scholarly journals Paper-based ELISA Diagnosis Technology for Human Brucellosis Based on a Multiepitope Fusion Protein

2021 ◽  
Author(s):  
Dehui Yin ◽  
Qiongqiong Bai ◽  
Xiling Wu ◽  
Han Li ◽  
Jihong Shao ◽  
...  
Keyword(s):  
Fusion Protein ◽  
B Cell ◽  
Predictive Value ◽  
Outer Membrane Proteins ◽  
Diagnostic Technique ◽  
Human Brucellosis ◽  
B Cell Epitopes ◽  
Whatman Filter Paper ◽  
Nano Zinc Oxide ◽  
Nano Zinc

Abstract Background: At present, as a serious zoonotic infectious disease, the incidence of brucellosis is increasing each year worldwide, exhibiting signs of resurgence. Brucellosis seriously threatens the health of humans, and it is necessary to strengthen the methods utilized for its rapid and accurate diagnosis.Methods: Bioinformatic technology was used to predict B-cell epitopes of the main outer membrane proteins of Brucella and subsequently verified the antigenicity of these epitopes. Prepared a Brucella multiepitope fusion protein and verified the antigenicity of the protein by indirect ELISA. Whatman filter paper was then modified with nano-zinc oxide to construct a paper-based ELISA (p-ELISA) technology for the diagnosis of brucellosis.Results: A total of 22 linear B cell epitopes were predicted. Each epitope could recognize some brucellosis sera. The constructed multiepitope fusion protein had good antigenicity and significantly reduced cross-reaction compared with LPS. The sensitivity and specificity of the method were 92.38% and 98.35%, the positive predictive value was 98.26%, and the negative predictive value was 91.67%.Conclusions: A multiepitope fusion protein of Brucella was successfully prepared, and a rapid diagnostic technique for brucellosis was established. This technology has potential application value and can be used for the rapid diagnosis of brucellosis.

scholarly journals Paper-based ELISA diagnosis technology for human brucellosis based on a multiepitope fusion protein

2021 ◽  
Vol 15 (8) ◽  
pp. e0009695
Author(s):  
Dehui Yin ◽  
Qiongqiong Bai ◽  
Xiling Wu ◽  
Han Li ◽  
Jihong Shao ◽  
...  
Keyword(s):  
Fusion Protein ◽  
B Cell ◽  
Sensitivity And Specificity ◽  
Predictive Value ◽  
Outer Membrane Proteins ◽  
Diagnostic Methods ◽  
Human Brucellosis ◽  
Testing Method ◽  
Animal Populations ◽  
Linear Epitopes

Background Brucellosis, as a serious zoonotic infectious disease, has been recognized as a re-emerging disease in the developing countries worldwide. In china, the incidence of brucellosis is increasing each year, seriously threatening the health of humans as well as animal populations. Despite a quite number of diagnostic methods currently being used for brucellosis, innovative technologies are still needed for its rapid and accurate diagnosis, especially in area where traditional diagnostic is unavailable. Methodology/Principal findings In this study, a total of 22 B cell linear epitopes were predicted from five Brucella outer membrane proteins (OMPs) using an immunoinformatic approach. These epitopes were then chemically synthesized, and with the method of indirect ELISA (iELISA), each of them displayed a certain degree of capability in identifying human brucellosis positive sera. Subsequently, a fusion protein consisting of the 22 predicted epitopes was prokaryotically expressed and used as diagnostic antigen in a newly established brucellosis testing method, nano-ZnO modified paper-based ELISA (nano-p-ELISA). According to the verifying test using a collection of sera collected from brucellosis and non-brucellosis patients, the sensitivity and specificity of multiepitope based nano-p-ELISA were 92.38% and 98.35% respectively. The positive predictive value was 98.26% and the negative predictive value was 91.67%. The multiepitope based fusion protein also displayed significantly higher specificity than Brucella lipopolysaccharide (LPS) antigen. Conclusions B cell epitopes are important candidates for serologically testing brucellosis. Multiepitope fusion protein based nano-p-ELISA displayed significantly sensitivity and specificity compared to Brucella LPS antigen. The strategy applied in this study will be helpful to develop rapid and accurate diagnostic method for brucellosis in human as well as animal populations.

scholarly journals A Multi-Epitope Fusion Protein-Based p-ELISA Method for Diagnosing Bovine and Goat Brucellosis

2021 ◽  
Vol 8 ◽  
Author(s):  
Dehui Yin ◽  
Qiongqiong Bai ◽  
Xiling Wu ◽  
Han Li ◽  
Jihong Shao ◽  
...  
Keyword(s):  
Fusion Protein ◽  
B Cell ◽  
Sensitivity And Specificity ◽  
Outer Membrane Proteins ◽  
Economic Losses ◽  
Enzyme Linked Immunosorbent Assay ◽  
Specific Method ◽  
Roc Curve Analysis ◽  
Predictive Values ◽  
B Cell Epitopes

In recent years, the incidence of brucellosis has increased annually, causing tremendous economic losses to animal husbandry in a lot of countries. Therefore, developing rapid, sensitive, and specific diagnostic techniques is critical to control the spread of brucellosis. In this study, bioinformatics technology was used to predict the B cell epitopes of the main outer membrane proteins of Brucella, and the diagnostic efficacy of each epitope was verified by an indirect enzyme-linked immunosorbent assay (iELISA). Then, a fusion protein containing 22 verified epitopes was prokaryotically expressed and used as an antigen in paper-based ELISA (p-ELISA) for serodiagnosis of brucellosis. The multi-epitope-based p-ELISA was evaluated using a collection of brucellosis-positive and -negative sera collected from bovine and goat, respectively. Receiver operating characteristic (ROC) curve analysis showed that the sensitivity and specificity of detection-ELISA in diagnosing goat brucellosis were 98.85 and 98.51%. The positive and the negative predictive values were 99.29 and 98.15%, respectively. In diagnosing bovine brucellosis, the sensitivity and specificity of this method were 97.85 and 96.61%, with the positive and negative predictive values being identified as 98.28 and 97.33%, respectively. This study demonstrated that the B cell epitopes contained in major antigenic proteins of Brucella can be a very useful antigen source in developing a highly sensitive and specific method for serodiagnosis of brucellosis.

scholarly journals Identification of Immunogenic Linear B-Cell Epitopes in C. burnetii Outer Membrane Proteins Using Immunoinformatics Approaches Reveals Potential Targets of Persistent Infections

Pathogens ◽  
2021 ◽  
Vol 10 (10) ◽  
pp. 1250
Author(s):  
Sílvia da Silva Fontes ◽  
Fernanda de Moraes Maia ◽  
Laura Santa’Anna Ataides ◽  
Fernando Paiva Conte ◽  
Josué da Costa Lima-Junior ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
B Cell ◽  
Outer Membrane ◽  
Q Fever ◽  
Outer Membrane Proteins ◽  
Humoral Response ◽  
Neglected Diseases ◽  
B Cell Epitopes ◽  
Wide Range ◽  
Linear B

Coxiella burnetii is a global, highly infectious intracellular bacterium, able to infect a wide range of hosts and to persist for months in the environment. It is the etiological agent of Q fever—a zoonosis of global priority. Currently, there are no national surveillance data on C. burnetii’s seroprevalence for any South American country, reinforcing the necessity of developing novel and inexpensive serological tools to monitor the prevalence of infections among humans and animals—especially cattle, goats, and sheep. In this study, we used immunoinformatics and computational biology tools to predict specific linear B-cell epitopes in three C. burnetii outer membrane proteins: OMP-H (CBU_0612), Com-1 (CBU_1910), and OMP-P1 (CBU_0311). Furthermore, predicted epitopes were tested by ELISA, as synthetic peptides, against samples of patients reactive to C. burnetii in indirect immunofluorescence assay, in order to evaluate their natural immunogenicity. In this way, two linear B-cell epitopes were identified in each studied protein (OMP-H(51–59), OMP-H(91–106), Com-1(57–76), Com-1(191–206), OMP-P1(197–209), and OMP-P1(215–227)); all of them were confirmed as naturally immunogenic by the presence of specific antibodies in 77% of studied patients against at least one of the identified epitopes. Remarkably, a higher frequency of endocarditis cases was observed among patients who presented an intense humoral response to OMP-H and Com-1 epitopes. These data confirm that immunoinformatics applied to the identification of specific B-cell epitopes can be an effective strategy to improve and accelerate the development of surveillance tools against neglected diseases.

scholarly journals Shotgun Immunoproteomic Approach for the Discovery of Linear B-Cell Epitopes in Biothreat Agents Francisella tularensis and Burkholderia pseudomallei

2021 ◽  
Vol 12 ◽  
Author(s):  
Patrik D’haeseleer ◽  
Nicole M. Collette ◽  
Victoria Lao ◽  
Brent W. Segelke ◽  
Steven S. Branda ◽  
...  
Keyword(s):  
Immune Responses ◽  
B Cell ◽  
Francisella Tularensis ◽  
Burkholderia Pseudomallei ◽  
Outer Membrane Proteins ◽  
Screening Method ◽  
Cost Effective ◽  
Immune Memory ◽  
B Cell Epitopes ◽  
Linear Epitopes

Peptide-based subunit vaccines are coming to the forefront of current vaccine approaches, with safety and cost-effective production among their top advantages. Peptide vaccine formulations consist of multiple synthetic linear epitopes that together trigger desired immune responses that can result in robust immune memory. The advantages of linear compared to conformational epitopes are their simple structure, ease of synthesis, and ability to stimulate immune responses by means that do not require complex 3D conformation. Prediction of linear epitopes through use of computational tools is fast and cost-effective, but typically of low accuracy, necessitating extensive experimentation to verify results. On the other hand, identification of linear epitopes through experimental screening has been an inefficient process that requires thorough characterization of previously identified full-length protein antigens, or laborious techniques involving genetic manipulation of organisms. In this study, we apply a newly developed generalizable screening method that enables efficient identification of B-cell epitopes in the proteomes of pathogenic bacteria. As a test case, we used this method to identify epitopes in the proteome of Francisella tularensis (Ft), a Select Agent with a well-characterized immunoproteome. Our screen identified many peptides that map to known antigens, including verified and predicted outer membrane proteins and extracellular proteins, validating the utility of this approach. We then used the method to identify seroreactive peptides in the less characterized immunoproteome of Select Agent Burkholderia pseudomallei (Bp). This screen revealed known Bp antigens as well as proteins that have not been previously identified as antigens. Although B-cell epitope prediction tools Bepipred 2.0 and iBCE-EL classified many of our seroreactive peptides as epitopes, they did not score them significantly higher than the non-reactive tryptic peptides in our study, nor did they assign higher scores to seroreactive peptides from known Ft or Bp antigens, highlighting the need for experimental data instead of relying on computational epitope predictions alone. The present workflow is easily adaptable to detecting peptide targets relevant to the immune systems of other mammalian species, including humans (depending upon the availability of convalescent sera from patients), and could aid in accelerating the discovery of B-cell epitopes and development of vaccines to counter emerging biological threats.

scholarly journals Identification of linear epitopes in SjSP-13 of Schistosoma japonicum using a GST-peptide fusion protein microplate array

2019 ◽  
Vol 12 (1) ◽  
Author(s):  
Li Ma ◽  
Wenrong Zhao ◽  
Xunya Hou ◽  
Mengmeng Liu ◽  
Yanna Li ◽  
...  
Keyword(s):  
Fusion Protein ◽  
B Cell ◽  
Schistosomiasis Japonica ◽  
Glutathione S Transferase ◽  
B Cell Epitopes ◽  
Linear Epitopes ◽  
Novel Method ◽  
Auc Value ◽  
Diagnostic Sensitivity And Specificity ◽  
Linear B

Abstract Background The identification and characterization of epitopes facilitate the discovery and development of new therapeutics, vaccines and diagnostics for infectious diseases. In this study, we developed a glutathione S-transferase (GST)-peptide fusion protein microplate array for the identification of linear B-cell epitopes and applied this novel method to the identification of linear B-cell epitopes of SjSP-13, an immunodiagnostic biomarker of schistosomiasis japonica. Methods SjSP-13 was divided into 17 overlapped peptides (p1-17), and the coding sequence of each peptide was obtained by annealing two complementary oligonucleotides. SjSP-13 peptides were expressed by fusion with an N-terminal GST tag and a C-terminal 6xHis tag. The GST-peptide-His fusion protein was specifically bound to the Immobilizer Glutathione MicroWell 96-well plates without purification. SjSP-13 peptides and core epitopes that could be recognized by sera from schistosomiasis patients were identified by ELISA and confirmed by Western blot analysis. The receiver operating characteristic (ROC) analysis was performed to determine the diagnostic validity of the identified peptide. Results Full-length GST-peptide-His fusion proteins were successfully expressed and specifically bound to the Immobilizer Glutathione MicroWell 96-well plates. Two adjacent peptides (p7 and p8) were found to be highly immunogenic in humans. The core epitope of p7 and p8 is an 11-aa peptide (80KCLDVTDNLPE90) and an 8-aa peptide (90EKIIQFAE97), respectively. The area under the ROC curve (AUC) value of the peptide which contains the two identified epitopes is 0.947 ± 0.019. The diagnostic sensitivity and specificity of the peptide is 76.7% (95% CI: 68.8–84.5%) and 100%, respectively. Conclusions 90EKIIQFAE97 and 80KCLDVTDNLPE90 are the two linear epitopes of SjSP-13 recognized by patient sera, and could be potential serological markers for schistosomiasis japonica.

scholarly journals In Silico Epitope-Based Peptide Vaccine Design against Invasive Non Typhoidal Salmonella (iNTS) Through Immunoinformatic Approaches

2020 ◽  
Author(s):  
Md. Chayan Ali ◽  
Sultana Israt Jahan ◽  
Mst. Shanzeda Khatun ◽  
Raju Das ◽  
Md Mafizur Rahman ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
B Cell ◽  
Outer Membrane ◽  
Vaccine Candidate ◽  
Binding Free Energy ◽  
Outer Membrane Proteins ◽  
Peptide Vaccine ◽  
Dynamics Simulation ◽  
Mhc I ◽  
B Cell Epitopes

Salmonella, especially invasive non-typhoidal Salmonella (iNTS) are responsible for developing various invasive diseases, and possess higher mortality rate, due to their higher antibiotic resistance profile than the other bacteria. Therefore, the present study was concerned to develop epitope based peptide vaccine against iNTS species as a successive and substitute protective measures. The study considered comprehensive Immunoinformatic approaches, followed by molecular docking and molecular dynamics simulation to predict the efficient vaccine candidate T cell and B cell epitopes, based on the outer membrane proteins. The study identified two best epitopes YGIFAITAL and KVLYGIFAI from total iNTS outer membrane proteins, which showed higher immunity, non-allergenicity, non-toxicity and also showed higher conservancy and population coverage values. Both epitopes showed higher binding affinity and stability towards HLA-C* 03:03. The MM-PBSA binding free energy showed the YGIFAITAL epitope binds more tightly with both MHC-I and MHC-II molecules. The total contact, H-bond analysis and RMSF results also validate the efficiency of these epitopes as vaccine candidate. The projected B cell epitopes AAPVQVGEAAGS, TGGGDGSNT and TGGGDGSNTGTTTT showed higher antigenicity. Overall, the study concluded that these epitopes can be considered as the potential vaccine candidate to make a successive vaccine against iNTS species. However, this result further needs to be validate by wet lab research to make successive vaccine with these projected epitopes.

scholarly journals Shotgun Immunoproteomic Approach for the Discovery of Linear B Cell Epitopes in Biothreat Agents Francisella tularensis and Burkholderia pseudomallei

2021 ◽  
Author(s):  
Patrik D'haeseleer ◽  
Nicole M Collette ◽  
Victoria Lao ◽  
Brent W Segelke ◽  
Steven S Branda ◽  
...  
Keyword(s):  
Immune Responses ◽  
B Cell ◽  
Francisella Tularensis ◽  
Burkholderia Pseudomallei ◽  
Outer Membrane Proteins ◽  
Screening Method ◽  
Immune Memory ◽  
B Cell Epitopes ◽  
Peptide Epitopes ◽  
Linear Epitopes

Peptide-based subunit vaccines are coming to the forefront of current vaccine approaches, with safety and cost-effective production among their top advantages. Peptide vaccine formulations consist of multiple synthetic linear epitopes that together trigger desired immune responses that can result in robust immune memory. The advantages of peptide epitopes are their simple structure, ease of synthesis, and ability to stimulate immune responses by means that do not require complex 3D conformation. Identification of linear epitopes is currently an inefficient process that requires thorough characterization of previously identified full-length protein antigens, or laborious techniques involving genetic manipulation of organisms. In this study, we apply a newly developed generalizable screening method that enables efficient identification of B cell epitopes in the proteomes of pathogenic bacteria. As a test case, we used this method to identify epitopes in the proteome of Francisella tularensis (Ft), a Select Agent with a well-characterized immunoproteome. Our screen identified many peptides that map to known antigens, including verified and predicted outer membrane proteins and extracellular proteins, validating the utility of this approach. We then used the method to identify seroreactive peptides in the less characterized immunoproteome of Select Agent Burkholderia pseudomallei (Bp). This screen revealed known Bp antigens as well as proteins that have not been previously identified as antigens. The present workflow is easily adaptable to detecting peptide targets relevant to the immune systems of other mammalian species, including humans (depending upon the availability of convalescent sera from patients), and could aid in accelerating the discovery of B cell epitopes and development of vaccines to counter emerging biological threats.

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