scholarly journals Two new murine monoclonal antibodies rised against human IgG

2001 ◽  
Vol 66 (5) ◽  
pp. 323-330 ◽  
Author(s):  
Marijana Petricevic ◽  
Aleksandra Inic ◽  
Ratko Jankov ◽  
Ljiljana Dimitrijevic
Keyword(s):  
Monoclonal Antibodies ◽  
Solid Phase ◽  
Proteolytic Enzymes ◽  
Cross Reactivity ◽  
Dot Blot ◽  
Human Igg ◽  
Western Blots ◽  
Binding Characteristics ◽  
Hybridoma Technology ◽  
Murine Monoclonal Antibodies

Many pathological conditions are accompanied with changes in the concentration of the total IgG or some of its fraction. For this reason there is great interest in the production of reagents specific for IgG. In this paper, the binding characteristics of two new murine monoclonal antibodies (MoAb), assigned MoAb 15 and MoAb 22, are reported. These MoAbs were produced by hybridoma technology. By performing ELISAs and Western blots analyzes, it was demonstrated that both MoAbs interact specifically with human IgG. Cross reactivity with other sera proteins was not observed. In order to precisely localize the epitopes recognized by MoAb 15 and MoAb 22, the Western blots interactions of these MoAbs with electrophoreticaly separated IgG-fragments, obtained by the action of proteolytic enzymes (papain, pepsin, trypsin), were analyzed. According to the results of these experiments, both MoAbs interacted with epitopes in the C 3 domain. The affinity constants, calculated from Scatchard plots of binding ofMoAb15 and MoAb22 to human IgG, wereKa15 = 1.71 106M-1 andKa22 = 2.15 109M-1. According to all these findings,MoAb 15 and MoAb 22 could be used in standard immunochemical techniques. However, the experiments showed that both MoAbs had bad immunoprecipitating properties. In solid phase techniques (ELISAs, Western blot, dot-blot, etc.), their application gave excellent results that highly recommended them for use in these types of analyzes.

Use of monoclonal and polyclonal antibodies to detect prostatic acid phosphatase by immunoblotting.

1986 ◽  
Vol 32 (10) ◽  
pp. 1832-1835 ◽  
Author(s):  
P C Patel ◽  
L Aubin ◽  
J Côte
Keyword(s):  
Monoclonal Antibodies ◽  
Acid Phosphatase ◽  
Western Blot ◽  
Polyclonal Antibodies ◽  
Prostatic Acid Phosphatase ◽  
The Other ◽  
Dot Blot ◽  
Western Blots ◽  
Dot Blot Assay ◽  
Polyclonal Antisera

Abstract We investigated two techniques of immunoblotting--the Western blot and the dot blot--for use in detecting prostatic acid phosphatase (PAP, EC 3.1.3.2). We used polyclonal antisera to human PAP, produced in rabbits by hyperimmunization with purified PAP, and PAP-specific monoclonal antibodies in the immunoenzymatic protocols. We conclude that PAP can be readily detected by Western blots with use of polyclonal antisera, but not with monoclonal antibodies. On the other hand, using a dot blot assay, we could easily detect PAP with both polyclonal and monoclonal antibodies.

scholarly journals Screening and development of monoclonal antibodies for identification of ferret T follicular helper cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Wenbo Jiang ◽  
Julius Wong ◽  
Hyon-Xhi Tan ◽  
Hannah G. Kelly ◽  
Paul G. Whitney ◽  
...  
Keyword(s):  
Animal Model ◽  
Monoclonal Antibodies ◽  
Lymph Node ◽  
Cross Reactivity ◽  
Tfh Cells ◽  
Follicular Helper ◽  
Lymph Node Cells ◽  
Human Viruses ◽  
Humoral Responses ◽  
Murine Monoclonal Antibodies

AbstractThe ferret is a key animal model for investigating the pathogenicity and transmissibility of important human viruses, and for the pre‐clinical assessment of vaccines. However, relatively little is known about the ferret immune system, due in part to a paucity of ferret‐reactive reagents. In particular, T follicular helper (Tfh) cells are critical in the generation of effective humoral responses in humans, mice and other animal models but to date it has not been possible to identify Tfh in ferrets. Here, we describe the screening and development of ferret-reactive BCL6, CXCR5 and PD-1 monoclonal antibodies. We found two commercial anti-BCL6 antibodies (clone K112-91 and clone IG191E/A8) had cross-reactivity with lymph node cells from influenza-infected ferrets. We next developed two murine monoclonal antibodies against ferret CXCR5 (clone feX5-C05) and PD-1 (clone fePD-CL1) using a single B cell PCR-based method. We were able to clearly identify Tfh cells in lymph nodes from influenza infected ferrets using these antibodies. The development of ferret Tfh marker antibodies and the identification of ferret Tfh cells will assist the evaluation of vaccine-induced Tfh responses in the ferret model and the design of novel vaccines against the infection of influenza and other viruses, including SARS-CoV2.

scholarly journals Characterization of two monoclonal antibodies against cytochrome b558 of human neutrophils

Blood ◽  
1989 ◽  
Vol 73 (6) ◽  
pp. 1686-1694 ◽  
Author(s):  
AJ Verhoeven ◽  
BG Bolscher ◽  
LJ Meerhof ◽  
R van Zwieten ◽  
J Keijer ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Transmembrane Protein ◽  
Autosomal Gene ◽  
Human Neutrophils ◽  
Western Blots ◽  
Binding Characteristics ◽  
Cytochrome B558 ◽  
Spectrophotometric Methods ◽  
Membrane Bound

Abstract Monoclonal antibodies (MoAbs) were raised against cytochrome b558, a membrane-bound component of the NADPH:O2 oxidoreductase in human neutrophils. This cytochrome consists of a low-molecular-weight (low- mol-wt) subunit of 22 to 23 Kd, probably encoded by an autosomal gene, and a high-mol-wt subunit of 75 to 90 Kd, encoded on the X-chromosome. MoAb 449 reacts with the low-mol-wt subunit and MoAb 48 with the high- mol-wt subunit on Western blots of purified cytochrome b558 and on blots of whole neutrophil extracts. In extracts of neutrophils from patients with chronic granulomatous disease (CGD) in which cytochrome b558 is not detectable by spectrophotometric methods, the low-mol-wt subunit is present, albeit in a much smaller amount. The high-mol-wt subunit is not detected by MoAb 48 in neutrophils of patients with X- linked CGD and in neutrophils of patients with the autosomal, cytochrome-b558-negative form of the disease. These results can be explained by a marked instability of these subunits when the synthesis of either of the two is disturbed. In differentiated HL-60 cells, the high-mol-wt subunit appears to be present in a different form. Cloning of the low-mol-wt subunit with the help of MoAb 449 suggests the presence of a heme-binding site on this subunit. By comparison of the binding characteristics of MoAb 449 to intact and permeabilized neutrophils with those of MoAb 7D5, recently isolated by Nakamura et al (Blood 69:1404, 1987), the low-mol-wt subunit was established as a transmembrane protein.

scholarly journals Generation of mid‐region murine monoclonal antibodies against multiple structural isoforms of amyloid‐beta by hybridoma technology

2020 ◽  
Vol 16 (S4) ◽  
Author(s):  
Julia Stockmann ◽  
Léon Beyer ◽  
Sandy Galkowski ◽  
Nathalie Woitzik ◽  
Jörn Güldenhaupt ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Amyloid Beta ◽  
Hybridoma Technology ◽  
Murine Monoclonal Antibodies

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.

scholarly journals Monoclonal antibodies against a C-terminal peptide of human brain acetylcholinesterase distinguish between erythrocyte and brain acetylcholinesterases

1996 ◽  
Vol 42 (1) ◽  
pp. 19-23 ◽  
Author(s):  
N Boschetti ◽  
U Brodbeck ◽  
S P Jensen ◽  
C Koch ◽  
B Nørgaard-Pedersen
Keyword(s):  
Monoclonal Antibodies ◽  
Human Brain ◽  
Solid Phase ◽  
Bovine Brain ◽  
Mammalian Brain ◽  
Dot Blot ◽  
Electric Eel ◽  
Terminal Amino ◽  
Dot Blot Analysis ◽  
Brain Acetylcholinesterase

Abstract Monoclonal antibodies (mAbs) were raised against a peptide of the 10 C-terminal amino acids of human brain acetylcholinesterase (AChE): H-Tyr-Ser-Lys-Gln-Asp-Arg-Cys-Ser-Asp-Leu-OH. Two positive clones (mAbs 190-1 and 190-2) were selected and tested for their ability to distinguish between mammalian brain and erythrocyte AChEs. In a solid-phase enzyme antigen immunoassay as well as by Western- and dot-blot analysis, both antibodies showed clear binding to AChE from human and bovine brain but not to AChE from erythrocytes. MAbs 190-1 and 190-2 reacted with neither AChE from electric eel nor butyrylcholinesterase from human serum. Both antibodies were used in a quantitative assay for AChE in amniotic fluids, where AChE activity could be found only in samples from open neural tube-defect pregnancies, but not in fluids from normal pregnancies or in artificially blood-contaminated samples.

scholarly journals Aglycosylation of human IgG1 and IgG3 monoclonal antibodies can eliminate recognition by human cells expressing FcγRI and/or FcγRII receptors

1989 ◽  
Vol 259 (2) ◽  
pp. 347-353 ◽  
Author(s):  
M R Walker ◽  
J Lund ◽  
K M Thompson ◽  
R Jefferis
Keyword(s):  
Monoclonal Antibodies ◽  
K562 Cells ◽  
Red Cells ◽  
Rosette Formation ◽  
Human Monoclonal Antibodies ◽  
Human Igg ◽  
Igg Binding ◽  
Human Igg1 ◽  
Murine Monoclonal Antibodies ◽  
Human Red Cells

Aglycosylated human IgG1 and IgG3 monoclonal anti-D (Rh) and human IgG1 and IgG3 chimaeric anti-5-iodo-4-hydroxy-3-nitrophenacetyl (anti-NIP) monoclonal antibodies produced in the presence of tunicamycin have been compared with the native glycosylated proteins with respect to recognition by human Fc gamma RI and/or Fc gamma RII receptors on U937, Daudi or K562 cells. Human red cells sensitized with glycosylated IgG3 form rosettes via Fc gamma RI with 60% of U937 cells. Inhibition of rosette formation required greater than 35-fold concentrated more aglycosylated than glycosylated human monoclonal anti-D (Rh) antibody. Unlabelled polyclonal human IgG and glycosylated monoclonal IgG1 and anti-D (Rh) antibody inhibited the binding of 125I-labelled monomeric human IgG binding by U937 Fc gamma RI at concentrations greater than 50-fold lower than the aglycosylated monoclonal IgG1 anti-D (Rh) (K50 approximately 3 x 10(-9) M and approximately 6 x 10(-7) M respectively). Similar results were obtained using glycosylated and aglycosylated monoclonal human IgG1 or IgG3 chimaeric anti-NIP antibody-sensitized red cells rosetting with Fc gamma RI-/Fc gamma RII+ Daudi and K562 cells. Rosette formation could be inhibited by the glycosylated form (at greater than 10(-6) M) but not by the aglycosylated form. Haemagglutination analysis using a panel of murine monoclonal antibodies specific for epitopes located on C gamma 2, C gamma 3 or C gamma 2/C gamma 3 interface regions did not demonstrate differences in Fc conformation between the glycosylated or aglycosylated human monoclonal antibodies. These data suggest that the Fc gamma RI and Fc gamma RII sites on human IgG are highly conformation-dependent and that the carbohydrate moiety serves to stabilize the Fc structure rather than interacting directly with Fc receptors.

scholarly journals Evaluation of Diagnostic Applications of Monoclonal Antibodies against Avian Influenza H7 Viruses

2010 ◽  
Vol 17 (9) ◽  
pp. 1398-1406 ◽  
Author(s):  
Ming Yang ◽  
Alfonso Clavijo ◽  
Jill Graham ◽  
John Pasick ◽  
James Neufeld ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Influenza A ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dot Blot ◽  
Western Blots ◽  
Positive Antibody ◽  
Dot Blot Assay ◽  
Diagnostic Applications ◽  
Ha Protein ◽  
H7 Subtype

ABSTRACT A panel of monoclonal antibodies (MAbs) was generated from mice immunized with binary ethylenimine (BEI)-inactivated H7N1 (A/TK/ON/18-2/00) virus. Using a dot blot assay, six of seven MAbs reacted with viruses of the H7 subtype, but not with any of the other 15 hemagglutinin (HA) subtypes tested. Four of the seven MAbs reacted with 14 different H7 isolates, indicating that the MAbs binding epitopes are conserved among viruses of the H7 subtype. The binding epitopes of all seven MAbs were conformational and reacted with the HA1 fraction of the HA protein in Western blots under nonreducing conditions. Applications of these MAbs in the development of rapid tests for H7 subtype viruses were evaluated. The MAbs demonstrated reactivity with AI virus H7 antigen in immunofluorescence and immunohistochemistry assays. Monoclonal antibody 3 showed a very strong immunostaining in the formalin-fixed and paraffin-embedded tissue from the H7N3 virus-infected chicken. A double-antibody sandwich (DAS) enzyme-linked immunosorbent assay (ELISA) was developed using two of the MAbs. The DAS ELISA specifically detected all H7 strains tested in this study. A competitive ELISA (cELISA) for the detection of H7-specific antibodies was evaluated using one MAb and BEI-inactivated H7N1 virus as the antigen. All infected birds showed positive antibody responses at 7 days postinfection. The sensitivity of this cELISA was comparable with that of an influenza A nucleoprotein-based cELISA. This panel of MAbs is valuable in the development of various immunoassays.

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