O-Polysaccharide Epitopic Heterogeneity at the Surface of Brucella spp. Studied by Enzyme-Linked Immunosorbent Assay and Flow Cytometry

ABSTRACT Smooth Brucella strains are classified into three serotypes, i.e., A+M−, A−M+, and A+M+, according to slide agglutination with A and M monospecific polyclonal sera. The epitopes involved have been located on the O-polysaccharide (O-PS) moiety of the smooth lipopolysaccharide (S-LPS), which represents the most exposed antigenic structure on the surface ofBrucella spp. By use of monoclonal antibodies (MAbs) a number of epitope specificities on the O-PS have been reported: A, M, and epitopes shared by both A and M dominant strains, which have been named common (C) epitopes. The latter have been further subdivided, according to relative MAb binding in enzyme-linked immunosorbent assays (ELISA) to A- and M-dominant Brucella strains and to cross-reacting Yersinia enterocolitica O:9, into five epitopic specificities: C (M>A), C (M=A), C/Y (M>A), C/Y (M=A), and C/Y (A>M). In the present study, we studied the occurrence of these epitopes at the surface of representatives of all Brucellaspecies and biovars including the live vaccine strains by analyzing the levels of MAb binding to whole Brucella cells in ELISA and flow cytometry assays. In ELISA, the level of MAb binding correlated well with the previously defined epitope specificity and the serotype defined by polyclonal sera for each Brucella species, biovar, or strain. However, MAbs to the C (M=A) and C (M>A) epitopes showed insignificant binding to B. suis biovar 2 strains and bound at lower titers to B. suis biovar 3 and B. neotomae than to the other Brucella strains. Some of the flow cytometry results were contradictory to those obtained by ELISA. In fact, it appeared by flow cytometry that all O-PS epitopes, including the A and M epitopes, are shared to different degrees byBrucella spp. which nevertheless show a high degree of O-PS heterogeneity according to MAb binding intensities. The subdivision of MAb specificities and Brucella serotypes was therefore less evident by flow cytometry than by ELISA. Whereas in ELISA the MAb specific for the A epitope showed insignificant binding to Y. enterocolitica O:9, this MAb bound strongly to Y. enterocolitica O:9 in flow cytometry. One of the two MAbs specific to the C (M=A) epitope also bound at a low but significant level to B. suis biovar 2 strains. However, as in ELISA the MAb specific for the C (M>A) epitope did not bind at all to B. suis biovar 2 strains in flow cytometry. Flow cytometry provided new information regarding specificity of the MAbs and may further explain some aspects of the capacity of passive protection of some MAbs against smooth Brucella infection in mice. As shown in the present study the occurrence of Brucella strains apparently completely devoid of one specific C O-PS epitope (e.g., B. suis biovar 2 devoid of the C [M>A] epitope) offers the possibility of obtaining vaccine strains devoid of a diagnostic O-PS epitope, which could further help to resolve the problem of discriminating infected from vaccinated animals that remains a major goal in brucellosis research.

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Monoclonal Antibodies Generated against Glycoconjugates Recognize Chemical Linkers

Antibodies ◽

10.3390/antib9030048 ◽

2020 ◽

Vol 9 (3) ◽

pp. 48

Author(s):

Jessica Ramadhin ◽

Vanessa Silva-Moraes ◽

Thomas Norberg ◽

Donald Harn

Keyword(s):

Drug Delivery ◽

Flow Cytometry ◽

Monoclonal Antibodies ◽

Structure And Function ◽

Enzyme Linked Immunosorbent Assay ◽

Incomplete Freund’S Adjuvant ◽

Delivery Platform ◽

Igg1 Mabs ◽

Human Milk Oligosaccharide ◽

And Function

Monoclonal antibodies (mAbs) that recognize glycans are useful tools to assess carbohydrates’ structure and function. We sought to produce IgG mAbs to the human milk oligosaccharide (HMO), lacto-N-fucopentaose III (LNFPIII). LNFPIII contains the Lewisx antigen, which is found on the surface of schistosome parasites. mAbs binding the Lewisx antigen are well-reported in the literature, but mAbs recognizing HMO structures are rare. To generate mAbs, mice were immunized with LNFPIII-DEX (P3DEX) plus CpGs in VacSIM®, a novel vaccine/drug delivery platform. Mice were boosted with LNFPIII-HSA (P3HSA) plus CpGs in Incomplete Freund’s Adjuvant (IFA). Splenocytes from immunized mice were used to generate hybridomas and were screened against LNFPIII conjugates via enzyme-linked immunosorbent assay (ELISA). Three positive hybridomas were expanded, and one hybridoma, producing IgG and IgM antibodies, was cloned via flow cytometry. Clone F1P2H4D8D5 was selected because it produced IgG1 mAbs, but rescreening unexpectedly showed binding to both LNFPIII and lacto-N-neotetraose (LNnT) conjugates. To further assess the specificity of the mAb, we screened it on two glycan microarrays and found no significant binding. This finding suggests that the mAb binds to the acetylphenylenediamine (APD) linker-spacer structure of the conjugate. We present the results herein, suggesting that our new mAb could be a useful probe for conjugates using similar linker spacer structures.

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Monoclonal Antibodies Reactive with Immunorecessive Epitopes of Glucuronoxylomannan, the Major Capsular Polysaccharide of Cryptococcus neoformans

Clinical and Diagnostic Laboratory Immunology ◽

10.1128/cdli.10.5.903-909.2003 ◽

2003 ◽

Vol 10 (5) ◽

pp. 903-909 ◽

Cited By ~ 18

Author(s):

Suzanne Brandt ◽

Peter Thorkildson ◽

Thomas R. Kozel

Keyword(s):

Monoclonal Antibody ◽

Immune Response ◽

Monoclonal Antibodies ◽

Cryptococcus Neoformans ◽

Capsular Polysaccharide ◽

Passive Immunization ◽

The Other ◽

Serotype A ◽

Humoral Immune ◽

New Information

ABSTRACT Cryptococcus neoformans is surrounded by an antiphagocytic capsule whose primary constituent is glucuronoxylomannan (GXM). An epitope shared by GXM serotypes A, B, C, and D is immunodominant when mice are immunized with serotype A GXM. In contrast, an epitope shared only by serotypes A and D is immunodominant when mice are immunized with serotype D. Hybridomas secreting antibodies reactive with subdominant epitopes were identified through a positive-negative screening procedure in which antibody-secreting colonies were characterized by reactivity with both the immunizing polysaccharide and GXMs from each of the four major serotypes. In this manner, a monoclonal antibody (MAb) that was reactive with an epitope shared only by serotypes A and B was identified and designated F10F5. Such an epitope has not been described previously. Immunization of mice with de-O-acetylated serotype A GXM generated a hybridoma that secreted an antibody, designated F12D2, that was reactive with all four serotypes. Unlike previously described monoclonal and polyclonal panspecific antibodies, the reactivity of MAb F12D2 was not altered by de-O-acetylation of GXM. These results indicate that there are at least two panspecific GXM epitopes; one epitope is dependent on O acetylation for antibody reactivity, and the other is independent of O acetylation. This study identifies strategies for production of MAbs that are reactive with subdominant or cryptic GXM epitopes and provides new information regarding the antigenic makeup and the humoral immune response to GXM, an essential virulence factor that is a target for active and passive immunization.

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Production of species-specific monoclonal antibodies that react with surface components on zoospores and cysts of Phytophthora nicotianae

Canadian Journal of Microbiology ◽

10.1139/w98-120 ◽

1998 ◽

Vol 44 (12) ◽

pp. 1161-1170 ◽

Cited By ~ 11

Author(s):

A V Robold ◽

A R Hardham

Keyword(s):

Monoclonal Antibodies ◽

Cell Lines ◽

Phytophthora Cinnamomi ◽

Phytophthora Nicotianae ◽

The Other ◽

Hybridoma Cells ◽

Enzyme Linked Immunosorbent Assay ◽

Specific Antibodies ◽

Hybridoma Cell Lines ◽

Species Specific

Monoclonal antibodies were generated against components on the surface of zoospores and cysts of the Oomycete, Phytophthora nicotianae, with the aim of obtaining antibodies diagnostic for this species of plant pathogen. A dipstick version of an enzyme-linked immunosorbent assay was used to screen hybridoma cell lines produced by following a coimmunization protocol in which a mouse was immunized with Phytophthora nicotianae cysts mixed with murine antisera raised against cysts of Phytophthora cinnamomi and Phytophthora cryptogea. Of the nine hybridoma cells lines which remained positive, five produced antibodies that reacted with species-specific epitopes on the surface of the spores. Immunofluorescence, immunogold, and immunoblot labelling showed that three of the five species-specific antibodies reacted with a polypeptide of relative molecular mass greater than 205 kDa which was distributed over the entire zoospore surface, including that of the two flagella. These antibodies also labelled the surface of cysts to varying degrees. The other two species-specific antibodies bound to the shaft of tubular mastigonemes that form two rows on the anterior flagellum. In immunoblots, one of these antibodies recognised a 40-kDa glycoprotein. Antibodies produced by the other four hybridoma cell lines reacted with all Phytophthora and Pythium species tested. The results (i) showed that the coimmunization technique effectively produced antibodies directed towards components specific for Phytophthora nicotianae in the presence of antigens common to many Phytophthora species, and (ii) revealed for the first time the biochemical nature of molecular constituents of flagellar mastigonemes in the Oomycetes.Key words: cell surface, flagella, immunodiagnostics, mastigonemes, monoclonal antibodies.

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Monoclonal antibodies against plasma protease inhibitors: II. Production and characterization of 25 monoclonal antibodies against human α1-antitrypsin. Correlation between antigenic structure and functional sites

Bioscience Reports ◽

10.1007/bf01120310 ◽

1984 ◽

Vol 4 (2) ◽

pp. 139-147 ◽

Cited By ~ 10

Author(s):

P. Hérion ◽

D. Siberdt ◽

M. Francotte ◽

J. Urbain ◽

A. Bollen

Keyword(s):

Monoclonal Antibody ◽

Monoclonal Antibodies ◽

Protease Inhibitors ◽

Antigenic Structure ◽

Light Chains ◽

Enzyme Linked Immunosorbent Assay ◽

Functional Sites ◽

Heavy Chains ◽

Low Levels

Twenty-five hybridomas secreting monoclonal antibodies against human α1-antitrypsim have been produced by the cell-fusion techmque (Kóhler and Milstein, 1976). All antibodies are specific for α1-antitrypsim and carry γ1-antitrypsim heavy chains and κ light chains. Inhibition experiments showed that these monoclonal antibodies define three independent antigenic regions on the α1-antitrypsim molecule; one of these domains appears to be involved in the interaction between α1-antitrypsim and trypsin. In addition, one monoclonal antibody, AATY39, was used to develop an enzyme-linked immunosorbent assay capable of detecting low levels of α1-antitrypsim in the range of 1 to 2 ng/ml.

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Monoclonal antibodies against plasma protease inhibitors: I. Production and characterization of 23 monoclonal antibodies against human α2

Bioscience Reports ◽

10.1007/bf01120822 ◽

1984 ◽

Vol 4 (1) ◽

pp. 39-48 ◽

Cited By ~ 5

Author(s):

P. Herion ◽

D. Siberdt ◽

G. Garduno Soto ◽

J. Urbain ◽

A. Bollen

Keyword(s):

Monoclonal Antibodies ◽

Protease Inhibitors ◽

Light Chains ◽

Enzyme Linked Immunosorbent Assay ◽

High Avidity ◽

Fusion Technique ◽

Epitope Specificity ◽

Fast Acting ◽

Inhibition Pattern

23 hybridomas secreting monoclonal antibodies against human α2, the fast-acting inhibitor of plasmin present in plasma, have been produced by the cell-fusion technique. Isotyping of the monoclonal antibodies has revealed that 14 monoclonal antibodies belong to the class IgG1, 6 to the class IgG2a, and 3 to the class tgG2b. All light chains belong to the κ group. The specificity and relative avidity of these monoclonals have been determined Using an indirect enzyme-linked immunosorbent assay. 13 monoclonals exhibit a relatively high avidity for α2, 5 are of intermediate avidity, and 5 of low avidity. The epitope specificity of these 23 rnonoclonal antibodies, originating from a single mouse, have been examined in inhibition experiments. A group of 10 monoclonal antibodies exhibit a very similar inhibition pattern. Partial inhibition effects displayed by 10 other antibodies define partially overlapping antigenic regions. The binding of these antibodies seems to produce a conformational change in the α2 molecule, reducing the binding of two other antibodies. The last antibody defines an independent epitope.

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

Clinical and Vaccine Immunology ◽

10.1128/cvi.00001-14 ◽

2014 ◽

Vol 22 (1) ◽

pp. 99-107 ◽

Cited By ~ 8

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.

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MONOCLONAL ANTIBODIES AGAINST THROHBIN-ANTITHROMBIN III COMPLEX: EPITOPE SPECIFICITY AND EFFECT ON THROMBIN-ANTITHROMBIN III INTERACTION

10.1055/s-0038-1643673 ◽

1987 ◽

Author(s):

S Asakura ◽

N Yoshida ◽

M Matsuda

Keyword(s):

Monoclonal Antibodies ◽

Antithrombin Iii ◽

Normal Serum ◽

Reactive Site ◽

High Affinity ◽

Epitope Specificity ◽

New Information ◽

Sds Page ◽

Human Thrombin ◽

Immunoglobulin Subclasses

Among monoclonal antibodies (MCA´s) raised against human thrombin (T)-antithrombin m (AT) complex (TAT), two MCA´s designated as JITAT-16 and 17 with high affinity, Kd = 4.6nMand 4.1 nfi, respectively, were selected and characterized for specificity and functions. Their respective immunoglobulin subclasses are IgGi and IgG2a, and epitopes were found to be different from each Dther as shown by crisscross inhibition experiments. Immuno-alotting of normal plasma and serum electrophoresed on non-SDS aolyacrylamide gel showed that these antibodies reacted with normal serum but not with plasma. This was verified by an anzyme-linked differential antibody immunosorbent assay using aither one of the MCA´s as the first antibody and the other MCA labeled with peroxidase as the second one. By immunoblotting after SDS-PAGE, we found that both antibodies reacted with TAT, nut not with its respective nascent constituent, AT or T. However, they reacted with reactive site-cleaved AT (or thrombin-nodified AT, ATM) and also a complex of AT with activated factor K (Xa-AT). These results indicate that both of these antibodies recognize enzyme-treated forms of AT, including AT molecules :omplexed with enzymes reversibly or irreversibly as well as ATM. Jpon incubation of T with AT in the presence of JITAT-16, T activity remained nearly unchanged and formation of irreversible rAT did not proceed as expected. Moreover, AT was preferentially :onverted to ATM. When JITAT-16 was added after completion of FAT formation, however, neither recovery of T activity nor generation of ATM was observed. These findings were not obtained vhen JITAT-17 had been substituted for JITAT-16. These data suggest that JITAT-16 may have converted AT from an inhibitor to a substrate for T after having recognized a possible intermediate reversible complex of AT with T. Undoubtedly, in the presence of a polyclonal antibody against AT, neither TAT formation nor ATM neneration was observed at all. The mechanism of the unique Function of JITAT-16 has not been fully clarified as yet, but this antibody seems to give us new information on the kinetic study of TAT formation and ATM generation when AT was allowed to react with enzymes.

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Monoclonal Antibodies to Immunodominant Epitope of Tropheryma whipplei

Clinical and Vaccine Immunology ◽

10.1128/cdli.9.1.156-159.2002 ◽

2002 ◽

Vol 9 (1) ◽

pp. 156-159 ◽

Cited By ~ 2

Author(s):

Zhongxing Liang ◽

Bernard La Scola ◽

Didier Raoult

Keyword(s):

Monoclonal Antibodies ◽

Biopsy Specimen ◽

Tropheryma Whipplei ◽

Duodenal Biopsy ◽

The Other ◽

Enzyme Linked Immunosorbent Assay ◽

Whipple’S Disease ◽

Immunodominant Epitope ◽

Whipple's Disease ◽

The Common

ABSTRACT Recent isolation of Tropheryma whipplei (formerly Trophyrema whippelii), the agent of Whipple’s disease, from the cardiac valve of a patient with Whipple’s disease endocarditis now allows the detection of reactive epitopes that could be used in a serological assay. In order to propose an enzyme-linked immunosorbent assay (ELISA) that uses recombinant T. whipplei antigen, we first determined by Western blotting of human, mouse, and rabbit antisera that the common immunodominant epitope is an 84-kDa protein. We then produced 13 monoclonal antibodies (MAbs) against T. whipplei, 12 of which recognize this immunodominant epitope. These MAbs did not react with phylogenetically closely related bacteria or bacteria previously shown to be cross-reactive with T. whipplei, but they did react with two other strains of T. whipplei isolated, one from an ocular sample and the other from a duodenal biopsy specimen. By confocal microscopy, the MAbs allowed detection of T. whipplei within infected fibroblasts. The identification of the 84-kDa antigen with our MAbs will make it possible to develop a diagnostic antigen for use in a diagnostic ELISA for Whipple’s disease.

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Characterization and Formulation of Multiple Epitope-Specific Neutralizing Monoclonal Antibodies for Passive Immunization against Cryptosporidiosis

Infection and Immunity ◽

10.1128/iai.68.5.2608-2616.2000 ◽

2000 ◽

Vol 68 (5) ◽

pp. 2608-2616 ◽

Cited By ~ 33

Author(s):

Deborah A. Schaefer ◽

Beth A. Auerbach-Dixon ◽

Michael W. Riggs

Keyword(s):

Monoclonal Antibodies ◽

Cryptosporidium Parvum ◽

Polyclonal Antibodies ◽

Passive Immunization ◽

Infection Process ◽

Enzyme Linked Immunosorbent Assay ◽

Intestinal Infection ◽

Coccidian Parasite ◽

Epitope Specificity ◽

Significant Efficacy

ABSTRACT The coccidian parasite Cryptosporidium parvum causes diarrhea in humans, calves, and other mammals. Neither immunization nor parasite-specific pharmaceuticals that are consistently effective against this organism are available. While polyclonal antibodies against whole C. parvum reduce infection, their efficacy and predictability are suboptimal. We hypothesized that passive immunization against cryptosporidiosis could be improved by using neutralizing monoclonal antibodies (MAbs) targeting functionally defined antigens on the infective stages. We previously reported that the apical complex and surface-exposed zoite antigens CSL, GP25-200, and P23 are critical in the infection process and are therefore rational targets. In the present study, a panel of 126 MAbs generated against affinity-purified CSL, GP25-200, and P23 was characterized to identify the most efficacious neutralizing MAb formulation targeting each antigen. To identify neutralizing MAbs, sporozoite infectivity following exposure to individual MAbs was assessed by enzyme-linked immunosorbent assay. Of 126 MAbs evaluated, 47 had neutralizing activity. These were then evaluated individually in oocyst-challenged neonatal mice, and 14 MAbs having highly significant efficacy were identified for further testing in formulations. Epitope specificity assays were performed to determine if candidate MAbs recognized the same or different epitopes. Formulations of two or three neutralizing MAbs, each recognizing distinct epitopes, were then evaluated. A formulation of MAbs 3E2 (anti-CSL [αCSL]), 3H2 (αGP25-200), and 1E10 (αP23) provided highly significant additive efficacy over that of either individual MAbs or combinations of two MAbs and reduced intestinal infection by 86 to 93%. These findings indicate that polyvalent neutralizing MAb formulations targeting epitopes on defined antigens may provide optimal passive immunization against cryptosporidiosis.

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Monoclonal Antibodies Specific for Immunorecessive Epitopes of Glucuronoxylomannan, the Major Capsular Polysaccharide of Cryptococcus neoformans, Reduce Serotype Bias in an Immunoassay for Cryptococcal Antigen

Clinical and Vaccine Immunology ◽

10.1128/cvi.05052-11 ◽

2011 ◽

Vol 18 (8) ◽

pp. 1292-1296 ◽

Cited By ~ 28

Author(s):

Ann Percival ◽

Peter Thorkildson ◽

Thomas R. Kozel

Keyword(s):

Monoclonal Antibodies ◽

Cryptococcus Neoformans ◽

Capsular Polysaccharide ◽

Polyclonal Antibodies ◽

Sub Saharan Africa ◽

Enzyme Linked Immunosorbent Assay ◽

Content Type ◽

Screening Strategies ◽

Sub Saharan ◽

High Degree

ABSTRACTImmunoassay for detection of glucuronoxylomannan (GXM), the major capsular polysaccharide ofCryptococcus neoformans, is an important tool for diagnosis of cryptococcosis. However, immunoassays that are based solely or in part on detection with polyclonal antibodies may show serotype bias in detection of GXM, particularly limited sensitivity for serotype C. In this study, we describe detection of GXM in an antigen capture sandwich enzyme-linked immunosorbent assay (ELISA) that used a cocktail of two monoclonal antibodies (MAbs). MAb F12D2 was previously produced by immunization with GXM that had been treated to removeO-acetyl groups, a major source of serotype specificity. MAb F12D2 has a high degree of reactivity with GXM of serotypes A, B, C, and D, but the reactivity with serotype D was less than was found with other MAbs. MAb 339 is highly reactive with GXM of serotypes A and D. Use of a combination of the two MAbs produced an immunoassay that had the best properties of both MAbs, including good reactivity with serotype C, which is an emerging threat in sub-Saharan Africa. These results suggest that next-generation immunoassays for diagnosis of cryptococcosis may be formulated by (i) use of immunization and hybridoma screening strategies that are designed to prospectively meet the needs of immunoassay performance and (ii) careful selection of MAbs that span the expected polysaccharide serotypes in the subject patient population.

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