scholarly journals Inter-relatedness of some isoenzymes of cytochrome P-450 from rat, rabbit and human, determined with monoclonal antibodies

1986 ◽  
Vol 236 (2) ◽  
pp. 569-577 ◽  
Author(s):  
D Sesardic ◽  
A R Boobis ◽  
J McQuade ◽  
S Baker ◽  
E A Lock ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Western Blotting ◽  
Epitope Mapping ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Peptide Fragments ◽  
Structural Relationships ◽  
Homologous Antigen ◽  
Complex Structural ◽  
Cytochrome P 450

Monoclonal antibodies have been raised to rat liver cytochromes P-450 b and c, and rabbit liver cytochrome P-450 form 4. A total of six antibodies have been studied. Each antibody reacted strongly both with its homologous antigen and with microsomal fractions selectively enriched with that antigen by treatment of animals with inducing compounds. However, several of the antibodies showed cross-reactivity, either within or between species. A combination of enzyme-linked immunosorbent assay, immunoadsorption, Western blotting and competitive radioimmunoassay revealed that each of the antibodies reacted with a different epitope. Proteolytic digestion of antigen followed by Western blotting of the peptide fragments enabled antibodies, otherwise identical in their reactivity, to be distinguished. It is concluded that complex structural relationships exist amongst the different isoenzymes of cytochrome P-450 and that epitope mapping will help in characterizing both animal and human cytochromes P-450.

scholarly journals Epitope Mapping of Monoclonal Antibodies to Calreticulin Reveals That Charged Amino Acids Are Essential for Antibody Binding

Antibodies ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 31
Author(s):  
Ann Christina Bergmann ◽  
Cecilie Kyllesbech ◽  
Rimantas Slibinskas ◽  
Evaldas Ciplys ◽  
Peter Højrup ◽  
...  
Keyword(s):  
Amino Acids ◽  
Monoclonal Antibodies ◽  
Epitope Mapping ◽  
Biological Function ◽  
Specific Binding ◽  
Enzyme Linked Immunosorbent Assay ◽  
Potential Therapeutic Target ◽  
Charged Amino Acids ◽  
Chaperone Protein ◽  
Terminal Amino

Calreticulin is a chaperone protein, which is associated with myeloproliferative diseases. In this study, we used resin-bound peptides to characterize two monoclonal antibodies (mAbs) directed to calreticulin, mAb FMC 75 and mAb 16, which both have significantly contributed to understanding the biological function of calreticulin. The antigenicity of the resin-bound peptides was determined by modified enzyme-linked immunosorbent assay. Specific binding was determined to an 8-mer epitope located in the N-terminal (amino acids 34–41) and to a 12-mer peptide located in the C-terminal (amino acids 362–373). Using truncated peptides, the epitopes were identified as TSRWIESK and DEEQRLKEEED for mAb FMC 75 and mAb 16, respectively, where, especially the charged amino acids, were found to have a central role for a stable binding. Further studies indicated that the epitope of mAb FMC 75 is assessable in the oligomeric structure of calreticulin, making this epitope a potential therapeutic target.

scholarly journals Investigation of major amino acid residues of anti-norfloxacin monoclonal antibodies responsible for binding with fluoroquinolones

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Patamalai Boonserm ◽  
Songchan Puthong ◽  
Thanaporn Wichai ◽  
Sajee Noitang ◽  
Pongsak Khunrae ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Amino Acid ◽  
3D Structure ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Amino Acid Residues ◽  
Variable Regions ◽  
Complementarity Determining Regions ◽  
Crucial Part ◽  
Major Amino Acid

AbstractIt is important to understand the amino acid residues that govern the properties of the binding between antibodies and ligands. We studied the binding of two anti-norfloxacins, anti-nor 132 and anti-nor 155, and the fluoroquinolones norfloxacin, enrofloxacin, ciprofloxacin, and ofloxacin. Binding cross-reactivities tested by an indirect competitive enzyme-linked immunosorbent assay indicated that anti-nor 132 (22–100%) had a broader range of cross-reactivity than anti-nor 155 (62–100%). These cross-reactivities correlated with variations in the numbers of interacting amino acid residues and their positions. Molecular docking was employed to investigate the molecular interactions between the fluoroquinolones and the monoclonal antibodies. Homology models of the heavy chain and light chain variable regions of each mAb 3D structure were docked with the fluoroquinolones targeting the crucial part of the complementarity-determining regions. The fluoroquinolone binding site of anti-nor 155 was a region of the HCDR3 and LCDR3 loops in which hydrogen bonds were formed with TYR (H:35), ASN (H:101), LYS (H:106), ASN (L:92), and ASN (L:93). These regions were further away in anti-nor 132 and could not contact the fluoroquinolones. Another binding region consisting of HIS (L:38) and ASP (H:100) was found for norfloxacin, enrofloxacin, and ciprofloxacin, whereas only ASP (H:100) was found for ofloxacin.

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.

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.

scholarly journals Development, Characterization, and Application of Monoclonal Antibodies against Severe Acute Respiratory Syndrome Coronavirus Nucleocapsid Protein

2010 ◽  
Vol 17 (12) ◽  
pp. 2033-2036 ◽  
Author(s):  
Dipankar Das ◽  
Sriram Kammila ◽  
Mavanur R. Suresh
Keyword(s):  
Monoclonal Antibodies ◽  
Severe Acute Respiratory Syndrome ◽  
Western Blotting ◽  
Nucleocapsid Protein ◽  
Epitope Mapping ◽  
High Specificity ◽  
Diagnostic Assays ◽  
Hybridoma Technology ◽  
Recombinant Nucleocapsid Protein

ABSTRACT Five monoclonal antibodies (MAbs) against recombinant nucleocapsid protein (NP) of severe acute respiratory syndrome (SARS)-causing coronavirus (CoV) were developed by hybridoma technology. Epitope mapping by Western blotting showed that these anti-SARS-CoV NP MAbs bind to distinct domains of NP. These anti-SARS-CoV NP MAbs, with their high specificity, are potentially ideal candidates for developing early and sensitive diagnostic assays for SARS-CoV.

Production and characterization of monoclonal antibodies to the sapstaining fungus Ophiostoma piceae

1994 ◽  
Vol 40 (1) ◽  
pp. 35-44 ◽  
Author(s):  
Srabani Banerjee ◽  
Judy Little ◽  
Maria Chan ◽  
Brian T. Luck ◽  
Colette Breuil ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Cell Wall ◽  
Monoclonal Antibodies ◽  
Light And Electron Microscopy ◽  
Cross Reactivity ◽  
Cell Wall Protein ◽  
Enzyme Linked Immunosorbent Assay ◽  
Enzymatic Modification ◽  
Ophiostoma Piceae

A sensitive immunological tool has been developed to detect the sapstaining fungus Ophiostoma piceae 3871, which plagues the wood industry. Monoclonal antibodies (1F3(1), 4G3(14), 4G2(4), and 2B6(24)) produced against cell wall protein extracts of this fungus were specific. Specificity was estimated by enzyme linked immunosorbent assay, western blotting, and light and electron microscopy using the immunogold technique. Electron microscopy revealed gold particles localized on the outer surface of the cell wall. When screened against 24 biological control fungi the antibodies showed pratically no cross-reactivity (< 4%). When tested against 19 other staining fungi, the antibodies recognized three strains of Ophiostoma piceae, 1F3(1) recognized Phialophora botulispora, and the antibodies showed less than 5% reactivity with the other fungi. Chemical and enzymatic modification of the antigen revealed that the epitopes recognized by the monoclonal antibodies were glycospecific. Although the antibodies were produced against the cell wall protein extracts of the fungus grown in liquid culture, they also recognized the fungus growing in wood and therefore can be employed to investigate wood colonization by this fungus.Key words: Ophiostoma piceae, monoclonal antibodies, glycoprotein.

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 Investigation of Anti-Toxocara Antibodies in Epileptic Patients and Comparison of Two Methods: ELISA and Western Blotting

2013 ◽  
Vol 2013 ◽  
pp. 1-5 ◽  
Author(s):  
Mohammad Zibaei ◽  
Farzaneh Firoozeh ◽  
Parviz Bahrami ◽  
Seyed Mahmoud Sadjjadi
Keyword(s):  
Western Blotting ◽  
Case Reports ◽  
Immunoblot Analysis ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Parasitic Infections ◽  
Case Control Studies ◽  
Epileptic Patients ◽  
Group 3 ◽  
Patients With Epilepsy

The relationship between Toxocara infection and epilepsy was previously demonstrated by several case-control studies and case reports. These previous studies were often based on the enzyme-linked immunosorbent assay (ELISA) using Toxocara excretory-secretory antigens, which are not specific due to cross-reactivity with other parasitic infections such as ascariasis, trichuriasis, and anisakiasis. An immunoblot analysis is highly specific and can detect low levels of Toxocara antibodies. Therefore, this assay may be useful in the identification of toxocariasis in epileptic patients. We examined patients who had epilepsy and healthy subjects for seropositivity for Toxocara infection by ELISA and Western blotting. Out of 85 epileptic patients, 10 (11.8%) and 3 (3.5%) persons exhibited Toxocara immunoglobulin G (IgG) antibodies responses by ELISA and by both techniques, respectively. Moreover, in the healthy group (), 3 (3.5%) persons were positive by ELISA, but none was detected by Western blotting. This study indicates that Toxocara infection is a risk factor for epilepsy in Iran. These findings strongly suggest the need to perform Western blotting immunodiagnosis, as well as the ELISA using Toxocara excretory-secretory antigens, to improve diagnosis of human toxocariasis in patients with epilepsy.

scholarly journals Combined Use of Indirect ELISA and Western Blotting with Recombinant Hepatocellular Carcinoma-Associated Antigen 59 Is a Potential Immunodiagnostic Tool for the Detection of Prepatent Haemonchus contortus Infection in Goat

Animals ◽  
2019 ◽  
Vol 9 (8) ◽  
pp. 548 ◽  
Author(s):  
Muhammad Ali-ul-Husnain Naqvi ◽  
Sana Zahra Naqvi ◽  
Muhammad Ali Memon ◽  
Kalibixiati Aimulajiang ◽  
Muhammad Haseeb ◽  
...  
Keyword(s):  
Western Blot ◽  
Western Blotting ◽  
Haemonchus Contortus ◽  
Indirect Elisa ◽  
Fasciola Hepatica ◽  
False Negative ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Combined Use

Haemonchus contortus is recognized as one of the important health problems in small ruminants, leading to reduced production and economic loss for farmers worldwide. Prepatent diagnosis of H. contortus infection is crucial to improve control strategies as this helminth may remove up to one-fifth of total erythrocytes and may cause anemia, edema, diarrhea, and ultimately death in young animals. In this study, one of the excretory and secretory products, rHc-HCA59, was purified and used as antigen to detect specific antibodies in H. contortus infected goats during prepatent stage of infection using indirect enzyme linked immunosorbent assay (ELISA) as screening test. All goats (n = 38) were housed indoor, experimentally infected with 8000 infective larvae (L3) of H. contortus, and serum samples were collected prior to infection and at 14th day of infection. Immunoblotting was performed to confirm the results of indirect ELISA, evaluate the cross reactivity against rHc-HCA59 in sera of most common co-infecting parasites and rectify the false negative samples. Furthermore, three different batches of rHc-HCA59 were produced to evaluate the repeatability of ELISA. No eggs were detected in feces of all goats collected at 7th and 14th day of infection but, H. contortus eggs were detected at 21 days post infection in the feces. Indirect ELISA performed in this study showed 87% sensitivity and 100% specificity. The western blot analysis confirmed immunoreactivity in serum samples which scored positive in indirect ELISA and recognized the samples as negative which had OD450 lower than negative cut-off value in indirect ELISA. Furthermore, all false negative sera (n = 5) that had OD450 value between positive and negative cut-off value in rHc-HCA59 based ELISA were clearly positive in western blot. Moreover, no cross-reactivity was detected in ELISA and western blotting against rHc-HCA59 in positive sera of Toxoplasma gondii, Fasciola hepatica, and Trichinella spiralis. The results of this study concluded that combined use of indirect ELISA and western blotting with rHc-HCA59 is a potential immunodiagnostic tool for the detection of H. contortus infection during prepatent period in goats.

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