scholarly journals A Rapid Extraction Method Combined with a Monoclonal Antibody-Based Immunoassay for the Detection of Amatoxins

Toxins ◽  
2019 ◽  
Vol 11 (12) ◽  
pp. 724 ◽  
Author(s):  
Candace S. Bever ◽  
Robert M. Hnasko ◽  
Luisa W. Cheng ◽  
Larry H. Stanker
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Rapid Method ◽  
Extraction Method ◽  
Extraction Procedure ◽  
Detection Methods ◽  
Enzyme Linked Immunosorbent Assay ◽  
Mushroom Species ◽  
Rapid Extraction ◽  
Amanita Mushrooms

Amatoxins (AMAs) are lethal toxins found in a variety of mushroom species. Detection methods are needed to determine the occurrence of AMAs in mushroom species suspected in mushroom poisonings. In this manuscript, we report the generation of novel monoclonal antibodies (mAbs, AMA9G3 and AMA9C12) and the development of a competitive, enzyme-linked immunosorbent assay (cELISA) that is sensitive at 1 ng mL−1 and shows selectivity for α-amanitin (α-AMA) and γ-amanitin (γ-AMA), and less for β-amanitin (β-AMA). In order to decrease the overall time needed for analysis, the extraction procedure for mushrooms was also simplified. A rapid (1 min) extraction procedure of AMAs using solvents as simple as water alone was successfully demonstrated using Amanita mushrooms. Together, the extraction method and the mAb-based ELISA represent a simple and rapid method that readily detects AMAs extracted from mushroom samples.

scholarly journals Unreliable Measurement of Basophil Maximum Leukotriene Release with the Bühlmann CAST 2000 Enzyme-Linked Immunosorbent Assay Kit

2006 ◽  
Vol 13 (3) ◽  
pp. 420-422 ◽  
Author(s):  
S. E. Burastero ◽  
C. Paolucci ◽  
D. Breda ◽  
G. Monasterolo ◽  
R. E. Rossi ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Immunosorbent Assay ◽  
False Positive ◽  
Positive Control ◽  
Enzyme Linked Immunosorbent Assay ◽  
Positive Results

ABSTRACT The Bühlmann CAST 2000 enzyme-linked immunosorbent assay is a potentially useful assay for measuring sulfidoleukotrienes released in vitro by allergen-challenged basophils. However, we observed that the positive-control reagent yielded positive signals in cell-free systems. These false-positive results depended on using a mouse anti-FcεRI monoclonal antibody and were prevented by degranulation-inducing reagents other than mouse monoclonal antibodies.

Monoclonal antibodies specific for prothrombin fragment 1.2 and their use in a quantitative enzyme-linked immunosorbent assay

1993 ◽  
Vol 39 (4) ◽  
pp. 583-591 ◽  
Author(s):  
M J Hursting ◽  
B T Butman ◽  
J P Steiner ◽  
B M Moore ◽  
M C Plank ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Human Plasma ◽  
Sandwich Elisa ◽  
Peptide Sequence ◽  
Carboxyl Terminus ◽  
Enzyme Linked Immunosorbent Assay ◽  
Thrombotic Risk ◽  
Prothrombin Fragment ◽  
Amino Terminal

Abstract Prothrombin fragment 1.2 (F1.2) is an activation peptide generated during a critical event of blood coagulation, the conversion of prothrombin to thrombin. As a marker of thrombin generation, F1.2 has clinical potential in assessing thrombotic risk and monitoring anticoagulant therapy. In developing a highly specific, monoclonal antibody-based immunoassay of human plasma F1.2, we generated six murine anti-F1.2 monoclonal antibodies, using as immunogen a synthetic peptide (sequence: CGSD-RAIEGR) similar to the unique carboxyl terminus of F1.2. Each antibody bound F1.2 but not prothrombin. Epitope mapping studies with one antibody (5-3B) showed that optimum binding required six to eight amino acids plus a terminal arginine to emulate the F1.2 carboxyl terminus. A quantitative sandwich ELISA for human plasma F1.2 was configured with monoclonal antibody 5-3B as the capture antibody and peroxidase-labeled polyclonal antibodies to the F1.2 amino-terminal region as detector antibodies. Calibrators were prepared by adding purified F1.2, 0-10 nmol/L, to F1.2-depleted plasma. Assay characteristics included the following: mean (+/- SD) analytical recovery of 98% +/- 13%; no interference from lipemia, hemolysis, icterus, or thrombolytic agents; 0.08 nmol/L sensitivity; and mean intra- and interassay imprecision (three lots) < 12% at both low and high concentrations of F1.2.

Generation of Human Monoclonal Anti-idiotypic Antibodies with Specificity to the Murine Monoclonal Anti-CA 125 Antibody B43.13

1996 ◽  
Vol 11 (4) ◽  
pp. 211-215
Author(s):  
J.B. Oltrogge ◽  
B. Donnerstag ◽  
R.P. Baum ◽  
A.A. Noujaim ◽  
L. Träger
Keyword(s):  
Ovarian Cancer ◽  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Peripheral Blood Lymphocytes ◽  
Cross Reactivity ◽  
Tumor Burden ◽  
Ca 125 ◽  
Enzyme Linked Immunosorbent Assay ◽  
Human Monoclonal Antibodies ◽  
Ebv Transformation

Two human monoclonal antibodies, HID-7E7 and ROB-6F2, were produced by EBV transformation of peripheral blood lymphocytes (PBL). PBL were obtained from a patient with ovarian cancer who had been exposed several times to a Tc-99m labeled murine monoclonal anti-CA 125 antibody (B43.13, Biomira, Edmonton) for immunoscintigraphy. The HID-7E7 and ROB-6F2 producing B-cells were cloned with a limiting dilution technique and have shown stable immunoglobulin secretion within a period of three years. The human monoclonal antibodies HID-7E7 and ROB-6F2 are of the IgG isotype, and bind with significant affinity to the murine monoclonal antibody B43.13, which was used for immunoscintigraphy. Binding affinity of ROB-6F2 to other murine antibodies could not be detected. Cross reactivity of HID-7E7 to a murine anti-CEA monoclonal antibody was observed. In order to verify the anti-idiotypic character of the generated human antibodies, the ability of HID-7E7 and ROB-6F2, respectively, to inhibit the formation of the CA125/B43.13 complex is demonstrated via an enzyme-linked immunosorbent assay. These human anti-idiotypic antibodies are possible candidates for immunotherapy of ovarian cancer in patients with a small tumor burden following surgery and/or chemotherapy.

Monoclonal antibodies against plasma protease inhibitors: II. Production and characterization of 25 monoclonal antibodies against human α1-antitrypsin. Correlation between antigenic structure and functional sites

1984 ◽  
Vol 4 (2) ◽  
pp. 139-147 ◽  
Author(s):  
P. Hérion ◽  
D. Siberdt ◽  
M. Francotte ◽  
J. Urbain ◽  
A. Bollen
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Protease Inhibitors ◽  
Antigenic Structure ◽  
Light Chains ◽  
Enzyme Linked Immunosorbent Assay ◽  
Functional Sites ◽  
Heavy Chains ◽  
Low Levels

Twenty-five hybridomas secreting monoclonal antibodies against human α1-antitrypsim have been produced by the cell-fusion techmque (Kóhler and Milstein, 1976). All antibodies are specific for α1-antitrypsim and carry γ1-antitrypsim heavy chains and κ light chains. Inhibition experiments showed that these monoclonal antibodies define three independent antigenic regions on the α1-antitrypsim molecule; one of these domains appears to be involved in the interaction between α1-antitrypsim and trypsin. In addition, one monoclonal antibody, AATY39, was used to develop an enzyme-linked immunosorbent assay capable of detecting low levels of α1-antitrypsim in the range of 1 to 2 ng/ml.

Development of the ELISAs for detection of hormone-disrupting chemicals

2000 ◽  
Vol 42 (7-8) ◽  
pp. 81-88 ◽  
Author(s):  
Y. Goda ◽  
A. Kobayashi ◽  
K. Fukuda ◽  
S. Fujimoto ◽  
M. Ike ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Phthalate Esters ◽  
Hybridoma Cells ◽  
Enzyme Linked Immunosorbent Assay ◽  
Spleen Cells ◽  
Myeloma Cells ◽  
Structural Resemblance ◽  
Reaction Test ◽  
Mouse Myeloma

Six kinds of enzyme-linked immunosorbent assay (ELISA) systems were developed for the quantitative analysis of hormone-disrupting chemicals (HDCs), such as estrogen (ES: the total amount of estrone (E1), 17 β-estra (E2) and estriol (E3)), E2, bisphenol A (BPA), alkylphenol (AP), phthalate esters (PE) and chlorophenols (CP). To generate specific monoclonal antibodies against BPA, AP, PE, CP, hybridoma cells were produced by the fusion of mouse myeloma cells and spleen cells from mice immunized with carboxylated derivatives, while anti E2 monoclonal antibody was selected from those available on the market, and anti ES monoclonal antibody was purchased from Teikoku Hormone Mfg Co. Ltd. The detection limits of ES, E2, BPA, AP, PE and CP ELISAs were 0.1, 0.1, 5, 10, 200, 10 μg/L, when E2, E2, BPA, Nonylphenol (NP), Dibutylphthalate (DBP), 2,4-CP were used as standard, respectively, and the specificity of each ELISA was confirmed with the cross-reaction test using several compounds which have structural resemblance to the compounds of interest.

scholarly journals Comparative study of adenoviruses with monoclonal antibodies

1992 ◽  
Vol 34 (1) ◽  
pp. 19-26 ◽  
Author(s):  
Terezinha Maria de Paiva ◽  
Sueko Takimoto ◽  
María Akíko Ishida ◽  
María Candida Oliveira de Souza ◽  
Tuneo Ishimaru ◽  
...  
Keyword(s):  
Cell Culture ◽  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Comparative Study ◽  
Neutralization Test ◽  
Human Adenovirus ◽  
Enzyme Linked Immunosorbent Assay ◽  
Viral Neutralization ◽  
Species Specific ◽  
Viral Neutralization Test

The obtainment of monoclonal antibodies for adenovirus species 4(Ad4) is described.The specificities of selected monoclonal antibodies were determined by means of viral neutralization test in cell culture, immunofluorescence and Enzyme-Linked Immunosorbent Assay (ELISA), in the presence of the following species of human adenovirus: 1, 2, 5 (subgenus C), 4 (subgenus E), 7 and 16 (subgenus B) and 9 (subgenus D). Two monoclonal antibodies species specific to adenovirus 4 (1CIII and 3DIII) and one monoclonal antibody that cross reacted with adenovirus species 4 and 7 (2HIII) were obtained.

Surface antigens present on vegetative Rhizobium meliloti cells may be diminished or absent when cells are in the bacteroid form

1992 ◽  
Vol 38 (6) ◽  
pp. 506-509 ◽  
Author(s):  
Perry Olsen ◽  
Mandy Collins ◽  
Wendell Rice
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Rhizobium Meliloti ◽  
Fluorescent Antibody ◽  
Surface Antigens ◽  
Complete Loss ◽  
Enzyme Linked Immunosorbent Assay ◽  
Vegetative Cells ◽  
Antibody Staining ◽  
Specific Antigens

The results of fluorescent antibody staining of three Rhizobium meliloti strains using three "strain-cross-reactive" and three "strain-specific" monoclonal antibodies indicated that strain-specific antigens present on surfaces of laboratory-cultured vegetative cells were diminished or absent on surfaces of bacteroid cells extracted from alfalfa nodules formed by the respective strains. In contrast, the monoclonal antibody cross-reactive antigens appeared to be generally conserved during the transition from vegetative cells to bacteroids. These results offer a basis for observed reductions or complete loss of capability of some "strain-specific" monoclonal antibodies developed against vegetative Rhizobium cells to detect the strains in nodule squash material by enzyme-linked immunosorbent assay. Key words: Rhizobium, monoclonal antibody, bacteroid, surface antigen, fluorescent antibody.

scholarly journals Identification of Genogroup I and Genogroup II Broadly Reactive Epitopes on the Norovirus Capsid

2005 ◽  
Vol 79 (12) ◽  
pp. 7402-7409 ◽  
Author(s):  
Tracy Dewese Parker ◽  
Noritoshi Kitamoto ◽  
Tomoyuki Tanaka ◽  
Anne M. Hutson ◽  
Mary K. Estes
Keyword(s):  
Monoclonal Antibody ◽  
Immune Response ◽  
Monoclonal Antibodies ◽  
Capsid Protein ◽  
Enzyme Linked Immunosorbent Assay ◽  
Linear Epitope ◽  
Norwalk Virus ◽  
Diagnostic Assays ◽  
Separate Genus ◽  
The Family

ABSTRACT Norwalk virus, a member of the family Caliciviridae, is an important cause of acute epidemic nonbacterial gastroenteritis. Norwalk and related viruses are classified in a separate genus of Caliciviridae called Norovirus, which is comprised of at least three genogroups based on sequence differences. Many of the currently available immunologic reagents used to study these viruses are type specific, which limits the identification of antigenically distinct viruses in detection assays. Identification of type-specific and cross-reactive epitopes is essential for designing broadly cross-reactive diagnostic assays and dissecting the immune response to calicivirus infection. To address this, we have mapped the epitopes on the norovirus capsid protein for both a genogroup I-cross-reactive monoclonal antibody and a genogroup II-cross-reactive monoclonal antibody by use of norovirus deletion and point mutants. The epitopes for both monoclonal antibodies mapped to the C-terminal P1 subdomain of the capsid protein. Although the genogroup I-cross-reactive monoclonal antibody was previously believed to recognize a linear epitope, our results indicate that a conformational component of the epitope explains the monoclonal antibody's genogroup specificity. Identification of the epitopes for these monoclonal antibodies is of significance, as they are components in a commercially available norovirus-diagnostic enzyme-linked immunosorbent assay.

Sensitive Measurement of Thrombopoietin by a Monoclonal Antibody Based Sandwich Enzyme-linked Immunosorbent Assay

1997 ◽  
Vol 78 (04) ◽  
pp. 1262-1267 ◽  
Author(s):  
Claudia C Folman ◽  
Albert E G K von dem Borne ◽  
Irma H J A M Rensink ◽  
Winald Gerritsen ◽  
C Ellen van der Schoot ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Immunosorbent Assay ◽  
Platelet Transfusion ◽  
Large Scale ◽  
Limit Of Detection ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Reliable Tool

SummaryIn this report a sensitive enzyme-linked immunosorbent assay (ELISA) for the measurement of plasma thrombopoietin (Tpo) is described that is solely based on monoclonal antibodies (MoAbs).The assay has an intra and inter-assay variance of 5-7% and 7-13%, respectively. Native and recombinant human Tpo (rhTpo) were recognized equally well, no cross reactivity with other cytokines was found and rhTpo added to plasma and serum was completely recovered. With the ELISA, Tpo concentrations in EDTA-anticoagulated plasma of all controls (n = 193) could be determined, since the limit of detection (2 ± 0.8 A.U./ml, mean ± sd) was lower than the concentration found in controls (11 ± 8 A.U./ml, mean ± sd; 2.5th-97.5th percentile: 4-32 A.U./ml). Tpo levels in serum were on average 3.4 times higher than in plasma.We showed in vivo that Tpo is bound by platelets, as in thrombocytopenic patients (n = 5) a platelet transfusion immediately led to a drop in plasma Tpo level, whereas in patients receiving chemotherapy the induced thrombocytopenia was followed by a rise in plasma Tpo levels.In summary, these results indicate that this ELISA is a reliable tool for Tpo measurements and is applicable for large scale studies.

Production of Sensitive Monoclonal Antibodies to Aflatoxin B1and Aflatoxin M1 and Their Application to ELISA of Naturally Contaminated Foods

1988 ◽  
Vol 51 (3) ◽  
pp. 201-204 ◽  
Author(s):  
D. E. DIXON-HOLLAND ◽  
J. J. PESTKA ◽  
B. A. BIDIGARE ◽  
W. L. CASALE ◽  
R. L. WARNER ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Solid Phase ◽  
Direct Detection ◽  
Aflatoxin M1 ◽  
Enzyme Linked Immunosorbent Assay ◽  
Spleen Cells ◽  
Direct Elisa ◽  
Simple Extraction ◽  
Hybridoma Cell Lines

Two new hybridoma Cell lines capable of secreting sensitive monoclonal antibodies for aflatoxin B1 (AFB1) and aflatoxin M1 (AFM1), were produced by fusing NS-1 myeloma cells with spleen cells of BALB/c female mice immunized with AFB1- and AFM1-carboxymethyloxime bovine serum albumin conjugates, respectively. Detection limits for these antibodies in the direct enzyme-linked immunosorbent assay (ELISA) were 0.5 ng/ml for AFB1 and 0.25 ng/ml for AFM1 Concentrations of AFB1 analogs (ng/ml) required to inhibit 50% binding of AFB1,-perioxidase conjugate to AFB1 monoclonal antibody solid phase in direct ELISA were: AFB1, 2.6; AFB2, 13; AFG1, 8; AFB2, 15; AFM1, 23. Analog concentrations (ng/ml) required to inhibit 50% binding of AFB1,-perioxidase conjugate to AFM1 monoclonal antibody solid phase were: AFM1,0.8; AFM2, 700; AFB1, 0.5; AFB2, 35; AFB2a, >10,000; AFG1, 12; AFG2a, 12; AFP1, 16; and AFQ1, 9.2. These new monoclonal antibodies were applicable to both the ELISA detection AFB1 in corn, cottonseed, cottonseed meal, and mixed feed following a simple extraction in 55% methanol as well as the direct detection of AFM1 in milk.

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