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.

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.

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.

Production of species-specific monoclonal antibodies that react with surface components on zoospores and cysts of Phytophthora nicotianae

1998 ◽  
Vol 44 (12) ◽  
pp. 1161-1170 ◽  
Author(s):  
A V Robold ◽  
A R Hardham
Keyword(s):  
Monoclonal Antibodies ◽  
Cell Lines ◽  
Phytophthora Cinnamomi ◽  
Phytophthora Nicotianae ◽  
The Other ◽  
Hybridoma Cells ◽  
Enzyme Linked Immunosorbent Assay ◽  
Specific Antibodies ◽  
Hybridoma Cell Lines ◽  
Species Specific

Monoclonal antibodies were generated against components on the surface of zoospores and cysts of the Oomycete, Phytophthora nicotianae, with the aim of obtaining antibodies diagnostic for this species of plant pathogen. A dipstick version of an enzyme-linked immunosorbent assay was used to screen hybridoma cell lines produced by following a coimmunization protocol in which a mouse was immunized with Phytophthora nicotianae cysts mixed with murine antisera raised against cysts of Phytophthora cinnamomi and Phytophthora cryptogea. Of the nine hybridoma cells lines which remained positive, five produced antibodies that reacted with species-specific epitopes on the surface of the spores. Immunofluorescence, immunogold, and immunoblot labelling showed that three of the five species-specific antibodies reacted with a polypeptide of relative molecular mass greater than 205 kDa which was distributed over the entire zoospore surface, including that of the two flagella. These antibodies also labelled the surface of cysts to varying degrees. The other two species-specific antibodies bound to the shaft of tubular mastigonemes that form two rows on the anterior flagellum. In immunoblots, one of these antibodies recognised a 40-kDa glycoprotein. Antibodies produced by the other four hybridoma cell lines reacted with all Phytophthora and Pythium species tested. The results (i) showed that the coimmunization technique effectively produced antibodies directed towards components specific for Phytophthora nicotianae in the presence of antigens common to many Phytophthora species, and (ii) revealed for the first time the biochemical nature of molecular constituents of flagellar mastigonemes in the Oomycetes.Key words: cell surface, flagella, immunodiagnostics, mastigonemes, monoclonal antibodies.

Detection of total bile acids in biological samples using an indirect competitive ELISA based on four monoclonal antibodies

2017 ◽  
Vol 9 (4) ◽  
pp. 625-633 ◽  
Author(s):  
Shuchen Liu ◽  
Yue Zhang ◽  
Baoping Qu ◽  
Gaofeng Qin ◽  
Jinjun Cheng ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Clinical Practice ◽  
Biological Samples ◽  
Competitive Elisa ◽  
Routine Clinical Practice ◽  
Enzyme Linked Immunosorbent Assay ◽  
Indirect Competitive Elisa ◽  
Different Types ◽  
Total Bile Acids

We investigated a newly developed indirect competitive enzyme-linked immunosorbent assay for the determination of 5 major components of TBA, which works efficiently in different types of biological samples, and may be suitable for routine clinical practice.

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.

scholarly journals An enzyme-linked immunosorbent assay for erythropoietin using monoclonal antibodies, tetrameric immune complexes, and substrate amplification

Blood ◽  
1989 ◽  
Vol 74 (2) ◽  
pp. 622-628 ◽  
Author(s):  
AW Wognum ◽  
PM Lansdorp ◽  
AC Eaves ◽  
G Krystal
Keyword(s):  
Monoclonal Antibodies ◽  
Immunosorbent Assay ◽  
Biological Fluids ◽  
Normal Serum ◽  
Detection Sensitivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Response Curves ◽  
Human Erythropoietin ◽  
Healthy Donors ◽  
Noncovalent Labeling

We recently reported the development of several monoclonal antibodies (MoAbs) to native human erythropoietin (Ep). In the present study we have used the two antibodies with highest affinity to develop a two- sided or sandwich enzyme-linked immunosorbent assay (ELISA) to measure Ep in human serum. In this assay Ep is incubated in microtiter wells precoated with the first (IgE) anti-Ep antibody. Assay wells are then incubated with the second (IgG1) anti-Ep antibody, which is labeled noncovalently with the enzyme alkaline phosphatase (AP) by means of bispecific tetrameric antibody complexes consisting of IgG1 anti-Ep cross-linked to IgG1 anti-AP using rat MoAbs specific for mouse IgG1. Application of this noncovalent labeling procedure, in combination with substrate amplification, results in a detection sensitivity of 0.5 to 1.0 mU/sample (5 to 10 mU/mL), which makes this assay suitable for measuring normal serum Ep levels. The validity of this ELISA for quantitating Ep in biological fluids was demonstrated by the parallelism obtained between pure recombinant Ep dose-response curves and those obtained with plasma and serum from healthy donors and patients with various hematologic disorders. Normal plasma Ep levels detected with this ELISA ranged from 9 to 101 mU/mL with a mean of 32 +/- 23 (SD) mU/mL. Ep levels in sera from patients with polycythemia vera were in the low to normal range, whereas Ep levels in sera from patients with secondary polycythemia and patients with aplastic anemia were moderately to strongly elevated. These results demonstrate that the Ep-ELISA is a sensitive, reliable, and nonradioactive immunologic method for quantitating Ep levels and should prove useful in a variety of clinical and laboratory settings.

Assay of Human Tissue-Type Plasminogen Activator (t-PA) with an Enzyme-Linked Immunosorbent Assay (ELISA) Based on Three Murine Monoclonal Antibodies to t-PA

1985 ◽  
Vol 54 (03) ◽  
pp. 684-687 ◽  
Author(s):  
Paul Holvoet ◽  
Hugo Cleemput ◽  
Désiré Collen
Keyword(s):  
Monoclonal Antibodies ◽  
Plasminogen Activator ◽  
Immunosorbent Assay ◽  
Human Tissue ◽  
Biological Fluids ◽  
Human Subjects ◽  
Tissue Type ◽  
Enzyme Linked Immunosorbent Assay ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator

SummaryAn enzyme-linked immunosorbent assay (ELISA) for the measurement of human tissue-type plasminogen activator (t-PA) was developed. Microtiter plates were coated with a mixture of two monoclonal antibodies and bound t-PA was quantitated with a third monoclonal antibody linked to peroxidase. The lower limit of sensitivity of the assay was 0.2 ng of t-PA per ml. The concentration of antigen in citrated plasma of human subjects was found to be 3.4 ± 0.8 ng/ml. The assay had a good reproducibility with values of 3.8, 6.5 and 4.9 percent respectively for the intra-, inter-assay and inter-dilution variation coefficients. The results of the ELISA assay on plasma samples from patients during thrombolytic therapy with t-PA correlated very well, over a wide concentration range, with those obtained with a previously described two-site immuno-radiometric assay (r = 0.96). This ELISA with monoclonal antibodies constitutes a stable and reproducible set of reagents for the measurement of t-PA antigen in biological fluids, avoiding the disadvantages of the use of radioisotopes and of polyclonal antibodies.

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.

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