scholarly journals Development and Application of a Novel Rapid and Throughput Method for Broad-Spectrum Anti-Foodborne Norovirus Antibody Testing

2021 ◽  
Vol 12 ◽  
Author(s):  
Yueting Zuo ◽  
Liang Xue ◽  
Junshan Gao ◽  
Yingyin Liao ◽  
Yueting Jiang ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Broad Spectrum ◽  
High Throughput Screening ◽  
Indirect Elisa ◽  
Polyclonal Antibodies ◽  
Enzyme Linked Immunosorbent Assay ◽  
Elisa Assay ◽  
Antibody Testing ◽  
Coefficients Of Variation ◽  
P Proteins

Foodbone norovirus (NoV) is the leading cause of acute gastroenteritis worldwide. Candidate vaccines are being developed, however, no licensed vaccines are currently available for managing NoV infections. Screening for stimulated antibodies with broad-spectrum binding activities can be performed for the development of NoV polyvalent vaccines. In this study, we aimed to develop an indirect enzyme-linked immunosorbent assay (ELISA) for testing the broad spectrum of anti-NoV antibodies. Capsid P proteins from 28 representative NoV strains (GI.1–GI.9 and GII.1–GII.22 except GII.11, GII.18, and GII.19) were selected, prepared, and used as coating antigens on one microplate. Combined with incubation and the horseradish peroxidase chromogenic reaction, the entire process for testing the spectrum of unknown antibodies required 2 h for completion. The intra-assay and inter-assay coefficients of variation were less than 10%. The new method was successfully performed with monoclonal antibodies and polyclonal antibodies induced by multiple antigens. In conclusion, the indirect ELISA assay developed in this study had a good performance of reliability, convenience, and high-throughput screening for broad-spectrum antibodies.

Monoclonal antibodies can precipitate low-density lipoprotein. I. Characterization and use in determining apolipoprotein B.

1985 ◽  
Vol 31 (10) ◽  
pp. 1654-1658 ◽  
Author(s):  
S Marcovina ◽  
D France ◽  
R A Phillips ◽  
S J Mao
Keyword(s):  
Monoclonal Antibodies ◽  
Apolipoprotein B ◽  
Polyclonal Antibodies ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Radial Immunodiffusion ◽  
Enzyme Linked Immunosorbent Assay ◽  
Low Density ◽  
Apo B ◽  
Blotting Technique

Abstract We produced 20 mouse monoclonal antibodies against human plasma low-density lipoprotein (LDL). Individually they failed to precipitate LDL in agarose gel by the double-immunodiffusion technique; collectively they did, or as few as two combined monoclonal antibodies could do so. To mimic polyclonal antibodies in determination of apolipoprotein B (apo B) by radial immunodiffusion, a combination of four particular monoclonal antibodies (clones A, B, C, and D) was necessary. We characterized these four clones with respect to temperature dependency, affinity, total binding to 125I-labeled LDL, and specificity to the different species of apolipoprotein B. Two monoclonal antibodies (B and C) bound 100% of 125I-labeled LDL; clones A and D bound 80% and 87%, respectively. All four clones bound maximally to LDL at 4 degrees C. The affinity constants for clones A, B, C, and D were 0.6, 2.1, 3.8, and 2.3 X 10(9) L/mol, respectively. By the Western blotting technique, the four monoclonal antibodies all reacted with the species B-100 and B-74 of apolipoprotein B, and to various degrees with B-48 and B-26. Radial immunodiffusion (chi) and direct enzyme-linked immunosorbent assay (y) with a mixture of the four monoclonal antibodies gave almost identical results for 70 patients: y = 0.921 chi-2.58; r = 0.933.

scholarly journals Development of Murine Monoclonal Antibodies for the Immunohistochemical Diagnosis of Systemic Bovine Aspergillosis

1996 ◽  
Vol 8 (1) ◽  
pp. 68-75 ◽  
Author(s):  
H. E. Jensen ◽  
B. Aalbaek ◽  
P. Lind ◽  
H. V. Krogh ◽  
P. L. Frandsen
Keyword(s):  
Monoclonal Antibodies ◽  
Polyclonal Antibodies ◽  
Fungal Species ◽  
Cross Reactivity ◽  
Water Soluble ◽  
Enzyme Linked Immunosorbent Assay ◽  
Immunohistochemical Techniques ◽  
Immunohistochemical Diagnosis ◽  
Murine Monoclonal Antibodies

Murine monoclonal antibodies (MAbs) against water-soluble somatic antigens (WSSA) and the wall fraction (WF) from Aspergillus fumigatus were produced by fusion of splenocytes from immunized BALB/c mice with mouse myeloma X63-Ag 8.653 cells. The supernatants of in vitro cultured hybridomas were initially screened for reactivity with the WSSA and the WF from A. fumigatus and WSSA of other fungi in an enzyme-linked immunosorbent assay (ELISA). Supernatants reacting only with A. fumigatus antigens were subsequently screened for homologous and heterologous reactivity with immunohistochemical techniques using formalin-fixed, paraffin-embedded tissues from experimentally infected mice. Because of a high immunohistochemical reactivity with homologous fungi, 4 MAbs raised against A. fumigatus WSSA and WF were selected for a further evaluation of cross-reactivity (diagnostic specificity) in immunohistochemical and immunoblotting assays. In immunohistochemical assays, all MAbs raised against WSSA cross-reacted heavily with a number of other fungal species. All 4 MAbs (MAb-WF-AF-1-4) raised against the WF reacted strongly with hyphae of Aspergillus spp.; hyphae of Scedosporium apiospermum were also strongly labeled by MAb-WF-AF-3 and-4. The 2 specifically reacting MAbs (MAb-WF-AF-1 and-2) were of the IgM biotype and were precipitating, and in immunoblotting experiments both bound to a 106-kD antigen of the WF, whereas they did not bind to WSSA of A. fumigatus. One of the 2 aspergillosis-specific MAbs (MAb-WF-AF-1) was used to screen 145 mycotic lesions of cattle. The diagnoses on bovine lesions obtained by MAb-WF-AF-1 were compared with results based on reactivity with heterologously absorbed polyclonal antibodies and, for some lesions, to culture results. In the vast majority of lesions ( n = 133), the MAb-WF-AF-1 and the polyclonal anti-Aspergillus antibodies reacted in a similar pattern, i.e., positively in 41 aspergillosis lesions and negatively in 92 zygomycotic lesions. Hyphae in 3 of 12 lesions that were not stained by the polyclonal antibodies reacted with the specific MAb-WF-AF-1; i.e., aspergillosis was diagnosed. The characteristics of the 2 MAbs (MAb-WF-AF-1 and-2) raised against the WF of A. fumigatus in ELISA and immunoblotting and immunohistochemical assays justify their application for the in situ diagnosis of systemic aspergillosis of cattle.

Development of Enzyme Immunoassay for Captan and Its Degradation Product Tetrahydrophthalimide in Foods

1993 ◽  
Vol 76 (2) ◽  
pp. 381-386 ◽  
Author(s):  
W Harvey Newsome ◽  
Jupiter M Yeung ◽  
Peter G Collins
Keyword(s):  
Detection Limit ◽  
Human Serum Albumin ◽  
Degradation Product ◽  
Protein Concentration ◽  
Polyclonal Antibodies ◽  
Enzyme Linked Immunosorbent Assay ◽  
Test Compound ◽  
Coefficients Of Variation ◽  
Spacer Arm ◽  
Related Compounds

Abstract A simple, sensitive, and precise enzyme-linked immunosorbent assay (ELISA) is described for the quantitation of captan as its degradation product tetrahydrophthalimide (THPI) in foods using polyclonal antibodies. Three hapten analogues of THPI with different alky I spacer arm lengths were synthesized. Immunogens and coating proteins were prepared by coupling these haptens to human serum albumin and ovalbumin, respectively. A 5-carbon spacer arm appeared to be optimum for the production of antibodies. Heterologous coating proteins did not improve the sensitivity, but reduction of homologous coating protein concentration did improve the sensitivity, resulting in a concentration of test compound required to inhibit binding by 50% of 15.5 ng/mL The antiserum is specific for captan, captafol, and THPI, but not other structurally related compounds. The minimum detection limit was 1 ng/mL; the linearity was 1-200 ng/mL. The overall recoveries of captan and THPI from 11 commodities spiked at 4 levels were 92 and 100%, respectively. The intra-assay and interassay coefficients of variation were 9.1 and 16.8% for apple blanks and 5.9 and 4.2% for apple spiked with 3 ppm THPI, respectively. The ELISA described is suitable for measuring captan and THPI at levels comparable to those typically found in fruit.

scholarly journals Generation and diagnostic application of monoclonal antibodies against Seneca Valley virus

2011 ◽  
Vol 24 (1) ◽  
pp. 42-50 ◽  
Author(s):  
Ming Yang ◽  
Rebekah van Bruggen ◽  
Wanhong Xu
Keyword(s):  
Monoclonal Antibodies ◽  
Indirect Elisa ◽  
Foot And Mouth Disease ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dot Blot ◽  
Specific Antibody Response ◽  
Dot Blot Assay ◽  
Infected Cells ◽  
Seneca Valley Virus ◽  
Vesicular Disease

Seneca Valley virus (SVV), a member of the Picornaviridae family, was implicated in a suspicious vesicular disease discovered in pigs from Canada in 2007. Because any outbreak of vesicular disease in pigs is assumed to be foot-and-mouth disease (FMD) until confirmed otherwise, a test for diagnosing the presence of SVV would be a very useful tool. To develop the diagnostic tests for SVV infection, 5 monoclonal antibodies (mAbs) were produced from mice immunized with binary ethylenimine (BEI)-inactivated SVV. Using a dot blot assay, the reactivity of the mAbs was confirmed to be specific for SVV, not reacting with any of the other vesicular disease viruses tested. The mAbs demonstrated reactivity with SVV antigen in infected cells by an immunohistochemistry assay. An SVV-specific competitive enzyme-linked immunosorbent assay (cELISA) was developed using BEI-inactivated SVV antigen and a mAb for serodiagnosis. The cELISA results were compared to the indirect isotype (immunoglobulin [Ig]M and IgG) ELISA and the virus neutralization test. All SVV experimentally inoculated pigs exhibited a positive SVV-specific antibody response at 6 days postinoculation, and the sera remained positive until the end of the experiment on day 57 (>40% inhibition) using the cELISA. The cELISA reflected the profile of the indirect ELISA for both IgM and IgG. This panel of SVV-specific mAbs is valuable for the identification of SVV antigen and the serological detection of SVV-specific antibodies.

Detection of Abrin in Food Using Enzyme-Linked Immunosorbent Assay and Electrochemiluminescence Technologies

2008 ◽  
Vol 71 (9) ◽  
pp. 1868-1874 ◽  
Author(s):  
ERIC A. E. GARBER ◽  
JENNIFER L. WALKER ◽  
THOMAS W. O'BRIEN
Keyword(s):  
Monoclonal Antibodies ◽  
Immunosorbent Assay ◽  
Dairy Products ◽  
Polyclonal Antibodies ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Ribosome Inactivating Protein ◽  
Limits Of Detection ◽  
Abrus Precatorius ◽  
A Priority

Abrin is a toxic ribosome-inactivating protein present in beans of Abrus precatorius, also known as rosary peas. The possibility that abrin could be used to adulterate food has made the development of assays for the detection of abrin a priority. Rabbit-derived polyclonal antibodies and mouse monoclonal antibodies were prepared against a mixture of abrin isozymes. The specificity and cross-reactivity of the antibodies were evaluated against a challenge library of 40 grains, nuts, legumes, and foods. An enzyme-linked immunosorbent assay (ELISA) and an electrochemiluminescence (ECL)–based assay were assembled and optimized. Polyclonal (capture) and polyclonal (detection) ELISAs, polyclonal and monoclonal ELISAs, and polyclonal and monoclonal ECL assays had limits of detection (LODs) of 0.1 to 0.5 ng/ml for abrin in buffer. The LOD for abrin dissolved into juices, dairy products, soda, chocolate drink, and condiments and analyzed with the ECL assay ranged from 0.1 to 0.5 ng/ml in the analytical sample. In contrast, the LODs for the ELISAs ranged from 0.5 to 10 ng/ml in the analytical sample.

scholarly journals Development of an Enzyme-Linked Immunosorbent Assay for Serotyping Ureaplasma urealyticum Strains Using Monoclonal Antibodies

2001 ◽  
Vol 8 (1) ◽  
pp. 52-57 ◽  
Author(s):  
Fedoua Echahidi ◽  
Gaëtan Muyldermans ◽  
Sabine Lauwers ◽  
Anne Naessens
Keyword(s):  
Monoclonal Antibodies ◽  
Immunosorbent Assay ◽  
Ureaplasma Urealyticum ◽  
Polyclonal Antibodies ◽  
Enzyme Linked Immunosorbent Assay ◽  
Elisa Method ◽  
Cross Reactions ◽  
Antigenic Preparation ◽  
Negative Controls ◽  
Reference Strains

ABSTRACT Ureaplasma urealyticum comprises 14 serotypes. The existing serotyping methods all use polyclonal antibodies. These methods are time-consuming and labor-intensive, and they cannot always be performed on primary isolates; in addition, the results are difficult to interpret. We developed a new enzyme-linked immunosorbent assay (ELISA) method using serotype-specific monoclonal antibodies (MAbs) to enable the serotyping of U. urealyticum isolates from primary broth cultures. Each of the 14 serotype reference strains was tested against 14 selected MAbs. Homologous reactions were very strong, while heterologous reactions were negligible. Three cross-reactions were observed: MAb 5 cross-reacted with serotype 2, MAb 14 cross-reacted with serotype 3, and MAb 8 cross-reacted with serotype 13. Despite the cross-reactions observed, all the serotype reference strains ofU. urealyticum could be identified and differentiated using a combination of MAbs. Reproducibility was analyzed with a fractionated antigenic preparation and with several freshly prepared antigens of the same strain. No significant interrun variation was found with the fractionated antigen, but significant variations in optical density (OD) values were found when freshly prepared antigens were tested. However, the variation in OD values did not influence the overall interpretation of the ELISA: reactions with homologous MAbs were always prominent compared to those of the negative controls. This newly developed ELISA using MAbs seems promising for serotyping of U. urealyticum clinical isolates.

scholarly journals Production of Monoclonal Antibodies Specific for the i and 1,2 Flagellar Antigens of Salmonella typhimurium and Characterization of Their Respective Epitopes

1998 ◽  
Vol 64 (12) ◽  
pp. 5033-5038 ◽  
Author(s):  
N. de Vries ◽  
K. A. Zwaagstra ◽  
J. H. J. Huis in’t Veld ◽  
F. van Knapen ◽  
F. G. van Zijderveld ◽  
...  
Keyword(s):  
Amino Acids ◽  
Monoclonal Antibodies ◽  
Salmonella Typhimurium ◽  
Immunosorbent Assay ◽  
Specific Antibody ◽  
Polyclonal Antibodies ◽  
Enzyme Linked Immunosorbent Assay ◽  
Specific Antibodies ◽  
Minimal Area

ABSTRACT Salmonella typhimurium expresses two antigenically distinct flagellins, each containing a different H antigen (i and 1,2), the combination of which is highly specific for this serotype. In this study, overlapping recombinant flagellin fragments were constructed from the fliC(H:i) and fljB (H:1,2) flagellin genes, and the expression products were tested for binding to H antigen-specific monoclonal and polyclonal antibodies. A minimal area, 86 amino acids for H:i and 102 amino acids for H:1,2, located in the central variable domain of each flagellin was required for the binding of serotype-specific antibodies, providing further evidence for the presence of a discontinuous H epitope. Two peptides comprising these areas were shown to be highly suitable for application as antigens in an enzyme-linked immunosorbent assay detecting S. typhimurium-specific antibody.

Immunostick Colorimetric Assay for Detection of Pseudomonas spp. in Refrigerated Meat and Milk

1997 ◽  
Vol 60 (8) ◽  
pp. 908-911 ◽  
Author(s):  
ROSALBA GUTIÉRREZ ◽  
TERESA GARCÍA ◽  
ISABEL GONZÁLEZ ◽  
BERNABÉ SANZ ◽  
PABLO E. HERNÁNDEZ ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Pseudomonas Fluorescens ◽  
Colorimetric Assay ◽  
Detection Threshold ◽  
Live Cells ◽  
Enzyme Linked Immunosorbent Assay ◽  
Elisa Assay ◽  
Pseudomonas Spp ◽  
Milk Samples ◽  
Elisa Format

Monoclonal antibodies generated against live cells of Pseudomonas fluorescens have been used in an immunostick enzyme-linked immunosorbent assay (ELISA) format for the detection of Pseudomonas spp. in refrigerated meat and milk. The detection threshold for the immunostick ELISA assay developed in this work is 104 CPU cm−2 for meat and 105 CPU ml−1 for milk samples.

scholarly journals Determination of Egg Proteins in Food Products by Enzyme Immunoassay

2000 ◽  
Vol 83 (1) ◽  
pp. 139-143 ◽  
Author(s):  
Jupiter M Yeung ◽  
W Harvey Newsome ◽  
Michael A Abbott
Keyword(s):  
Detection Limit ◽  
Enzyme Immunoassay ◽  
Polyclonal Antibodies ◽  
Enzyme Linked Immunosorbent Assay ◽  
Antigen Binding ◽  
Food Ingredients ◽  
Coefficients Of Variation ◽  
Egg Products ◽  
Whole Egg

Abstract An enzyme-linked immunosorbent assay (ELISA) was developed to determine the presence of egg proteins in foods. The polyclonal antibodies developed were specific to whole egg proteins and did not cross-react with any of the 38 nuts, legumes, or other common food ingredients tested. The concentrations of egg proteins that will inhibit 50% of antibody–antigen binding, IC50, were 3–7 ng/mL, and the linear range was 0.5–62.5 ng/mL. The detection limit was 0.2 ppm for various foods. Recoveries ranged from 67 to 96%. The intra- and inter-assay coefficients of variation in this procedure were 10–13% for ice cream spiked at 0.8 and 1.6 ppm. The ELISA has been applied to ice creams, noodles, pasta, and breads. Egg proteins were identified in all declared egg products, and no false positives were found.

scholarly journals Characterization and Formulation of Multiple Epitope-Specific Neutralizing Monoclonal Antibodies for Passive Immunization against Cryptosporidiosis

2000 ◽  
Vol 68 (5) ◽  
pp. 2608-2616 ◽  
Author(s):  
Deborah A. Schaefer ◽  
Beth A. Auerbach-Dixon ◽  
Michael W. Riggs
Keyword(s):  
Monoclonal Antibodies ◽  
Cryptosporidium Parvum ◽  
Polyclonal Antibodies ◽  
Passive Immunization ◽  
Infection Process ◽  
Enzyme Linked Immunosorbent Assay ◽  
Intestinal Infection ◽  
Coccidian Parasite ◽  
Epitope Specificity ◽  
Significant Efficacy

ABSTRACT The coccidian parasite Cryptosporidium parvum causes diarrhea in humans, calves, and other mammals. Neither immunization nor parasite-specific pharmaceuticals that are consistently effective against this organism are available. While polyclonal antibodies against whole C. parvum reduce infection, their efficacy and predictability are suboptimal. We hypothesized that passive immunization against cryptosporidiosis could be improved by using neutralizing monoclonal antibodies (MAbs) targeting functionally defined antigens on the infective stages. We previously reported that the apical complex and surface-exposed zoite antigens CSL, GP25-200, and P23 are critical in the infection process and are therefore rational targets. In the present study, a panel of 126 MAbs generated against affinity-purified CSL, GP25-200, and P23 was characterized to identify the most efficacious neutralizing MAb formulation targeting each antigen. To identify neutralizing MAbs, sporozoite infectivity following exposure to individual MAbs was assessed by enzyme-linked immunosorbent assay. Of 126 MAbs evaluated, 47 had neutralizing activity. These were then evaluated individually in oocyst-challenged neonatal mice, and 14 MAbs having highly significant efficacy were identified for further testing in formulations. Epitope specificity assays were performed to determine if candidate MAbs recognized the same or different epitopes. Formulations of two or three neutralizing MAbs, each recognizing distinct epitopes, were then evaluated. A formulation of MAbs 3E2 (anti-CSL [αCSL]), 3H2 (αGP25-200), and 1E10 (αP23) provided highly significant additive efficacy over that of either individual MAbs or combinations of two MAbs and reduced intestinal infection by 86 to 93%. These findings indicate that polyvalent neutralizing MAb formulations targeting epitopes on defined antigens may provide optimal passive immunization against cryptosporidiosis.

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