scholarly journals Double immunocytochemical staining in the study of antibody-producing cells in vivo. Detection of specific antibody-producing cells in the spleen and simultaneous determination whether or not they produce immunoglobulin G antibodies.

1984 ◽  
Vol 32 (7) ◽  
pp. 677-680 ◽  
Author(s):  
N Van Rooijen ◽  
N Kors ◽  
R Van Nieuwmegen
Keyword(s):  
Alkaline Phosphatase ◽  
Immunocytochemical Staining ◽  
Igg Antibodies ◽  
Specific Antibodies ◽  
In Vivo Detection ◽  
Rabbit Igg ◽  
Present Technique ◽  
Antibody Class ◽  
Ig G

Rabbits were primed intravenously with human serum albumin (HSA) and boosted with the same antigen 2 months later. Cells producing specific antibodies against HSA could be detected in vivo and it could be determined whether or not they belonged to the immunoglobulin (Ig) G class using a combined peroxidase (HRP) and alkaline phosphatase (AP) immunocytochemical technique. HRP-HSA conjugate was used for detection of anti-HSA-producing cells and AP-sheep anti-rabbit IgG (SRIgG) was used to determine the IgG class of the antibodies produced by these cells in the same spleen section. After performing both HRP and AP cytochemistry, cells with a red-stained cytoplasm represent anti-HSA-producing cells not stained for their antibody class and cells with a blue-stained cytoplasm represent cells producing IgG antibodies not directed against HSA. Cells with a double-stained cytoplasm represent cells producing anti-HSA antibodies belonging to the IgG class. We also attempted to determine whether or not part of the anti-HSA-producing cells belonged to the IgM class using AP-sheep anti-rabbit IgM (SRIgM). In this case no double-stained cells were detected, indicating that the affinity of intracellular IgM-anti-HSA antibodies is too low to allow detection using the present technique.

scholarly journals Broadly Neutralizing Hemagglutinin Stalk-Specific Antibodies Induce Potent Phagocytosis of Immune Complexes by Neutrophils in an Fc-Dependent Manner

mBio ◽  
2016 ◽  
Vol 7 (5) ◽  
Author(s):  
Caitlin E. Mullarkey ◽  
Mark J. Bailey ◽  
Diana A. Golubeva ◽  
Gene S. Tan ◽  
Raffael Nachbagauer ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Fc Receptor ◽  
Igg Antibodies ◽  
Specific Antibodies ◽  
Effector Functions ◽  
Iga Antibodies ◽  
Specific Igg ◽  
Optimal Protection ◽  
Specific Igg Antibodies

ABSTRACTBroadly neutralizing antibodies that recognize the conserved hemagglutinin (HA) stalk have emerged as exciting new biotherapeutic tools to combat seasonal and pandemic influenza viruses. Our general understanding of the mechanisms by which stalk-specific antibodies achieve protection is rapidly evolving. It has recently been demonstrated that broadly neutralizing HA stalk-specific IgG antibodies require Fc-Fcγ receptor (FcγR) interactions for optimal protectionin vivo. Here we examine the neutrophil effector functions induced by stalk-specific antibodies. As the most abundant subset of blood leukocytes, neutrophils represent a critical innate effector cell population and serve an instrumental role in orchestrating downstream adaptive responses to influenza virus infection. Yet, the interplay of HA stalk-specific IgG, Fc-FcγR engagement, and neutrophils has remained largely uncharacterized. Using anin vitroassay to detect the production of reactive oxygen species (ROS), we show that human and mouse monoclonal HA stalk-specific IgG antibodies are able to induce the production of ROS by neutrophils, while HA head-specific antibodies do not. Furthermore, our results indicate that the production of ROS is dependent on Fc receptor (FcR) engagement and phagocytosis. We went on to assess the ability of monoclonal HA stalk-specific IgA antibodies to induce ROS. Consistent with our findings for monoclonal IgGs, only HA stalk-specific IgA antibodies elicited ROS production by neutrophils. This induction is dependent on the engagement of FcαR1. Taken together, our findings describe a novel FcR-dependent effector function induced by HA stalk-specific IgG and IgA antibodies, and importantly, our studies shed light on the mechanisms by which HA stalk-specific antibodies achieve protection.IMPORTANCEThe present study provides evidence that broadly neutralizing HA stalk-specific antibodies induce downstream Fc-mediated neutrophil effector functions. In addition to their ability to neutralize, this class of antibodies has been shown to rely on Fc-Fc receptor interactions for optimal protectionin vivo. Curiously, neutralizing antibodies that bind the HA head domain do not require such interactions. Our findings build on these previous observations and provide a more complete picture of the relationship between stalk-specific antibodies and cells of the innate immune compartment. Furthermore, our data suggest that the ability of HA stalk-specific antibodies to mediate Fc-Fc receptor engagement is epitope dependent. Overall, this work will inform the rational design of improved influenza virus vaccines and therapeutics.

scholarly journals Double immunocytochemical staining in the study of antibody-producing cells in vivo. Combined detection of antigen specificity (anti-TNP) and (sub)class of intracellular antibodies.

1985 ◽  
Vol 33 (3) ◽  
pp. 175-178 ◽  
Author(s):  
N Van Rooijen ◽  
N Kors
Keyword(s):  
Keyhole Limpet Hemocyanin ◽  
Staining Technique ◽  
Immunocytochemical Staining ◽  
Antigen Specificity ◽  
Specific Antibodies ◽  
Igm Antibodies ◽  
Rabbit Class ◽  
Spleen And Lymph Nodes ◽  
Combined Detection

Mice and rabbits were immunized with trinitrophenyl (TNP)-conjugated keyhole limpet hemocyanin (KLH). Cells producing specific antibodies against the hapten TNP were detected in vivo in spleen and lymph nodes using a TNP--alkaline phosphatase (AP) conjugate. Using horseradish peroxidase (HRP)-conjugated anti-mouse (sub)class (IgG2A, IgG2B, IgM) antibodies and anti-rabbit class (IgG, IgM) antibodies and a double immunocytochemical staining technique for simultaneous demonstration of the enzymes AP and HRP, we were able to determine both the antigen specificity (anti-TNP) and the (sub)class of intracellular antibodies produced by individual antibody-forming cells in vivo.

scholarly journals Infection with Anaplasma phagocytophilum in a young dog: a case report

2008 ◽  
Vol 52 (No. 5) ◽  
pp. 207-212 ◽  
Author(s):  
O. Melter ◽  
I. Stehlik ◽  
H. Kinska ◽  
I. Volfova ◽  
V. Ticha ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Case Report ◽  
Dna Sequence ◽  
Anaplasma Phagocytophilum ◽  
Clinical Symptoms ◽  
Acute Onset ◽  
Blood Analysis ◽  
Golden Retriever ◽  
Igg Antibodies ◽  
Specific Antibodies

An 11-months-old male Golden Retriever occasionally found to have <i>Ixodes ricinus</i> ticks attached to the skin developed the acute onset of fever, lameness and inappetence followed by rapidly progressive depression, ataxia and reluctance to move. Inclusions (morulae) were observed in granulocytes. The blood analysis revealed severe thrombocytopoenia, lymphopoenia, eosinopoenia, elevation of alkaline phosphatase and hypercholesterolaemia, mostly suggestive of an <i>Anaplasma phagocytophilum</i> infection. The amplification of a DNA sequence specific for Anaplasma phagocytophilum and detection of specific antibodies supported the diagnosis. <i>Borrelia burgdorferi</i>, another tick-borne pathogen, or specific antiborrelial IgG antibodies were not detected. The dog was treated with oral doxycycline for 14 days: clinical symptoms resolved within six days.

scholarly journals Double immunocytochemical staining in the study of antibody-producing cells in vivo. Simultaneous detection of cells producing monospecific antibodies and cells producing cross-reacting antibodies in the spleen of rabbits after injection of two related antigens.

1984 ◽  
Vol 32 (8) ◽  
pp. 844-848 ◽  
Author(s):  
N Van Rooijen ◽  
N Kors ◽  
R Van Nieuwmegen
Keyword(s):  
Gamma Globulin ◽  
Simultaneous Detection ◽  
Immunocytochemical Staining ◽  
Secondary Immune Response ◽  
Spleen Tissue ◽  
Specific Antibodies ◽  
Similarities And Differences ◽  
Human Gamma Globulin ◽  
Monospecific Antibodies

Rabbits were injected simultaneously with both human gamma globulin (HGG) and bovine gamma globulin (BGG). Sections of spleen tissue were prepared from spleen biopsies taken during the primary or secondary immune response, and incubated simultaneously with horseradish peroxidase (HRP)-HGG conjugate and alkaline phosphatase (AP)-BGG conjugate in order to detect cells containing specific antibodies against one or both of the antigens. After both HRP and AP cytochemistry, cells with a red-stained cytoplasm, cells with a blue-stained cytoplasm, and cells with a violet-stained cytoplasm were detected in the spleen. The red-stained cells had bound the HRP-HGG conjugate, indicating that these cells contained anti-HGG antibodies. The blue-stained cells had bound the AP-BGG conjugate, indicating that these cells contained anti-BGG antibodies. The violet-stained cells had obviously bound both the HRP-HGG conjugate and the AP-BGG conjugate, indicating that these cells contained antibodies cross-reacting with both antigens. Results are compared with earlier studies on the antigenic similarities and differences between HGG and BGG when used as antigens in rabbits.

scholarly journals Tissue localization of lymphocyte surface antigens and receptors for immunoglobulin G Fc and complement in the chicken.

1982 ◽  
Vol 30 (3) ◽  
pp. 201-206 ◽  
Author(s):  
S Thunold ◽  
R Boyd ◽  
K Schauenstein ◽  
G Wick
Keyword(s):  
B Lymphocytes ◽  
B Lymphocyte ◽  
Surface Antigens ◽  
Mononuclear Phagocytes ◽  
Carbon Particles ◽  
Tissue Sections ◽  
Lymphocyte Surface ◽  
Rabbit Igg ◽  
Ig G

Frozen sections of chicken lymphoid organs were examined for lymphocyte surface antigens by antisera to T and B lymphocytes (ATS;ABS), and for the presence of immunoglobulin (Ig) G Fc and complement receptors (FcR;CR) by hemadsorption with sheep erythrocytes (E) coated with chicken IgG(EA), and E coated with rabbit IgG and chicken complement (EAC). In the spleen FcR positive cells were confined to the periellipsoidal sheaths and the germinal centers. CR positive cells were found in the same spleen areas, as well as in the medulla of bursal follicles. These lymphoid areas reacted strongly with ABS, but they also stained with neutral alpha-naphthyl butyrate esterase, and phagocytosed carbon particles were found in the periellipsoidal sheaths. Furthermore, in vivo treatment with cyclophosphamide, which resulted in pronounced B-lymphocyte depletion, did not affect FcR activity, but reduced CR activity significantly. These data indicate that the FcR activity demonstrated in tissue sections is mainly confined to mononuclear phagocytes, while the CR positive cells are mainly B lymphocytes.

Effects of a Monoclonal Antibody to Glycoprotein IIb/IIIa (P256) and of Enzymically Derived Fragments of P256 on Human Platelets

1991 ◽  
Vol 65 (04) ◽  
pp. 432-437 ◽  
Author(s):  
A W J Stuttle ◽  
M J Powling ◽  
J M Ritter ◽  
R M Hardisty
Keyword(s):  
Monoclonal Antibody ◽  
Flow Cytometry ◽  
Platelet Aggregation ◽  
Calcium Ions ◽  
Human Platelet ◽  
Human Platelets ◽  
In Vivo Detection ◽  
Fibrinogen Binding ◽  
Specific Antagonist

SummaryThe anti-platelet monoclonal antibody P256 is currently undergoing development for in vivo detection of thrombus. We have examined the actions of P256 and two fragments on human platelet function. P256, and its divalent fragment, caused aggregation at concentrations of 10−9−3 × 10−8 M. A monovalent fragment of P256 did not cause aggregation at concentrations up to 10−7 M. P256–induced platelet aggregation was dependent upon extracellular calcium ions as assessed by quin2 fluorescence. Indomethacin partially inhibited platelet aggregation and completely inhibited intracellular calcium mobilisation. Apyrase caused partial inhibition of aggregation. Aggregation induced by the divalent fragment was dependent upon fibrinogen and was inhibited by prostacyclin. Aggregation induced by the whole antibody was only partially dependent upon fibrinogen, but was also inhibited by prostacyclin. P256 whole antibody was shown, by flow cytometry, to induce fibrinogen binding to indomethacin treated platelets. Monovalent P256 was shown to be a specific antagonist for aggregation induced by the divalent forms. In–111–labelled monovalent fragment bound to gel-filtered platelets in a saturable and displaceable manner. Monovalent P256 represents a safer form for in vivo applications

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