Self-enhancement lateral flow immunoassay for COVID-19 diagnosis.

Equipment-free colorimetric-based lateral flow immunoassay (LFIA) is the most convenient and popular tool for various applications, including diagnostic tools requiring high sensitivity for the detection of pathogens. Thus, improvements and developments of LFIA are constantly being reported. Herein, we enriched the sensitivity of LFIA using the gold enhancement principle, emphasizing needlessly complicated apparatus, only one step for the strip test operation, and typical time incubation (15 min) process. Self-enhanced LFIA was then executed for subsequent flows by overlapping the additionally enhanced pad composed of gold ions and reducing agent on the conjugate pad and the sample pad. Self-enhanced LFIA was performed to detect SARS-CoV-2 antigens in saliva. The obtained result depicted that the achieved sensitivity was up to tenfold compared with that of conventional LFIA by visual measurements. The detection limits of self-enhanced LFIA detecting nucleocapsid protein antigens in the saliva sample was 0.50 and 0.10 ng/mL employed by naked eye detection and calibration curve-based calculation, respectively. When the proposed device was applied to 207 human saliva samples, the diagnostic performance presented a 96.10 % sensitivity and 99.23 % specificity. This self-enhanced LFIA could be implemented in large-scale production and demonstrates higher sensitivity with effortless use, which meets the requirements for point-of-care testing and on-field mass screening.

Development of a triple antibody sandwich enzyme-linked immunosorbent assay for cassava mosaic disease detection using a monoclonal antibody to Sri Lankan cassava mosaic virus.

BACKGROUND: Cassava mosaic disease (CMD) is one of the most devastating viral diseases for cassava production in Africa and Asia. Accurate yet affordable diagnostics are one of the fundamental tools supporting successful CMD management, especially in developing countries. This study aimed to develop an antibody-based immunoassay for the detection of Sri Lankan cassava mosaic virus (SLCMV), the only cassava mosaic begomovirus currently causing CMD outbreaks in Southeast Asia (SEA). METHODS: Monoclonal antibodies (MAbs) against the recombinant coat protein of SLCMV were generated using hybridoma technology. MAbs were characterized and used to develop a triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) for SLCMV detection in cassava leaves and stems. Assay specificity, sensitivity and efficiency for SLCMV detection was investigated and compared to those of a commercial ELISA test kit and PCR, the gold standard. RESULTS: A TAS-ELISA for SLCMV detection was successfully developed using the newly established MAb 29B3 and an in-house polyclonal antibody (PAb) against begomoviruses, PAb PK. The assay was able to detect SLCMV in leaves, green bark from cassava stem tips, and young leaf sprouts from stem cuttings of SLCMV-infected cassava plants without cross-reactivity to those derived from healthy cassava controls. Sensitivity comparison using serial dilutions of SLCMV-infected cassava sap extracts revealed that the assay was 256-fold more sensitive than a commercial TAS-ELISA kit and 64-fold less sensitive than PCR using previously published SLCMV-specific primers. In terms of DNA content, our assay demonstrated a limit of detection of 2.21 to 4.08 × 106 virus copies as determined by quantitative real-time PCR (qPCR). When applied to field samples (n = 490), the TAS-ELISA showed high accuracy (99.6%), specificity (100%), and sensitivity (98.2%) relative to the results obtained by the reference PCR. SLCMV infecting chaya (Cnidoscolus aconitifolius) and coral plant (Jatropha multifida) was also reported for the first time in SEA. CONCLUSIONS: Our findings suggest that the TAS-ELISA for SLCMV detection developed in this study can serve as an attractive tool for efficient, inexpensive and high-throughput detection of SLCMV and can be applied to CMD screening of cassava stem cuttings, large-scale surveillance, and screening for resistance.

Double antibody pairs sandwich-ELISA (DAPS-ELISA) detects Acidovorax citrulli serotypes with broad coverage.

Acidovorax citrulli, a seedborne bacterium and quarantine pest, causes the devastating bacterial fruit blotch disease in cucurbit plants. Immunological assays such as ELISA are widely used in routine field inspections for this bacterium. However, to the best of our knowledge, none of the currently available monoclonal antibodies (MAbs) can detect all common A. citrulli strains. We therefore aimed to produce a panel of MAbs and to develop an ELISA-based method capable of detecting all A. citrulli strains. We used a high-throughput bead array technique to screen and characterize A. citrulli-specific MAbs produced from hybridoma clones. The hybridoma library was simultaneously screened against five A. citrulli strains (PSA, KK9, SQA, SQB and P) and the closely related bacterium, Delftia acidovorans. Three MAbs exhibiting different binding patterns to A. citrulli were used to develop an ELISA-based method called "double antibody pairs sandwich ELISA" (DAPS-ELISA). DAPS-ELISA employing mixtures of MAbs was able to specifically detect all 16 A. citrulli strains tested without cross-reactivity with other bacteria. By contrast, our previously developed MAb capture-sandwich ELISA (MC-sELISA) and a commercial test kit detected only 15 and 14 of 16 strains, respectively. The sensitivity of the DAPS-ELISA ranged from 5×105 to 1×106 CFU/mL, while those of the MC-sELISA and the commercial test kit ranged from 5×104 to 1×107 CFU/mL and 5×104 to 5×105 CFU/mL, respectively. DAPS-ELISA thus represents an alternative method enabling rapid, accurate, and inexpensive detection of all A. citrulli strains. The method can be applied to seed testing prior to planting as well as to routine field inspections.

Significant Sensitivity Improvement for Camera-Based Lateral Flow Immunoassay Readers.

Recent developments in smartphone-based strip readers have further improved the performances of lateral flow test kits. Most smartphone cameras encode an unaltered and nonlinear power-law transfer function that maps the light intensity to a pixel value; this poses some limitations for camera-based strip readers. For faint-color test lines which are almost as white such as with nitrocellulose pads, the slope of the transfer function is low. Therefore, it is difficult to differentiate between the faint test lines and the white background. We show that by manually setting the camera exposure time-instead of using the automatic settings-to the high-slope region of the transfer function, the reader's sensitivity can be improved. We found that the sensitivity and the limit of detection of the Acidovorax avenae subsp. citrulli (Aac) test kit were enhanced up to 3-fold and 5-fold, respectively, when using the readers at the optimal camera settings, compared to the automatic mode settings. This simple technique can be readily applied to any existing camera-based colorimetric strip reader to significantly improve its performance.

Development and application of triple antibody sandwich enzyme-linked immunosorbent assays for begomovirus detection using monoclonal antibodies against Tomato yellow leaf curl Thailand virus.

BACKGROUND: Tomato yellow leaf curl Thailand virus, TYLCTHV, is a begomovirus that causes severe losses of tomato crops in Thailand as well as several countries in Southeast and East Asia. The development of monoclonal antibodies (MAbs) and serological methods for detecting TYLCTHV is essential for epidemiological studies and screening for virus-resistant cultivars. METHODS: The recombinant coat protein (CP) of TYLCTHV was expressed in Escherichia coli and used to generate MAbs against TYLCTHV through hybridoma technology. The MAbs were characterized and optimized to develop triple antibody sandwich enzyme-linked immunosorbent assays (TAS-ELISAs) for begomovirus detection. The efficiency of TAS-ELISAs for begomovirus detection was evaluated with tomato, pepper, eggplant, okra and cucurbit plants collected from several provinces in Thailand. Molecular identification of begomoviruses in these samples was also performed through PCR and DNA sequence analysis of the CP gene. RESULTS: Two MAbs (M1 and D2) were generated and used to develop TAS-ELISAs for begomovirus detection. The results of begomovirus detection in 147 field samples indicated that MAb M1 reacted with 2 begomovirus species, TYLCTHV and Tobacco leaf curl Yunnan virus (TbLCYnV), whereas MAb D2 reacted with 4 begomovirus species, TYLCTHV, TbLCYnV, Tomato leaf curl New Delhi virus (ToLCNDV) and Squash leaf curl China virus (SLCCNV). Phylogenetic analyses of CP amino acid sequences from these begomoviruses revealed that the CP sequences of begomoviruses recognized by the narrow-spectrum MAb M1 were highly conserved, sharing 93% identity with each other but only 72-81% identity with MAb M1-negative begomoviruses. The CP sequences of begomoviruses recognized by the broad-spectrum MAb D2 demonstrated a wider range of amino acid sequence identity, sharing 78-96% identity with each other and 72-91% identity with those that were not detected by MAb D2. CONCLUSIONS: TAS-ELISAs using the narrow-specificity MAb M1 proved highly efficient for the detection of TYLCTHV and TbLCYnV, whereas TAS-ELISAs using the broad-specificity MAb D2 were highly efficient for the detection of TYLCTHV, TbLCYnV, ToLCNDV and SLCCNV. Both newly developed assays allow for sensitive, inexpensive, high-throughput detection of begomoviruses in field plant samples, as well as screening for virus-resistant cultivars.

An accurate, specific, sensitive, high-throughput method based on a microsphere immunoassay for multiplex detection of three viruses and bacterial fruit blotch bacterium in cucurbits.

To employ a microsphere immunoassay (MIA) to simultaneously detect multiple plant pathogens (potyviruses, Watermelon silver mottle virus, Melon yellow spot virus, and Acidovorax avenae subsp. citrulli) in actual plant samples, several factors need to be optimized and rigorously validated. Here, a simple extraction method using a single extraction buffer was successfully selected to detect the four pathogens in various cucurbit samples (cucumber, cantaloupe, melon, and watermelon). The extraction method and assay performance were validated with inoculated and field cucurbit samples. The MIA showed 98-99% relative accuracy, 97-100% relative specificity and 92-100% relative sensitivity when compared to commercial ELISA kits and reverse transcription PCR. In addition, the MIA was also able to accurately detect multiple-infected field samples. The results demonstrate that one common extraction method for all tested cucurbit samples could be applied to detect multiple pathogens; avoiding the need for multiple protocols to be employed. This multiplex method can therefore be instrumental for high-throughput screening of multiple plant pathogens with many advantages such as a shorter assay time (2.5h) with single assay format, a lower cost of detection ($5 vs $19.7 for 4 pathogens/sample) and less labor requirement. Its multiplex capacity can also be expanded to detect up to 50 different pathogens upon the availability of specific antibodies.

Development of a protocol for the identification of tospoviruses and thrips species in individual thrips.

A protocol for identifying tospovirus and thrips species in an individual thrips sample was successfully developed. First, an individual thrips was soaked in an RNA stabilization solution to preserve protein and nucleic acids and ground in a carbonate buffer containing 0.2% sodium diethyldithiocarbamate. Initially, the thrips extracts were screened for tospovirus infection by dot blot analysis using antibodies to nucleocapsid (N) proteins of tospoviruses. Thrips extracts with positive results by dot blot analysis were further subjected to RNA extraction. Next, tospovirus species were identified by reverse transcription-polymerase chain reaction (RT-PCR) using species-specific primers for the N genes of four tospoviruses known to occur in Thailand, including Capsicum chlorosis virus (CaCV), Melon yellow spot virus (MYSV), Tomato necrotic ringspot virus (TNRV) and Watermelon silver mottle virus (WSMoV). The residual genomic DNA in the thrips RNA extract was used as a template to identify thrips species by PCR with species-specific primers to the internal transcribed spacer 2 regions of the rRNA of Ceratothripoides claratris, Frankliniella intonsa, Scirtothrips dorsalis and Thrips palmi. This protocol was initially validated against laboratory-reared thrips and then used to determine the occurrence of viruliferous thrips species collected from tomato, pepper, watermelon and cucumber fields in Thailand.

Antibody array in a multiwell plate format for the sensitive and multiplexed detection of important plant pathogens.

The global seed market is considered to be an important industry with a total value of $10,543 million US dollars in 2012. Because plant pathogens such as bacteria and viruses cause a significant economic loss to both producers and exporters, the seed export industry urgently requires rapid, sensitive, and inexpensive testing for the pathogens to prevent disease spreading worldwide. This study developed an antibody array in a multiwell plate format to simultaneously detect four crucial plant pathogens, namely, a bacterial fruit blotch bacterium Acidovorax avenae subsp. citrulli (Aac), Chilli veinal mottle virus (ChiVMV, potyvirus), Watermelon silver mottle virus (WSMoV, tospovirus serogroup IV), and Melon yellow spot virus (MYSV, tospovirus). The capture antibodies specific to the pathogens were immobilized on each well at preassigned positions by an automatic microarrayer. The antibodies on the arrays specifically captured the corresponding pathogens present in the sample extracts. The presence of pathogens bound on the capture antibodies was subsequently detected by a cocktail of fluorescently conjugated secondary antibodies. The limits of detection of the developed antibody array for the detection of Aac, ChiVMV, WSMoV, and MYSV were 5 × 10(5) CFU/mL, 30 ng/mL, 1000 ng/mL, and 160 ng/mL, respectively, which were very similar to those of the conventional ELISA method. The antibody array in a multiwell plate format accurately detected plant pathogens in single and multiple detections. Moreover, this format enables easy handling of the assay at a higher speed of operation.

Development of a multiplex RT-PCR-ELISA to identify four distinct species of tospovirus.

In this study, a multiplex RT-PCR-ELISA was developed to detect and differentiate four tospovirus species found in Thailand, namely Capsicum chlorosis virus (CaCV), Melon yellow spot virus (MYSV), Tomato necrotic ringspot virus (TNRV), and Watermelon silver mottle virus (WSMoV). In this system, nucleocapsid (N) gene fragments of four tospoviruses were simultaneously amplified and labeled with digoxigenin (DIG) in a single RT-PCR reaction using a pair of degenerate primers binding to the same conserved regions in all four tospovirus N genes. The DIG-labeled amplicons were distinguished into species by four parallel hybridizations to species-specific biotinylated probes in streptavidin-coated microtiter wells followed by ELISA detection using a peroxidase-conjugated anti-DIG antibody. Results indicated that the multiplex RT-PCR-ELISA assay could specifically identify each of these four tospoviruses without cross-reactivity between species or reactivity to healthy plant negative controls. Assay sensitivity was 10- to 1000-fold higher than conventional RT-PCR. When applied to naturally infected plants, all samples yielded concordant results between RT-PCR-ELISA and the reference RT-PCR. In conclusion, the multiplex RT-PCR-ELISA developed in this study has superior specificity, sensitivity, and high-throughput capacity compared to conventional RT-PCR and is an attractive alternative for the identification of different tospovirus species.

Multiplex detection of plant pathogens using a microsphere immunoassay technology.

Plant pathogens are a serious problem for seed export, plant disease control and plant quarantine. Rapid and accurate screening tests are urgently required to protect and prevent plant diseases spreading worldwide. A novel multiplex detection method was developed based on microsphere immunoassays to simultaneously detect four important plant pathogens: a fruit blotch bacterium Acidovorax avenae subsp. citrulli (Aac), chilli vein-banding mottle virus (CVbMV, potyvirus), watermelon silver mottle virus (WSMoV, tospovirus serogroup IV) and melon yellow spot virus (MYSV, tospovirus). An antibody for each plant pathogen was linked on a fluorescence-coded magnetic microsphere set which was used to capture corresponding pathogen. The presence of pathogens was detected by R-phycoerythrin (RPE)-labeled antibodies specific to the pathogens. The assay conditions were optimized by identifying appropriate antibody pairs, blocking buffer, concentration of RPE-labeled antibodies and assay time. Once conditions were optimized, the assay was able to detect all four plant pathogens precisely and accurately with substantially higher sensitivity than enzyme-linked immunosorbent assay (ELISA) when spiked in buffer and in healthy watermelon leaf extract. The assay time of the microsphere immunoassay (1 hour) was much shorter than that of ELISA (4 hours). This system was also shown to be capable of detecting the pathogens in naturally infected plant samples and is a major advancement in plant pathogen detection.

Comparison of techniques to screen and characterize bacteria-specific hybridomas for high-quality monoclonal antibodies selection.

Antibodies are very important materials for diagnostics. A rapid and simple hybridoma screening method will help in delivering specific monoclonal antibodies. In this study, we systematically developed the first antibody array to screen for bacteria-specific monoclonal antibodies using Listeria monocytogenes as a bacteria model. The antibody array was developed to expedite the hybridoma screening process by printing hybridoma supernatants on a glass slide coated with an antigen of interest. This screening method is based on the binding ability of supernatants to the coated antigen. The bound supernatants were detected by a fluorescently labeled anti-mouse immunoglobulin. Conditions (slide types, coating, spotting, and blocking buffers) for antibody array construction were optimized. To demonstrate its usefulness, antibody array was used to screen a sample set of 96 hybridoma supernatants in comparison to ELISA. Most of the positive results identified by ELISA and antibody array methods were in agreement except for those with low signals that were undetectable by antibody array. Hybridoma supernatants were further characterized with surface plasmon resonance to obtain additional data on the characteristics of each selected clone. While the antibody array was slightly less sensitive than ELISA, a much faster and lower cost procedure to screen clones against multiple antigens has been demonstrated.

Novel and Highly Specific Monoclonal Antibody to Acidovorax citrulli and Development of ELISA-Based Detection in Cucurbit Leaves and Seed.

A novel monoclonal antibody (MAb) specific to the seedborne bacterium Acidovorax citrulli was produced. MAb 11E5 reacted specifically with 19 strains of A. citrulli but not with three closely related bacteria in the family Comamonadaceae (i.e., A. facilis, Comamonas acidovorans, and C. testosteroni) and another seven phytopathogenic bacteria. Moreover, this MAb detected a strain of A. citrulli that was not detected by a commercial enzyme-linked immunosorbent assay (ELISA)-based kit and a commercial immunochromatographic strip test. In Western blot analysis, MAb 11E5 reacted with an A. citrulli protein of a molecular mass >170 kDa. MAb 11E5 was employed to develop two sandwich ELISA systems: MAb captured-sandwich ELISA (MC-sELISA) and polyclonal antibody captured-sandwich ELISA (PC-sELISA). MC-sELISA was 10 times more sensitive than PC-sELISA for detection of A. citrulli in cucurbit leaf and seed extracts. The detection limit of the MC-sELISA was 5 × 104 CFU/ml. Detection of A. citrulli in naturally infected cucurbit leaves, fruit, and seed was also feasible using MC-sELISA. The newly established MCsELISA provides another alternative for specific detection of A. citrulli in cucurbits and can be applied for routine field inspection.

Tomato necrotic ringspot virus, a new tospovirus isolated in Thailand.

A new tospovirus isolated from naturally infected tomato plants grown in Nakhon Pathom province (Thailand) was characterized. Infected plants showed symptoms consisting of necrotic spots, necrotic ringspots and stem necrosis. This virus was detected using general antibodies that could recognize watermelon silver mottle virus (WSMoV), capsicum chlorosis virus (CaCV) and melon yellow spot virus (MYSV). However, it did not react with specific monoclonal antibodies (MAbs) to WSMoV and CaCV or a specific MAb to MYSV. The complete nucleotide sequences of S and M RNAs of the virus were determined. They were 3,023 and 4,716 nucleotides in length, respectively, and contained two ORFs in an ambisense arrangement. Sequence analysis indicated that amino acid sequence of the N protein shared 58.2%, 56.0% and 51.8% identity with those of CaCV, WSMoV and MYSV, respectively. The virus was experimentally transmitted by Thrips palmi and Ceratothripoides claratris. Based on our results, we conclude that this tospovirus isolate should be considered a member of a new species. The name tomato necrotic ringspot virus (TNRV) is proposed for this tospovirus.

Development of surface plasmon resonance imaging for detection of Acidovorax avenae subsp. citrulli (Aac) using specific monoclonal antibody.

An immunosensor based on surface plasmon resonance imaging (SPR imaging) using a specific monoclonal antibody 11E5 (MAb 11E5) was developed for the detection of the seed-borne bacterium Acidovorax avenae subsp. citrulli (Aac), which causes fruit blotch in watermelons and cantaloupes, and compared to the conventional ELISA technique. The 1:40 mixed self-assembled monolayer (mixed SAM) surface was used for the immobilized MAb 11E5 on sensor surface for the detection of Aac. Both whole cells and broken cells of Aac were tested by using direct and sandwich detection assay. The limit of detection (LOD) of Aac using the SPR imaging technique and a direct detection assay was 10(6)cfu/ml and a subsequent amplification of the SPR signal using a polyclonal antibody (PAb) lowered the LOD to 5×10(5) cfu/ml. The LOD for the ELISA technique was 5×10(4) cfu/ml for the detection of Aac, which was slightly better than that for the SPR technique. However, the sensor surface based on SPR imaging offered a major advantage in terms of surface regeneration, allowing at least five cycles with a shorter time assay, multi-channel analysis with an application on multiplex detection, and an ease of the surface usage for the detection of Aac in the naturally infected plant. The surface was tested against the naturally infected sample and showed good selectivity toward the Aac bacteria.

Development of antibody array for simultaneous detection of foodborne pathogens.

Pathogenic bacterial contaminations present serious problems for food industry and public health. Rapid, accurate and affordable assays are needed. In this study, antibody arrays to simultaneously detect two foodborne pathogenic bacteria (Escherichia coli O157:H7 and Salmonella spp.) have been developed using chemiluminescent detecting system. Solid supports using nitrocellulose membrane and poly-l-lysine (PLL) glass slide were compared and optimized for antibody array construction. Many parameters including optimal concentrations of antibodies, blocking reagents, assay time, storage time, sensitivity and cross-reactivity were considered during optimization. This study revealed that the PLL slide was a more suitable support due to highly accurate results and the absence of non-specific background. Phosphate-buffered saline (PBS, pH 7.2) and 3% skim milk in PBS buffer were optimal spotting and blocking reagents, respectively. With the same sensitivity for bacterial detection as in a conventional ELISA (10(5)-10(6)CFU/ml for the E. coli O157:H7 and 10(6)-10(7)CFU/ml for Salmonella detections), this antibody array has advantages of a much shorter assay time of 1h and much lower required amounts of antibodies. Moreover, there was no cross-reactivity in the detection among bacteria tested in this study. Bacteria detection in food sample was feasible as demonstrated using bacteria-added milk.

Biological and molecular characterization of tospoviruses in Thailand.

Twenty-eight isolates of tospoviruses associated with tomato, pepper, cucurbits, peanut, and Physalis plants collected from fields in different regions of Thailand were characterized. On the basis of N gene and protein sequence relationships, three tospoviruses were identified, namely Watermelon silver mottle virus (WSMoV), Capsicum chlorosis virus (CaCV), and Melon yellow spot virus (MYSV). CLUSTAL analysis of selected N protein sequences showed different isolates of CaCV in three distinct clades. Based on necrosis symptoms on tomato and their 93% identity to CaCV isolates in the other two clades, CaCV-TD8, CaCV-AIT and CaCV-KS16-Thailand tomato tospovirus were designated as CaCV-tomato necrosis strain. A phylogenetic tree based on the 413-amino-acid Gc fragment of the CaCV-Pkk isolate supported the existence of three distinct CaCV clades. Vigna unguiculata produced concentric rings useful for discriminating the Thai CaCV peanut isolates from tomato or pepper isolates. By using reverse transcription polymerase chain reaction with species-specific primers, the three tospoviruses could be detected in mixed infections in watermelon and Physalis, as well as in the bodies of thrips vectors, Thrips palmi and Scirtothrips dorsalis, collected from fields.