scholarly journals Rigid monoclonal antibodies improve detection of SARS-CoV-2 nucleocapsid protein

2021 ◽  
Author(s):  
Curtis D. Hodge ◽  
Daniel J. Rosenberg ◽  
Mateusz Wilamowski ◽  
Andrzej Joachimiak ◽  
Greg L. Hura ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Nucleocapsid Protein ◽  
Point Of Care ◽  
Sandwich Elisa ◽  
Epidemiological Studies ◽  
Detection Sensitivity ◽  
Linear Arrangement ◽  
X Ray Scattering ◽  
Biological Phenomena ◽  
Flow Devices

AbstractMonoclonal antibodies (mAbs) are the basis of treatments and diagnostics for pathogens and other biological phenomena. We conducted a structural characterization of mAbs against the N-terminal domain of nucleocapsid protein (NPNTD) from SARS-CoV-2 using small angle X-ray scattering (SAXS). Our solution-based results distinguished the mAbs’ flexibility and how this flexibility impacts the assembly of multiple mAbs on an antigen. By pairing two mAbs that bind different epitopes on the NPNTD, we show that flexible mAbs form a closed sandwich-like complex. With rigid mAbs, a juxtaposition of the Fabs is prevented, enforcing a linear arrangement of the mAb pair, which facilitates further mAb polymerization. In a modified sandwich ELISA, we show the rigid mAb-pairings with linear polymerization led to increased NPNTD detection sensitivity. These enhancements can expedite the development of more sensitive and selective antigen-detecting point-of-care lateral flow devices (LFA), key for early diagnosis and epidemiological studies of SARS-CoV-2 and other pathogens.

Rapid Detection of Salmonella spp. in Foods by Combination of a New Selective Enrichment and a Sandwich ELISA Using Two Monoclonal Antibodies against Dulcitol 1-Phosphate Dehydrogenase

1996 ◽  
Vol 59 (11) ◽  
pp. 1158-1163 ◽  
Author(s):  
HUAIZE TIAN ◽  
TAKAHISA MIYAMOTO ◽  
TAKASHI OKABE ◽  
YOICHIRO KURAMITSU ◽  
KEN-ICHI HONJOH ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Rapid Detection ◽  
Enrichment Culture ◽  
Sandwich Elisa ◽  
False Negative ◽  
Detection Sensitivity ◽  
Elisa Method ◽  
Salmonella Spp ◽  
Selective Enrichment ◽  
Raw Foods

A rapid-detection method was developed for food-borne dulcitol-positive Salmonella spp. in foods that involves a new preenrichment and selective enrichment system and a sandwich ELISA using two monoclonal antibodies against dulcitol 1-phosphate dehydrogenase. Preenrichment and selective enrichment were in Enterobacteriaceae enrichment mannitol (EEM) broth at 42°C for 6 h and in a new dulcitol-magnesium chloride-pyridinesulfonic acid brilliant green-novobiocin (DMPBN) medium at 42°C for 27 h, respectively. The cells were collected from the selective enrichment culture and suspended in 0.1 ml of 1 N NaOH for 2 min. The solution was neutralized with 0.1 ml of 2 M Tris-HCl buffer (pH 7.5) and the mixture was used as a sample for ELISA. The detection sensitivity of the ELISA was 105 CFU of Salmonella spp. per ml of culture. Competing non-Salmonella organisms in raw food did not interfere with the detection of Salmonella cells even when present at 107: 1 (non-Salmonella: Salmonella ratio) in food. Nonmotile Salmonella gallinarum was detected by the ELISA. The minimum detectable number of initial inoculum of Salmonella typhimurium was 0.69 CFU/25 g of raw chicken after the preenrichment in EEM broth and the selective enrichment in DMPBN medium. The present ELISA method required a total analysis time of 36 h including the preenrichment and selective enrichment periods. The ELISA method was compared with a conventional cultural method for the detection of Salmonella cells in 130 samples of raw foods. Of the samples tested, 16 were Salmonella-positive and 114 samples were negative by both methods. False-positive and false-negative results were not encountered.

Antibody Screening Results for Anti-Nucleocapsid Antibodies Towards the Development of a SARS-CoV-2 Nucleocapsid Protein Antigen Detecting Lateral Flow Assay

2021 ◽  
Author(s):  
David Cate ◽  
Helen Hsieh ◽  
Veronika Glukhova ◽  
Joshua D Bishop ◽  
H Gleda Hermansky ◽  
...  
Keyword(s):  
Nucleocapsid Protein ◽  
Point Of Care ◽  
Lateral Flow ◽  
Screening Methods ◽  
Antibody Screening ◽  
Liquid Handling ◽  
Large Numbers ◽  
Accurate Performance ◽  
Further Development ◽  
Assay Conditions

<p></p><p>The global COVID-19 pandemic has created an urgent demand for large numbers of inexpensive, accurate, rapid, point-of-care diagnostic tests. Analyte-based assays are suitably inexpensive and can be rapidly mass-produced, but for sufficiently accurate performance they require highly optimized antibodies and assay conditions. We used an automated liquid handling system, customized to handle arrays of lateral flow immunoassay (LFA) tests in a high-throughput screen, to identify anti-nucleocapsid antibodies that will perform optimally in an LFA. We tested 1021 anti-nucleocapsid antibody pairs as LFA capture and detection reagents with the goal of highlighting pairs that have the greatest affinity for unique epitopes of the nucleocapsid protein of SARS-CoV-2 within the LFA format. In contrast to traditional antibody screening methods (e.g., ELISA, bio-layer interferometry), the method described here integrates real-time reaction kinetics with transport in, and immobilization directly onto, nitrocellulose. We have identified several candidate antibody pairs that are suitable for further development of an LFA for SARS-CoV-2.</p><p></p>

Ferromagnetic Resonance Biosensor for Homogeneous and Volumetric Detection of DNA

2017 ◽  
Author(s):  
Bo Tian ◽  
Peter Svedlindh ◽  
Mattias Strömberg ◽  
Erik Wetterskog
Keyword(s):  
Magnetic Nanoparticles ◽  
Ferromagnetic Resonance ◽  
Limit Of Detection ◽  
Point Of Care ◽  
Rolling Circle Amplification ◽  
Resonance Field ◽  
Isothermal Amplification ◽  
Detection Sensitivity ◽  
Serum Samples ◽  
Rolling Circle

In this work, we demonstrate for the first time, a ferromagnetic resonance (FMR) based homogeneous and volumetric biosensor for magnetic label detection. Two different isothermal amplification methods, <i>i.e.</i>, rolling circle amplification (RCA) and loop-mediated isothermal amplification (LAMP) are adopted and combined with a standard electron paramagnetic resonance (EPR) spectrometer for FMR biosensing. For RCA-based FMR biosensor, binding of RCA products of a synthetic Vibrio cholerae target DNA sequence gives rise to the formation of aggregates of magnetic nanoparticles. Immobilization of nanoparticles within the aggregates leads to a decrease of the net anisotropy of the system and a concomitant increase of the resonance field. A limit of detection of 1 pM is obtained with an average coefficient of variation of 0.16%, which is superior to the performance of other reported RCA-based magnetic biosensors. For LAMP-based sensing, a synthetic Zika virus target oligonucleotide is amplified and detected in 20% serum samples. Immobilization of magnetic nanoparticles is induced by their co-precipitation with Mg<sub>2</sub>P<sub>2</sub>O<sub>7</sub> (a by-product of LAMP) and provides a detection sensitivity of 100 aM. The fast measurement, high sensitivity and miniaturization potential of the proposed FMR biosensing technology makes it a promising candidate for designing future point-of-care devices.<br>

scholarly journals Development of a Colloidal Gold Immunochromatographic Assay for Duck Enteritis Virus Detection Using Monoclonal Antibodies

Pathogens ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 365
Author(s):  
Fengli Liu ◽  
Yanxin Cao ◽  
Maokai Yan ◽  
Mengxu Sun ◽  
Qingshui Zhang ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Colloidal Gold ◽  
Preventive Intervention ◽  
Virus Detection ◽  
Cross Reactivity ◽  
Duck Enteritis Virus ◽  
Detection Sensitivity ◽  
Immunochromatographic Assay ◽  
Efficient Detection ◽  
Enteritis Virus

Duck viral enteritis is a highly contagious and fatal disease of commercial waterfowl flocks. The disease occurs sporadically or epizootically in mainland China due to insufficient vaccinations. Early and rapid diagnosis is important for preventive intervention and the control of epizootic events in clinical settings. In this study, we generated two monoclonal antibodies (MAbs) that specifically recognized the duck enteritis virus (DEV) envelope glycoprotein B and tegument protein UL47, respectively. Using these MAbs, a colloidal gold-based immunochromatographic assay (ICA) was developed for the efficient detection of DEV antigens within 15 min. Our results showed that the detection limit of the developed ICA strip was 2.52 × 103 TCID50/mL for the virus infected cell culture suspension with no cross-reactivity with other pathogenic viruses commonly encountered in commercially raised waterfowl. Using samples from experimentally infected ducks, we demonstrated that the ICA detected the virus in cloacal swab samples on day three post-infection, demonstrating an 80% concordance with the PCR. For tissue homogenates from ducks succumbing to infection, the detection sensitivity was 100%. The efficient and specific detection by this ICA test provides a valuable, convenient, easy to use and rapid diagnostic tool for DVE under both laboratory and field conditions.

scholarly journals Diagnosis of Herpes Simplex Virus: Laboratory and Point-of-Care Techniques

2021 ◽  
Vol 13 (2) ◽  
pp. 518-539
Author(s):  
Peuli Nath ◽  
Md Alamgir Kabir ◽  
Somaiyeh Khoubafarin Doust ◽  
Aniruddha Ray
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Point Of Care ◽  
Diagnostic Techniques ◽  
Nucleic Acid Amplification ◽  
Physical Contact ◽  
Detection Sensitivity ◽  
Detection Techniques ◽  
Advantages And Disadvantages ◽  
Simplex Virus

Herpes is a widespread viral infection caused by the herpes simplex virus (HSV) that has no permanent cure to date. There are two subtypes, HSV-1 and HSV-2, that are known to cause a variety of symptoms, ranging from acute to chronic. HSV is highly contagious and can be transmitted via any type of physical contact. Additionally, viral shedding can also happen from asymptomatic infections. Thus, early and accurate detection of HSV is needed to prevent the transmission of this infection. Herpes can be diagnosed in two ways, by either detecting the presence of the virus in lesions or the antibodies in the blood. Different detection techniques are available based on both laboratory and point of care (POC) devices. Laboratory techniques include different biochemical assays, microscopy, and nucleic acid amplification. In contrast, POC techniques include microfluidics-based tests that enable on-spot testing. Here, we aim to review the different diagnostic techniques, both laboratory-based and POC, their limits of detection, sensitivity, and specificity, as well as their advantages and disadvantages.

Evaluation of Human Fecal Calprotectin Detection Kits and Their Potential use as Non-Invasive Intestinal Biomarker of Infectious Enterocolitis in Pigs

2021 ◽  
Author(s):  
Jéssica Barbosa ◽  
Lucas Rodrigues ◽  
Daniel Columbus ◽  
Juan Aguirre ◽  
John Harding ◽  
...  
Keyword(s):  
Intestinal Inflammation ◽  
Point Of Care ◽  
Sandwich Elisa ◽  
Fecal Calprotectin ◽  
Field Application ◽  
Dipstick Test ◽  
Infectious Colitis ◽  
Fecal Samples ◽  
Non Invasive ◽  
Infectious Enterocolitis

Abstract Background: Fecal calprotectin is largely applied as a non-invasive intestinal inflammation biomarker in human medicine. Previous studies in pigs investigated the levels of fecal calprotectin in healthy animals only. Thus, there is a knowledge gap regarding its application during infectious diarrhea. This study investigated the usefulness of fecal calprotectin as a biomarker of intestinal inflammation in Brachyspira hyodysenteriae and Salmonella Typhimurium infected pigs. Results: Fecal samples from pigs with colitis (n=18) were collected from animals experimentally inoculated with B. hyodysenteriae G44 or from sham-inoculated controls. Fecal samples from pigs with enteritis (n=14) were collected from animals inoculated with Salmonella enterica serovar Typhimurium or from sham-inoculated controls. For both groups, fecal samples were scored as: 0 = normal; 1 = soft, wet cement; 2 = watery feces; 3 = mucoid diarrhea; and 4 = bloody diarrhea. Fecal calprotectin levels were assayed using a sandwich ELISA, a turbidimetric immunoassay and a point-of-care dipstick test. Fecal calprotectin levels were greater in colitis samples scoring 4 versus ≤ 4 using ELISA, and in feces scoring 3 and 4 versus ≤ 1 using immunoturbidimetry (P < 0.05). No differences were found in calprotectin concentration among fecal scores for enteritis samples, regardless of the assay used. All samples were found below detection limits using the dipstick method.Conclusions: Fecal calprotectin is a potential non-invasive biomarker of infectious colitis, but it is not suitable for detection of enteritis. While practical, the use of commercially available human presents sensitivity limitations. Further studies are needed to validate the field application of calprotectin as a marker.

SARS Coronavirus Nucleocapsid Protein Monoclonal Antibodies Developed Using a Prokaryotic Expressed Protein

Hybridoma ◽  
2011 ◽  
Vol 30 (5) ◽  
pp. 481-485 ◽  
Author(s):  
Juan Zhang ◽  
Ding Wang ◽  
Yue Li ◽  
Qing Zhao ◽  
Ailong Huang ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Nucleocapsid Protein ◽  
Sars Coronavirus

Preparation of monoclonal antibodies against KHV and establishment of an antigen sandwich ELISA for KHV detection

2019 ◽  
Vol 128 ◽  
pp. 36-40 ◽  
Author(s):  
Yingying Li ◽  
Qing Wang ◽  
Sven M. Bergmann ◽  
Weiwei Zeng ◽  
Yingying Wang ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Sandwich Elisa

scholarly journals Monoclonal Antibodies against Nucleocapsid Protein of SARS-CoV-2 Variants for Detection of COVID-19

2021 ◽  
Vol 22 (22) ◽  
pp. 12412
Author(s):  
Ruei-Min Lu ◽  
Shih-Han Ko ◽  
Wan-Yu Chen ◽  
Yu-Ling Chang ◽  
Hsiu-Ting Lin ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Nucleocapsid Protein ◽  
Limit Of Detection ◽  
Wide Spectrum ◽  
Mitigation Strategies ◽  
Respiratory Pathogens ◽  
Wild Type ◽  
Human Coronavirus ◽  
Spectrum Detection ◽  
Clinical Detection

Mitigation strategies of the coronavirus disease 2019 (COVID-19) pandemic have been greatly hindered by the continuous emergence of SARS-CoV-2 variants. New sensitive, rapid diagnostic tests for the wide-spectrum detection of viral variants are needed. We generated a panel of 41 monoclonal antibodies against the SARS-CoV-2 nucleocapsid protein (NP) by using mice hybridoma techniques. Of these mAbs, nine exhibited high binding activities and were applied in latex-based lateral flow immunoassays (LFIAs). The LFIAs utilizing NP-mAb-7 and -40 had the best sensitivity and lowest limit of detection: 8 pg for purified NP and 625 TCID50/mL for the authentic virus (hCoV-19/Taiwan/4/2020). The specificity tests showed that the NP-mAb-40/7 LFIA strips did not cross-react with five human coronavirus strains or 20 other common respiratory pathogens. Importantly, we found that 10 NP mutants, including alpha (B.1.1.7), beta (B.1.351), gamma (P.1), and delta (B.1.617.2) variants, could be detected by NP-mAb-40/7 LFIA strips. A clinical study (n = 60) of the NP-mAb-40/7 LFIA strips demonstrated a specificity of 100% and sensitivity of 90% in infected individuals with cycle threshold (Ct) values < 29.5. These anti-NP mAbs have strong potential for use in the clinical detection of SARS-CoV-2 infection, whether the virus is wild-type or a variant of concern.

scholarly journals Antibody Screening Results for Anti-Nucleocapsid Antibodies Towards the Development of a SARS-CoV-2 Nucleocapsid Protein Antigen Detecting Lateral Flow Assay

2021 ◽  
Author(s):  
David Cate ◽  
Helen Hsieh ◽  
Veronika Glukhova ◽  
Joshua D Bishop ◽  
H Gleda Hermansky ◽  
...  
Keyword(s):  
Nucleocapsid Protein ◽  
Point Of Care ◽  
Lateral Flow ◽  
Screening Methods ◽  
Antibody Screening ◽  
Liquid Handling ◽  
Large Numbers ◽  
Accurate Performance ◽  
Further Development ◽  
Assay Conditions

<p></p><p>The global COVID-19 pandemic has created an urgent demand for large numbers of inexpensive, accurate, rapid, point-of-care diagnostic tests. Analyte-based assays are suitably inexpensive and can be rapidly mass-produced, but for sufficiently accurate performance they require highly optimized antibodies and assay conditions. We used an automated liquid handling system, customized to handle arrays of lateral flow immunoassay (LFA) tests in a high-throughput screen, to identify anti-nucleocapsid antibodies that will perform optimally in an LFA. We tested 1021 anti-nucleocapsid antibody pairs as LFA capture and detection reagents with the goal of highlighting pairs that have the greatest affinity for unique epitopes of the nucleocapsid protein of SARS-CoV-2 within the LFA format. In contrast to traditional antibody screening methods (e.g., ELISA, bio-layer interferometry), the method described here integrates real-time reaction kinetics with transport in, and immobilization directly onto, nitrocellulose. We have identified several candidate antibody pairs that are suitable for further development of an LFA for SARS-CoV-2.</p><p></p>

Sign in / Sign up

Export Citation Format

Share Document