Antigenic analysis of testicular cytochromes c using monoclonal antibodies

1986 ◽  
Vol 64 (12) ◽  
pp. 1211-1217 ◽  
Author(s):  
In C. Kim ◽  
Hector Nolla
Keyword(s):  
Monoclonal Antibodies ◽  
Amino Acid ◽  
Cross Reactivity ◽  
Amino Acid Sequences ◽  
Antigenic Structure ◽  
Enzyme Linked Immunosorbent Assay ◽  
Antigenic Analysis ◽  
Cyt C ◽  
Primary Amino ◽  
Species Specific

Testicular cytochrome c (cyt ct) was isolated from testes of sexually mature, rat, mouse, rabbit, and bull, among which rat testis is highly rich in cyt c1. By fusion of NS-1 myeloma cells and spleen cells of mice immunized with rat cyt ct, 11 stable mouse hybridoma cell lines were established. Using an enzyme-linked immunosorbent assay, it was determined that 4 of the 11 anti-rat cyt ct monoclonal antibodies (McAb) did not bind to somatic cyt c (cyt cs) of vertebrates nor to cyt ct of mouse, rabbit, and bull. Four other McAb showed no binding to cyt cs but showed different patterns of cross-reactivity with these four cyt ct. Therefore, these McAb appear to be very sensitive and useful probes for the discrimination or identification of extremely similar isocytochromes c. Although the primary amino acid sequences between cyt cs of rat and mouse are identical, the antigenic structure of cyt ct of rat and mouse are clearly distinct with regard to cross-reactivity with some anti-rat cyt ct McAb. Furthermore, these McAb also reveal that the primary amino acid sequences of cyt ct, which reflect differences in the surface conformation of the molecule, are probably species specific.

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 Erythropoietin structure-function relationships: high degree of sequence homology among mammals

Blood ◽  
1993 ◽  
Vol 82 (5) ◽  
pp. 1507-1516 ◽  
Author(s):  
D Wen ◽  
JP Boissel ◽  
TE Tracy ◽  
RH Gruninger ◽  
LS Mulcahy ◽  
...  
Keyword(s):  
Amino Acid ◽  
Structure Function ◽  
Disulfide Bridge ◽  
Cross Reactivity ◽  
Amino Acid Sequences ◽  
Exon 2 ◽  
Cdna Sequences ◽  
Intron 1 ◽  
Species Specific ◽  
High Degree

Abstract To investigate structure-function relationships of erythropoietin (Epo), we have obtained cDNA sequences that encode the mature Epo protein of a variety of mammals. A first set of primers, corresponding to conserved nucleotide sequences between mouse and human DNAs, allowed us to amplify by polymerase chain reaction (PCR) intron 1/exon 2 fragments from genomic DNA of the hamster, cat, lion, dog, horse, sheep, dolphin, and pig. Sequencing of these fragments permitted the design of a second generation of species-specific primers. RNA was prepared from anemic kidneys and reverse-transcribed. Using our battery of species-specific 5′ primers, we were able to successfully PCR- amplify Epo cDNA from Rhesus monkey, rat, sheep, dog, cat, and pig. Deduced amino acid sequences of mature Epo proteins from these animals, in combination with known sequences for human, Cynomolgus monkey, and mouse, showed a high degree of homology, which explains the biologic and immunological cross-reactivity that has been observed in a number of species. Human Epo is 91% identical to monkey Epo, 85% to cat and dog Epo, and 80% to 82% to pig, sheep, mouse, and rat Epos. There was full conservation of (1) the disulfide bridge linking the NH2 and COOH termini; (2) N-glycosylation sites; and (3) predicted amphipathic alpha- helices. In contrast, the short disulfide bridge (C29/C33 in humans) is not invariant. Cys33 was replaced by a Pro in rodents. Most of the amino acid replacements were conservative. The C-terminal part of the loop between the C and D helices showed the most variation, with several amino acid substitutions, deletions, and/or insertions. Calculations of maximum parsimony for intron 1/exon 2 sequences as well as coding sequences enabled the construction of cladograms that are in good agreement with known phylogenetic relationships.

scholarly journals Erythropoietin structure-function relationships: high degree of sequence homology among mammals

Blood ◽  
1993 ◽  
Vol 82 (5) ◽  
pp. 1507-1516 ◽  
Author(s):  
D Wen ◽  
JP Boissel ◽  
TE Tracy ◽  
RH Gruninger ◽  
LS Mulcahy ◽  
...  
Keyword(s):  
Amino Acid ◽  
Structure Function ◽  
Disulfide Bridge ◽  
Cross Reactivity ◽  
Amino Acid Sequences ◽  
Exon 2 ◽  
Cdna Sequences ◽  
Intron 1 ◽  
Species Specific ◽  
High Degree

To investigate structure-function relationships of erythropoietin (Epo), we have obtained cDNA sequences that encode the mature Epo protein of a variety of mammals. A first set of primers, corresponding to conserved nucleotide sequences between mouse and human DNAs, allowed us to amplify by polymerase chain reaction (PCR) intron 1/exon 2 fragments from genomic DNA of the hamster, cat, lion, dog, horse, sheep, dolphin, and pig. Sequencing of these fragments permitted the design of a second generation of species-specific primers. RNA was prepared from anemic kidneys and reverse-transcribed. Using our battery of species-specific 5′ primers, we were able to successfully PCR- amplify Epo cDNA from Rhesus monkey, rat, sheep, dog, cat, and pig. Deduced amino acid sequences of mature Epo proteins from these animals, in combination with known sequences for human, Cynomolgus monkey, and mouse, showed a high degree of homology, which explains the biologic and immunological cross-reactivity that has been observed in a number of species. Human Epo is 91% identical to monkey Epo, 85% to cat and dog Epo, and 80% to 82% to pig, sheep, mouse, and rat Epos. There was full conservation of (1) the disulfide bridge linking the NH2 and COOH termini; (2) N-glycosylation sites; and (3) predicted amphipathic alpha- helices. In contrast, the short disulfide bridge (C29/C33 in humans) is not invariant. Cys33 was replaced by a Pro in rodents. Most of the amino acid replacements were conservative. The C-terminal part of the loop between the C and D helices showed the most variation, with several amino acid substitutions, deletions, and/or insertions. Calculations of maximum parsimony for intron 1/exon 2 sequences as well as coding sequences enabled the construction of cladograms that are in good agreement with known phylogenetic relationships.

scholarly journals Location of antigenic sites recognized by monoclonal antibodies in the influenza A virus nucleoprotein molecule

2009 ◽  
Vol 90 (7) ◽  
pp. 1730-1733 ◽  
Author(s):  
Natalia L. Varich ◽  
Konstantin S. Kochergin-Nikitsky ◽  
Evgeny V. Usachev ◽  
Olga V. Usacheva ◽  
Alexei G. Prilipov ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Amino Acid ◽  
Influenza A Virus ◽  
Influenza A ◽  
Antigenic Variation ◽  
Strain Differences ◽  
Antigenic Structure ◽  
Amino Acid Residues ◽  
Mutant Proteins ◽  
Antigenic Sites

The locations of amino acid positions relevant to antigenic variation in the nucleoprotein (NP) of influenza virus are not conclusively known. We analysed the antigenic structure of influenza A virus NP by introducing site-specific mutations at amino acid positions presumed to be relevant for the differentiation of strain differences by anti-NP monoclonal antibodies. Mutant proteins were expressed in a prokaryotic system and analysed by performing ELISA with monoclonal antibodies. Four amino acid residues were found to determine four different antibody-binding sites. When mapped in a 3D X-ray model of NP, the four antigenically relevant amino acid positions were found to be located in separate physical sites of the NP molecule.

Rapid, sensitive two-site ELISA for detection of cows' milk in goats' or ewes' milk using monoclonal antibodies

1994 ◽  
Vol 61 (1) ◽  
pp. 91-99 ◽  
Author(s):  
Didier Levieux ◽  
Annie Venien
Keyword(s):  
Monoclonal Antibodies ◽  
Sandwich Elisa ◽  
High Specificity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Factors Affecting ◽  
Assay Performance ◽  
Capture Antibody ◽  
Constant Quality ◽  
Species Specific ◽  
Β Lactoglobulin

SummaryA sandwich ELISA (enzyme-linked immunosorbent assay) of the two-site type has been successfully developed for the detection of cows' milk in goats' or ewes' milk. The assay uses two monoclonal antibodies (MAb) raised in mice against cows' β-lactoglobulin (β-lg). These MAb recognize different epitopes of the β-lg, which are sufficiently distinct to allow simultaneous binding of the corresponding antibodies. One of the MAb recognizes a species-specific epitope of the bovine β-lg and was adsorbed to a plastic microtitration plate (capture antibody). The second MAb was labelled with peroxidase and used to detect the captured cows' β-lg. Factors affecting assay performance were investigated. The optimized assay is highly specific, reproducible (intra- and inter-assay CV were 8 and 13% respectively) and sensitive: as little as 5 ng β-lg/ml or 1 part cows' milk per 100000 parts goats' or ewes' milk can be detected. The technique is robust, cheap, rapid, reliable and suitable for high sample throughput, semi-automation and screening surveys. The MAb used guarantee the high specificity of the assay and indefinite reagent supply of constant quality once approved by collaborative national or international trials.

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 Identification of Mimotope Peptides Which Bind to the Mycotoxin Deoxynivalenol-Specific Monoclonal Antibody

1999 ◽  
Vol 65 (8) ◽  
pp. 3279-3286 ◽  
Author(s):  
Qiaoping Yuan ◽  
James J. Pestka ◽  
Brandon M. Hespenheide ◽  
Leslie A. Kuhn ◽  
John E. Linz ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Alkaline Phosphatase ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Synthetic Peptide ◽  
Amino Acid Sequences ◽  
In Vitro Translation ◽  
Enzyme Linked Immunosorbent Assay ◽  
Translation System

ABSTRACT Monoclonal antibody 6F5 (mAb 6F5), which recognizes the mycotoxin deoxynivalenol (DON) (vomitoxin), was used to select for peptides that mimic the mycotoxin by employing a library of filamentous phages that have random 7-mer peptides on their surfaces. Two phage clones selected from the random peptide phage-displayed library coded for the amino acid sequences SWGPFPF and SWGPLPF. These clones were designated DONPEP.2 and DONPEP.12, respectively. The results of a competitive enzyme-linked immunosorbent assay (ELISA) suggested that the two phage displayed peptides bound to mAb 6F5 specifically at the DON binding site. The amino acid sequence of DONPEP.2 plus a structurally flexible linker at the C terminus (SWGPFPFGGGSC) was synthesized and tested to determine its ability to bind to mAb 6F5. This synthetic peptide (designated peptide C430) and DON competed with each other for mAb 6F5 binding. When translationally fused with bacterial alkaline phosphatase, DONPEP.2 bound specifically to mAb 6F5, while the fusion protein retained alkaline phosphatase activity. The potential of using DONPEP.2 as an immunochemical reagent in a DON immunoassay was evaluated with a DON-spiked wheat extract. When peptide C430 was conjugated to bovine serum albumin, it elicited antibody specific to peptide C430 but not to DON in both mice and rabbits. In an in vitro translation system containing rabbit reticulocyte lysate, synthetic peptide C430 did not inhibit protein synthesis but did show antagonism toward DON-induced protein synthesis inhibition. These data suggest that the peptides selected in this study bind to mAb 6F5 and that peptide C430 binds to ribosomes at the same sites as DON.

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