Preparation of Anti-Ciguatoxin Monoclonal Antibodies Using Synthetic Haptens: Sandwich ELISA Detection of Ciguatoxins

2014 ◽  
Vol 97 (2) ◽  
pp. 373-379 ◽  
Author(s):  
Takeshi Tsumuraya ◽  
Ikuo Fujii ◽  
Masahiro Hirama
Keyword(s):  
Monoclonal Antibodies ◽  
Sandwich Elisa ◽  
Food Poisoning ◽  
Cross Reactivity ◽  
Protein Conjugates ◽  
Natural Toxins ◽  
Ciguatera Fish Poisoning ◽  
Fish Poisoning ◽  
Brevetoxin A ◽  
Channel Activator

Abstract Ciguatera fish poisoning (CFP) is a form of food poisoning caused by the consumption of fish that have accumulated a type of sodium channel activator toxin called ciguatoxins (CTXs), which are produced by dinoflagellates of the genus Gambierdiscus through the food chain. CFP affects more than 50 000 people each year. The extremely low level of CTXs in tainted fish has hampered the development of antibodies for the detection of these toxins. Monoclonal antibodies (mAbs) specific against major congeners of CTX3C, 51-hydroxyCTX3C, CTX1B, and 54-deoxyCTX1B were prepared by immunization of mice with protein conjugates of rationally designed synthetic haptens in place of the natural toxins. We found that haptenic groups possessing a surface area larger than 400 Å2 were required to produce mAbs that can bind strongly to CTXs. Direct sandwich ELISA utilizing two different monoclonal antibodies that bind specifically to one of the two wings of a CTX were established to detect CTXs. No cross-reactivity was observed against the other marine toxins tested, including brevetoxin A, brevetoxin B, okadaic acid, and maitotoxin.

scholarly journals Rationally Designed Synthetic Haptens to Generate Anti-Ciguatoxin Monoclonal Antibodies, and Development of a Practical Sandwich ELISA to Detect Ciguatoxins

Toxins ◽  
2019 ◽  
Vol 11 (9) ◽  
pp. 533 ◽  
Author(s):  
Takeshi Tsumuraya ◽  
Masahiro Hirama
Keyword(s):  
Monoclonal Antibodies ◽  
Limit Of Detection ◽  
Sandwich Elisa ◽  
Food Poisoning ◽  
Enzyme Linked Immunosorbent Assay ◽  
Fda Guidance ◽  
Fish Poisoning ◽  
Highly Sensitive ◽  
Taste And Smell ◽  
Reliable Methods

“Ciguatera” fish poisoning (CFP) is one of the well-known food poisoning caused by the ingestion of fish that have accumulated trace amounts of ciguatoxins (CTXs). CFP affects more than 50,000 individuals annually. The difficulty in preventing CFP comes from the lack of reliable methods for analysis of CTXs in contaminated fish, together with the normal appearance, taste, and smell of CTX-contaminated fish. Thus, a sensitive, accurate, routine, and portable analytical method to detect CTXs is urgently required. Monoclonal antibodies (mAbs) specific against either wing of major CTX congeners (CTX1B, 54-deoxyCTX1B, CTX3C, and 51-hydroxyCTX3C) were generated by immunizing mice with rationally designed synthetic haptens-KLH conjugates instead of the CTXs. Haptenic groups with a surface area greater than 400 Å2 are required to produce mAbs that can strongly bind to CTXs. Furthermore, a highly sensitive fluorescence-based sandwich enzyme-linked immunosorbent assay (ELISA) was developed. This protocol can detect and quantify four major CTX congeners (CTX1B, 54-deoxyCTX1B, CTX3C, and 51-hydroxyCTX3C) with a limit of detection (LOD) of less than 1 pg/mL. The LOD determined for this sandwich ELISA is sufficient to detect CTX1B-contaminated fish at the FDA guidance level of 0.01 ppb.

scholarly journals Development of Vasoinhibin-Specific Monoclonal Antibodies

2021 ◽  
Vol 12 ◽  
Author(s):  
Nils Müller ◽  
Juan Pablo Robles ◽  
Magdalena Zamora ◽  
Johannes Ebnet ◽  
Hülya Markl-Hahn ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Serum Proteins ◽  
Limit Of Detection ◽  
Sandwich Elisa ◽  
Vascular Diseases ◽  
High Specificity ◽  
Cross Reactivity ◽  
Dimensional Structure ◽  
Serum Samples ◽  
Limit Of Quantitation

Vasoinhibin is a protein hormone with antiangiogenic, antivasodilatatory, and antivasopermeability effects generated by the proteolytic cleavage of prolactin. The discovery of its role in diabetic retinopathy and peripartum cardiomyopathy led to the evaluation of new pharmacological treatments in clinical interventional trials. However, the quantitative evaluation of vasoinhibin in biological samples from patients has not been possible due to the lack of vasoinhibin-specific antibodies. Recently, loop 1 of vasoinhibin was identified to have a different three-dimensional structure compared to PRL, and thus to contain vasoinhibin-specific epitopes. Here, we report the development of two sets of vasoinhibin-specific monoclonal antibodies against two neighboring regions of the vasoinhibin loop 1. An experimental sandwich ELISA with two monoclonal anti-vasoinhibin antibodies was developed, which had no cross-reactivity to recombinant human full-length prolactin. The ELISA had a quantitation limit of 100 ng/ml, and intra-assay- and inter-assay coefficients of variation of 12.5% and 14%, respectively. The evaluation of 15 human serum samples demonstrated concentrations of below limit of detection (n=3), below limit of quantitation (n=1) and between 0.23 µg/ml (230 ng/ml) to 605 µg/ml (n=12) in the quantifiable range. Despite the high specificity of the monoclonal-monoclonal antibody sandwiches which discriminate vasoinhibin from PRL, there might be cross-reactivities by serum proteins other than vasoinhibin. A fully established vasoinhibin ELISA may support diagnostic and therapeutic measures in vascular diseases.

scholarly journals Monoclonal antibodies for the S2 subunit of spike of SARS-CoV-1 cross-react with the newly-emerged SARS-CoV-2

2020 ◽  
Vol 25 (28) ◽  
Author(s):  
Zhiqiang Zheng ◽  
Vanessa Marthe Monteil ◽  
Sebastian Maurer-Stroh ◽  
Chow Wenn Yew ◽  
Carol Leong ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Common Cold ◽  
Sandwich Elisa ◽  
Cell Receptor ◽  
Cross Reactivity ◽  
S Protein ◽  
Mammalian Cell Lines ◽  
Infected Cells ◽  
The Cross ◽  
Novel Coronavirus

Background A novel coronavirus, SARS-CoV-2, which emerged at the end of 2019 and causes COVID-19, has resulted in worldwide human infections. While genetically distinct, SARS-CoV-1, the aetiological agent responsible for an outbreak of severe acute respiratory syndrome (SARS) in 2002–2003, utilises the same host cell receptor as SARS-CoV-2 for entry: angiotensin-converting enzyme 2 (ACE2). Parts of the SARS-CoV-1 spike glycoprotein (S protein), which interacts with ACE2, appear conserved in SARS-CoV-2. Aim The cross-reactivity with SARS-CoV-2 of monoclonal antibodies (mAbs) previously generated against the S protein of SARS-CoV-1 was assessed. Methods The SARS-CoV-2 S protein sequence was aligned to those of SARS-CoV-1, Middle East respiratory syndrome (MERS) and common-cold coronaviruses. Abilities of mAbs generated against SARS-CoV-1 S protein to bind SARS-CoV-2 or its S protein were tested with SARS-CoV-2 infected cells as well as cells expressing either the full length protein or a fragment of its S2 subunit. Quantitative ELISA was also performed to compare binding of mAbs to recombinant S protein. Results An immunogenic domain in the S2 subunit of SARS-CoV-1 S protein is highly conserved in SARS-CoV-2 but not in MERS and human common-cold coronaviruses. Four murine mAbs raised against this immunogenic fragment could recognise SARS-CoV-2 S protein expressed in mammalian cell lines. In particular, mAb 1A9 was demonstrated to detect S protein in SARS-CoV-2-infected cells and is suitable for use in a sandwich ELISA format. Conclusion The cross-reactive mAbs may serve as useful tools for SARS-CoV-2 research and for the development of diagnostic assays for COVID-19.

Differential titration of plasmin and plasminogen in milk using sandwich ELISA with monoclonal antibodies

1997 ◽  
Vol 64 (1) ◽  
pp. 77-86 ◽  
Author(s):  
DIDIER DUPONT ◽  
CHRISTELLE BAILLY ◽  
JEANNE GROSCLAUDE ◽  
JEAN-CLAUDE COLLIN
Keyword(s):  
Monoclonal Antibodies ◽  
Proteinase Inhibitors ◽  
Sandwich Elisa ◽  
Bovine Milk ◽  
Milk Proteins ◽  
Cross Reactivity ◽  
The Other ◽  
Milk Samples ◽  
Skimmed Milk ◽  
Mastitic Milk

Two sandwich enzyme-linked immunosorbent assays (ELISA) have been developed for quantitation of bovine milk plasminogen and plasmin. The assays used two monoclonal antibodies, one specific for plasminogen and the other specific for plasminogen plus plasmin. Plasmin concentration was obtained by subtracting the first concentration from the second. The assays were sensitive (linear range, 5–75 ng/ml), repeatable (CV, 8 and 5% for plasmin and plasminogen titration respectively), specific (no cross reactivity with the major milk proteins) and directly applicable to skimmed milk with no particular pretreatment of the sample. However, ELISA did not permit complete titration of milk plasminogen and plasmin (only 60–90% could be quantified). This lack of accuracy was due to casein interfering with the plasmin–plasminogen titration by ELISA. Results obtained with ELISA or an enzymic technique on 20 milk samples collected from individual cows milked throughout pregnancy showed that the ELISA was particularly suitable for analysis of late lactation or mastitic milk where proteinase inhibitors interfered with enzymic quantitation.

scholarly journals Detection of spring viraemia of carp virus (SVCV) with monoclonal antibodies

2008 ◽  
Vol 52 (No. 7) ◽  
pp. 308-316 ◽  
Author(s):  
S. Reschova ◽  
D. Pokorova ◽  
Z. Nevorankova ◽  
J. Hulova ◽  
T. Vesely
Keyword(s):  
Monoclonal Antibodies ◽  
Severe Disease ◽  
Sandwich Elisa ◽  
Infectious Pancreatic Necrosis Virus ◽  
Cross Reactivity ◽  
Necrosis Virus ◽  
Sandwich Elisa Assay ◽  
Pancreatic Necrosis Virus ◽  
Set Up ◽  
Low Sensitivity

Monoclonal antibodies (MAbs) against spring viraemia of carp virus (SVCV), a severe disease in cyprinid fish, were prepared. Nine MAbs were characterised using Western blotting (WB) where all reacted with glycoprotein G, except for MAb 2E1, which showed no reactivity in WB. All nine MAbs showed specificity in an immunoperoxidase test. In ELISA assays, their titres ranged between 1:32 000 and 1:128 000. A panel of SVCV isolates from different European regions were set up and examined by sandwich ELISA assay using the MAbs at a concentration of 15 &mu;g/ml. Only MAb 4C12/3C8 showed low sensitivity in most of the isolates at an absorbance of A<sub>450</sub> the other MAbs, even the lowest absorbance value measured exceeded cut-off for evaluation of the whole reaction. No cross-reaction with the infectious haematopoietic necrosis virus (IHNV), viral haemorrhagic septicaemia virus (VHSV) or infectious pancreatic necrosis virus (IPNV) was demonstrated. 2E1 did not show cross-reactivity with PFRV classified in genogroup III&minus;IV and reacted with a Czech SVCV isolate; its identity was confirmed by means of RT PCR assay. The others MAbs reacted positively with PFRV F4 reference strain, isolated from <i>Esox lucius</i> L. (genogroup III).

scholarly journals Development of a Double Nanobody-Based Sandwich Immunoassay for the Detecting Staphylococcal Enterotoxin C in Dairy Products

Foods ◽  
2021 ◽  
Vol 10 (10) ◽  
pp. 2426
Author(s):  
Yanwei Ji ◽  
Lili Chen ◽  
Yingying Wang ◽  
Kaihui Zhang ◽  
Haofen Wu ◽  
...  
Keyword(s):  
Phage Display ◽  
Dairy Products ◽  
Sandwich Elisa ◽  
Protein A ◽  
Food Poisoning ◽  
High Specificity ◽  
Cross Reactivity ◽  
Staphylococcal Enterotoxin ◽  
Enzyme Linked Immunosorbent Assay ◽  
Staphylococcal Protein A

Staphylococcal enterotoxins (SEs) represent the leading reason for staphylococcal food poisoning (SFP) and various other diseases. Reports often indicate Staphylococcal enterotoxin C (SEC) as the most frequently found enterotoxin in dairy products. To minimize consumer exposure to SEC, this paper aimed to create a sandwich enzyme-linked immunosorbent assay (ELISA) based on nanobodies (sandwich Nbs-ELISA) to accurately detect SEC in dairy products without the influence of staphylococcal protein A (SpA). Therefore, after inoculating a Bactrian camel with SEC, a phage display Nb library was created. Eleven Nbs against SEC were identified in three biopanning steps. Based on their affinity and pairing level, a sandwich Nbs-ELISA was developed using the C6 anti-SEC Nb as the capture antibody, while the detection antibody was represented by the C11 phage display anti-SEC Nb. In optimal conditions, the quantitative range of the present sandwich ELISA was 4-250 ng/mL with a detection limit (LOD) of 2.47 ng/mL, obtained according to the blank value plus three standard deviations. The developed technique was subjected to specific measurements, revealing minimal cross-reactivity with Staphylococcus aureus (S. aureus), Staphylococcal enterotoxin A (SEA), Staphylococcal enterotoxin B (SEB), and SpA. The proposed method exhibited high specificity and an excellent recovery rate of 84.52~108.06% in dairy products. Therefore, the sandwich Nbs-ELISA showed significant potential for developing a specific, sensitive technique for SEC detection in dairy products.

scholarly journals Monoclonal antibodies to bovine coronavirus and their use in enzymoimmunoanalysis and immunochromatography

2001 ◽  
Vol 46 (No. 5) ◽  
pp. 125-131 ◽  
Author(s):  
S. Reschová ◽  
D. Pokorová ◽  
Z. Nevoránková ◽  
J. Franz
Keyword(s):  
Monoclonal Antibodies ◽  
Sandwich Elisa ◽  
Haemagglutination Inhibition ◽  
Cross Reactivity ◽  
Immunochromatographic Test ◽  
Structural Protein ◽  
Bovine Coronavirus ◽  
Specificity And Sensitivity ◽  
Faecal Samples ◽  
Golden Standard

Two monoclonal antibodies (MAb) to the outer structural protein E2 (spike peplomeric protein) and two MAb to the inner capsid protein N of bovine coronavirus (BCV) were prepared and identified by Western blotting to be used for increasing the specificity and sensitivity of BCV detection. The MAb were checked by the haemagglutination inhibition test and immunoperoxidase tests and no cross reactivity with rotavirus was demonstrated by the immunoperoxidase test and ELISA. A mixture of all the four MAb at predetermined optimum concentrations was first used in sandwich ELISA and then, in combination with an anti‑coronavirus polyclonal antibody, for the development of a simple and rapid immunochromatographic test (ICT). The results of which can be read visually within 10 min. The inclusion of MAb into ELISA and ICT allows the detection of both intact and incomplete BCV virions. ELISA and ICT were used in the examination of a set of 74 faecal samples collected from calves suffering from diarrhoea. ELISA, used as the golden standard verified by electron microscopy, detected BCV in 15 samples (20.3%) and ICT in 16 samples. Three of the ICT‑positive samples were negative by ELISA. On the other hand, two of the 58 ICT‑negative samples were positive by ELISA. Sensitivity and specificity of ICT were 94.9% and 86.7%, respectively

scholarly journals Anthrax Spore Detection by a Luminex Assay Based on Monoclonal Antibodies That Recognize Anthrose-Containing Oligosaccharides

2010 ◽  
Vol 17 (9) ◽  
pp. 1446-1451 ◽  
Author(s):  
Marco Tamborrini ◽  
Marcelle Holzer ◽  
Peter H. Seeberger ◽  
Nadia Schürch ◽  
Gerd Pluschke
Keyword(s):  
Monoclonal Antibodies ◽  
Sandwich Elisa ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
First Line ◽  
Luminex Assay ◽  
Detection Systems ◽  
Highly Sensitive ◽  
Spore Detection ◽  
Assay Detection

ABSTRACT The similarity of endospore surface antigens between bacteria of the Bacillus cereus group complicates the development of selective antibody-based anthrax detection systems. The surface of B. anthracis endospores exposes a tetrasaccharide containing the monosaccharide anthrose. Anti-tetrasaccharide monoclonal antibodies (MAbs) and anti-anthrose-rhamnose disaccharide MAbs were produced and tested for their fine specificities in a direct spore enzyme-linked immunosorbent assay (ELISA) with inactivated spores of a broad spectrum of B. anthracis strains and related species of the Bacillus genus. Although the two sets of MAbs had different fine specificities, all of them recognized the tested B. anthracis strains and showed only a limited cross-reactivity with two B. cereus strains. The MAbs were further tested for their ability to be implemented in a highly sensitive and specific bead-based Luminex assay. This assay detected spores from different B. anthracis strains and two cross-reactive B. cereus strains, correlating with the results obtained in direct spore ELISA. The Luminex assay (detection limit 103 to 104 spores per ml) was much more sensitive than the corresponding sandwich ELISA. Although not strictly specific for B. anthracis spores, the developed Luminex assay represents a useful first-line screening tool for the detection of B. anthracis spores.

scholarly journals SUN-LB124 Novel Elisa Assays Demonstrate Specificity of Islet and Intestinal Processing of Proglucagon

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
David A D’Alessio ◽  
Ajay Kumar ◽  
Bhanu Kalra ◽  
Shivani Mistry ◽  
Jenny Tong
Keyword(s):  
Monoclonal Antibodies ◽  
Sandwich Elisa ◽  
Cross Reactivity ◽  
Plasma Glucagon ◽  
Tissue Specific ◽  
Genetic Ablation ◽  
L Cells ◽  
Healthy Humans ◽  
Significant Change ◽  
Meal Ingestion

Abstract Circulating proglucagon peptides (PGP) are produced in islet α-cells, enteroendocrine L-cells. Release of PGP is thought to be tissue specific, e.g. α-cells make glucagon and L-cells make GLP-1 through predominant actions of proconvertases 2 and 1/3 (PC2 and PC1/3). However, this dichotomous model has recently been challenged. To address the contribution of the gut and pancreas to plasma PGP we developed 4 novel sandwich ELISA assays and applied them in studies with PGP stimulation from the islet (IV arginine) and intestine (meal). Monoclonal antibodies were raised in mice with genetic ablation of proglucagon transcription. Clones were screened and selected for affinity and specificity, and assays for glucagon, GLP-1, glicentin and oxyntomodulin developed. Eight healthy humans received 5 g arginine intravenously after a 12 hour fast and had blood sampled for 15 minutes; an additional 10 consumed a liquid mixed nutrient meal and prandial blood was taken for 180 minutes. None of the assays registered signal in plasma from proglucagon null mice, and specificity, background and cross-reactivity were acceptable in each. In response to IV arginine plasma glucagon increased 4-fold, and GLP-1 1.5-fold, with significant increases in 15-minute AUC; there was no significant change in either glicentin or oxyntomodulin. In response to meal ingestion there was no change in circulating glucagon, but oxyntomodulin, GLP-1 and glicentin increased 2, 3, and 4-fold respectively. These findings are generally compatible with PC1/3 dominant processing of PGP in the gut, but raise the possibility that α-cells produce both PC2 (glucagon) and PC1/3 (GLP-1) products.

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.

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