scholarly journals Epitope Mapping of the Diphtheria Toxin and Development of an ELISA-Specific Diagnostic Assay

Vaccines ◽  
2021 ◽  
Vol 9 (4) ◽  
pp. 313
Author(s):  
Salvatore Giovanni De-Simone ◽  
Larissa Rodrigues Gomes ◽  
Paloma Napoleão-Pêgo ◽  
Guilherme Curty Lechuga ◽  
Jorge Soares de Pina ◽  
...  
Keyword(s):  
B Cell ◽  
Diphtheria Toxin ◽  
High Performance ◽  
Catalytic Domain ◽  
Elisa Test ◽  
Antigenic Determinants ◽  
Enzyme Linked Immunosorbent Assay ◽  
Spot Synthesis ◽  
Humoral Immune

Background: The diphtheria toxoid antigen is a major component in pediatric and booster combination vaccines and is known to raise a protective humoral immune response upon vaccination. Although antibodies are considered critical for diphtheria protection, little is known about the antigenic determinants that maintain humoral immunity. Methods: One-hundred and twelve 15 mer peptides covering the entire sequence of diphtheria toxin (DTx) protein were prepared by SPOT synthesis. The immunoreactivity of membrane-bound peptides with sera from mice immunized with a triple DTP vaccine allowed mapping of continuous B-cell epitopes, topological studies, multiantigen peptide (MAP) synthesis, and Enzyme-Linked Immunosorbent Assay (ELISA) development. Results: Twenty epitopes were identified, with two being in the signal peptide, five in the catalytic domain (CD), seven in the HBFT domain, and five in the receptor-binding domain (RBD). Two 17 mer (CB/Tx-2/12 and CB/DTx-4–13) derived biepitope peptides linked by a Gly-Gly spacer were chemically synthesized. The peptides were used as antigens to coat ELISA plates and assayed with human (huVS) and mice vaccinated sera (miVS) for in vitro diagnosis of diphtheria. The assay proved to be highly sensitive (99.96%) and specific (100%) for huVS and miVS and, when compared with a commercial ELISA test, demonstrated a high performance. Conclusions: Our work displayed the complete picture of the linear B cell IgG response epitope of the DTx responsible for the protective effect and demonstrated sufficient specificity and eligibility for phase IIB studies of some epitopes to develop new and fast diagnostic assays.

scholarly journals Epitope Mapping of the Diphtheria Toxin and Development of an ELISA-Specific Diagnostic Assay

2021 ◽  
Author(s):  
Salvatore Giovanni De-Simone ◽  
Larissa Rodrigues Gomes ◽  
Paloma Napoleão-Pêgo ◽  
Guilherme Curty Lechuga ◽  
Jorge Soares de Pina ◽  
...  
Keyword(s):  
B Cell ◽  
High Performance ◽  
Elisa Test ◽  
Antigenic Determinants ◽  
Spot Synthesis ◽  
Diagnostic Assays ◽  
Humoral Immune ◽  
Entire Sequence ◽  
Membrane Bound

(1) Background: The diphtheria toxoid antigen is a major component in pediatric and booster combination vaccines and is known to raise a protective humoral immune response upon vaccination. Although antibodies are considered critical for diphtheria protection, little is known about the antigenic determinants that maintain humoral immunity. (2) Methods: One hundred twelve 15-mer peptides covering the entire sequence of DTx protein were prepared by Spot-synthesis. Membrane-bound peptides immunoreactivity with sera from mice immunized with triple DTP vaccine allowed mapping of continuous B-cell epitopes, topological studies, MAPs synthesis, and ELISA development. (3) Results: Twenty epitopes were identified, being 2 in the signal peptide, 5 in the CD, 7 in the HBFT domain, and 5 in the RBD. Two 17-mer (CB/Tx-2/12 and CB/DTx-4-13) derived bi-epitope peptides linked by a Gly-Gly spacer were chemically synthesized. The peptides were used as antigens to coat ELISA plates and assayed with human (huVS) and mice vaccined sera (miVS) for in vitro diagnosis of diphtheria. The assay proved to be highly sensitive (99.96%) and specific (100%) either for huVS and miVS and when compared with a commercial ELISA test, demonstrate high performance. (4) Conclusions: Our work displayed the complete picture of the linear B cell IgG response epitope of the DTx responsible for the protective effect and demonstrated the specificity and eligibility to enter phase IIB studies of some epitopes to develop new and fast diagnostic assays.

scholarly journals Monoclonal antibodies to the 140,000 mol wt glycoprotein of B lymphocyte membranes (CR2 receptor) initiates proliferation of B cells in vitro

Blood ◽  
1985 ◽  
Vol 66 (4) ◽  
pp. 824-829
Author(s):  
BS Wilson ◽  
JL Platt ◽  
NE Kay
Keyword(s):  
Monoclonal Antibodies ◽  
B Cells ◽  
B Cell ◽  
Peripheral Blood ◽  
B Lymphocyte ◽  
Human Peripheral Blood ◽  
Raji Cell ◽  
Lymphoid Cells ◽  
Humoral Immune

Several mouse monoclonal IgG antibodies (AB1, AB2, AB3, and AB5) were developed that reacted with a 140,000 mol wt glycoprotein on the surface of cultured RAJI B lymphoid cells. The antibodies reacted with purified normal human peripheral blood B cells and CLL Ig+ B cells and showed specific germinal center and mantle zone staining in tissue sections of secondary lymphoid organs. Immunodepletion studies using 125I surface-labeled Raji cell membrane antigens demonstrated that the antigen identified by AB5 is the same 140,000 mol wt glycoprotein detected by anti-B2 that has recently been shown to react with the C3d fragment or CR2 receptor. (Iida et al: J Exp Med 158:1021, 1983). Addition of the AB series and anti-B2 monoclonal antibodies to cultures of purified human peripheral blood B cells resulted in the uptake of 3H- thymidine at two to six times background control levels provided that irradiated autologous T cells were added to the culture. Stimulation was not evoked by other monoclonal antibodies to B cell surface molecules (ie, B1, BA-1, BA-2, and HLA-DR). Pepsin-generated F(ab')2 fragments of anti-CR2 antibodies were essentially as effective as the intact IgG molecule in stimulating B cells. Induction of B cell proliferation by antibody binding to CR2 suggests that the C3d receptor may have an integral role in regulation of humoral immune response.

scholarly journals Attenuation of Apoptosis Underlies B Lymphocyte Stimulator Enhancement of Humoral Immune Response

2000 ◽  
Vol 192 (7) ◽  
pp. 953-964 ◽  
Author(s):  
Richard K.G. Do ◽  
Eunice Hatada ◽  
Hayyoung Lee ◽  
Michelle R. Tourigny ◽  
David Hilbert ◽  
...  
Keyword(s):  
Immune Response ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Cell Survival ◽  
B Lymphocyte ◽  
Humoral Immune ◽  
B Cell Survival ◽  
B Lymphocyte Stimulator

B lymphocyte stimulator (BLyS) is a newly identified monocyte-specific TNF family cytokine. It has been implicated in the development of autoimmunity, and functions as a potent costimulator with antiimmunoglobulin M in B cell proliferation in vitro. Here we demonstrate that BLyS prominently enhances the humoral responses to both T cell–independent and T cell–dependent antigens, primarily by attenuation of apoptosis as evidenced by the prolonged survival of antigen-activated B cells in vivo and in vitro. BLyS acts on primary splenic B cells autonomously, and directly cooperates with CD40 ligand (CD40L) in B cell activation in vitro by protecting replicating B cells from apoptosis. Moreover, although BLyS alone cannot activate the cell cycle, it is sufficient to prolong the survival of naive resting B cells in vitro. Attenuation of apoptosis by BLyS correlates with changes in the ratios between Bcl-2 family proteins in favor of cell survival, predominantly by reducing the proapoptotic Bak and increasing its prosurvival partners, Bcl-2 and Bcl-xL. In either resting or CD40L-activated B cells, the NF-κB transcription factors RelB and p50 are specifically activated, suggesting that they may mediate BLyS signals for B cell survival. Together, these results provide direct evidence for BLyS enhancement of both T cell–independent and T cell–dependent humoral immune responses, and imply a role for BLyS in the conservation of the B cell repertoire. The ability of BLyS to increase B cell survival indiscriminately, at either a resting or activated state, and to cooperate with CD40L, further suggests that attenuation of apoptosis underlies BLyS enhancement of polyclonal autoimmunity as well as the physiologic humoral immune response.

scholarly journals Cumulus Cell Expansion, Its Role in Oocyte Biology and Perspectives of Measurement: A Review

2015 ◽  
Vol 45 (4) ◽  
pp. 212-225 ◽  
Author(s):  
J. Nevoral ◽  
M. Orsák ◽  
P. Klein ◽  
J. Petr ◽  
M. Dvořáková ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Hyaluronic Acid ◽  
High Performance ◽  
Uv Light ◽  
Cumulus Cell ◽  
Developmental Competence ◽  
Enzyme Linked Immunosorbent Assay ◽  
Cumulus Expansion

Abstract Cumulus expansion of the cumulus-oocyte complex is necessary for meiotic maturation and acquiring developmental competence. Cumulus expansion is based on extracellular matrix synthesis by cumulus cells. Hyaluronic acid is the most abundant component of this extracellular matrix. Cumulus expansion takes place during meiotic oocyte maturation under in vivo and in vitro conditions. Quantification and measurement of cumulus expansion intensity is one possible method of determining oocyte quality and optimizing conditions for in vitro cultivation. Currently, subjective methods of expanded area and more exact cumulus expansion measurement by hyaluronic acid assessment are available. Among the methods of hyaluronic acid measurement is the use of radioactively labelled synthesis precursors. Alternatively, immunological and analytical methods, including enzyme-linked immunosorbent assay (ELISA), spectrophotometry, and high-performance liquid chromatography (HPLC) in UV light, could be utilized. The high sensitivity of these methods could provide a precise analysis of cumulus expansion without the use of radioisotopes. Therefore, the aim of this review is to summarize and compare available approaches of cumulus expansion measurement, respecting special biological features of expanded cumuli, and to suggest possible solutions for exact cumulus expansion analysis.

scholarly journals Intersection of the complement and immune systems: a signal transduction complex of the B lymphocyte-containing complement receptor type 2 and CD19.

1991 ◽  
Vol 173 (1) ◽  
pp. 55-64 ◽  
Author(s):  
A K Matsumoto ◽  
J Kopicky-Burd ◽  
R H Carter ◽  
D A Tuveson ◽  
T F Tedder ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Immune Response ◽  
B Cell ◽  
B Lymphocyte ◽  
Protein Complexes ◽  
Immunoglobulin Superfamily ◽  
Immune Systems ◽  
Humoral Immune ◽  
Membrane Complex

The complement system augments the humoral immune response, possibly by a mechanism that involves the B lymphocyte membrane receptor, CR2, which binds the C3dg fragment of C3 and triggers several B cell responses in vitro. The present study demonstrates that CR2 associates with a complex of membrane proteins that may mediate signal transduction by ligated CR2. Monoclonal antibodies to CR2 immunoprecipitated from digitonin lysates of Raji B lymphoblastoid cells a membrane complex containing CR2, approximately equimolar amounts of CD19, which is a member of the immunoglobulin superfamily, and three unidentified components: p130, p50, and p20. The complex, which was immunoprecipitated also with anti-CD19, could be dissociated by Nonidet P-40, accounting for its absence in previous studies of CR2. Expression of recombinant CR2 and CD19 in K562 erythroleukemia cells led to formation of a complex that contained not only these two proteins but also p130, p50, and p20, and another component, p14. These unidentified components of the CR2/CD19 complex coimmunoprecipitated with CD19 and not with CR2 from singly transfected cells, indicating primary association with the former. CD19 replicated the capacity of CR2 to interact synergistically with mIgM for increasing free intracellular Ca2+, suggesting that the complex mediates this function of CR2. Therefore, CR2 associates directly with CD19 to become a ligand-binding subunit of a pre-existing signal transduction complex of the B cell that may be representative of a family of membrane protein complexes. This interaction between the complement and immune systems differs from that between immunoglobulin and Clq by involving membrane rather than plasma proteins, and by having complement involved in the afferent phase of the immune response.

scholarly journals Role of a nonimmunoglobulin cell surface determinant in the activation of B lymphocytes by thymus-independent antigens.

1979 ◽  
Vol 149 (2) ◽  
pp. 495-506 ◽  
Author(s):  
B Subbarao ◽  
D E Mosier ◽  
A Ahmed ◽  
J J Mond ◽  
I Scher ◽  
...  
Keyword(s):  
Cell Surface ◽  
B Cell ◽  
B Lymphocytes ◽  
Selective Inhibition ◽  
Antibody Responses ◽  
Antigenic Determinants ◽  
Functional Assay ◽  
Induce Antibody

Lyb 5 is a B-cell alloantigen which is expressed on 50-60% of B cells. It was defined originally on the basis of cytotoxicity. We have described a new reactivity within the anti-Lyb 5 serum on the basis of selective inhibition of antibody responses in vitro by this antiserum in the absence of complement. This inhibitory activity of anti-Lyb 5.1 serum appears to be due to recognition of antigenic determinants different from the prototype antigens detected in the cytotoxicity assay. Anti-Lyb 5 serum incorporated into spleen cell cultures selectively inhibits antibody responses to a class of thymus-independent antigens (TI-2) previously characterized by their failure to elicit antibody formation in immature mice or in the defective CBA/N strain. Responses to optimal concentrations of TI-1 antigens, which can induce antibody synthesis in these mice, are unaffected by the addition of anti-Lyb 5.1 serum. The B-cell alloantigen defined by this functional assay is designated tentatively Lyb 7 and it is shown to be distinct from cell surface immunoglobulins. Lyb 7 appears to have a role in the activation of B lymphocytes by the TI-2 class of thymus-independent antigens.

scholarly journals Seroprevalence of brucellosis in sheep and goats in the Arabian Gulf region

2020 ◽  
Vol 13 (8) ◽  
pp. 1495-1509
Author(s):  
M. Ebid ◽  
A. El Mola ◽  
F. Salib
Keyword(s):  
Arabian Gulf ◽  
Elisa Test ◽  
Enzyme Linked Immunosorbent Assay ◽  
Gulf Region ◽  
Time Saving ◽  
Tissue Samples ◽  
Sheep And Goats ◽  
Serological Examination ◽  
Accessory Glands

Background and Aim: Brucellosis is a zoonosis that occurs worldwide. There were more efforts to control brucellosis in all countries. This study was performed to determine the seroprevalence of brucellosis in sheep and goats in some areas in the Arabian Gulf. Materials and Methods: The study analyzed 8500 sera from non-vaccinated sheep and goats. Animals included 6441 sheep (3420 from farms and 3021 from quarantine) and 2059 goats (1580 from farms and 479 from quarantine). Sera were tested using the Rose Bengal Plate Test (RBPT) and confirmed with an indirect enzyme-linked immunosorbent assay (i-ELISA) test. Final confirmation analyzed blood samples from confirmed infected animals (n=30, 23 sheep and seven goats) using polymerase chain reaction (PCR) and culture. Results: The serological examination showed that 62/8500 of animals (0.729%, confidence interval [CI] 95% 0.57-0.94) were seropositive for brucellosis. Thirteen of 5000 (0.26%, CI 95% 0.15-0.45) and 49/3500 (1.4%, CI 95% 1.1-1.8) of animals from farms and quarantine were seropositive, respectively. Out of the 6441, 46 (0.71%) sheep and 16/2059 (0.78%) goats were seropositive. i-ELISA confirmed 41/62 RBPT-positive animals – 41/8500 (0.482%, CI 95% 0.36-0.65). Eight of 5000 of these animals (0.16%, CI 95% 0.08-0.32) and 33/3500 (0.94%, CI 95% 0.67-1.3) were confirmed positive in farms and quarantine, respectively. Thirty of 6441 (0.466%) and 11/2059 (0.534%) cases were positive in sheep and goats, respectively. PCR confirmed 18 of 41 positive animals (0.212% of all sera, CI 95% 0.13-0.34) identified by both RBPT and i-ELISA. Three of 5000 (0.06%, CI 95% 0.019-0.19) and 15/3500 (0.429%, CI 95% 0.26-0.71) from farms and quarantine were confirmed positive. Tissue samples (uterine, supra-mammary, testicular, and accessory glands lymph node) were collected from positive animals, as detected by RBPT and i-ELISA, at culling or slaughtering. Using in vitro culture, 14/30 were confirmed positive – 3/7 from farms (two sheep and one goat) and 11/23 from quarantine (nine sheep and two goats). Biovar 1 was dominant. PCR confirmed 23/30 tissue samples, 4/7 from farms (three sheep and one goat), and 19/23 from quarantine (15 sheep and four goats). Conclusion: The overall brucellosis rate in sheep and goats is 0.48%, with fewer animals from farms testing positive (0.16%) in this area of the Arabian Gulf. The infection appears to be well controlled, and continuous effort is still needed to maintain control and completely eradicate brucellosis. Additional support is needed for testing and slaughterhouse monitoring. In quarantine (imported animals), brucellosis infection in the slaughterhouse (0.94%) could pose a risk for transmission and spread of infection. The effort is needed to monitor this threat, and PCR is a sensitive and time-saving test for brucellosis diagnosis. All 14 confirmed positive samples were Biovar 1 dominant.

scholarly journals Stability of Alteplase for Catheter-Directed, Ultrasound-Facilitated Thrombolysis

2021 ◽  
Author(s):  
John Fanikos ◽  
Kathleen Marquis ◽  
Leo Francis Buckley ◽  
Lena Kim Tran ◽  
Kevin C McLaughlin ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
High Performance ◽  
In Vitro Study ◽  
Bleeding Risk ◽  
Reversed Phase ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rp Hplc ◽  
Catheter Directed Thrombolysis ◽  
Over Time

Introduction Ultrasound-facilitated catheter-directed thrombolysis is used with low-dose alteplase to treat pulmonary embolism. This reduces the bleeding risk that accompanies systemic administration of higher alteplase doses. While studies suggest that alteplase given over 2 to 6 hours is safe and effective, few data exist to support alteplase stability under these conditions. Therefore, we undertook this in vitro study to determine the duration of alteplase stability. Methods Alteplase was prepared in solutions of 8 mg in 100 mL, 6 mg in 150 mL, and 8 mg in 200 mL. Solutions were administered through the EkoSonicTM Endovascular System with and without ultrasound, to simulate administration over 2, 4, and 6 hours. Alteplase was assessed with reversed-phase high-performance liquid chromatography (RP-HPLC). Assays were performed at time 0 and at 30-minute intervals during simulated infusion. An enzyme-linked immunosorbent assay (ELISA) assay was used to measure alteplase concentrations that were at time 0 and at 15-minute intervals during simulated infusion. Results Using RP-HPLC, in the absence of ultrasound, the alteplase concentration remained within 1% of the original concentration through 120, 240, and 360 minutes of infusion. Using RP-HPLC for measurement, alteplase, in the presence of ultrasound, degraded steadily over time to approximately 90%, 80%, and 70% of its original amounts in 120, 240, and 360 minutes, respectively. Alteplase that remained was available for enzymatic activity. Conclusions Alteplase solutions of 0.04 and 0.08 mg/mL degraded steadily over time during simulated ultrasound-facilitated catheter-directed administration. Alteplase that did not degrade remained available for enzymatic activity.

scholarly journals The analysis of the monoclonal immune response to influenza virus. II. The antigenicity of the viral hemagglutinin.

1976 ◽  
Vol 144 (4) ◽  
pp. 985-995 ◽  
Author(s):  
W Gerhard
Keyword(s):  
Immune Response ◽  
Influenza Virus ◽  
Monoclonal Antibodies ◽  
Influenza Viruses ◽  
Antigenic Determinants ◽  
Humoral Immune ◽  
Viral Hemagglutinin ◽  
Or Groups ◽  
Virus Strains

The antigenicity of the hemagglutinins (HA) of five influenza viruses of the A0 and A1 subtypes has been analyzed by means of monoclonal antibodies of murine origin produced in vitro. Secondary monoclonal anti-HA(PR8) antibodies were able to differentiate 14 antigenic determinants (or groups of determinants) on the HA of five influenza virus strains of the A0 and A1 subtypes. Taking into account that certain pairs of determinants delineated on heterologous HA may reflect the heterogeneity of the humoral immune response to a single homologous determinant, the presence of at least eight determinants (host cell-derived determinants not included) on the homologous HA of PR8 and probably on the HA of influenza viruses in general is postulated. Three types of HA-determinants of A0 and A1 influenza virus strains could be distinguished: strain-specific, partially shared, and determinant(s) common to all five virus strains tested. Roughly 40, 55, and 5%, respectively, of the secondary anti-PR8 antibodies of BALB/c mice were directed against determinants belonging to either of the three types.

scholarly journals Monoclonal antibodies to the 140,000 mol wt glycoprotein of B lymphocyte membranes (CR2 receptor) initiates proliferation of B cells in vitro

Blood ◽  
1985 ◽  
Vol 66 (4) ◽  
pp. 824-829 ◽  
Author(s):  
BS Wilson ◽  
JL Platt ◽  
NE Kay
Keyword(s):  
Monoclonal Antibodies ◽  
B Cells ◽  
B Cell ◽  
Peripheral Blood ◽  
B Lymphocyte ◽  
Human Peripheral Blood ◽  
Raji Cell ◽  
Lymphoid Cells ◽  
Humoral Immune

Abstract Several mouse monoclonal IgG antibodies (AB1, AB2, AB3, and AB5) were developed that reacted with a 140,000 mol wt glycoprotein on the surface of cultured RAJI B lymphoid cells. The antibodies reacted with purified normal human peripheral blood B cells and CLL Ig+ B cells and showed specific germinal center and mantle zone staining in tissue sections of secondary lymphoid organs. Immunodepletion studies using 125I surface-labeled Raji cell membrane antigens demonstrated that the antigen identified by AB5 is the same 140,000 mol wt glycoprotein detected by anti-B2 that has recently been shown to react with the C3d fragment or CR2 receptor. (Iida et al: J Exp Med 158:1021, 1983). Addition of the AB series and anti-B2 monoclonal antibodies to cultures of purified human peripheral blood B cells resulted in the uptake of 3H- thymidine at two to six times background control levels provided that irradiated autologous T cells were added to the culture. Stimulation was not evoked by other monoclonal antibodies to B cell surface molecules (ie, B1, BA-1, BA-2, and HLA-DR). Pepsin-generated F(ab')2 fragments of anti-CR2 antibodies were essentially as effective as the intact IgG molecule in stimulating B cells. Induction of B cell proliferation by antibody binding to CR2 suggests that the C3d receptor may have an integral role in regulation of humoral immune response.

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