Double-antibody Sandwich Enzyme-linked Immunosorbent Assay Research Articles

First Report of Mosaic Disease of Crape Myrtle Caused by Watermelon Mosaic Virus in Jiangsu Province in China

The crape myrtle ( Lagerstroemia indica) is a deciduous shrub that has become a popular landscape tree on public and private properties. However, crape myrtle is constantly subjected to multiple pathogen attacks, which severely affect the yield and quality and cause economic losses. In June 2019, foliar virus-like symptoms of chlorosis and mosaic were observed on crape myrtle in Yangzhou city, Jiangsu Province, East China. Leaf samples were collected from symptomatic plants. All samples were assayed by western blotting using a polyclonal antiserum to cucumber mosaic virus (CMV) and tobacco mosaic virus (TMV) and a double-antibody sandwich-ELISA assay for watermelon mosaic virus (WMV). The results revealed that all samples reacted positively with the WMV but negatively with CMV and TMV. RT-PCR was employed to confirm the obtained serological results. An amplicon of 288 bp was obtained from all the samples. Nucleotide BLAST analysis revealed that the sequence had 98.25 to 100% nucleotide identity to eight WMV isolate sequences available in GenBank. A pathogenicity assay was conducted using virus-free Nicotiana benthamiana seedlings inoculated with sap extracted from symptomatic crape myrtle leaves. Systemic infection of WMV was also confirmed in N. benthamiana by RT-PCR. On the basis of the data from serological and molecular analyses, the virus was identified as WMV. To our knowledge, this is the first report of WMV on crape myrtle in Jiangsu Province in China.

Determination of Some Virus Diseases on Gerbera Plants in Antalya Province

Gerbera spp., which is in the Asteraceae family, is the most widely cultivated cut flower ornamental plant in Türkiye after carnation. Approximately 74% of Türkiye's production is met from Antalya province. Virus diseases are a problem in the cultivation of Gerbera as they significantly reduce the yield and quality. In this study, which was carried out to detect some virus diseases seen in Gerbera production areas in Antalya province, surveys were carried out in commercial production areas in the 2021-2022 production season. For this purpose, a total of 112 leaf samples, which are thought to be infected with viruses, were collected from 100 Gerbera plants showing symptoms similar to virus disease symptoms and 12 selected weeds regardless of the presence of symptoms. These leaf samples, which were later brought to the laboratory, then were tested by the Double Antibody Sandwich Enzyme-linked Immunosorbent Assay (DAS-ELISA) method to detect infections of Tomato spotted wilt virus (TSWV), Cucumber mosaic virus (CMV), Tobacco streak virus (TSV) and Impatiens necrotic spot virus (INSV). As a result of the tests, TSWV (41.9%), CMV (1.7%) and TSV (37.5%) infections were detected in Gerbera plant samples. In addition, TSWV (25%) infections were most common in weed samples, followed by TSV (16%) and CMV (8.3%) infections. The presence of INSV virus was not detected in any of the leaf samples tested by DAS-ELISA. With this study, TSWV, TSV and CMV infections in Gerbera production areas in Türkiye were revealed for the first time.

Konya İli Ereğli İlçesi Domates Ekim Alanlarında Domateste Yaygın Olarak Bulunan Üç Virüsün Serolojik Tespiti

In this study, it was aimed to reveal some viruses that are a problem in tomato production areas in the Ereğli district of Konya, where tomato production is common. For this reason, in the 2019 production season, leaf samples were taken from 160 tomato plants showing symptoms similar to virus disease symptoms such as mosaic, deformations, yellowing, blight on the leaves and stunted plants. Possible infections of Cucumber mosaic virus (CMV), Tomato spotted-wilt virus (TSWV) and Tomato mosaic virus (ToMV) on these leaves were tested by serological-based DAS-ELISA (Double Antibody Sandwich- Enzyme Linked Immuno Sorbent Assay) method. According to DAS-ELISA results, it was determined that 66.25% of the samples were infected with at least one of these three viruses. In addition, TSWV was detected as the most common virus with a 48.75% relative infection rate in the samples, followed by ToMV (28.75%) and CMV (26.25%). Mixed infections of the viruses were also detected in the leaf samples tested. CMV + TSWV double infection was detected in 22 samples, ToMV + TSWV double infection was detected in 14 samples, and CMV + ToMV + TSWV triple infection was detected in 12 plant samples.

Konya İli Park ve Ev Bahçelerinde Peyzaj Amaçlı Yetiştirilen Dahlia Türlerinde Enfeksiyon Yapan Bazı Virüs Hastalıklarının Serolojik Yöntemlerle Belirlenmesi

In this study, it was aimed to reveal some viruses that are a problem in Dahlia plants grown for ornamental plants in parks and home gardens in Selçuklu, Karatay and Meram districts of Konya. For this reason, in the 2019 summer, leaf samples were taken from 152 Dahlia plants showing symptoms similar to virus disease symptoms such as mosaic, deformations, yellowing, blight on the leaves and stunted plants. Possible infections of Cucumber mosaic virus (CMV), Tomato spotted-wilt virus (TSWV) and Impatiens necrotic spot virus, (INSV) on these leaves were tested by serological-based DAS-ELISA (Double Antibody Sandwich- Enzyme Linked Immuno Sorbent Assay) method. According to DAS-ELISA results, it was determined that 46.71% of the samples were infected with at least one of these three viruses. In addition, TSWV was detected as the most common virus with a 38.82% relative incidence in the samples, followed by CMV (27.63%). Mixed infections of the viruses were also detected in the leaf samples tested. As a result of the tests, only CMV + TSWV double infection was detected in a total of 30 samples. On the other hand, INSV infection was not detected in any of the leaf samples tested. As a result of survey studies, the average of prevalence rate of virus diseases in Dahlia plants grown in Konya was calculated as 69.56%. Moreover, it was determined that virus diseases were most common in Selçuklu district (75.47%). This is the first report of CMV and TSWV on Dahlia spp. in Türkiye.

Türkiye'de Biberde Enfeksiyon Oluşturan Tobacco mild green mosaic virus İzolatlarının Belirlenmesi ve Moleküler Karakterizasyonu

In this study, Tobacco mild green mosaic virus (TMGMV) infection was investigated in pepper crops from two regions having high economical importance in Turkey. A total of 397 leaf and fruit samples showing mosaic, yellowing, mottling, and pitting symptoms were collected to test by double antibody sandwich ELISA (DAS-ELISA) using polyclonal antiserum. DAS-ELISA results indicated that 97 out of all tested samples (24.4%) were found to be infected with TMGMV. Samples resulting positive for TMGMV infection were used to amplify the virus coat protein (CP) gene sequences with specific primers by RT-PCR for further molecular characterization of the virus isolates. A comparison of the CP sequences of the virus isolates revealed that the identity of nucleotides ranged between 97.2 and 100%, and the amino acid similarity ranged between 96.8 and 100% among themselves. To the best of our knowledge, this is the first report of TMGMV infection in Capsicum annuum in Turkey.

Development of an ELISA Assay for the Determination of SARS-CoV-2 Protein Subunit Vaccine Antigen Content.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) protein subunit vaccine is one of the mainstream technology platforms for the development of COVID-19 vaccines, and most R&D units use the receptor-binding domain (RBD) or spike (S) protein as the main target antigen. The complexity of vaccine design, sequence, and expression systems makes it urgent to establish common antigen assays to facilitate vaccine development. In this study, we report the development of a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) to determine the antigen content of SARS-CoV-2 protein subunit vaccines based on the United States Pharmacopeia <1220> and ICH (international conference on harmonization) Q14 and Q2 (R2) requirements. A monoclonal antibody (mAb), 20D8, was identified as the detection antibody based on its high RBD binding activity (EC50 = 8.4 ng/mL), broad-spectrum anti-variant neutralizing activity (EC50: 2.7−9.8 ng/mL for pseudovirus and EC50: 9.6−127 ng/mL for authentic virus), good in vivo protection, and a recognized linear RBD epitope (369−379 aa). A porcine anti-RBD polyclonal antibody was selected as the coating antibody. Assay performance met the requirements of the analytical target profile with an accuracy and precision of ≥90% and adequate specificity. Within the specification range of 70−143%, the method capability index was >0.96; the misjudgment probability was <0.39%. The method successfully detected SARS-CoV-2 protein subunit vaccine antigens (RBD or S protein sequences in Alpha, Beta, Gamma, or Delta variants) obtained from five different manufacturers. Thus, we present a new robust, reliable, and general method for measuring the antigenic content of SARS-CoV-2 protein subunit vaccines. In addition to currently marketed and emergency vaccines, it is suitable for vaccines in development containing antigens derived from pre-Omicron mutant strains.

Preoperative and postoperative urinary NGAL levels in children with bladder exstrophy.

Urinary NGAL (neutrophil gelatinase-associated lipocalin) levels have been shown to predict renal damage in various medical conditions. The present study was conducted to study the role of urinary NGAL levels in children with bladder exstrophy-epispadias complex post single-stage total reconstruction (SSTR) as markers of early renal functionreduction. Urine samples were collected from children with bladder exstrophy before SSTR (Group A, n = 11), 5years post SSTR (Group B, n = 40) and controls (Group C, n = 41) and stored at - 20°C. NGAL levels were estimated using double antibody sandwich ELISA. Mean NGAL levels in Groups A, B and C were 1.39, 34.24 and 2.58ng/ml, respectively. Mean NGAL levels among Group B subjects with glomerular filtration rate (GFR) ≥ 80ml/min/1.73m2 body surface area (BSA) was 29.8ng/ml, while it was 31.74ng/ml in those with GFR < 80ml/min. Urine samples were also evaluated 6months post SSTR. Mean NGAL at 6months was 6.76ng/ml, while at 12months it was 30.3ng/ml, remaining > 30ng/ml at 18 and 24months. Dimercaptosuccinic acid (DMSA) scans did not show any scarring, and GFRon diethylenetriamine pentaacetate (DTPA) scans remained stable. Increasing levels of urinary NGAL following bladder-exstrophyandepispadias complex repair suggest that NGAL detects the earliest signs of renal damage even before any deterioration is observed in DMSA and/or DTPA-GFR scans. Further studies with an adequate sample size and periodic measurement of NGAL need to be performed before any definitive conclusion can be drawn.

Hatay ilinde yetişen Cucurbitaceae familyasına ait kültür bitkilerinde bazı virüslerin DAS-ELISA ve RT-PCR yöntemleriyle saptanması ve moleküler karakterizasyonu

Amaç: Bu çalışmada, Hatay ilinde yetiştirilen hıyar, kabak ve kavun bitkilerinde cucurbit yellow stunting disorder virus (CYSDV), cucurbit chlorotic yellows virus (CCYV) ve beet pseudo-yellows virus (BPYV)’lerinin Double Antibody Sandwich-Enzyme Linked Immuno Sorbent Assay (DAS-ELISA) ve Reverse Transcription-Polymerase Chain Reaction (RT-PCR) yöntemleriyle belirlenmesi, elde edilen virüs izolatlarının filogenetik ilişkilerinin ortaya konulması amaçlanmıştır. Yöntem ve Bulgular: Hatay ilinden simptomlu ve simptomsuz 90 adet hıyar, kabak ve kavun örneği toplanmıştır. CCYV’nin varlığını araştırmak için DAS-ELISA ve RT-PCR yöntemleri, CYSDV ve BPYV’lerinin varlığını araştırmak için ise RT-PCR yöntemi kullanılmıştır. Gözlenen en yaygın simptomlar yapraklarda küçülme, deformasyon, yaprak kırışıklıkları, kıvrılmalar ve damar açılmalarıdır. DAS-ELISA testi sonuçlarına göre testlenen örneklerde CCYC bulunamamıştır. RT-PCR analizleri sonucunda testlenen örneklerde BPYV ve CCYV bulunamamış ancak CYSDV’nin heat shock protein 70h (HSP70h) genini çoğaltan 364 bp büyüklüğünde PCR amplikonları 11 örnekte (9 kabak, 2 kavun) elde edilmiştir. Bu ürünlerden 10 tanesinin doğrudan iki yönlü sekans analizi sonucunda, elde edilen nükleotit dizilerinin gen bankasında kayıtlı CYSDV izolatları ile yüksek oranda (%99) benzerlik gösterdiği saptanmıştır. Bu çalışma ile CYSDV ilk defa Hatay ilinde kabak ve kavun bitkilerinde rapor edilmiştir. Genel Yorum: Hatay ili kabak ve kavun alanlarından toplanan örneklerde CYSDV ilk kez tespit edilmiştir. Testlenen örneklerinin hiçbirinde BPYV ve CCYV belirlenememiştir. Testlenen örneklerin %12.22 oranında CYSDV ile enfekteli bulunması, sağlıklı bitki materyallerinin kullanılmasının önemini göstermektedir. Çalışmanın Önemi ve Etkisi: Hatay ilinde ilk kez bu çalışma ile kabak ve kavun bitkilerinde CYSDV’ünün varlığı, RT-PCR analizleri ile kanıtlanmıştır.

Inhibitory Effects of Pepper Mild Mottle Virus Infection by Supernatants of Five Bacterial Cultures in Capsicum annuum L.

Pepper mild mottle virus (PMMoV), one of the most prevalent viruses in chili pepper (Capsicum annuum L.) is a non-enveloped, rod-shaped, single-stranded positive-sense RNA virus classified in the genus Tobamovirus. The supernatants of five bacterial cultures (Pseudomonas putida [PP], Bacillus licheniformis [BLI], P. fluorescens [PF], Serratia marcescens [SER], and B. amyloliquifaciens [BA]) were analyzed to find novel antiviral agents to PMMoV in chili pepper. Foliar spraying with supernatants (1:1, v/v) obtained from Luria-Bertani broth cultures of PP, BLI, PF, SER, and BA inhibited PMMoV infection of chili pepper if applied before the PMMoV inoculation. Double-antibody sandwich enzyme-linked immunosorbent assay showed that treatments of five supernatants resulted in 51-66% reductions in PMMoV accumulation in the treated chili pepper. To identify key compounds in supernatants of PP, BLI, PF, SER, and BA, the supernatants were subjected to gas chromatography-mass spectrometry. The 24 different types of compounds were identified from the supernatants of PP, BLI, PF, SER, and BA. The compounds vary from supernatants of one bacterial culture to another which includes simple compounds-alkanes, ketones, alcohols, and an aromatic ring containing compounds. The compounds triggered the inhibitory effect on PMMoV propagation in chili pepper plants. In conclusion, the cultures could be used to further conduct tissue culture and field trial experiments as potential bio-control agents.

A sensitive double antibodies sandwich ELISA for the diagnosis and therapeutic evaluation of cervical cancer

BackgroundVascular endothelial growth factor (VEGF) is a highly specific factor for tumors growth. However, the study on the mechanism of VEGF in cervical cancer, and the correlation between the expression level of VEGF and the therapeutic evaluation, prognosis of cervical cancer is not clear till now. MethodsIn this study, RT-qPCR and IHC were used to evaluate the abnormal expression of VEGF in cervical cancer. The survival plots of the VEGF expression related to OS were observed by using the KM plotter. The mAbs against VEGF were screened and identified by ELISA addicted test, indirect ELISA, Western-blot, and dot-ELISA. We designed and prepared the overlapping truncations (V1, V2, V3) of VEGF to identify the B cell epitopes. Then, the epitopes recognized by anti-VEGF mAbs were mapped and displayed on a 3D structure of VEGF by using the PyMOL software. The highly specific and sensitive sandwich ELISA was established to detect the total VEGF quantification in 206 clinical sera samples, thus to evaluate the changes of VEGF before and after chemoradiotherapy in cervical cancer patients. ResultsThe VEGF was high expressed in cervical cancer tissues and cells, resulting a poor prognosis of cervical cancer. The mAbs 2E5 and 6D9 were selected with the titer of 1:256000 and 1:128000 respectively. The mAbs both had strong ability to combine with VEGF protein within 15 min and were identified as subclass IgG1 with κ-type light chains. 2E5 bound to V1 and V2, recognizing the N-terminal (1–121 aa) of VEGF, however 6D9 bound to V3, recognizing the C-terminal (116–174 aa) of VEGF. The 206 clinical samples were tested with the established VEGF-DAS-ELISA and calculated according to the equation (y = 0.0042088× + 0.105109, R2 = 0.998). The results indicated that the expression levels of VEGF in cervical cancer samples were positively higher than those in normal samples. Importantly, we found the expression level of sera VEGF in cervical cancer patients decreased significantly after chemoradiotherapy. Therefore, the variable of VEGF levels in cervical cancer patients before and after treatment can be used as a new indicator of efficacy evaluation to guide the clinical treatment of cervical cancer. ConclusionA sensitive DAS-ELISA was established successfully, using which we can track the VEGF to evaluate the efficacy and estimate prognosis of cervical cancer. It is helpful for the diagnosis, therapeutic evaluation and prognosis of cervical cancer.

Effect of Chitosan Nanoparticles (CS-NPs) on In Vitro Regeneration Response and Production of Potato virus Y (PVY)-Free Plants of Potato

Potato virus Y (PVY) causes serious loss in the yield and quality of potatoes. The effect of chitosan nanoparticles (CS-NPs) on the regeneration response and production of PVY-free plants under in vitro conditions was studied. Murashige and Skoog (MS) medium supplemented with 0.1 mg L−1 Gibberellic acid (GA3), 0.1 mg L−1 Naphthalene acetic acid (NAA) and 500 mg L−1 malt extract was used for regeneration of plantlets from sprouts. Double Antibody Sandwich Enzyme-Linked Immunosorbent Assay (DAS-ELISA) and Reverse Transcript Polymerase Chain Reaction (RT-PCR) was used for virus indexing of the mother plant and in vitro-regenerated plantlets. Explants of PVY positive potato plants were cultured on same medium amended with 100, 200, 250, and 300 mg L−1 of (CS-NPs). Shoot regeneration decreased from 100 to 200 mg L−1 as the concentrations of antiviral (CS-NPs) up to 250 mg L−1. It was decreased with the increase in the concentration of the antiviral (CS-NPs) up to 300 mg L−1. Antiviral (CS-NPs) at the concentration of 250 mg L−1 showed a positive effect on shoot regeneration. In vitro-regenerated plantlets were virus free and tested negative in both ELISA and RT-PCR. The level of 100 mg L−1 of (CS_NPs) produced 38.8% PVY-free plants and 30.50% of cultures showed shoot regeneration. The level of 200 mg L−1 of (CS-NPs) produced 49.6% PVY-free plants while 46.8% of cultures showed shoot regeneration. The level of 250 mg L−1 of (CS-NPs) was the most effective and produced 100.0% PVY-free plants and 100.0% of cultures showed shoot regeneration. Histopathological changes simultaneously with elucidation of resistance and growth enhancement were evident in the treated plants with (CS-NPs) than those untreated control. In conclusion, (CS-NPs) treatment is an effective tool to produce PVY-free sprouts explants and has potential for producing virus-free planting material for the potato industry.

Chitosan-functionalized graphene oxide as adjuvant in HEV P239 vaccine

Searching appropriate adjuvants for vaccine is a potent method to intense the immune efficacy. In the present study, we developed a novel Hepatitis E virus (HEV) vaccine by utilizing chitosan modified nano-graphene oxide (GO-CS) as an adjuvant to support HEV antigen P239 protein (GO/CS/P239). The characterization of GO/CS/P239 was observed by atomic force microscope. The safety of GO/CS/P239 was measured by CCK-8 method, hemolysis test and acute challenge test. The anti-HEV titers and cytokines production were analyzed by double antibody sandwich ELISA. As the results showed, by contrast with a vaccine that contained only the P239 protein, GO/CS/P239 vaccine can promote immune cells to produce more IgG antibodies and cytokines, which were able to stimulate the organism to produce stronger both cellular and humoral immunity. Collectively, GO/CS/P239 particles have been demonstrated to be safe both in vitro and in vivo, and can facilitate sufficient immune response to protect organisms from virus infection, which suggested that our exploration offers a promising alternative vaccine that can control HEV infection.

First Report of Bacterial Leaf Scald of Plum Caused by Xylella fastidiosa in Texas.

Xylella fastidiosa is the etiological agent of Plum Leaf Scald (Greco et al. 2021). The disease was first reported in Argentina (Fernandez-Valiela et al. 1954) and then Brazil and Paraguay (French et al. 1978). In the USA, Plum Leaf Scald has been reported in the Southeastern United States (Wells et al. 1981a) and California (Hernandez-Martinez et al. 2009). In August 2021, during the Stone Fruit Survey of FY2020, plum trees (Mexican variety, Prunus mexicana) with symptoms of leaf scald, were observed in a Central Texas orchard with approximately 7% of trees exhibiting symptoms. Leaf margins were asymmetrically scorched, with necrotic areas that transitioned into chlorotic and healthy green tissues. To detect the presence of the pathogen, leaf sample petioles were tested using a double-antibody sandwich (DAS) ELISA® with X. fastidiosa specific antiserum (Agdia Inc., Elkhart, IN) according to manufacturer's guidelines. X. fastidiosa was detected in 20 of the 35 symptomatic samples. To confirm ELISA results, total DNA was extracted from the plant samples using the Plant DNeasy® kit (Qiagen Co. Hilden, Germany) following the manufacturer's protocol. All 20 ELISA-positive samples tested positive in a X. fastidiosa-specific real time PCR assay, using the primers XF1F and XF1R and probe XF1p (Schaad et al. 2002). Moreover, the ELISA-negative samples were also negative for PCR assay. Symptomatic samples were used to isolate the pathogen. Samples were debarked, surface-sterilized and xylem fluid collected. The fluid was gently imprinted on buffered charcoal yeast extract (BCYE) (Wells et al. 1981b) or periwinkle wilt modified (PWM) agar plates (Summer et al. 2010). After 10 days of incubation, individual colonies were observed. The colonies were slightly convex, white, opalescent, mucoid, circular with entire margins and with smooth surfaces on both media plates. Isolated colonies were triple-streak single colony purified and archived. Genomic DNA was extracted from four purified isolates using the DNeasy Blood and Tissue Qiagen® Kit, to conduct conventional PCR using HL5/HL6 (Francis et al. 2006), which identified the isolates as X. fastidiosa. Using the 16S rRNA primer pair U3/U4 (James 2010), amplicons were sequenced and compared against the NCBI database using the BLASTn algorithm. Comparative sequence analysis of amplicons from the four isolates were identical and indicated that the isolates were 100% identical to X. fastidiosa subsp. multiplex RIV5 (CP064326.1) from cherry plum, and IVIA5901 (CP047134.1) from almond. The sequences of all four isolates were deposited into NCBI GenBank, with the accession numbers OM617940 (467), OM617941 (470), OM617942 (471) and OM617943 (468). To our knowledge, this is the first report of X. fastidiosa associated with plum leaf scald in Texas, extending the geographical range of this important bacterial disease, in the Southern United States. This study highlights the importance of routine scouting of agricultural settings with a view to assessing and detecting early threats from either pests or disease and implementing relevant management strategies.

Preparation of monoclonal antibodies against Pinellia ternata lectin protein and establishment of double-antibody sandwich ELISA

To determine the content of endogenous toxic substance Pinellia ternata lectin(PTL) protein in Pinelliae Rhizoma and the related processed products, this study prepared specific monoclonal antibodies against PTL by hybridoma cell technology, and established a quantitative double-antibody sandwich enzyme linked immunosorbent assay(ELISA) for PTL antigen. The detection conditions were 2.5 μg·mL~(-1) working concentration of the captured antibody and 1∶450 of the dilution multiple of detected antibody. The coating condition was staying overnight at 4 ℃. The blocking time and incubation times of antigen and detected antibody were all 90 minutes. The incubation time of horseradish peroxidase conjugated streptavidin-horseradish peroxidase(SA-HRP) was 15 minutes. The quantitative limit of the method for PTL antigen was 0.375 ng·mL~(-1). The linear range was 75.000-4 800.000 pg·mL~(-1), and R~2=0.997 1. The recovery rate was 90.0%-110.0%, and the variation coefficients of intra-test and inter-test precision were 2.0%-3.0% and 2.0%-8.5%.The content of PTL in three batches of Pinelliae Rhizoma and the related processed products was determined by the method, and the average content of PTL in Pinelliae Rhizoma was 35.42 mg·g~(-1). The average content of PTL in Pinelliae Rhizoma Praeparatum Cum Alumine, Pinelliae Rhizoma Praeparatum, and Pinelliae Rhizoma Praeparatum Cum Zingibere Et Alumine were 1.15 mg·g~(-1), 16.53 μg·g~(-1), and 122.63 ng·g~(-1), respectively, indicating that the content of PTL decreased significantly after processing. The quantitative double-antibody sandwich ELISA for PTL antigen established in this paper had good linearity, sensitive response, and high accuracy, which provided a simple and effective monitoring method for the detection of PTL content in the processing of Pinelliae Rhizoma.

Evaluation of porcine GM-CSF during PRRSV infection in vitro and in vivo indicating a protective role of GM-CSF related with M1 biased activation in alveolar macrophage during PRRSV infection.

Granulocyte-macrophage colony stimulating factor (GM-CSF), participates in diverse biological processes associated with innate and adaptive immunity, has unknown effects during PRRSV infection. Here, a double-antibody sandwich ELISA for pGM-CSF was developed in-house for evaluation of pGM-CSF level during PRRSV infection both in vitro and in vivo. In in vitro assay, it was notable that PRRSV-infected porcine alveolar macrophages (PAMs) yielded inconsistent pGM-CSF protein- and mRNA-level, suggesting a post-transcriptional inhibition of pGM-CSF mRNA was employed by PRRSV. Meanwhile, concurrent analysis of pGM-CSF levels in serum samples from PRRSV-infected piglets suggested that effect of PRRSV infection demonstrated minimum effect on pGM-CSF levels regardless of PRRSV virulence phenotypes. Moreover, in vitro treatment of PAMs with pGM-CSF prior PRRSV inoculation did not inhibit PRRSV replication in PAMs although genes downstream of pGM-CSF in PAMs could be upregulated by pGM-CSF treatment. Meanwhile, knockdown of pGM-CSF using siRNA did not enhance PRRSV replication as well. Intriguingly, therapeutic antibody treatment of HP-PRRSV-infected piglets led to significantly increased serum pGM-CSF levels, thus aligning with low pneumonia incidence and low intracellular PRRSV-RNA levels in PAMs of therapeutic antibody treated piglets. Furthermore, transcriptome analysis of PAMs from infected piglets revealed increased serum pGM-CSF levels correlated with activation of downstream signal of pGM-CSF in PAMs as evidenced by a M1-like phenotypes of gene expression pattern, implying a potential host-protective role played by pGM-CSF for PRRSV infection in vivo. In conclusion, our results demonstrated developments of a highly sensitive and specific ELISA for pGM-CSF and revealed a potential protective role conferred by pGM-CSF during PRRSV infection.

First report of tobacco streak virus on Echinacea purpurea (Linn.) Moench in China.

Tobacco streak virus (TSV) is a member of the genus Ilarvirus in the family Bromoviridae (Vinodkumar et al. 2017). TSV is transmitted by thrips, seeds, pollen, and mechanical injury and has a broad host range, causing severe damage to several horticultural, oil and food crops including tobacco, sunflower, peanut, cotton, and soybean (Zambrana-Echevarría et al. 2021). TSV is now distributed mainly in the United States (McDaniel et al. 1992; Zambrana-Echevarría et al. 2021), India (Jain et al. 2008), Iran (Hosseini et al. 2012), Australia (Sharman et al. 2009) and Mexico (Silva-Rosales et al. 2013). Purple coneflower (Echinacea purpurea L.) is widely grown in China as an important herbal ornamental plant. In June 2020, Echinacea purpurea with the symptoms of necrosis lesions, malformation, and stunting were observed in the field of Haidian district, Beijing, China (40°2'69″ N, 116°28'28″ E) (Supplementary Fig. 1A). Total RNA of leaf tissue extracted using the hot borate method (Liang et al. 2020) was used for high-throughput sequencing on Illumina HiSeq X-10 platform at Biomarker Technologies (Beijing, China). Overall, 23,988,298 reads were generated. The final contigs assembled by Mega-Hit (v1.2.9) and Cap3 (Version Date: 02/10/15) were subjected to BLAST against GenBank using BLASTn and BLASTx algorithms. Of these contigs, 297 shared high nucleotide sequence similarities to the genomic sequence of broad bean wilt virus 2, while 9 contigs showed high nt sequence similarities (95-100%) to the genomic sequence of TSV. To confirm the presence of TSV, 30 randomly selected samples from Haidian district (40°2'69″ N, 116°28'28″ E) were tested by the double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) using a TSV specific monoclonal antibody (Agdia, SAR 25500/0500), where 18 samples were positive. In addition, total RNAs from 4 DAS-ELISA positive plants were extracted for TSV detection by reverse transcription-polymerase chain reactions (RT-PCR) using primer pair specific for the coat protein gene of TSV (TSV-CP-F, 5'-ATGAATACTTTGATCCAAGGTCC-3'; TSV-CP-R, 5'-TCAGTCTTGATTCACCAGAAAA-3'). The fragment with the expected size (~700 bp) was amplified in all 4 plants (Supplementary Fig. 1B) and subjected to direct Sanger sequencing. The CP gene of TSV CNB isolate was deposited in GenBank (MZ542767) and shared 100% sequence identity at the nucleotide level with the Gyp isolate infecting Ajuga reptans from Australia (JX463347.1). Furthermore, the local lesion host Chenopodium quinoa was used to purify and propagate TSV by mechanical inoculation with infected leaf sap. A pure culture of the TSV CNB isolate was obtained by single local lesion isolation after 3 serial passages on C. quinoa and back inoculated on E. purpurea seedlings. Systemic symptomology including leaf malformation was observed on E. purpurea three weeks post-inoculation (Supplementary Fig. 2A). The existence of TSV in two symptomatic leaf samples of E. purpurea was further verified by RT-PCR using specific primer pair (TSV-CP-F/R) (Supplementary Fig. 2B). In addition, the purified TSV CNB isolate was also inoculated to Nicotiana tabacum (Supplementary Fig. 2C). As previously reported (More et al. 2017), the Nicotiana tabacum plants infected with TSV developed typical streaks in systemic leaves. To the best of our knowledge, this is the first report of TSV on E. purpurea in China. This finding will assist further investigation into the epidemiology of diseases caused by TSV in China. Future studies are required to determine the incidence and impact that TSV might have on E. purpurea and other hosts in China.

Plasma and Peritoneal Fluid ZEB Levels in Patients with Endometriosis and Infertility.

Zinc finger E-box-binding homeobox 1 (ZEB1) and zinc finger E-box-binding homeobox 2 (ZEB2) are transcription factors that regulate epithelial–mesenchymal transformation (EMT). The aim of this study was to compare levels of ZEB1 and ZEB2 in the peritoneal fluid and plasma between patients with and without endometriosis in order to assess their utility in the diagnostic process. Plasma and peritoneal fluid samples were collected from 50 patients with and 48 without endometriosis during planned surgical procedures in eight clinical centers. Quantitative ZEB1 and ZEB2 levels analyses were performed using a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA). No significant differences were observed in ZEB1 levels in any of the subanalyses nor any differences regarding ZEB2 levels between patients with and without endometriosis. Plasma ZEB2 levels were significantly higher among patients with infertility compared to fertile women (16.07 ± 12.70 ng/L vs. 12.07 ± 11.92 ng/L; p < 0.04). Both ZEB1 and ZEB2 do not seem to have a significant value in the initial diagnosis of endometriosis as a single marker. The differences in ZEB2 plasma levels between patients with and without infertility indicate the possibility of EMT dysregulation in the pathogenesis of adverse fertility outcomes.

Plasma and Peritoneal Poly (ADP-Ribose) Polymerase Levels in Patients with Endometriosis.

The evidence of poly (ADP-ribose) polymerase (PARP) association with the immune response could be coherent with the immunological theory of endometriosis and suggests the possibility of a new research direction. The aim of the study was to evaluate the levels of PARP in plasma and peritoneal fluid of patients with and without endometriosis. It was a multicenter, cross-sectional study. Plasma and peritoneal fluid samples were collected from patients with and without endometriosis during planned laparoscopic procedures in eight clinical centers. In total, 84 samples of plasma and 84 samples of the peritoneal fluid were included in the final analyses. Double-antibody sandwich enzyme-linked immunosorbent assay was performed in order to assess levels of PARP in collected samples. No statistically significant differences regarding the detected levels of PARP in plasma and peritoneal fluid comparing patients with and without endometriosis were observed. Patients with a history of infertility had significantly higher plasma PARP concentrations (p = 0.04). We have not observed the potential role of PARP concentration levels in plasma nor peritoneal fluid as an endometriosis biomarker. We have determined an association between a higher plasma PARP concentration and a history of infertility.

Efficient Detection of Pre-Proinsulin by Double Antibody Sandwich ELISA

To detect residual pre-proinsulin (PPI) in recombinant human insulin production, an analytical method based on double-antibody sandwich ELISA was developed in this study. The BALB/c mice were immunized with PPI, and the hybridomas secreting anti-PPI monoclonal antibodies were obtained using the conventional cell fusion technique and ELISA screening. We purified the antibody using a Protein G gel column and identified its purity by SDS-PAGE. The sandwich ELISA was used to explore the pairing effect, and the specificity of the paired antibody was determined. We selected a paired antibody with relatively good specificity to establish sandwich ELISA, constructed a quantitative curve, and evaluated the accuracy and sensitivity of the method. Six anti-PPI monoclonal antibodies were obtained, named P1, P2, P3, P4, P5 and P6, of which P5 had the highest titer value. The sandwich ELISA method was established with P5 for plating and P2 as detection antibodies. The linear range of the quantitative curve of PPI by sandwich ELISA was 0.645 to 82.5 pg/mL, the recovery was 95% and the detection limit was 3.06 pg/mL. In this study, we prepared six anti-PPI monoclonal antibodies and established the sandwich ELISA method to detect PPI in process control and product release control for recombinant human insulin production.

New Weed Hosts for Tomato Brown Rugose Fruit Virus in Wild Mediterranean Vegetation.

Tomato brown rugose fruit virus (ToBRFV; genus, Tobamovirus, family, Virgaviridae) was first reported in 2015 infecting tomatoes grown under protected cropping in the Jordan Valley. Since then, ToBRFV has been detected in tomatoes grown in both protected and open fields across Jordan. The increased incidence of ToBRFV prompted this investigation of the potential role of natural weed hosts in the dissemination of ToBRFV. A survey was conducted in the Jordan Valley and highlands to determine possible reservoir hosts of ToBRFV in fields and greenhouse complexes in which tomatoes were grown. Detection of ToBRFV infection was made by double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) and further confirmation by reverse-transcription polymerase chain reaction (RT-PCR), followed by DNA cloning and sequencing, and bioassays. Thirty weed species belonging to twenty-six genera from sixteen families were tested. Twelve species belonging to eight families were infected of which ten species are newly reported hosts for ToBRFV. Seed transmission of ToBRFV in Solanum nigrum was confirmed in a grow-out experiment. To our knowledge, this is the first report of the natural occurrence of ToBRFV on weed hosts. Identification of natural reservoirs of ToBRFV can help to develop management practices focused on weed plant species to prevent ToBRFV transmission. The extent to which ToBRFV survives in diverse alternate weed host species outside tomato growing seasons in different world regions requires further research in order to establish the risk associated with the possible contribution of weeds as a reservoir for primary infections in tomato crops.