Comparison of different methods for localizing antigenic regions in histone H2A

Muller, Sylviane; Plaué, S.; Couppez, Maurice; Regenmortel, M.H.V. Van

doi:10.1016/0161-5890(86)90095-7

Cited by 91 publications

(33 citation statements)

References 20 publications

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“…These simple dilution curves support the earlier results of double-diffusion assays, which suggested that polyclonal sera did not distinguish between centrifugal components of CPMV (Bruening & Agrawal, 1967). However, binding of antigen to microtitre plates promotes substantial changes in conformation and antigenic character of proteins (Friguet et al, 1984;Halk, 1986;Muller et al, 1986). To minimize the influence of these factors on measurement of antibody specificity, liquid phase competition assays were performed (McCullough et al, 1985).…”

supporting

confidence: 50%

Serological Differentiation between Top Component and Nucleoprotein Components of Comoviruses

Kalmar

Eastwell

1989

Journal of General Virology

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SUMMARYRabbit antisera produced against two comoviruses (cowpea mosaic virus and cowpea severe mosaic virus) were used in plate-trapped ELISA and liquid phase competition ELISA. In the latter, the top component competed against bound unfractionated virus more effectively than did nucleoprotein components. One murine monoclonal antibody elicited to cowpea mosaic virus and three monoclonal antibodies to cowpea severe mosaic virus exhibited differential binding to top component, relative to either middle or bottom components in plate-trapped or antibody-trapped ELISAs. Differential binding to centrifugal components of virus by two of these monoclonal antibodies was maintained in liquid phase competition assays, These data suggest that the encapsidation of RNA alters the configuration of the virion surface in the vicinity of the antibody epitopes. Ribonuclease digestion of intact or denatured virus did not affect the binding of monoclonal antibodies.Like other comoviruses, cowpea mosaic virus (CPMV) and cowpea severe mosaic virus (CPSMV) are resolved into three major fractions by density gradient centrifugation (Bruening, 1969). The slowest sedimenting top component consists of empty capsids whereas the middle and bottom components are nucleocapsids containing one molecule of either RNA 2 (Mr 1"37 x 106) or RNA 1 (Mr 2.02 x 106), respectively. PAGE does not show differences between the protein composition of the three components (Geelen et al., 1972;Wu & Bruening, 1971) and rabbit antisera prepared against unfractionated virus reacts with all components in Ouchterlony double-diffusion assays (Bruening & Agrawal, 1967). This paper reports that liquid phase competition assays utilizing rabbit antisera reveal a difference between the binding of antibodies to empty capsids and to nucleoprotein virions. Antigenic differences between top component and middle or bottom components were confirmed by assays with monoclonal antibodies.CPSMV-DG and CPMV-SB isolates were originally obtained from G. Bruening (Department of Plant Pathology, University of California, Davis, Ca., U.S.A.), propagated in cowpeas (Vigna unguiculata Walp. cv. California Black Eye) and isolated as previously described (Bruening, 1969). Approximately 5 mg virus was suspended in 0-5 ml gradient buffer (50 mM-KH2PO, pH 7.0, 2 mM-disodium EDTA) and layered onto 11 ml of 39 ~ (w/w) caesium chloride in gradient buffer. Gradients were centrifuged for 36 h at 36000 r.p.m, and 4 °C in an SW 40Ti rotor (Beckman) and fractionated by side-puncture. Fractions were diluted at least 10-fold in gradient buffer and pelleted by centrifugation at 45000r.p.m. for 2h. The pellets were resuspended in gradient buffer. Concentrations of virus components were calculated using A260 at 1 mg/ml of 6.2, 10-0 and 8.1 for middle component, bottom component and unfractionated virus and A2so at 1 mg/ml of 1.28 for top component (Geelen et al., 1972).The preparation of rabbit antisera and of the panel of monoclonal antibodies produced by hybridomas have been described (Kalmar & Eastwell,...

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confidence: 50%

Serological Differentiation between Top Component and Nucleoprotein Components of Comoviruses

Kalmar

Eastwell

1989

Journal of General Virology

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“…Yet 8.3% of these nonepitopes are reported as "epitopes" in the fBcpreds dataset (21). Thus, binary classification of antigen regions into epitopes or non-epitopes is problematic because all epitopes of most antigens are not known, and defining B-cell epitopes and nonepitopes is a challenging task due to the variability in epitope discovery assays (71) and the stochastic antibody responses to protein antigens (9) and their epitopes (2). Muller et al (71) found almost the entire histone 2A protein antigenic when they forced highest B-cell stimulation and antibody reactivity by excessive use of adjuvants and high antigen doses.…”

Section: Discussionmentioning

confidence: 99%

“…Thus, binary classification of antigen regions into epitopes or non-epitopes is problematic because all epitopes of most antigens are not known, and defining B-cell epitopes and nonepitopes is a challenging task due to the variability in epitope discovery assays (71) and the stochastic antibody responses to protein antigens (9) and their epitopes (2). Muller et al (71) found almost the entire histone 2A protein antigenic when they forced highest B-cell stimulation and antibody reactivity by excessive use of adjuvants and high antigen doses. In contrast, raising antisera in our study by experimental infection rather than by forced immunization very likely resulted in much lower adjuvantation and lower antigenic stimulus by physiologically processed native protein antigens (2).…”

Section: Discussionmentioning

confidence: 99%

Inadequate Reference Datasets Biased toward Short Non-epitopes Confound B-cell Epitope Prediction

Rahman

Chowdhury

Sachse

et al. 2016

Journal of Biological Chemistry

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X-ray crystallography has shown that an antibody paratope typically binds 15-22 amino acids (aa) of an epitope, of which 2-5 randomly distributed amino acids contribute most of the binding energy. In contrast, researchers typically choose for B-cell epitope mapping short peptide antigens in antibody binding assays. Furthermore, short 6 -11-aa epitopes, and in particular non-epitopes, are over-represented in published B-cell epitope datasets that are commonly used for development of B-cell epitope prediction approaches from protein antigen sequences. We hypothesized that such suboptimal length peptides result in weak antibody binding and cause false-negative results. We tested the influence of peptide antigen length on antibody binding by analyzing data on more than 900 peptides used for B-cell epitope mapping of immunodominant proteins of Chlamydia spp. We demonstrate that short 7-12-aa peptides of B-cell epitopes bind antibodies poorly; thus, epitope mapping with short peptide antigens falsely classifies many B-cell epitopes as non-epitopes. We also show in published datasets of confirmed epitopes and non-epitopes a direct correlation between length of peptide antigens and antibody binding. Elimination of short, <11-aa epitope/non-epitope sequences improved datasets for evaluation of in silico B-cell epitope prediction. Achieving up to 86% accuracy, protein disorder tendency is the best indicator of B-cell epitope regions for chlamydial and published datasets. For B-cell epitope prediction, the most effective approach is plotting disorder of protein sequences with the IUPred-L scale, followed by antibody reactivity testing of 16 -30-aa peptides from peak regions. This strategy overcomes the well known inaccuracy of in silico B-cell epitope prediction from primary protein sequences.Knowledge of B-cell epitopes of proteins is essential in many fields of applied biomedical research, such as antibody diagnostics and therapeutics, vaccines, as well basic research. Laboratory methods for identification of such epitopes are time-consuming and labor-intensive. Hence, any reduction in the need for discovery and confirmatory wet-lab research by epitope prediction algorithms is highly desirable. Among in silico predictive methods from primary sequence information, epitope prediction algorithms are distinguished for their lack of reliability (1). This underperformance prompted us to examine current approaches to B-cell epitope prediction by use of extensive data on epitopes and confirmed non-epitope regions of the Chlamydia spp. proteome, accumulated in research on chlamydial molecular serology (2).Recent three-dimensional antibody-antigen complex studies (3-7) show that about 15-22-aa 2 antigen peptide residues are structurally involved in binding of epitopes to ϳ17-aa residues in antibody complementarity-determining regions (CDRs; paratopes). Among these 15-22 structural epitope residues, about 2-5 aa, termed functional residues, contribute most of the total binding energy to antibodies (6). These functional residues lie...

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“…The coupled peptide was mixed (1:1) with complete Freund adjuvant and injected (2 ml/animal) as in Muller (1986). The antibody was obtained by (NH 4 ) 2 SO 4 precipitation at different bleedings followed by absorption on a protein A/G affinity column as in Andrew (1992).…”

Section: Polyclonal Anti-cellubrevin and Anti-nsf Antibody Productionmentioning

confidence: 99%

“…Polyclonal antibody directed against full-length NSF-His was raised in New Zealand White female rabbits as in Muller (1986). The recombinant protein was expressed and purified as in Morgan (1995).…”

Section: Polyclonal Anti-cellubrevin and Anti-nsf Antibody Productionmentioning

confidence: 99%

Endothelial Transcytotic Machinery Involves Supramolecular Protein–Lipid Complexes

Predescu

Palade

2001

MBoC

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We have demonstrated that the plasmalemmal vesicles (caveolae) of the continuous microvascular endothelium function as transcytotic vesicular carriers for protein molecules Ͼ20 Å and that transcytosis is an N-ethylmaleimide-sensitive factor (NSF)-dependent, N-ethylmaleimide-sensitive process. We have further investigated NSF interactions with endothelial proteins to find out 1) whether a complete set of fusion and targeting proteins is present in the endothelium; 2) whether they are organized in multimolecular complexes as in neurons; and 3) whether the endothelial multimolecular complexes differ from their neuronal counterparts, because of their specialized role in transcytosis. To generate the complexes, we have used myc-NSF, cultured pulmonary endothelial cells, and rat lung cytosol and membrane preparations; to detect them we have applied coimmunoprecipitation with myc antibodies; and to characterize them we have used velocity sedimentation and cross-linking procedures. We have found that both cytosolic and membrane fractions contain complexes that comprise beside soluble NSF attachment proteins and SNAREs (soluble NSF attachment protein receptor), rab 5, dynamin, caveolin, and lipids. By immunogold labeling and negative staining we have detected in these complexes, myc-NSF, syntaxin, dynamin, caveolin, and endogenous NSF. Similar complexes are formed by endogenous NSF. The results indicate that complexes with a distinct protein-lipid composition exist and suggest that they participate in targeting, fusion, and fission of caveolae with the endothelial plasmalemma.

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Comparison of different methods for localizing antigenic regions in histone H2A

Cited by 91 publications

References 20 publications

Serological Differentiation between Top Component and Nucleoprotein Components of Comoviruses

Serological Differentiation between Top Component and Nucleoprotein Components of Comoviruses

Inadequate Reference Datasets Biased toward Short Non-epitopes Confound B-cell Epitope Prediction

Endothelial Transcytotic Machinery Involves Supramolecular Protein–Lipid Complexes

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