A robust dry reagent lateral flow assay for diagnosis of active schistosomiasis by detection of Schistosoma circulating anodic antigen

An earlier reported laboratory assay, performed in The Netherlands, to diagnose Schistosoma infections by detection of the parasite antigen CAA in serum was converted to a more user-friendly format with dry reagents. The improved assay requires less equipment and allows storage and worldwide shipping at ambient temperature. Evaluation of the new assay format was carried out by local staff at Ampath Laboratories, South Africa. The lateral flow (LF) based assay utilized fluorescent ultrasensitive up-converting phosphor (UCP) reporter particles, to be read by a portable reader (UPlink) that was also provided to the laboratory. Over a period of 18 months, about 2000 clinical samples were analyzed prospectively in parallel with a routinely carried out CAA–ELISA. LF test results and ELISA data correlated very well at CAA concentrations above 300 pg/mL serum. At lower concentrations the UCP–LF test indicates a better performance than the ELISA. The UCP–LF strips can be stored as a permanent record as the UCP label does not fade. At the end of the 18 months testing period, LF strips were shipped back to The Netherlands where scan results obtained in South Africa were validated with different UCP scanning equipment including a novel, custom developed, small lightweight UCP strip reader (UCP-Quant), well suited for testing in low resource settings.     

上转磷光技术在快速生物分析中的应用

上转磷光技术 (UPT)是基于上转磷光材料 (UCP)而发展起来的一种新型标记技术。UCP是由几种稀土金属元素掺杂于某些晶体的晶格中构成的纳米级颗粒。由于独特的结构 ,UCP可由红外光激发而发射可见光———上转磷光。这种特殊性质使UCP作为标记物在生物分析中 ,以无本底干扰、无淬灭、适于多重分析和定量分析等显著优势 ,从本质上有别于传统标记物。UCP将在快速免疫分析、微点阵、高通量药物筛选、基因组学研究、外科手术组织成像、食品环境检测以及生化战防御等多方面得到广泛地应用。     

Lateral-flow and up-converting phosphor reporters to detect single-stranded nucleic acids in a sandwich-hybridization assay

Up-converting Phosphor Technology (UPT) particles were used as reporters in lateral-flow (LF) assays to detect single-stranded nucleic acids. The 400-nm phosphor particles exhibit strong visible luminescence upon excitation with infrared (IR) light resulting in the total absence of background autofluorescence from other biological compounds. A sandwich-type hybridization assay was applied using two sequence-specific oligonucleotides. One of the oligonucleotides probes was covalently bound to the UPT particle (reporter) for direct labeling and detection, whereas the second oligonucleotide probe contained biotin for capture by avidin during LF. The whole procedure of hybridization, UPT-LF detection, and analysis required a minimum time of 20 min. Moreover, aiming at minimal equipment demands, the hybridization conditions were chosen such that the entire assay could be performed at ambient temperature. During lateral flow, only targets hybridized to both capture and detection oligonucleotide were trapped and detected at an avidin capture line on the LF strip. Analysis (IR scanning) of the strips was performed in an adapted microtiter plate reader provided with a 980-nm IR laser for excitation of the phosphor particles (a portable reader was also available). Visible luminescence was measured and presented as relative fluorescence units (RFU) allowing convenient quantitation of the phosphor signal. With the assay described here as little as 0.1 fmol of a specific single-stranded nucleic acid target was detected in a background of 10 microg fish sperm DNA.     

Highly Efficient Immunodiagnosis of Episomal Banana streak MY virus Using Polyclonal Antibodies Raised Against Recombinant Viral‐Associated Protein

Banana streak MY virus (BSMYV) is the causal agent of viral leaf streak disease of banana, which leads to considerable losses in banana production in most of the banana‐growing regions worldwide. Developing high‐throughput virus detection system is essential for managing viral diseases especially in vegetatively propagated crops like banana. In this study, viral‐associated protein (VAP) coded by ORF II of BSMYV was expressed in Escherichia coli, and polyclonal antibodies were raised against purified recombinant VAP (rVAP) fusion protein in rabbits. Specificity and sensitivity of resulting antibodies were tested in Western blot, immunosorbent electron microscopy (ISEM) and enzyme‐linked immunosorbent assays (ELISAs). In direct antigen‐coated (DAC)‐ELISA, antibodies reacted specifically to BSMYV in crude sap, up to 1 : 8000 dilutions, but not to healthy leaf extracts. Using this antiserum, an immunocapture polymerase chain reaction (IC‐PCR) assay was developed and compared with DAC‐ELISA. VAP antibody‐based IC‐PCR is highly specific and could differentiate episomal virus infection from the integrated endogenous BSV (eBSV) sequences. The recombinant antibodies were validated by testing with a large number of banana germplasm conserved in the field gene bank. Field samples collected during surveys and mother cultures used in tissue culture propagation suggest that antibodies generated against rVAP are sensitive and useful for large‐scale detection of BSMYV. To the best of our knowledge, this is the first report on the production of polyclonal antiserum against recombinant VAP of BSMYV and its suitability for serology‐based testing by ELISA and IC‐PCR. This VAP‐based immunodiagnosis can be applied in quarantine, germplasm exchange and certification programmes.     

Feasibility of a Lateral Flow Test for Neurocysticercosis Using Novel Up-Converting Nanomaterials and a Lightweight Strip Analyzer

Neurocysticercosis is a frequent parasitic infection of the human brain, occurring in most of the world, and requires imaging of the brain to diagnose. To determine the burden of disease and to simplify diagnosis, a field-friendly rapid lateral flow (LF) based antibody screening test was developed. The assay utilizes novel nano-sized up-converting phosphor (UCP) reporter particles in combination with a portable lightweight analyzer and detects antibodies in serum samples reactive with bacterial-expressed recombinant (r) T24H, a marker for detecting neurocysticercosis cases. Three sequential flow steps allow enrichment of antibodies on the Test (T) line and consecutive binding of protein-A coated UCP reporter particles. Antibody binding was determined by measuring 550 nm emission after excitation of the UCP label with a 980 nm infrared (IR) diode. Clinical sensitivity and specificity of the assay to detect cases of human neurocysticercosis with 2 or more viable brain cysts were 96% and 98%, respectively, using a sample set comprised of sera from 63 confirmed cases and 170 healthy parasite-naïve non-endemic controls. In conclusion: Proof-of-principle, of a rapid UCP-LF screening assay for neurocysticercosis was demonstrated. The assay utilized bacterial-expressed rT24H as a potential alternative for baculovirus-expressed rT24H. Performance of the UCP-LF assay was excellent, although further studies need to confirm that bacterial expressed antigen can entirely replace previously used baculovirus antigen. In addition, the increasing availability of commercial sources for UCP reporter materials as well as the accessibility of affordable semi-handheld scanners may allow UCP-based bioanalytical systems for point-of-care to evolve at an even faster pace.     

A Cocktail ELISA Semi‐nested RT‐PCR Assay to Detect Bean pod mottle virus in Soya bean Seeds

Bean pod mottle virus (BPMV) has been identified as an important pathogen for plant quarantine in China because large quantities of soya bean seeds (approximately 7 × 10⁷ tons) are imported annually. To develop a practical detection programme for BPMV, a cocktail enzyme‐linked immunosorbent assay (ELISA) nested RT‐PCR using a combination of serological and molecular methods was designed for soya bean seeds. The single‐vessel detection assay was performed in a 96‐well ELISA plate, which served as a carrier for the subsequent nested RT‐PCR assay. Assay specificity was demonstrated by the production of the expected 330‐ and 296‐bp bands using the external and internal primers, respectively. This method was 10⁴‐fold more sensitive than immunocapture‐RT‐PCR (IC‐RT‐PCR). In particular, it is important to note that this assay resulted in successful micro‐extraction from soya bean seeds and combined the advantages of each individual technique. The cocktail ELISA nested RT‐PCR is a specific, sensitive, rapid and economical procedure to rapidly identify and characterize BPMV and could be suitable for both primary‐level platforms and laboratories.     

Down‐regulation of miR‐10a‐5p in synoviocytes contributes to TBX5‐controlled joint inflammation

MicroRNAs are considered to play critical roles in the pathogenesis of human inflammatory arthritis, including rheumatoid arthritis (RA). The purpose of this study was to determine the relationship between miR‐10a‐5p and TBX5 in synoviocytes and evaluate their contribution to joint inflammation. The expression of miR‐10a‐5p and TBX5 in the synovium of RA and human synovial sarcoma cell line SW982 stimulated by IL‐1β was determined by RT‐qPCR and Western blotting. The direct interaction between miR‐10a‐5p and TBX5 3′UTR was determined by dual‐luciferase reporter assay in HeLa cells. Mimics and inhibitors of miR‐10a‐5p were transfected into SW982 cells. TBX5 was overexpressed by plasmid transfection or knocked down by RNAi. Proinflammatory cytokines and TLR3 and MMP13 expressions were determined by RT‐qPCR and Western blotting. Down‐regulated expression of miR‐10a‐5p and up‐regulation of TBX5 in human patients with RA were found compared to patients with OA. IL‐1β could reduce miR‐10a‐5p and increase TBX5 expression in SW982 cells in vitro. The direct target relationship between miR‐10a‐5p and 3′UTR of TBX5 was confirmed by luciferase reporter assay. Alterations of miR‐10‐5p after transfection with its mimic and inhibitor caused the related depression and re‐expression of TBX5 and inflammatory factors in SW982 cells. Overexpression of TBX5 after pCMV3‐TBX5 plasmid transfection significantly promoted the production of TLR3, MMP13 and various inflammatory cytokines, while this effect was rescued after knocking down of TBX5 with its specific siRNA. We conclude that miR‐10a‐5p in a relation with TBX5 regulates joint inflammation in arthritis, which would serve as a diagnostic and therapeutic target for RA treatment.     

Genetic fusion protein 3×STa‐ovalbumin is an effective coating antigen in ELISA to titrate anti‐STa antibodies

Heat‐stable toxin type I (STa)‐ovalbumin chemical conjugates are currently used as the only coating antigen in ELISA to titrate anti‐STa antibodies for ETEC vaccine candidates. STa‐ovalbumin chemical conjugation requires STa toxin purification, a process that can be carried out by only a couple of laboratories and often with a low yield. Alternative ELISA coating antigens are needed for anti‐STa antibody titration for ETEC vaccine development. In the present study, we genetically fused STa toxin gene (three copies) to a modified chicken ovalbumin gene for genetic fusion 3×STa‐ovalbumin, and examined application of this fusion protein as an alternative coating antigen of anti‐STa antibody titration ELISA. Data showed fusion protein 3×STa‐ovalbumin was effectively expressed and extracted, and anti‐STa antibody titration ELISA using this recombinant protein (25 ng per well) or STa‐ovalbumin chemical conjugates (10 ng/well) showed the same levels of sensitivity and specificity. Furthermore, mice immunized with this fusion protein developed anti‐STa antibodies; induced antibodies showed in vitro neutralization activity against STa toxin. These results indicate that recombinant fusion protein 3×STa‐ovalbumin is an effective ELISA coating antigen for anti‐STa antibody titration, enabling a reliable reagent supply to make standardization of STa antibody titration assay feasible and to accelerate ETEC vaccine development.

     

Synthesis and Biological Evaluation of Novel 1‐(4‐(Hydroxy(1‐oxo‐1,3‐dihydro‐2H‐inden‐2‐ylidene)methyl)phenyl)‐3‐phenylurea Derivatives

A series of novel phenylurea containing 2‐benzoylindan‐1‐one derivatives 3a  –  3j were synthesized from the reaction of phenylurea‐substituted acetophenones 1a  –  1j with phthalaldehyde 2 under mild reaction conditions in good yields. All synthesized compounds were characterized by spectroscopic methods. The obtained compounds ( 3a  –  3j ) were evaluated for anticancer activity against HeLa and C6 cell lines. Antiproliferative activity was determined by the BrdU proliferation ELISA assay, 3f and 3g were found to be most active compounds. The compounds were also screened for antimicrobial activity and all compounds showed remarkable activity against used microorganisms.     

Cloning,expression, and identification of anti‐carbofuran single chain Fv gene

Phage display method was used to clone anti‐carbofuran (CBF) single chain Fv (scFv) gene. The heavy chain and light chain variable region genes were amplified by the polymerase chain reaction from the CBF‐specific hybridoma cell lines 5D3 and assembled as a scFv DNA fragment with linker peptide (Gly₄Ser)₃. The scFv DNA fragment was cloned into M13 phagemid vector pCANTAB5E and the anti‐CBF antibody libraries were then constructed. After one round of panning with CBF‐ovalbumin (CBF‐OVA) as a conjugate, antigen‐binding positive recombinant phage clones were successfully selected by enzyme‐linked immunosorbent assay (ELISA). The positive phages were used to infect Escherichia coli HB2151 cells and the expression of the soluble scFv antibodies was then induced by IPTG. The scFv antibody was about 31 kDa by SDS‐PAGE and showed HRP‐anti‐E‐tag antibody‐recognized activity by Western blotting. The indirect competitive ELISA (icELISA) showed that the recombinant scFv antibody could competitively combine with CBF, with the IC₅₀ value of 1.07 ng/mL. The cross reactivity studies showed that the anti‐CBF scFv antibody, similar to the parent monoclonal antibody, poses high specificity to CBF and has little reactivity to the analogs. Taken together, these findings suggest that the recombinant scFv antibody can be used for further developing immunoassay method for CBF. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

口蹄疫病毒非结构蛋白3AB 的原核表达及应用

旨在建立一种检测口蹄疫病毒非结构蛋白抗体的敏感、特异的ELISA方法。克隆、表达了口蹄疫病毒非结构蛋白3AB基因,原核表达的重组蛋白经亲和层析法纯化及Western blotting鉴定后作为包被抗原,建立检测口蹄疫病毒非结构蛋白抗体的3AB间接ELISA方法,通过与商品化试剂盒3ABC-ELISA的比对试验对其进行评价。结果显示,重组蛋白3AB以包涵体形式表达;能与口蹄疫病毒感染血清发生特异性反应,而不能与疫苗免疫动物血清发生反应;在检测田间样品时,与3ABC-ELISA具有同样的特异性和敏感性 (P＞     

Tips on improving the efficiency of electrotransfer of target proteins from Phos‐tag SDS‐PAGE gel

The sensitivity of Western blotting analysis after Phos‐tag SDS‐PAGE is occasionally inferior to that after normal (Phos‐tag‐free) SDS‐PAGE under similar experimental conditions, possibly as a result of inefficient electrotransfer from the Phos‐tag gel to the blotting membrane. We therefore present tips on improving the efficiency of electrotransfer of proteins in semidry and wet‐tank blotting. When model samples containing several standard phosphoproteins were subjected to semidry blotting, their electrotransfer efficiencies after Phos‐tag SDS‐PAGE were markedly inferior to those of their dephosphorylated counterparts in the same gel. This was ameliorated by immersing the electrophoresed Phos‐tag gel in a transfer buffer containing 1 mM EDTA for 30 min before electroblotting. Similarly, phosphoproteomes in crude cell extracts were inefficiently transferred by semidry blotting, but the efficiencies of their electrotransfer were improved by pretreatment with EDTA. In contrast, the efficiencies of wet‐tank blotting of the same samples were not dependent on the degree of phosphorylation, and the efficiencies of electrotransfer of all proteins from Phos‐tag gels were similar to those from normal gels. In some cases involving the use of a Phos‐tag gel, addition of 0.1% w/v of SDS to the transfer buffer significantly improved the electrotransfer.     

Effect of budesonide on TGF‐β1‐enhanced VEGF production by lung fibroblasts

VEGF (vascular endothelial growth factor) is a potent proangiogenic cytokine, and vascular change is one of the characteristic features of airway remodelling. Since the glucocorticoids have shown antifibrosis properties, we sought to investigate whether budesonide, a widely used glucocorticoid in clinical practice, could attenuate TGF‐β1 (transforming growth factor‐β1)‐induced VEGF production by HFL‐1 (human lung fibroblasts). HFL‐1 fibroblasts were treated with various concentrations of budesonide (10^?11 M, 10^?9 M and 10^?7 M) in the absence or presence of TGF‐β1. Postculture media were collected for ELISA of VEGF at the indicated times. The cell lysates were subjected to Western blotting analysis to test TGF‐β1/Smad and MAP (mitogen‐activated protein) kinase signalling activation, respectively. The results suggested that budesonide pretreatment reduced the significant increase of VEGF release induced by TGF‐β1 in HFL‐1 fibroblasts in a dose‐dependent manner, and suppressed the increase of phospho‐Smad3 and phosphor‐ERK (extracellular signal‐regulated kinase) protein levels. In conclusion, budesonide may reduce TGF‐β1‐induced VEGF production in the lung, probably through the Smad/ERK signalling pathway and, thus, may provide new sight into the molecular mechanism underlying glucocorticoid therapy for airway inflammatory diseases.     

Specific and Rapid Detection of Lily symptomless virus and Arabis mosaic virus in Lily by Dual IC‐RT‐PCR

Lily symptomless virus (LSV) and Arabis mosaic virus (ArMV) cause severe losses of quantity and quality of lily flower and bulb production. Specificity, sensitivity and speed of detection methods for viruses need to be improved greatly to prevent LSV and ArMV from spreading from infected lilies. A dual IC‐RT‐PCR procedure for detection was developed in which the antibodies of LSV and ArMV were mixed and the mixture used to coat the PCR tubes. The particles of the two viruses were captured by the respective antibodies. Interference by other RNA viruses in infected lily was eliminated in the RT‐PCR. Also, an RNA extraction step was omitted. The dual IC‐RT‐PCR products of LSV and ArMV were 521 bp and 691 bp, respectively. The specificity of the method was validated; only LSV and ArMV of four viruses were detected by dual IC‐RT‐PCR. The sensitivity of the detection method is 1 mg leaf tissue and higher than DAS‐ELISA due to enrichment by dual immunocapture.     

基于脂蛋白P80的滑液支原体抗体ELISA检测方法的建立及应用

【目的】研究滑液支原体(Mycoplasma synoviae, MS)脂蛋白P80的免疫反应性及其在MS血清抗体ELISA检测中的应用。【方法】对MSP80的氨基酸序列进行生物信息学分析、原核表达和纯化,并用免疫印迹法分析其与6种不同MS分离株阳性血清的免疫反应性以及与其他禽病原血清的交叉反应性;运用纯化的MS P80表达蛋白作为包被抗原建立了MS血清抗体的间接ELISA检测方法,对其敏感性和重复性进行检测;比较检测了与美国爱德士检测试剂盒对50份临床血清样品的阳性符合率。【结果】生物信息学分析预测MS P80蛋白为脂蛋白且含有信号肽,其在MS种内同源性高达98%-100%,与其他种属P80蛋白同源性在25%-34%之间,成功表达和纯化了MS P80重组蛋白(rMS P80);Western blotting分析表明纯化的rMS P80具有良好的免疫反应性和特异性;运用rMS P80建立的MS血清ELISA抗体检测方法可对不同株MS阳性血清进行抗体效价检测,而对其他禽病原阳性血清均无交叉反应性;该检测方法的批内变异系数小于5%,批间变异系数小于10%,重复性良好;与美国IDEXX检测试剂盒比较,本文建立的ELISA抗体检测方法敏感性更高,阳性符合率为75%,阴性符合率为89.47%,总样本符合率为86%。【结论】MS P80具有较好的免疫反应性、种内保守性和种间特异,并且可用作MS抗体检测的靶标抗原。     

Hsa_circ_0079662 induces the resistance mechanism of the chemotherapy drug oxaliplatin through the TNF‐α pathway in human colon cancer

The aim of the study was to research the biological functions of circRNA (hsa_circ_0079662) and its underlying mechanism in colorectal cancer. Drug‐resistant cell lines (HT29‐LOHP, HCT116‐LOHP, HCT8‐LOHP) were separately dealt with oxaliplatin concentration gradient (0.1‐10 μmol/L). Real‐time PCR, Western blotting, dual‐luciferase assay, miRNA pull‐down assay, coimmunoprecipitation and ELASA were performed to explore the mechanism of chemotherapy drug oxaliplatin resistance in CRC. The results showed that the expression of hsa_circ_0079662 was increased in drug‐resistant cell lines by RT‐PCR. The expression of HOXA9, TRIP6, Vcam‐1, VEGFC, MMP3, MMP9 and MMP14 was higher by Western blotting. Interaction between HOXA9 and TRIP6 in CO‐IP detection. Additionally, the cytokines TNF‐α, IL‐1 and IL‐6 were also found. In conclusion, hsa_circ_0079662, as a ceRNA binding with hsa‐mir‐324‐5p, can regulate target gene HOXA9 and induced the mechanism of chemotherapy drug oxaliplatin resistance in CRC through the TNF‐α pathway in human colon cancer.     

Validation of an ELISA for urinary dopamine: applications in monitoring treatment of dopamine‐related disorders

Dopamine is a catecholamine that serves as a neurotransmitter in the central and peripheral nervous system. Non‐invasive, reliable, and high‐throughput techniques for its quantification are needed to assess dysfunctions of the dopaminergic system and monitor therapies. We developed and validated a competitive ELISA for direct determination of dopamine in urine samples. The method provides high specificity, good accuracy, and precision (average inter‐assay variation < 12%). The analysis is not affected by general urinary components and structurally related drugs and metabolites. The correlation between ELISA and LC‐MS/MS analyses was very good (r = 0.986, n = 28). The reference range was 64–261 μg/g Cr (n = 64). Week‐to‐week biological variations of second morning urinary dopamine under free‐living conditions were 23.9% for within‐ and 35.5% for between‐subject variation (n = 10). The assay is applied in monitoring Parkinson's disease patients under different treatments. Urinary dopamine levels significantly increase in a dose‐dependent manner for Parkinson's disease patients under l ‐DOPA treatment. The present ELISA provides a cost‐effective alternative to chromatographic methods to monitor patients receiving dopamine restoring treatment to ensure appropriate dosing and clinical efficacy. The method can be used in pathological research for the assessment of possible peripheral biological markers for disorders related to the dopaminergic system.     

Non‐invasive imaging of allogeneic transplanted skin graft by 131I‐anti‐TLR5 mAb

Although ¹⁸F‐fluorodeoxyglucose (¹⁸F‐FDG) uptake can be used for the non‐invasive detection and monitoring of allograft rejection by activated leucocytes, this non‐specific accumulation is easily impaired by immunosuppressants. Our aim was to evaluate a ¹³¹I‐radiolabelled anti‐Toll‐like receptor 5 (TLR5) mAb for non‐invasive in vivo graft visualization and quantification in allogeneic transplantation mice model, compared with the non‐specific radiotracer ¹⁸F‐FDG under using of immunosuppressant. Labelling, binding, and stability studies were performed. BALB/c mice transplanted with C57BL/6 skin grafts, with or without rapamycin treatment (named as allo‐treated group or allo‐rejection group), were injected with ¹³¹I‐anti‐TLR5 mAb, ¹⁸F‐FDG, or mouse isotype ¹³¹I‐IgG, respectively. Whole‐body phosphor‐autoradiography and ex vivo biodistribution studies were obtained. Whole‐body phosphor‐autoradiography showed ¹³¹I‐anti‐TLR5 mAb uptake into organs that were well perfused with blood at 1 hr and showed clear graft images from 12 hrs onwards. The ¹³¹I‐anti‐TLR5 mAb had significantly higher graft uptake and target‐to‐non‐target ratio in the allo‐treated group, as determined by semi‐quantification of phosphor‐autoradiography images; these results were consistent with ex vivo biodistribution studies. However, high ¹⁸F‐FDG uptake was not observed in the allo‐treated group. The highest allograft‐skin‐to‐native‐skin ratio (A:N) of ¹³¹I‐anti‐TLR5 mAb uptake was significantly higher than the ratio for ¹⁸F‐FDG (7.68 versus 1.16, respectively). ¹³¹I‐anti‐TLR5 mAb uptake in the grafts significantly correlated with TLR5 expression in the allograft area. The accumulation of ¹³¹I‐IgG was comparable in both groups. We conclude that radiolabelled anti‐TLR5 mAb is capable of detecting allograft with high target specificity after treatment with the immunosuppressive drug rapamycin.     

Severe Fever with Thrombocytopenia Syndrome Virus Antigen Detection Using Monoclonal Antibodies to the Nucleocapsid Protein

Background

Severe fever with thrombocytopenia syndrome (SFTS) is a tick-borne infectious disease with a high case fatality rate, and is caused by the SFTS virus (SFTSV). SFTS is endemic to China, South Korea, and Japan. The viral RNA level in sera of patients with SFTS is known to be strongly associated with outcomes. Virological SFTS diagnosis with high sensitivity and specificity are required in disease endemic areas.

Methodology/Principal Findings

We generated novel monoclonal antibodies (MAbs) against the SFTSV nucleocapsid (N) protein and developed a sandwich antigen (Ag)-capture enzyme-linked immunosorbent assay (ELISA) for the detection of N protein of SFTSV using MAb and polyclonal antibody as capture and detection antibodies, respectively. The Ag-capture system was capable of detecting at least 350–1220 TCID₅₀/100 μl/well from the culture supernatants of various SFTSV strains. The efficacy of the Ag-capture ELISA in SFTS diagnosis was evaluated using serum samples collected from patients suspected of having SFTS in Japan. All 24 serum samples (100%) containing high copy numbers of viral RNA (>10⁵ copies/ml) showed a positive reaction in the Ag-capture ELISA, whereas 12 out of 15 serum samples (80%) containing low copy numbers of viral RNA (<10⁵ copies/ml) showed a negative reaction in the Ag-capture ELISA. Among these Ag-capture ELISA-negative 12 samples, 9 (75%) were positive for IgG antibodies against SFTSV.

Conclusions

The newly developed Ag-capture ELISA is useful for SFTS diagnosis in acute phase patients with high levels of viremia.