plum pox potyvirus and use of ELISA for its evaluation1

The susceptibility to plum pox potyvirus (PPV) of 13 apricot cultivars and two hybrids was studied under isolated conditions in the experimental orchard of the Research Breeding Station at Vesele from 1989 to 1993 after artificial inoculation of the trees. The plants were inoculated in the nursery in 1988 by chip-budding with a local source of virus and then planted into the field trial. First symptoms of plum pox appeared on the leaves in 1990. Reinoculation of symptomless trees was done in summer 1991. By 1993, plum pox had totally invaded some apricot trees of the most susceptible cultivars (Madarska, Velkopavlovicka, Kisinevskij rannij, Ligeti orias, Bergeron, Vesna and Vestar). Uninoculated control trees remained symptomless despite the proximity of diseased trees and the fact that the orchard was not sprayed with insecticides during the period of experiment. Cvs Julskij, Veecot, Vegama and Veharda remained free from plum pox symptoms on leaves and fruits. In 1993-05/06, the trees were assayed for the presence of the PPV antigen by ELISA. Leaf samples from 33 trees showing symptoms were positive. Samples prepared from the healthy tissues of leaves with symptoms were negative. Other trees (indexed by ELISA) growing in the neighbourhood of diseased trees in the period of 5 years were visually and ELISA negative.     

水稻条纹病毒单克隆抗体的制备及检测应用

 用水稻条纹病毒(RSV)免疫的BALB/c小鼠脾细胞与SP2/0鼠骨髓瘤细胞融合,经筛选克隆,获得4株能稳定传代且分泌抗RSV单克隆抗体的杂交瘤细胞。各株单抗腹水的ELISA效价均在1:80000~1:5120000之间。Westernblot分析表明,4株单抗均与RSV的35kDa的外壳蛋白亚基有特异反应。建立了间接ELISA测定RSV的方法,4株单抗检测病汁液的稀释度能达到2560倍以上,与其它病毒无交叉反应。对江苏省部分县市的大田灰飞虱带毒率进行检测,结果显示灰飞虱的带毒率为12.5%~41.5%     

A monoclonal antibody‐based ELISA for the analysis of the insecticide flucythrinate in environmental and crop samples

A competitive enzyme‐linked immunosorbent assay (ELISA) has been developed for the detection of the insecticide flucythrinate in environmental and food samples. Two types of haptens, the acid moiety that is the hydrolyzed product of flucythrinate, and the carboxylated propyl derivative of the alcohol moiety, were used to prepare monoclonal antibodies (MAbs). Five MAbs, which raised against the former hapten, were reactive with flucythrinate. Among them, MAb F1A27‐4 showed the highest activity toward flucythrinate, and did not cross‐react with other pyrethroids such as cycloprothrin, fenvalerate, fluvalinate, etofenprox and silafluofen. The assay conditions of indirect competitive ELISA with MAb F1A27‐4 were studied to optimize the detection of flucythrinate in environmental and food samples. Incubation at 4 °C in the assay buffer, pH 8, with 300 mM sodium chloride improved the sensitivity. The addition of rabbit serum albumin or rabbit antiserum and the presence of 50 ml litre^?1 of methanol reduced matrix effects of the samples. Under optimized conditions, the ELISA detected flucythrinate spiked in water, soil, and extracts of apple and tea samples down to 10 mg litre^?1, 0.2 mg litre^?1, 0.3 mg litre^?1 and 0.3 mg litre^?1, respectively. The mean recovery and CV ranged from 91% to 120% and from 5% to 12%, respectively. The ELISA results in apple samples correlated well with those from LC–MS analysis (r² = 0.99, n = 12). © 2001 Society of Chemical Industry     

Production of plants of Nicotiana benthamiana and Prunus domestica transgenic for plum pox potyvirus coat protein,and demonstration of PPV resistance in transformed N. benthamiana1

Leaf discs of Nicotiana benthamiana plants were transformed with Agrobacterium tumefaciens and transgenic plants expressing plum pox potyvirus (PPV) coat protein (CP) were generated. Homozygous R₂ progeny from these plants were inoculated with PPV. Plants were scored for the appearance of symptoms and tested for infection by DAS-ELISA. Various levels of resistance were obtained after an initial stage in which PPV was able to multiply in all the transgenic plants. Within 2–3 weeks after inoculation, the transgenic resistant plants fully recovered from virus infection. Conversely, control and susceptible transgenic lines developed severe symptoms and high virus titres. Prunus domestica (plum) was transformed by inoculating hypocotyl slices with A. tumefaciens containing a binary plasmid which included the NPTII, GUS, and PPV CP genes within its T-DNA region. Transgenic shoots were rooted and established in the glasshouse. Analysis of selected transformants by PCR showed that the engineered foreign genes had been integrated, including that for PPV CP. Histological assays on young leaves of these putative transformants gave a positive reaction. This suggests that all genes transferred are expressed in these transformed plums.     

Production of monoclonal antibodies against synthetic peptides of the N-terminal region of Potato virus Y coat protein and their use in PVY strain differentiation

Antibodies were prepared against two synthetic peptides, P19 and P11, derived from the coat protein N-terminal region of two pepper isolates of Potato virus Y from Tunisia (PVY-P21 and PVY-P2, respectively). The peptides were selected by comparing the predicted amino acid sequences of three pepper and four potato PVY isolates on the basis of their polymorphism and hydrophilicity. Sera with high titres were only obtained against P19. Three MAbs, raised in response to P19, reacted with the homologous virus (PVY-P21) in TAS-ELISA. When tested against a broad range of PVY isolates and related viruses, MAb 3C5 proved to be PVY species specific, whereas MAbs 8A4 and 1D6 reacted specifically with standard isolates of PVY^O, PVY^C and PVY^N-W strains, but not with other PVY isolates. Consequently, epitope(s) recognized by 8A4 and 1D6 MAbs may be specific to a PVY group comprising all serologically PVY^non–N isolates. Surprisingly, and unlike isolate PVY-P21, many Tunisian field pepper isolates did not carry this epitope(s), thus revealing serological heterogeneity within the PVY pepper group. As PVY is one of the most economically important plant pathogens in a range of crops, including pepper, these MAbs will provide a useful tool for practical diagnosis and strain identification of PVY.     

Genetic diversity and molecular epidemiology of the T strain of Plum pox virus

Very limited information is available on the origin, diversity and evolution of Plum pox virus (PPV) ‘Turkey’ (T) strain. Phylogenetic analyses based on partial sequences of 421 isolates and complete genome sequences of 57 isolates, representing the geographical distribution of PPV-T in Turkey, revealed the existence of several monophyletic and, in some cases, geographically limited groups within the PPV-T strain (Ankara-Konya1-Kayseri, Ankara-Balkan, Istanbul, Konya2 and Balkan). PPV-T diversity (0.018%) was found to be greater than that of PPV strains D and Rec but lower than that of the M strain when including the newly described and divergent M-Istanbul isolates, suggesting a long evolutionary history for PPV-T. The European part of Turkey in the Balkans, close to Bulgaria where PPV was identified for the first time, appears as a likely centre of origin for PPV-T isolates. The colonization of various parts of Turkey by diverse isolates from that region, followed by secondary local spread, is the most likely scenario for the diffusion of PPV-T in Turkey.