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.

Solid-phase iodothyronine-5′-deiodinase (5′-D) assays applied in production of monoclonal antibodies against 5′-D

1988 ◽  
Vol 118 (3) ◽  
pp. 439-445
Author(s):  
N. Boye ◽  
H. Frøkiaer ◽  
K. Kaltoftt ◽  
P. Laurberg
Keyword(s):  
Monoclonal Antibodies ◽  
Microsomal Fraction ◽  
Solid Phase ◽  
Rat Kidney ◽  
Enzyme Linked Immunosorbent Assay ◽  
Cortical Tissue ◽  
Spleen Cells ◽  
Hybridoma Cell Lines ◽  
Mouse Myeloma

ABSTRACT Characterization of iodothyronine-deiodinating enzymes has been difficult due to loss of enzyme activity during purification. To obtain a new tool for studying these enzymes we investigated the possibility of developing monoclonal antibodies (MAbs) against iodothyronine-5′-deiodinase (5′-D). Two specific and sensitive solid-phase microassays were developed for screening hybridoma supernatants for the presence of antibodies inhibiting rat kidney 5′-D. and antibodies binding to but not inhibiting the enzyme. BALB/c mice were immunized with a 3-((3-cholamidopropyl) -dimethylammonio) -1- propanesulphonate (CHAPS)-solubilized 5′-D-rich membrane preparation from rat kidney cortical tissue. Spleen cells were fused with NSI-Ag 4/1 mouse myeloma cells by means of polyethylene glycol. Two hybridoma cell lines (AF5 and BE8) secreting MAbs specifically binding to without inhibiting 5′-D were produced. The AF5 antibody was of the IgG2a subclass and the BE8 antibody of the IgG2b subclass. Binding of one of the antibodies to the enzyme inhibited binding of the other in both an enzyme-linked immunosorbent assay (ELISA) and a specific enzymebinding assay. CHAPS-solubilized kidney microsomal fraction was chromatographed on a Sepharose 6B column. Elution profiles of 5′-D activity and MAb-binding antigens, as measured by ELISA with both AF5 and BE8, were identical. Monoclonal antibodies should be valuable probes in the further elucidation of the nature of the iodothyronine-deiodinating activity in various tissues. J. Endocr. (1988) 118, 439–445

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 Generation of Rat Monoclonal Antibody to Detect Hydrogen Sulfide and Polysulfides in Biological Samples

Antioxidants ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 1160
Author(s):  
Shingo Kasamatsu ◽  
Yuki Kakihana ◽  
Taisei Koga ◽  
Hisashi Yoshioka ◽  
Hideshi Ihara
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Hydrogen Sulfide ◽  
Biological Samples ◽  
Biological Fluids ◽  
Enzyme Linked Immunosorbent Assay ◽  
Biological Processes ◽  
Pathological Conditions ◽  
Colorimetric Immunoassay ◽  
Hybridoma Cell Lines

Hydrogen sulfide (H2S) is endogenously produced by enzymes and via reactive persulfide/polysulfide degradation; it participates in a variety of biological processes under physiological and pathological conditions. H2S levels in biological fluids, such as plasma and serum, are correlated with the severity of various diseases. Therefore, development of a simple and selective H2S measurement method would be advantageous. This study aimed to generate antibodies specifically recognizing H2S derivatives and develop a colorimetric immunoassay for measuring H2S in biological samples. We used N-ethylmaleimide (NEM) as an H2S detection agent that forms a stable bis-S-adduct (NEM-S-NEM). We also prepared bis-S-heteroadduct with 3-maleimidopropionic acid, which, in conjugation with bovine serum albumin, was to immunize Japanese white rabbits and Wistar rats to enable generation of polyclonal and monoclonal antibodies, respectively. The generated antibodies were evaluated by competitive enzyme-linked immunosorbent assay. We could obtain two stable hybridoma cell lines producing monoclonal antibodies specific for NEM-S-NEM. By immunoassay with the monoclonal antibody, the H2S level in mouse plasma was determined as 0.2 μM, which was identical to the level detected by mass spectrometry. Taken together, these monoclonal antibodies can be a useful tool for a simple and highly selective immunoassay to detect H2S in biological samples.

scholarly journals Use of a sensitive bioimmunoabsorbent assay to isolate and characterize monoclonal antibodies to biologically active human erythropoietin

Blood ◽  
1988 ◽  
Vol 71 (6) ◽  
pp. 1731-1737
Author(s):  
AW Wognum ◽  
PM Lansdorp ◽  
CJ Eaves ◽  
G Krystal
Keyword(s):  
Monoclonal Antibodies ◽  
Specific Binding ◽  
Biologically Active ◽  
Enzyme Linked Immunosorbent Assay ◽  
Spleen Cells ◽  
Active Growth ◽  
Fixed Amount ◽  
Human Erythropoietin ◽  
Hybridoma Cell Lines ◽  
Culture Supernatants

At present, one of the most sensitive assays for human erythropoietin (Ep) is a bioassay that measures the Ep-dependent proliferation of spleen cells from phenylhydrazine-treated mice after 24 hours in culture. We describe how this assay can be used as the basis of a very sensitive method for detecting mouse antibodies to biologically active human Ep. In this procedure, microtiter wells are first coated with goat anti-mouse Ig antibody, then treated with mouse antibodies (serum or hybridoma culture supernatants), and finally incubated with a fixed amount of pure human Ep. Specific binding of anti-Ep antibodies is detected by adding spleen cells from phenylhydrazine-treated mice to the wells and measuring the ability of the cells to incorporate 3H- thymidine 24 hours later. This bioimmunosorbent assay (BISA) revealed the presence of anti-EP antibodies in sera from mice immunized with either pure human urinary Ep or a synthetic dodecapeptide corresponding to the aminoterminal region of Ep and in the culture supernatants from three of eight stable anti-Ep antibody-producing hybridoma cell lines that we have isolated. The three monoclonal antibodies showed similar reactivities in the BISA, but showed different affinities for Ep, with Kd values of approximately 0.7, 8, and 240 nmol/L, respectively. Further studies showed that all antibodies were capable of neutralizing Ep bioactivity and of binding 125I-labeled Ep in a radioimmunosorbent assay (RIA) but were virtually unreactive to Ep adsorbed to the bottom of enzyme-linked immunosorbent assay (ELISA) wells. Our results suggest that the BISA strategy may be an important complement to conventional RIA and ELISA techniques for identification of monoclonal antibodies specific for biologically active growth factors.

scholarly journals Comparison of Monoclonal Antibodies and Polymerase Chain Reaction Assays for the Typing of Isolates Belonging to the D and M Serotypes of Plum Pox Potyvirus

1998 ◽  
Vol 88 (3) ◽  
pp. 198-204 ◽  
Author(s):  
T. Candresse ◽  
M. Cambra ◽  
S. Dallot ◽  
M. Lanneau ◽  
M. Asensio ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Polymerase Chain Reaction ◽  
Monoclonal Antibodies ◽  
Large Scale ◽  
Direct Detection ◽  
Enzyme Linked Immunosorbent Assay ◽  
Polymorphism Analysis ◽  
Specific Monoclonal Antibody ◽  
Chain Reaction ◽  
Polymerase Chain

Plum pox potyvirus (PPV) isolates may be divided into four groups separated by serological, molecular, and epidemiological differences. Monoclonal antibodies specific for the two major groups of isolates, represented by the D and M serotypes of the virus, have been obtained. Polymerase chain reaction (PCR)-based assays allowing the direct detection and differentiation of PPV isolates have also been developed. We now report on a large-scale comparison of these two typing approaches. The results obtained show an overall excellent correlation between the results obtained in indirect double-antibody sandwich enzyme-linked immunosorbent assay using PPV-D— and PPV-M—specific monoclonal antibodies and those derived from either specific PCR assays or restriction fragment length polymorphism analysis of PCR fragments. Without exception, all isolates reacting positively with the PPV-M—specific monoclonal antibody were found to belong to the M serotype using the PCR-based assays, while 51 out of 53 isolates recognized by the D-specific monoclonal antibodies belonged to the D serotype according to the PCR typing results. However, failure to react with a specific monoclonal antibody did not prove as effective a predictor of the serotype of the isolate analyzed. In a few cases, the results obtained with the various techniques diverged, indicating low level variability of the epitopes recognized by the serotype-specific monoclonal antibodies. Isolates belonging to the two minor groups of PPV (El Amar and Cherry) also gave divergent results, indicating that the current typing assays are not suited for the analysis of such isolates.

scholarly journals Human megakaryocytes. V. Changes in the phenotypic profile of differentiating megakaryocytes.

1985 ◽  
Vol 161 (3) ◽  
pp. 457-474 ◽  
Author(s):  
R B Levene ◽  
J M Lamaziere ◽  
H E Broxmeyer ◽  
L Lu ◽  
E M Rabellino
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Solid Phase ◽  
Blood Platelets ◽  
Platelet Glycoprotein ◽  
Antigenic Determinants ◽  
Enzyme Linked Immunosorbent Assay ◽  
Phenotypic Profile ◽  
Phenotypic Changes

Human megakaryocytes were studied for phenotypic changes occurring throughout differentiation using a panel of monoclonal antibodies raised against marrow megakaryocytes and blood platelets. 11 monoclonal antibody preparations were selected for restricted specificity against megakaryocytes and/or platelets after screening by immunofluorescence, complement-mediated cytolysis, and solid phase enzyme-linked immunosorbent assay. The expression of the cellular epitopes recognized by these reagents enabled the identification of three levels of megakaryocyte maturation characterized by distinct immunologic phenotypes. Based upon their reactivities against megakaryocytic cells at different ontogenetic levels, monoclonal antibodies were operationally categorized into three groups. Group A consisted of six different monoclonal antibodies that recognized antigens on the colony-forming unit-megakaryocyte (CFU-Mk), in vitro grown colony megakaryocytes, and early immature marrow megakaryocytes, only, and did not detect their respective epitopes on either mature megakaryocytes or platelets. A monoclonal antibody categorized in group B detected a cell antigen expressed by megakaryocytic cells at all maturational levels, but which is lost or suppressed during terminal differentiation and is not expressed on blood platelets. Group C included four different monoclonal antibodies raised against platelets that recognized antigenic determinants expressed on the CFU-Mk, colony megakaryocytes, early and mature megakaryocytes, and platelets. Three group C monoclonal antibodies (PC-1, PC-3, and PC-4) were specific for platelet glycoprotein IIb/IIIa. Additionally, group C monoclonal antibody PC-2 was unique in that it showed partial reactivity against the clonable progenitor for the erythroid series (BFU-E). Recognition of discrete phenotypic changes in differentiating megakaryocytes will enable multiparameter analyses of these cells as well as the study of factors regulating the dynamics of megakaryocytopoiesis in health and disease.

scholarly journals Use of a sensitive bioimmunoabsorbent assay to isolate and characterize monoclonal antibodies to biologically active human erythropoietin

Blood ◽  
1988 ◽  
Vol 71 (6) ◽  
pp. 1731-1737 ◽  
Author(s):  
AW Wognum ◽  
PM Lansdorp ◽  
CJ Eaves ◽  
G Krystal
Keyword(s):  
Monoclonal Antibodies ◽  
Specific Binding ◽  
Biologically Active ◽  
Enzyme Linked Immunosorbent Assay ◽  
Spleen Cells ◽  
Active Growth ◽  
Fixed Amount ◽  
Human Erythropoietin ◽  
Hybridoma Cell Lines ◽  
Culture Supernatants

Abstract At present, one of the most sensitive assays for human erythropoietin (Ep) is a bioassay that measures the Ep-dependent proliferation of spleen cells from phenylhydrazine-treated mice after 24 hours in culture. We describe how this assay can be used as the basis of a very sensitive method for detecting mouse antibodies to biologically active human Ep. In this procedure, microtiter wells are first coated with goat anti-mouse Ig antibody, then treated with mouse antibodies (serum or hybridoma culture supernatants), and finally incubated with a fixed amount of pure human Ep. Specific binding of anti-Ep antibodies is detected by adding spleen cells from phenylhydrazine-treated mice to the wells and measuring the ability of the cells to incorporate 3H- thymidine 24 hours later. This bioimmunosorbent assay (BISA) revealed the presence of anti-EP antibodies in sera from mice immunized with either pure human urinary Ep or a synthetic dodecapeptide corresponding to the aminoterminal region of Ep and in the culture supernatants from three of eight stable anti-Ep antibody-producing hybridoma cell lines that we have isolated. The three monoclonal antibodies showed similar reactivities in the BISA, but showed different affinities for Ep, with Kd values of approximately 0.7, 8, and 240 nmol/L, respectively. Further studies showed that all antibodies were capable of neutralizing Ep bioactivity and of binding 125I-labeled Ep in a radioimmunosorbent assay (RIA) but were virtually unreactive to Ep adsorbed to the bottom of enzyme-linked immunosorbent assay (ELISA) wells. Our results suggest that the BISA strategy may be an important complement to conventional RIA and ELISA techniques for identification of monoclonal antibodies specific for biologically active growth factors.

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.

scholarly journals Studies on the syngeneic mixed lymphocyte reaction. III. Development of a monoclonal antibody with specificity for autoreactive T cells.

1983 ◽  
Vol 158 (4) ◽  
pp. 1307-1318 ◽  
Author(s):  
P B Hausman ◽  
C E Moody ◽  
J B Innes ◽  
J J Gibbons ◽  
M E Weksler
Keyword(s):  
Monoclonal Antibody ◽  
T Cells ◽  
Monoclonal Antibodies ◽  
Proliferative Response ◽  
Normal Mouse ◽  
Mixed Lymphocyte Reaction ◽  
Mouse Strains ◽  
Spleen Cells ◽  
Autoreactive T Cells

Monoclonal antibodies with specificity for autoreactive murine T cells have been developed. These antibodies inhibit proliferative response of splenic T cells activated by syngeneic spleen cells. These antibodies have no effect on the proliferative response of T cells activated by allogeneic spleen cells or PHA. The number of splenic T cells that react with these monoclonal antibodies is comparable in several normal mouse strains.

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