Group-specific Monoclonal Antibody Research Articles

Designing a size exclusion-based hapten and the development of a quantitative and visual time-resolved fluorescence immunochromatography assay strip for detecting dimethomorph and flumorph in a group-specific manner

Based on the size and surface properties of dimethomorph and flumorph, we used a computer simulation-assisted size exclusion hapten design strategy to develop group-specific monoclonal antibodies that can simultaneously recognize dimethomorph and flumorph. For this, we performed quantitative and visual semi-quantitative time-resolved fluorescence immunochromatography (TRFICA) to simultaneously detect dimethomorph and flumorph in potatoes and apples. In potato samples, the visual limit of detection (vLOD) for dimethomorph and flumorph was 4 ng/mL and 8 ng/mL, respectively, whereas the quantitative limit of detection (qLOD) for dimethomorph and flumorph was 0.26 and 0.33 ng/mL, respectively. The vLOD of dimethomorph and flumorph in apple samples was 8 ng/mL, whereas the qLOD of dimethomorph and flumorph was 0.17 and 0.38 ng/mL, respectively. The average recovery of potato and apple samples ranged from 77.5% to 121.7%, which indicated that the method can be used to rapidly detect dimethomorph and flumorph in food samples.

Fluorescent microsphere immunochromatographic sensor for the detection of total fumonisins B1, B2, and B3 in grain samples

Fumonisin Bs (FBs) are the most common toxic and carcinogenic substances found among naturally occurring contaminants of cereals. A group-specific monoclonal antibody (mAb) 2H7 was prepared that equably recognized fumonisins B1 (FB1), B2 (FB2), and B3 (FB3) with a half-maximal inhibitory concentration (IC50) value for the three forms of 1.52, 2.93, and 3.85 ng/ml, respectively. On this basis, a fluorescent microsphere-based immunochromatographic strip (FM-ICS) was established for the rapid qualitative and quantitative detection of total FBs (FB1, FB2, and FB3) in corn, wheat, and rice. A recovery test showed that the recovery of FM-ICS was 92.8–108.3% with coefficients of variation of 1.2–7.9%. This method has the same reliability as seen with liquid chromatography-tandem mass spectrometry. And the accuracy was further confirmed by certified reference materials. Therefore, it can be used for the detection of FBs in cereal samples.

The role of acidification of the medium on the erythrocyte agglutinating and adsorbing properties of blood group-specific monoclonal antibodies.

Studies on the specificity of ABH antigen-antibody interactions at different pH values are rare. Therefore, the aim of this study was to estimate the effect of acidification of the reacting medium on the agglutinating ability of anti-A monoclonal antibodies (mABs) and their inhibition by glycoconjugates of red blood cell membranes. Anti-A mABs were obtained from the Fourth International Workshop on Monoclonal Antibodies Against Human Red Blood Cell and Related Antigens (on July 19-20, 2002, in Paris, France). The glycoconjugates were isolated from erythrocytes' membranes. The inhibition of the lipid and protein isotypes of the blood group A antigen was assessed. The acidic medium decreased the agglutinating ability of acid immunoglobulin M (IgM) anti-A mABs, in contrast to alkaline immunoglobulin G (IgG) mABs. Meanwhile, at pH 6.5, IgM antibodies exhibited a high adsorption capacity, while IgG antibodies demonstrated a strong adsorption capacity at an alkaline pH. mABs 2-19, 2-28, 2-22, and 2-8 were inhibited by the acidic lipid and protein glycotopes of erythrocyte membranes. However, mAB 2-8 was inhibited by both acidic and alkaline glycotope variants. The agglutinating and adsorbing abilities of mABs, which are revealed at opposing pH values, should be taken into account during studies of antigen-antibody interactions.

A highly sensitive lateral flow immunoassay based on a group-specific monoclonal antibody and amorphous carbon nanoparticles for detection of sulfonamides in milk.

To meet high-throughput screening of the residues of sulfonamides (SAs) with high sensitivity toward sulfamethazine (SM2) in milk samples, a new highly sensitive lateral flow immunoassay (LFA) based on amorphous carbon nanoparticles (ACNs) was developed. First, a group-specific monoclonal antibody 10H7 (mAb 10H7) that could recognize 25 SAs with high sensitivity toward SM2 (IC50 value of 0.18ng/mL) was prepared based on H1 as an immune hapten and H4 as a heterologous coating hapten. Then, mAb 10H7 was conjugated to ACNs as an immune probe for LFA development. Under the optimized conditions, the LFA could detect 25 SAs with the cut-off value toward SM2 of 2ng/mL, which could meet the requirement for detection of SAs. In addition, the LFA developed was also used for screening SAs' residues in real milk samples, withresults beingconsistent with HPLC-MS/MS. Thus, this LFA can be used as a high-throughput screening tool for detection of SAs.

ELISA-Based Method for Variant-Independent Detection of Total Microcystins and Nodularins via a Multi-immunogen Approach.

Required routine monitoring of microcystins (MCs) and nodularins (NODs) in water samples, as posed by U.S. EPA Unregulated Contaminant Monitoring Rule 4, demands cost-effective, reliable, and sensitive detection methods. To target as many MC and NOD variants as possible, we developed an indirect competitive enzyme-linked immunosorbent assay (ELISA) with group-specific monoclonal antibodies for variant-independent detection of total MCs and NODs. In this ELISA method, the mice monoclonal antibodies presenting both high affinities and broad-spectrum recognition capabilities against MCs and NODs were self-produced by designing MC hapten-based multi-immunogens to minimize specificity for the particular variant. Their high affinities and variant-independent binding capabilities against MCs and NODs were validated by both wet lab and in silico methods. The developed ELISA method achieved a limit of detection of below 0.3 μg/L for 13 MC/NOD variants, well with the reported best cross-reactivities of 60-127% relative to MC-LR. As a case study, this ELISA method was used to map the variations of intracellular and extracellular total MCs/NODs in the Luoma Lake drinking water source, China, in July, 2020. Its capability to measure total MCs/NODs with high sensitivity and high throughput in a simple and affordable way would truly be a disruptive technology capable of changing our understanding of bloom/toxin dynamics and having obvious implications for monitoring efforts.

Determination of aflatoxin and zearalenone analogs in edible and medicinal herbs using a group-specific immunoaffinity column coupled to ultra-high-performance liquid chromatography with tandem mass spectrometry

Six aflatoxins (AFs; AF B1, B2, G1, G2, M1 and M2) and six zearalenone (ZEN) analogs (ZEN, zearalanone, α-zeralanol, β-zeralanol, α-zearalenol, and β-zearalenol) were simultaneously extracted from edible and medicinal herbs using a group-specific immunoaffinity column (IAC) and then identified by ultra-high-performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS). The IAC was prepared by coupling N-hydroxysuccinimide-activated Sepharose 4B Fast Flow gel with two group-specific monoclonal antibodies. The column capacities to six AFs and six ZEN analogs ranged from 100.2 ng to 167.1 ng and from 59.5 ng to 244.4 ng, respectively. The IAC–UPLC–MS/MS method was developed and validated with three different matrices (Chinese yam [Dioscorea polystachya], Platycodon grandiflorum and coix seed [Semen Coicis]). Recoveries of twelve analytes from edible and medicinal herbs were in the range of 64.7%–112.1%, with relative standard deviations below 13.7%. The limits of quantification were in the range from 0.08 μg kg−1 to 0.2 μg kg−1. The method was proven to be sensitive and accurate, and suitable for the determination of real samples.

Ultrasensitive immunochromatographic strip for detection of cyproheptadine

ABSTRACT Using immunological and cell fusion technology, group-specific monoclonal antibodies were produced against cyproheptadine. The 50% inhibitory concentration of the antibody for cyproheptadine was 0.37 ng/mL, the linear range was 0.16–0.85 ng/mL, and recoveries of antibody in spiked negative pig urine were 80–120%. An ultrasensitive immunochromatographic strip, based on the antibody, was developed for the rapid detection of cyproheptadine in pig urine. The test strip had a cut-off value of 5 ng/mL.

Gold immunochromatographic sensor for the rapid detection of twenty-six sulfonamides in foods

A gold immunochromatographic sensor (GICS) was developed for the rapid detection of 26 sulfonamides in honey samples. The sensor was based on a group-specific monoclonal antibody (mAb) that can recognize all 26 sulfonamides. Three haptens (hapten 1 with a thiazole ring, hapten 2 with a benzene ring, and hapten 3 with a straight carbon chain) were used for antigen preparation. With hybridoma technology, a group-specific mAb was screened with a 50% maximal inhibitory concentration (IC50) against sulfathizole (STZ) and the other 25 analogues ranging from 0.08 to 90.18 ng/mL. Mono-dispersed gold nanoparticles were conjugated with the mAb to develop the lateral immunochromatographic strip. A labeled antibody concentration of 0.1 μg/mL and a coating antigen concentration of 0.2 μg/mL in the test line were chosen for strip preparation. Under optimized conditions, the visual limits of detection (vLOD) for the concentrations of STZ, sulfamethoxazole, sulfamethizole, sulfadiazine, sulfamerazine, sulfadimethoxine, sulfamonomethoxine, sulfameter, sulfamethoxypyridazine, and sulfachloropyridazine were 5, 0.25, 0.25, 10, 5, 10, 25, 2.5, 5, 0.25, and 10 μg/kg, respectively. Scanner analysis in honey samples revealed good performance for detection of the 26 sulfonamides. Commercial honey samples were tested with the sensor and positive results were confirmed with high-performance liquid chromatography. The proposed strip sensor provides a convenient method for the rapid and reliable determination of sulfonamides pollutants in honey samples.

Preparation of a Broadly Specific Monoclonal Antibody-Based Indirect Competitive ELISA for the Detection of Benzodiazepines in Edible Animal Tissues and Feed

Incorrect use of benzodiazepines could result in serious health problems. To monitor the illegal use of benzodiazepine compounds in animals, a group-specific monoclonal antibody (mAb) was prepared in this study. The obtained 3D7 mAb, which is an IgG1 isotype mAb, displayed an IC50 value of 8.9 ng mL−1 for diazepam and exhibited cross-reaction for diazepam (100 %), nitrazepam (49 %), nordiazepam (140 %), temazepam (32 %), oxazepam (17 %), estazolam (7.5 %), and alprazolam (2.4 %). Based on this mAb, for the first time in this study, an optimized indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) protocol that did not require complicated sample preparation and clean-up was developed. The detection limits of this ic-ELISA for benzodiazepines ranged from 1.2 to 3.3 μg kg−1 in muscle matrix and from 25.2 to 55.4 μg kg−1 in feed matrix. The recoveries ranged from 70.9 to 111.3 % with coefficients of variation below 15.0 %. Good correlations (r > 0.9494) between the results of the ic-ELISA and high-performance liquid chromatography were also observed. This simple method reduced the time required for sample preparation, ensured greater throughput, and met the requirements for benzodiazepine residue analyses. In conclusion, the proposed method is a sensitive and rapid multi-residue technique that offers a cost-effective alternative to current published procedures without any concession in the ability to detect benzodiazepine sedative misuse.

Isolation of a Bluetongue virus group-specific monoclonal antibody and application to a diagnostic competitive ELISA.

The Bluetongue virus (BTV) VP7 protein represents an important group-specific antigen that can serve as a basis for diagnostic tests. Here, we report the generation of a novel BTV group-specific monoclonal antibody (Mab; herein named 4H7) that recognizes a conformational epitope in the VP7 protein. We used a phage-displayed peptide screen and site-directed mutagenesis to define the VP7 amino acid residues that most strongly contribute to the conformational epitope recognized by Mab 4H7. Amino acid residues at positions 175, 185, 186, and 278 of the BTV VP7 protein strongly contributed to Mab 4H7 binding. These key amino acid residues are conserved among all BTV serotypes, whereas related Orbiviruses possess at least one amino acid substitution at these positions. We developed a competitive enzyme-linked immunosorbent assay (c-ELISA) using Mab 4H7 and recombinant BTV VP7 protein to detect serum antibodies against this BTV group-specific VP7 epitope. The c-ELISA was used to screen 833 clinical samples collected from animals in three provinces of China. BTV seroprevalence in the three provinces ranged from 25.42 to 47.45 %. This work provides the foundation for a new diagnostic c-ELISA that can be further applied to BTV surveillance activities and informs our understanding of the structure of the BTV VP7 protein.

Development of a Biotin-Avidin Mediated Immunoassay for Simultaneous Detection of Danofloxacin, Enrofloxacin and Ciprofloxacin in Milk

To generate a group-specific monoclonal antibody (McAb) against danofloxacin (DANO), enrofloxacin (ENR) and ciprofloxacin (CIP), glutaraldehyde was used to link the presentative hapten of CIP to the immunogen and coating antigen, respectively, leaving the common structure of these 3 drugs exposed in both conjugates as a major antigenic site. Consequently, a McAb (6D3) with high cross-reactivity to this three antibiotics has been obtained by using hybridomas technique. In a biotin-avidin mediated enzyme-linked immunosorbent assay, the IC50values for DANO, ENR and CIP were 5.1, 4.5 and 4.2 ng mL-1, respectively. The ELISA was used for the detection of spiked DANO and ENR+CIP in milk. The recoveries ranged from 74.1 to 92.4% and coefficients of variation were in a range of 6.6-11.9%. The accuracies and sensitivity of the method were good for simultaneous analysis of the 3 drugs in milk after a simple sample extraction process.

Monoclonal antibodies with group specificity toward sulfonamides: selection of hapten and antibody selectivity

Immunoassays based on the current available antibodies for large multi-sulfonamide screening programs have suffered from high selectivity for individual sulfonamides and a wide range of selectivities for different sulfonamides. In this study, five synthesized haptens, HS, BS, CS, SA10, and TS and two sulfonamides, SG and SMX were used as haptens, which may or may not contain a ring structure at the N1 position of the sulfonamides, were selected to evaluate the effectiveness for producing group-specific monoclonal antibodies (MAbs). Mice immunized with three different two-ring haptens were used for hybridoma production, which resulted in three unique MAbs recognizing 10, 13, and 15 sulfonamides showing 50 % inhibition (IC50) at concentrations below 100 ng mL(-1). MAb 4D11 derived from one novel immunizing hapten could recognize 12 sulfonamides with IC50 values ranging from 1.2 to 12.4 ng mL(-1), almost within 1 order of magnitude. These produced MAbs show lower IC50 values in addition to significantly improved group specificity compared with previously generated MAbs. This study clearly indicates that the careful selection of the immunizing hapten has an important effect on the specificity of the generated antibodies.

Production of a highly group-specific monoclonal antibody against zearalenone and its application in an enzyme-linked immunosorbent assay

A monoclonal antibody (mAb) against zearalenone (ZEN) was produced using ZEN-carboxymethoxylamine and -BSA conjugates. Antibody produced by one clone showing a very high binding ability was selected and found to have a higher affinity for ZEN compared to a commerciall ZEN antibody. We developed two direct competitive ELISA systems using the selected antibody (ZEN-coated and anti-ZEN antibody-coated ELISA). Quantitative ranges for the anti-ZEN antibody-coated ELISA and ZEN-coated ELISA were from 25 to 750 ppb and from 12.5 to 100 ppb, respectively. The detection limit of both methods as measured with standard solutions was 10 ppb. The intra-plate and inter-well variation of both ELISAs were less than 10%. The IC50 values for α-zearalenol, β-zearalenol, α-zearalanol, and β-zearalanol compared to ZEN were 108.1, 119.3, 114.1, and 130.3% for the ZEN-coated ELISA. These values were 100.7, 120.7, 121.6, and 151.6% for the anti-ZEN antibody-coated ELISA. According to the anti-ZEN antibody-coated ELISA, the average recovery rates of ZEN from spiked animal feed containing 150 to 600 ng/mL of ZEN ranged from 106.07 to 123.00% with 0.93 to 2.28% coefficients of variation. Our results demonstrate that the mAb developed in this study could be used to simultaneously screen for ZEN and its metabolites in feed.

Production of Group Specific Monoclonal Antibody to Aflatoxins and its Application to Enzyme-linked Immunosorbent Assay

Through the present study, we produced a monoclonal antibody against aflatoxin B1 (AFB1) using AFB1- carboxymethoxylamine BSA conjugates. One clone showing high binding ability was selected and it was applied to develop a direct competitive ELISA system. The epitope densities of AFB1-CMO against BSA and KLH were about 1 : 6 and 1 : 545, respectively. The monoclonal antibody (mAb) from cloned hybridoma cell was the IgG1 subclass with λ-type light chains. The IC50s of the monoclonal antibody developed for AFB1, AFB2, AFG1 and AFG2 were 4.36, 7.22, 6.61 and 29.41 ng/ml, respectively, based on the AFB1-KLH coated ELISA system and 15.28, 26.62, 32.75 and 56.67 ng/ml, respectively, based on the mAb coated ELISA. Cross-relativities of mAb to AFB1 for AFB2, AFG1 and AFG2 were 60.47, 65.97 and 14.83% in the AFB1-KLH coated ELISA, and 59.41, 46.66 and 26.97% in the mAb coated ELISA, respectively. Quantitative calculations for AFB1 from the AFB1-Ab ELISA and AFB1-Ag ELISA ranged from 0.25 to 25 ng/ml (R2 > 0.99) and from 1 to 100 ng/ml (R2 > 0.99), respectively. The intra- and inter-assay precision CVs were < 10% in both ELISA assay, representing good reproducibility of developed assay. Recoveries ranged from 79.18 to 91.27%, CVs ranged from 3.21 to 7.97% after spiking AFB1 at concentrations ranging from 5 to 50 ng/ml and following by extraction with 70% methanol solution in the Ab-coated ELISA. In conclusion, we produced a group specific mAb against aflatoxins and developed two direct competitive ELISAs for the detection of AFB1 in feeds based on a monoclonal antibody developed.

Development of a cell-based high throughput assay system and IN-house image analysis software for screening of active compounds against dengue virus

Dengue virus (DV) is a mosquito-borne pathogen capable of infecting multiple target organs in the human host. Infection with any one of the four dengue serotypes can cause an acute febrile illness known as Dengue Fever (DF), while subsequent infection with a heterologous serotype or a highly virulent strain can result in the more severe and sometimes lethal Dengue Hemorrhagic Fever (DHF) and Dengue Shock Syndrome (DSS). According to the World Health Organization, approximately 2.5 billion people live in dengue endemic countries, with an estimated 50 million cases occurring annually. The lack of an effective dengue vaccine has prompted the need to discover compounds that can inhibit DV infection. In this study, a cell-based, high-throughput assay system that utilizes an image-based analysis algorithm for evaluating active compounds that target DV infection is described. Using representative strains of DV1, DV2, DV3 serotypes, a human-derived hepatoma cell line (Huh7.5) and Aedes albopictus mosquito-derived cell line (clone C6/36) were inoculated with a high titer of each virus in a 384-well plate culture system in the presence of varying concentration of known active compounds. After 1-2 rounds of viral replication (48~72 h), infection was arrested by para-formaldehyde fixation. DV-infected cells were visualized by probing with D1-4G2-4-15 mAb, a flavivirus group-specific monoclonal antibody that targets the E protein, and a mouse IgG-specific AlexaFluor488™ secondary antibody. Images of the DV-infected cell culture are captured with an automated confocal microscope (Evotec Opera™), and analyzed using IM v3.0, a custom-based image analysis software developed by IP-Korea’s Image Mining Group. A 10-point dose-response curve was generated for each active compound and reproduced in several experiments. This newly developed HTA (High Throughput Assay) system for Dengue can be a useful tool to screen large compound libraries for active drugs that have inhibitory effects to DV infection.

Confirmation of Chlamydophila abortus in infected cell culture using Indirect Immunofluorescence technique

Chlamydophila abortus (C. abortus) is an important abortifacient agent in bovines and ovines. Clinical diagnosis of the disease is often difficult. An early diagnosis can be achieved based on direct demonstration of the organism in clinical material and through the cultural recovery of the organism in embryonated chicken egg. For confirmatory diagnosis antigen detection methods or serological techniques can be adopted. The present study is aimed at the confirmatory diagnosis of C. abortus infection by indirect immunofluorescence technique following the isolation of the organism in cell culture. Specific apple green fluorescing inclusions of C. abortus in McCoy cell lines was detected from 72 h to 96 h post infection employing anti-chlamydial group specific monoclonal antibodies. Thus, a confirmatory diagnosis of the infection was possible with this study.

Cucumber mosaic virus groups IA and II are represented among isolates from naturally infected lilies

Four cucumber mosaic virus isolates (named Cas, CB, P26 and Simp2) found in naturally infected lily plants were characterized on the basis of their serological properties and the results of analysis of RNA3 sequence fragments containing coat protein and movement protein genes. The properties of lily isolates were compared with those of eight other virus isolates originating from dahlia, delphinium, impatiens, honeysuckle, cucumber and redcurrant plants. On the basis of the reaction with group-specific monoclonal antibodies and RNA3 sequence analysis, two lily isolates (Cas and CB) were classified to group I of CMV, similarly to all previously reported virus isolates found in lily plants. Surprisingly, sequences of coat protein and movement protein genes of two other lily isolates (P26 and Simp2) showed more than 98% similarity to CMV group II isolates, and only 77% similarity to group I isolates. Results of ELISA with CMV group II-specific monoclonal antibodies confirmed the classification of isolate Simp2 as a member of group II. Isolate P26 reacted neither with CMV group I nor with group II-specific monoclonal antibodies. In order to explain the lack of reaction of P26 isolate with monoclonal antibodies, comparative analysis of predicted amino acid sequence of the coat protein was done. This revealed a mutation—change from alanine at position 138 to threonine, probably responsible for particular serological properties of isolate P26.

Development of an immunoassay for rapid screening of vardenafil and its potential analogues in herbal products based on a group specific monoclonal antibody

Vardenafil is a phosphodiesterase-5 (PDE-5) inhibitor for the treatment of erectile dysfunction (ED). Undeclared vardenafil and related analogues adulterated in herbal products are a threat to public health. To screen vardenafil and its analogues in herbal matrix rapidly, an immunoassay based on a group specific monoclonal antibody (McAb) was developed. Glutaraldehyde was used to link vardenafil to immunogen and coating-antigen, respectively. Through the assessment of the structural specificity of eight anti-vardenafil McAbs, the McAb of 4B9 was characterized as being specific to the common structure of vardenafil and its analogues. An indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was established based on this McAb, the limit of detection of vardenafil was 5.0 ng mL(-1), the calibration curve was linear from 5.0 to 40 ng mL(-1) (R(2)=0.952) with an IC(50) value of 18.2 ng mL(-1). In the extracts of 20 Chinese traditional drugs, the detection capability (CCbeta) of vardenafil was 0.08 mg g(-1), the recoveries were 76-116% and the coefficients of variation (CV%) were 9.7%-16.2%. The ic-ELISA was in good agreement with LC-UV when detected herbal products containing vardenafil and its analogue. The method is a suitable tool for screening vardenafil and its analogues as illegal additives in herbal products.

Production, Characterization and Assay Application of a Purified, Baculovirus-Expressed, Serogroup Specific Bluetongue Virus Antigen

The predominant serodiagnostic assay used in many countries to detect bluetongue virus (BTV) infections is a competitive enzyme-linked immunosorbent assay (c-ELISA) which employs two critical reagents: a cell culture-derived BTV antigen and group-specific monoclonal antibody (Mab). Ongoing difficulties have been reported by laboratories in the production and quality control of the native antigen reagent which relies on the presence of adequate molar quantities and appropriate presentation of the major BTV core protein VP7. To address this important issue, a recombinant baculovirus was constructed containing a cDNA copy of genome segment 7 of BTV serotype 11 and used to infect insect cells which, in turn, expressed high levels of theVP7 protein with an estimated molecular mass of 39 kDa. In its purified form, this recombinant protein could be detected by group-specific Mabs designated 3.17.A3 and 8A3B.6 produced against BTV serotypes 1 and 17, respectively, as well as by polyclonal bovine antibodies raised against North American and South African BTV serotypes. No reactivity was observed by Western blot analysis with these two Mabs suggesting that the common antigenic determinants, on the BTV VP7 protein, were mainly conformational. It was interesting to note that the purified recombinant VP7 protein demonstrated a greater degree of reactivity with Mab 8A3B.6 compared to that exhibited with Mab 3.17.A3 when evaluated in an ELISA. Due to its antigenic similarity to the native antigen, the recombinant protein was found to be a suitable replacement for use in a c-ELISA to detect BTV-specific antibodies with the added advantage that it could be consistently produced and was, therefore, amenable to quality control testing for purity, stability and other standards.

First Report of the Natural Infection of Coreopsis auriculata 'Nana' with Lettuce mosaic virus in the United States.

Perennial cultivars of Coreopsis, a genus native to the United States, are widely grown for aesthetics in home gardens and roadsides and are increasingly used in conservation projects and native-plant gardens. During the spring and summer of 2006 and 2007, Coreopsis auriculata 'Nana' plants with foliar symptoms showing chlorotic spots and rings were observed in wholesale and retail nurseries in Washington. Nicotiana benthamiana plants inoculated with crude sap extracts from symptomatic leaves of C. auriculata 'Nana' obtained from two different sources showed systemic mosaic mottling symptoms, indicating the presence of a virus. Symptomatic leaf samples from C. auriculata 'Nana' and N. benthamiana tested positive in antigen-coated plate-ELISA with potyvirus group-specific monoclonal antibodies (Agdia Inc., Elkhart, IN). Additional analysis by ELISA was positive for Lettuce mosaic virus (LMV; Agdia Inc.). To confirm these results, total RNA extracted from symptomatic N. benthamiana leaves was subjected to reverse transcription (RT)-PCR using potyvirus degenerate primers (PNIbF5: 5'-GCCAGCCCTCCACCGTNGTNGAYAA-3' and PCPR1: 5'-GGGGAGGTGCCGTTCTCDATRCACCA-3') covering the 3' end of the NIb gene and the 5' end of the CP gene (1). A single DNA band of approximately 1,000 bp amplified from symptomatic leaves of two independent plants was cloned separately into pCR2.1 (Invitrogen Corp., Carlsbad, CA). Two independent clones per amplicon were sequenced from both orientations. Pairwise comparison of these sequences with corresponding nucleotide sequences of potyviruses in GenBank showed 93 to 99% identity in the NIb/CP region with LMV sequences from France (GenBank Accession Nos. X97704, X65652, and X97705), China (GenBank Accession Nos. AJ306288 and AJ488153), and Brazil (GenBank Accession No. AJ278854). These results confirmed the presence of LMV in symptomatic leaves of N. benthamiana and C. auriculata 'Nana'. The occurrence of LMV has been reported in ornamental plants that included freeway daisy (Osteospermum fruticosum), lisianthus (Eustoma grandiflorum), and gazanias (Gazania spp.) (2-4). To our knowledge, this is the first documented evidence for the occurrence of LMV in Coreopsis, an economically important perennial ornamental widely grown in the United States. Although the origin of LMV in C. auriculata 'Nana' is not known, distribution of cuttings from LMV-infected C. auriculata 'Nana' plants to wholesale and retailers within Washington and across the country by movement of plant material could pose a risk to other ornamentals and crops like lettuce because of the broad host range of LMV and its potential transmission by several species of polyphagous aphids. Seed transmission as a potential means of dissemination of LMV in Coreopsis has not been examined, although the virus is seedborne in other plants such as lettuce.