scholarly journals In Vitro and In Vivo Neutralization of the Relapsing Fever Agent Borrelia hermsii with Serotype-Specific Immunoglobulin M Antibodies

2001 ◽  
Vol 69 (2) ◽  
pp. 1009-1015 ◽  
Author(s):  
Alan G. Barbour ◽  
Virgilio Bundoc
Keyword(s):  
Monoclonal Antibodies ◽  
Antibody Response ◽  
Immunoglobulin M ◽  
Enzyme Linked Immunosorbent Assay ◽  
Relapsing Fever ◽  
Borrelia Hermsii ◽  
Intact Cells ◽  
Whole Cells

ABSTRACT The antigenic variation of the relapsing fever agent Borrelia hermsii is associated with changes in the expression of the Vlp and Vsp outer membrane lipoproteins. To investigate whether these serotype-defining proteins are the target of a neutralizing and protective antibody response, monoclonal antibodies were produced from spleens of infected mice just after clearance of serotype 7 cells from the blood. Two immunoglobulin M monoclonal antibodies, H7-7 and H7-12, were studied in detail. Both antibodies specifically agglutinated serotype 7 cells and inhibited their growth in vitro. Administered to mice before or after infection, both antibodies provided protection against infection or substantially reduced the number of spirochetes in the blood of mice after infection. Whereas antibody H7-12 bound to Vlp7 in Western blotting, enzyme-linked immunosorbent assay, and immunoprecipitation assays, as well as to whole cells in other immunoassays, antibody H7-7 only bound to wet, intact cells of serotype 7. Antibody H7-7 selected against cells expressing Vlp7 in vitro and in vivo, an indication that Vlp7 was a conformation-sensitive antigen for the antibody. Vaccination of mice with recombinant Vlp7 with adjuvant elicited antibodies that bound to fixed whole cells of serotype 7 and to Vlp7 in Western blots, but these antibodies did not inhibit the growth of serotype 7 in vitro and did not provide protection against an infectious challenge with serotype 7. The study established that a Vlp protein was the target of a neutralizing antibody response, and it also indicated that the conformation and/or the native topology of Vlp were important for eliciting that immunity.

Characterization of monoclonal antibodies recognizing each extracellular loop domain of occludin

2019 ◽  
Vol 166 (4) ◽  
pp. 297-308 ◽  
Author(s):  
Yoshimi Shimizu ◽  
Yoshitaka Shirasago ◽  
Takeru Suzuki ◽  
Tomoyuki Hata ◽  
Masuo Kondoh ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Transmembrane Protein ◽  
Site Directed Mutagenesis ◽  
Enzyme Linked Immunosorbent Assay ◽  
Intact Cells ◽  
Flow Cytometric ◽  
Junction Protein ◽  
Fcm Analysis

Abstract The tight junction protein occludin (OCLN) is a four-pass transmembrane protein with two extracellular loops (ELs), and also functions as a co-receptor for hepatitis C virus (HCV). Recently, we reported the establishment of monoclonal antibodies (mAbs) recognizing each intact EL domain of OCLN that can strongly prevent HCV infection in vitro and in vivo, and these mAbs were applicable for flow cytometric (FCM) analysis, immunocytochemistry (ICC) and cell-based enzyme-linked immunosorbent assay. In the present study, we further examined the application of these anti-OCLN mAbs and characterized their binding properties. All four mAbs were available for immunoprecipitation. The three first EL (EL1)-recognizing mAbs were applicable for immunoblotting, but the second EL (EL2)-recognizing one was not. Using site-directed mutagenesis, we also determined residues of OCLN critical for recognition by each mAb. Our findings showed that the small loop between two cysteines of the EL2 domain is essential for the binding to one EL2-recognizing mAb and that the recognition regions by three EL1-recognizing mAbs overlap, but are not the same sites of EL1. To obtain a deeper understanding of OCLN biology and its potential as a therapeutic target, specific mAbs to detect or target OCLN in intact cells should be powerful tools for future studies.

scholarly journals A Novel Mouse Monoclonal Antibody C42 against C-Terminal Peptide of Alpha-1-Antitrypsin

2021 ◽  
Vol 22 (4) ◽  
pp. 2141
Author(s):  
Srinu Tumpara ◽  
Elena Korenbaum ◽  
Mark Kühnel ◽  
Danny Jonigk ◽  
Beata Olejnicka ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Flow Cytometry ◽  
Western Blotting ◽  
Complex Mixture ◽  
Immunoglobulin M ◽  
Confocal Laser ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dot Blot

The C-terminal-fragments of alpha1-antitrypsin (AAT) have been identified and their diverse biological roles have been reported in vitro and in vivo. These findings prompted us to develop a monoclonal antibody that specifically recognizes C-36 peptide (corresponding to residues 359–394) resulting from the protease-associated cleavage of AAT. The C-36-targeting mouse monoclonal Immunoglobulin M (IgM) antibody (containing κ light chains, clone C42) was generated and enzyme-linked immunosorbent assay (ELISA)-tested by Davids Biotechnologie GmbH, Germany. Here, we addressed the effectiveness of the novel C42 antibody in different immunoassay formats, such as dot- and Western blotting, confocal laser microscopy, and flow cytometry. According to the dot-blot results, our novel C42 antibody detects the C-36 peptide at a range of 0.1–0.05 µg and shows no cross-reactivity with native, polymerized, or oxidized forms of full-length AAT, the AAT-elastase complex mixture, as well as with shorter C-terminal fragments of AAT. However, the C42 antibody does not detect denatured peptide in SDS-PAGE/Western blotting assays. On the other hand, our C42 antibody, unconjugated as well as conjugated to DyLight488 fluorophore, when applied for immunofluorescence microscopy and flow cytometry assays, specifically detected the C-36 peptide in human blood cells. Altogether, we demonstrate that our novel C42 antibody successfully recognizes the C-36 peptide of AAT in a number of immunoassays and has potential to become an important tool in AAT-related studies.

scholarly journals Modulation of Polymorphonuclear Cell Interleukin-8 Secretion by Human Monoclonal Antibodies to Type 8 Pneumococcal Capsular Polysaccharide

2003 ◽  
Vol 71 (12) ◽  
pp. 6775-6783 ◽  
Author(s):  
Tamika Burns ◽  
Zhaojing Zhong ◽  
Michael Steinitz ◽  
Liise-anne Pirofski
Keyword(s):  
Monoclonal Antibodies ◽  
Capsular Polysaccharide ◽  
Immunoglobulin M ◽  
Interleukin 8 ◽  
Blue Staining ◽  
Enzyme Linked Immunosorbent Assay ◽  
Polymorphonuclear Cells ◽  
Human Monoclonal Antibodies ◽  
Classical Complement Pathway

ABSTRACT Pneumococcal capsular polysaccharide (PS) vaccines induce type-specific immunoglobulin M (IgM), IgG, and IgA. Type-specific IgG to the PS is sufficient to confer protection against the homologous serotype of the pneumococcus, but the efficacies of type-specific IgM and IgA are less well understood. We examined the in vitro activities and efficacies in mice of two human monoclonal antibodies (MAbs) to type 8 PS, NAD (IgA) and D11 (IgM). MAb-mediated opsonophagocytic killing was evaluated after coculture of type 8 pneumococci with human polymorphonuclear cells (PMNs), type-specific or control MAbs, and human complement sources. The effects of the MAbs on PMN interleukin-8 (IL-8) and IL-6 secretion were determined in supernatants from cocultures containing pneumococci and PMNs by enzyme-linked immunosorbent assay. MAb efficacy was determined in an intratracheal model of type 8 infection in mice with classical complement pathway deficiency. Both MAbs were protective in 100% of infected mice. Neither MAb promoted a significant amount of killing of type 8 pneumococci compared to its isotype control MAb. Both type-specific MAbs mediated complement-dependent modulation of PMN IL-8 secretion, with increased secretion at effector/target (E:T) ratios of 500:1 and 50:1 and reduced secretion at 1:5. Trypan blue staining revealed that PMNs cocultured with D11 were less viable at an E:T ratio of 1:5 than PMNs cocultured with the control MAb. PMN IL-6 secretion was increased by both type-specific and control MAbs. These results suggest that certain type-specific IgM and IgAs might contribute to host defense by modulation of the inflammatory response to pneumococci.

scholarly journals Avidity, Potency, and Cross-Reactivity of Monoclonal Antibodies to Pneumococcal Capsular Polysaccharide Serotype 6B

2001 ◽  
Vol 69 (1) ◽  
pp. 336-344 ◽  
Author(s):  
Yan Sun ◽  
Young-il Hwang ◽  
Moon H. Nahm
Keyword(s):  
Monoclonal Antibodies ◽  
Capsular Polysaccharide ◽  
Enzyme Linked Immunosorbent Assay ◽  
Antigen Binding ◽  
Strongly Correlated ◽  
Pneumococcal Infections ◽  
The Cross ◽  
Binding Titer

ABSTRACT Many pneumococcal capsular polysaccharides (PSs) are similar in structure, and a pneumococcal antibody often binds to all of the PSs with a similar structure. Yet, these cross-reactive antibodies may bind to the structurally related pneumococcal capsular PSs with an avidity too low to be effective. If memory B cells producing such weakly cross-reactive antibodies are elicited with pneumococcal conjugate vaccines, the memory cells for low-avidity antibodies could compromise the subsequent immune responses to the cross-reactive PS (original antigenic sin). To investigate these issues, we produced 14 hybridomas secreting monoclonal antibodies (MAbs) to the capsular PS ofStreptococcus pneumoniae serotype 6B by immunizing BALB/c mice with antigens containing 6B PS and studied their epitope, avidity, in vitro opsonizing capacity, in vivo protective capacity, and “antigen binding titer” by enzyme-linked immunosorbent assay (ELISA) of 6A and 6B capsular PSs. Six MAbs bound to the non-cross-reactive 6B-specific epitope, and seven MAbs bound to the cross-reactive epitope present in both 6A and 6B PSs One MAb (Hyp6BM6) revealed a novel epitope. This epitope was found on 6A PS in solution, but not on 6A PS adsorbed onto the plastic surface of the ELISA plates. The avidity of the MAb for 6A or 6B PS ranged from 7.8 × 106 M−1 to 4.1 × 1011M−1. No MAbs were weakly cross-reactive, since none of the cross-reactive MAbs showed any tendency toward having less avidity to 6A PS (the cross-reactive PS) than to 6B PS. Avidity influenced the results of several antibody assays. When all of the hybridomas were examined, avidity strongly correlated with the titer of a unit amount of MAb to bind antigen-coated ELISA plates (r = 0.91) or to opsonize pneumococci in vitro (r = −0.85). Because both assay results are avidity dependent, the ELISA and the opsonization assay results were strongly correlated (r= 0.91), regardless of avidity. Avidity also correlated with the potency of a MAb to passively protect mice against pneumococcal infections. When only the immunoglobulin G hybridomas were examined, little increase in opsonizing capacity and in vivo protective potency was observed above 109 M−1. Taken together, an ELISA measuring antigen binding titer may be an adequate measure of the protective immunity induced with pneumococcal vaccines, and the absence of a partially cross-reactive MAb suggests that antigenic sin may not be significant in responses to vaccines against the S. pneumoniae 6B serotype.

Antipeptide Monoclonal Antibodies to Defined Fibrinogen Aα Chain Regions: Anti-Aα 487-498, a Structural Probe for Fibrinogenolysis

Blood ◽  
1998 ◽  
Vol 91 (5) ◽  
pp. 1590-1598 ◽  
Author(s):  
Joan H. Sobel ◽  
Ilya Trakht ◽  
Nicolas Pileggi ◽  
Hong Qi Wu
Keyword(s):  
Monoclonal Antibodies ◽  
Degradation Products ◽  
Clinical Intervention ◽  
Molecular Forms ◽  
Enzyme Linked Immunosorbent Assay ◽  
Peptide Cleavage ◽  
Multiple Antigenic Peptide ◽  
Structural Probe

The fibrinogen αC domain (Aα 220-610) is one of the earliest targets attacked by plasmin following fibrinolytic system activation. Monoclonal antibodies (MoAbs) to defined sequences within the αC domain provide the opportunity to explore the structure-function relationships involved in plasmin's interaction with its Aα chain substrate at greater resolution and can serve as reagents with potential clinical use for detecting fibrinogenolysis in vivo. The MoAb F-104 was raised against a multiple antigenic peptide derivative modelled after the hydrophilic 12-residue sequence corresponding to Aα 487-498 within the αC domain. A sensitive solution phase competitive enzyme-linked immunosorbent assay (ELISA) was developed for MoAb F-104 that can be applied for the direct measurement of intact fibrinogen (purified or plasma; ED50%≈5 pmol Aα chain equivalents/mL), with negligible cross-reactive interference from peptide cleavage products released by plasmin from the COOH-terminal end of the Aα chain (<3%). Immunoblotting and ELISA studies to characterize the fate of the F-104 epitope during fibrinogenolysis in vitro indicated a rapid loss of fibrinogen-associated immunoreactivity that reflected the heterogeneity of plasmin cleavage sites within the αC domain; cleavage at the 493-494 arg-his bond destroyed the F-104 epitope, while cleavage at other sites released it in an altered, inaccessible, conformation within the structure of 35- to 40-kD and 17.5- to 18-kD Aα chain degradation products. Application of the F-104 ELISA to monitor the course of Aα chain proteolysis in a small study population of patients undergoing thrombolytic therapy for myocardial infarction (n = 14) showed that the loss of fibrinogen-associated F-104 immunoreactivity was a very early marker (within 15 to 30 minutes) of in vivo fibrinogenolysis. Additional data obtained suggest that MoAb F-104 may have promise as a reagent for evaluating the creation of an effective lytic state early during therapy, information that could help determine the need for further clinical intervention. Thus, these studies illustrate a rational, targeted, approach towards the development of a novel antifibrinogen MoAb whose application as a structural probe for the region Aα 487-498 in vitro and in vivo can provide new insights into the various molecular forms of fibrinogen that circulate under physiologic conditions and in disease.

scholarly journals Immunocapture Enzyme-Linked Immunosorbent Assay for Assessment of In Vitro Potency of Recombinant Hepatitis B Vaccines

2010 ◽  
Vol 17 (8) ◽  
pp. 1252-1260 ◽  
Author(s):  
Rajalakshmi Shanmugham ◽  
Nagarajan Thirumeni ◽  
Varaprasada Sankarashetty Rao ◽  
Vidyasagar Pitta ◽  
Saranyarevathy Kasthuri ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Hepatitis B ◽  
Hepatitis B Surface Antigen ◽  
Automated Analysis ◽  
Hepatitis B Vaccine ◽  
Enzyme Linked Immunosorbent Assay ◽  
Batch Release ◽  
Hepatitis B Vaccines

ABSTRACT Quantification of hepatitis B surface antigen (HBsAg) or relative in vitro potency in the final vaccines is a prerequisite for hepatitis B vaccine batch release. The commercial kit for automated analysis (AxSYM) is expensive, and an alternative is required for the estimation of HBsAg in hepatitis B vaccines. Mouse monoclonal antibodies (MAbs) specific for HBsAg were developed and characterized. One of the monoclonal antibodies (HBs06) was used in development of an immunocapture ELISA (IC-ELISA) as an unlabeled capture antibody and biotin-labeled detection antibody. The IC-ELISA was standardized and validated using experimental hepatitis B vaccine batches with various HBsAg concentrations per dose and commercial vaccines. The vaccine was treated with an alkaline solubilizer to desorb the HBsAg from Algel-adjuvanted vaccines before testing, and the sensitivity of the test was 5 ng/ml. A good correlation could be observed between the HBsAg estimates derived by both formats, except for the higher HBsAg concentration range, where the IC-ELISA format could estimate closer to the actual values than AxSYM. There was a significant correlation between the estimated relative potencies of the two methods. There was lack of correlation between the in vivo potency and the relative in vitro potency. However, the estimates of IC-ELISA were comparable to the in vivo values when compared with the estimates of AxSYM. The IC-ELISA can therefore be considered to be a reliable test for deriving in vitro relative potency and antigen concentration in vaccine batches for batch control and release.

scholarly journals Characterization of monoclonal antibodies against Naja naja oxiana neurotoxin I

1994 ◽  
Vol 303 (1) ◽  
pp. 163-170 ◽  
Author(s):  
B G Stiles ◽  
F W Sexton ◽  
S B Guest ◽  
M A Olson ◽  
D C Hack
Keyword(s):  
Monoclonal Antibodies ◽  
Enzyme Linked Immunosorbent Assay ◽  
Naja Naja ◽  
Naja Kaouthia ◽  
Laticauda Semifasciata ◽  
Alpha Bungarotoxin ◽  
Erabutoxin B ◽  
Long Chain

Seven monoclonal antibodies (mAbs) were developed against neurotoxin I (NT-1), a protein from central Asian cobra (Naja naja oxiana) venom which binds specifically to nicotinic acetylcholine receptor (AchR). All of the mAbs cross-reacted with another long-chain post-synaptic neurotoxin, Bungarus multicinctus alpha-bungarotoxin (alpha-BT), but not Naja naja kaouthia alpha-cobratoxin, in an enzyme-linked immunosorbent assay (e.l.i.s.a.). Short-chain post-synaptic neurotoxins like Naja naja atra cobrotoxin, Laticauda semifasciata erabutoxin b, or N. n. oxiana neurotoxin II did not cross-react with the NT-1 mAbs, but an antigen(s) found in Dendroaspis polylepis, Acanthophis antarcticus and Pseudechis australis venoms was immunoreactive. The e.l.i.s.a. readings for dithiothreitol-reduced NT-1 and NT-1 mAbs ranged from 13 to 27% of those for native toxin but reduced alpha-BT was not immunoreactive. Synthetic NT-1 peptides were used in epitope-mapping studies and two, non-contiguous regions (Cys15-Tyr23 and Lys25-Gly33 or Pro17-Lys25 and Asp29-Lys37) were recognized by the NT-1 mAbs. The NT-1 mAbs individually inhibited 31-71% of alpha-BT binding to AchR in vitro and afforded a slight protective effect in vivo with a toxin: antibody mole ratio of 1:1.5. This report is the first to describe mAbs which recognize and protect against a heterologous, long-chain, post-synaptic neurotoxin from snake venom.

scholarly journals Correlation betweenIn VitroComplement Deposition and Passive Mouse Protection of Anti-Pneumococcal Surface Protein A Monoclonal Antibodies

2014 ◽  
Vol 22 (1) ◽  
pp. 99-107 ◽  
Author(s):  
Naeem Khan ◽  
Raies Ahmad Qadri ◽  
Devinder Sehgal
Keyword(s):  
Monoclonal Antibodies ◽  
Bactericidal Activity ◽  
Age Groups ◽  
Enzyme Linked Immunosorbent Assay ◽  
Protein Vaccine ◽  
Hyperimmune Serum ◽  
Passive Protection ◽  
Clade 1

ABSTRACTThe shortcomings of the licensed polysaccharide-based pneumococcal vaccine are driving efforts toward development of a protein-based vaccine that is serotype independent and effective in all age groups. An opsonophagocytic killing assay (OPKA) is used to evaluate the antibody response against polysaccharide-based pneumococcal vaccines. However, the OPKA is not reliable for noncapsular antigens. Thus, there is a need to develop anin vitrosurrogate for protection for protein vaccine candidates like pneumococcal surface antigen A (PspA). PspA is a serologically variable cell surface virulence factor. Based on its sequence, PspA has been classified into families 1 (clade 1 and 2), 2 (clades 3, 4 and 5), and 3 (clade 6). Here, we report the characterization of 18 IgG anti-PspA monoclonal antibodies (anti-PspAhkR36AMAbs) generated from mice immunized with heat-killed strain R36A (clade 2). An enzyme-linked immunosorbent assay (ELISA)-based analysis of the reactivity of the MAbs with recombinant PspAs from the 6 clades indicated that they were family 1 specific. This was confirmed by flow cytometry using a hyperimmune serum generated against PspA from R36A. Eight MAbs that bind at least one clade 1- and clade 2-expressing strain were evaluated for complement deposition, bactericidal activity, and passive protection. The anti-PspAhkR36AMAb-dependent deposition of complement on pneumococci showed a positive correlation with passive protection against strain WU2 (r= 0.8783,P= 0.0041). All of our protective MAbs showed bactericidal activity; however, not all MAbs that exhibited bactericidal activity conferred protectionin vivo. The protective MAbs described here can be used to identify conserved protection eliciting B cell epitopes for engineering a superior PspA-based vaccine.

scholarly journals Variable major proteins of Borrellia hermsii.

1982 ◽  
Vol 156 (5) ◽  
pp. 1312-1324 ◽  
Author(s):  
A G Barbour ◽  
S L Tessier ◽  
H G Stoenner
Keyword(s):  
Molecular Weight ◽  
Monoclonal Antibodies ◽  
Antigenic Variation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Major Protein ◽  
Relapsing Fever ◽  
Western Blots

Borrelia hermsii, a relapsing fever agent, manifests antigenic variation in vivo and in vitro. We studied three mouse-passaged serotypes of strain HS1 (7, 14, and 21) and a HS1 derivative obtained after multiple in vitro passages (C serotype). All four serotypes had two major proteins in whole cell lysates fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. One major protein species (pII) had the same apparent subunit molecular weight (or approximately 3.9 X 10(4) in all the serotypes. In contrast, the other abundant protein in lysates, pI, had a different apparent molecular weight in each serotype. In one gel the molecular weights of pIc, pI7, pI14, and pI21 were 1.9, 4.2, 4.1, and 4.0 X 10(4), respectively. Serotype-specific mouse antisera bound to both hemologous and heterologous pIIs, to homologous pI, but not to heterologous pI in Western blots. Hybridomas were raised from spleens of mice infected with B. hermsii. Monoclonal antibodies were identified by immunofluorescence assays using whole organisms. Monoclonal antibodies specific for serotype 7 (H1826) or for serotype 21 (H3326) bound only to pI7 or pI21, respectively, in Western blots. The surface location of the pI was suggested not only by the immunofluorescence studies but also by the labeling of pI7 and pI21 when whole cells of serotypes 7 and 21 were incubated with 125I in the presence of Iodogen. Under the same circumstances, pII was relatively poorly labeled. These studies have identified the variable pI proteins of B. hermsii as serotype-specific antigens. A change from one pI to another may be the basis of antigenic variation of Borrelia species during relapsing fever.

scholarly journals The Borrelia hermsii Factor H Binding Protein FhbA Is Not Required for Infectivity in Mice or for Resistance to Human ComplementIn Vitro

2014 ◽  
Vol 82 (8) ◽  
pp. 3324-3332 ◽  
Author(s):  
Lindy M. Fine ◽  
Daniel P. Miller ◽  
Katherine L. Mallory ◽  
Brittney K. Tegels ◽  
Christopher G. Earnhart ◽  
...  
Keyword(s):  
Genetic Manipulation ◽  
Binding Protein ◽  
Factor H ◽  
Negative Regulator ◽  
Human Complement ◽  
Relapsing Fever ◽  
Borrelia Hermsii ◽  
Content Type

ABSTRACTThe primary causative agent of tick-borne relapsing fever in North America isBorrelia hermsii. It has been hypothesized thatB. hermsiievades complement-mediated destruction by binding factor H (FH), a host-derived negative regulator of complement.In vitro,B. hermsiiproduces a single FH binding protein designated FhbA (FH binding protein A). The properties and ligand binding activity of FhbA suggest that it plays multiple roles in pathogenesis. It binds plasminogen and has been identified as a significant target of a B1b B cell-mediated IgM response in mice. FhbA has also been explored as a potential diagnostic antigen forB. hermsiiinfection in humans. The ability to test the hypothesis that FhbA is a critical virulence factorin vivohas been hampered by the lack of well-developed systems for the genetic manipulation of the relapsing fever spirochetes. In this report, we have successfully generated aB. hermsiifhbAdeletion mutant (theB. hermsiiYORΔfhbAstrain) through allelic exchange mutagenesis. Deletion offhbAabolished FH binding by the YORΔfhbAstrain and eliminated cleavage of C3b on the cell surface. However, the YORΔfhbAstrain remained infectious in mice and retained resistance to killingin vitroby human complement. Collectively, these results indicate thatB. hermsiiemploys an FhbA/FH-independent mechanism of complement evasion that allows for resistance to killing by human complement and persistence in mice.

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