scholarly journals Generation and Evaluation of Chicken IgY-scFv for the Purposes of CPV Diagnosis and Therapy

2020 ◽  
Author(s):  
Shikun Ge ◽  
Long Xu ◽  
Ben Li ◽  
Fagang Zhong ◽  
XiaoYing Zhang
Keyword(s):  
Development Strategy ◽  
Antibody Fragment ◽  
Elisa Test ◽  
Cross Reactivity ◽  
Antibody Engineering ◽  
Clinical Samples ◽  
Similar Reaction ◽  
Single Chain ◽  
Diagnosis And Therapy ◽  
T7 Phage Display

Abstract Background: Canine parvovirus (CPV) can cause acute and highly contagious enteritis in dog, antibodies have been used for diagnosis and therapy of CPV disease, generation of functional antibody fragments by using novel antibody engineering platforms is promising in veterinary practice. Results: The IgY single chain fragment variables (scFv) were generated by T7 phage display system and expressed in E. coli system after immunizing hens with virus-like particles (VLP) of CPV-VP2. The titer of the primary scFv library reached to 1.5×10 6 pfu/mL, and 95% of the phages contained the target fragments. The CPV-VLP and CPV-VP2 protein showed similar reaction values to the purified scFv in the ELISA test, and the results of ELISA analysis using IgY-scFv toward CPV clinical samples were consistent with commercial immunochromatographic assay (ICA) and PCR detection, the scFv did not show cross reactivity with canine distemper virus (CDV) and canine coronavirus (CCV). IgY-scFv was successfully expressed in CRFK cells, and in the virus suppression assay, 55% of CPV infections were eliminated within 24 hours. Docking results demonstrated that the number of amino acids of the binding sides between scFv and VP2 were AA37 and AA40, respectively. Conclusions: This study revealed the feasibility of a novel functional antibody fragment development strategy by generating diversified avian IgY-scFv libraries towards the pathogenic target of interest for both detection and therapeutic purposes in veterinary medicine.

scholarly journals A blocking antibody against canine CSF-1R maturated by limited CDR mutagenesis

2020 ◽  
Vol 3 (3) ◽  
pp. 193-204
Author(s):  
Breno C B Beirão ◽  
Teresa P Raposo ◽  
Louise M Imamura ◽  
Max Ingberman ◽  
Ted Hupp ◽  
...  
Keyword(s):  
Inflammatory Diseases ◽  
Antibody Fragment ◽  
Cross Reactivity ◽  
Antibody Engineering ◽  
Model Systems ◽  
Native Form ◽  
Blocking Antibody ◽  
Macrophage Activity ◽  
Beneficial Effects ◽  
Variable Heavy

Abstract CSF-1R is a receptor mostly associated with the mononuclear phagocytic system. However, its expression within tumors has been linked with poor prognosis in both humans and dogs. Accordingly, several reports have demonstrated the beneficial effects of blocking CSF-1R in model systems of cancer. In this study, we generated a monoclonal antibody that could block CSF-1R in dogs as the first step to develop an anticancer drug for this species. Initially, an antibody was raised by the hybridoma methodology against the fragment responsible for receptor dimerization. mAb3.1, one of the resulting hybridoma clones, was able to bind macrophages in fixed tissues and was shown to inhibit cells of the mononuclear phagocytic line. Nevertheless, mAb 3.1 could not bind to some glycoforms of the receptor in its native form, while also demonstrating cross-reactivity with other proteins. To enhance binding properties of the mAb, five amino acids of the complementarity-determining region 2 of the variable heavy chain of mAb3.1 were mutated by PCR, and the variant scFv clones were screened by phage display. The selected scFv clones demonstrated improved binding to the native receptor as well as increased anti-macrophage activity. The resulting scFv antibody fragment presented here has the potential for use in cancer patients and in inflammatory diseases. Furthermore, this work provides insights into the use of such restricted mutations in antibody engineering.

scholarly journals Exploiting Cross-reactivity to Neutralize Two Different Scorpion Venoms with One Single Chain Antibody Fragment

2010 ◽  
Vol 286 (8) ◽  
pp. 6143-6151 ◽  
Author(s):  
Lidia Riaño-Umbarila ◽  
Gabriel Contreras-Ferrat ◽  
Timoteo Olamendi-Portugal ◽  
Citlalli Morelos-Juárez ◽  
Gerardo Corzo ◽  
...  
Keyword(s):  
Antibody Fragment ◽  
Cross Reactivity ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Scorpion Venoms ◽  
Single Chain Antibody Fragment

scholarly journals Generation of a Broadly Cross-Neutralizing Antibody Fragment against Several Mexican Scorpion Venoms

Toxins ◽  
2019 ◽  
Vol 11 (1) ◽  
pp. 32 ◽  
Author(s):  
Lidia Riaño-Umbarila ◽  
Ilse Gómez-Ramírez ◽  
Luis Ledezma-Candanoza ◽  
Timoteo Olamendi-Portugal ◽  
Everardo Rodríguez-Rodríguez ◽  
...  
Keyword(s):  
Recombinant Antibody ◽  
Antibody Fragment ◽  
Cross Reactivity ◽  
Site Directed Mutagenesis ◽  
Significant Advance ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Promising Alternative ◽  
Neutralization Capacity ◽  
Scorpion Venoms

The recombinant antibody fragments generated against the toxic components of scorpion venoms are considered a promising alternative for obtaining new antivenoms for therapy. Using directed evolution and site-directed mutagenesis, it was possible to generate a human single-chain antibody fragment with a broad cross-reactivity that retained recognition for its original antigen. This variant is the first antibody fragment that neutralizes the effect of an estimated 13 neurotoxins present in the venom of nine species of Mexican scorpions. This single antibody fragment showed the properties of a polyvalent antivenom. These results represent a significant advance in the development of new antivenoms against scorpion stings, since the number of components would be minimized due to their broad cross-neutralization capacity, while at the same time bypassing animal immunization.

scholarly journals Characterization of a novel mCH3 conjugated anti-PcrV scFv molecule

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Samira Komijani ◽  
Elham Bayat ◽  
Elham Rismani ◽  
Soma Hosseini ◽  
Reza Moazzami ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biological Activity ◽  
Antibody Fragment ◽  
In Silico Analysis ◽  
Elisa Test ◽  
Lung Epithelial Cells ◽  
Single Chain ◽  
Serum Stability ◽  
Stereochemical Quality ◽  
Scfv Molecule

AbstractPseudomonas aeruginosa (PA) is a leading cause of nosocomial infections and death in cystic fibrosis patients. The study was conducted to evaluate the physicochemical structure, biological activity and serum stability of a recombinant anti-PcrV single chain variable antibody fragment genetically attached to the mCH3cc domain. The stereochemical properties of scFv-mCH3 (YFL001) and scFv (YFL002) proteins as well as molecular interactions towards Pseudomonas aeruginosa PcrV were evaluated computationally. The subcloned fragments encoding YFL001 and YFL002 in pET28a were expressed within the E. coli BL21-DE3 strain. After Ni–NTA affinity chromatography, the biological activity of the proteins in inhibition of PA induced hemolysis as well as cellular cytotoxicity was assessed. In silico analysis revealed the satisfactory stereochemical quality of the models as well as common residues in their interface with PcrV. The structural differences of proteins through circular dichroism spectroscopy were confirmed by NMR analysis. Both proteins indicated inhibition of ExoU positive PA strains in hemolysis of red blood cells compared to ExoU negative strains as well as cytotoxicity effect on lung epithelial cells. The ELISA test showed the longer serum stability of the YFL001 molecule than YFL002. The results were encouraging to further evaluation of these two scFv molecules in animal models.

scholarly journals Analysis of a Routinely Used Commercial Anti-Chikungunya IgM ELISA Reveals Cross-Reactivities with Dengue in Brazil: A New Challenge for Differential Diagnosis?

Diagnostics ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 819
Author(s):  
Monique da Rocha Queiroz Lima ◽  
Raquel Curtinhas de Lima ◽  
Elzinandes Leal de Azeredo ◽  
Flavia Barreto dos Santos
Keyword(s):  
Disease Surveillance ◽  
Endemic Area ◽  
Chikungunya Virus ◽  
Elisa Test ◽  
Cross Reactivity ◽  
Immunoglobulin M ◽  
Diagnostic Tools ◽  
Healthy Individuals ◽  
Denv Serotypes ◽  
Igm Elisa

In Brazil, chikungunya emerged in 2014, and by 2016, co-circulated with other arbovirosis, such as dengue and zika. ELISAs (Enzyme-Linked Immunosorbent Assays) are the most widely used approach for arboviruses diagnosis. However, some limitations include antibody cross reactivities when viruses belong to the same genus, and sensitivity variations in distinct epidemiological scenarios. As chikungunya virus (CHIKV) is an alphavirus, no serological cross reactivity with dengue virus (DENV) should be observed. Here, we evaluated a routinely used chikungunya commercial IgM (Immunoglobulin M) ELISA test (Anti-Chikungunya IgM ELISA, Euroimmun) to assess its performance in confirming chikungunya in a dengue endemic area. Samples (n = 340) representative of all four DENV serotypes, healthy individuals and controls were tested. The Anti-CHIKV IgM ELISA test had a sensitivity of 100% and a specificity of 25.3% due to the cross reactivities observed with dengue. In dengue acute cases, the chikungunya test showed an overall cross-reactivity of 31.6%, with a higher cross-reactivity with DENV-4. In dengue IgM positive cases, the assay showed a cross-reactivity of 46.7%. Serological diagnosis may be challenging and, despite the results observed here, more evaluations shall be performed. Because distinct arboviruses co-circulate in Brazil, reliable diagnostic tools are essential for disease surveillance and patient management.

scholarly journals Transmissible Viral Proventriculitis Caused by Chicken Proventricular Necrosis Virus Displaying Serological Cross-Reactivity with IBDV

Animals ◽  
2020 ◽  
Vol 11 (1) ◽  
pp. 8
Author(s):  
Marcin Śmiałek ◽  
Michał Gesek ◽  
Daria Dziewulska ◽  
Jowita Samanta Niczyporuk ◽  
Andrzej Koncicki
Keyword(s):  
Body Weight ◽  
Broiler Chickens ◽  
Elisa Test ◽  
Cross Reactivity ◽  
Infected Group ◽  
Feed Conversion ◽  
Necrosis Virus ◽  
Bursal Disease ◽  
Group A ◽  
Potential Factor

Transmissible viral proventriculitis (TVP) of chickens is manifested in decreased body weight gains, poor feed conversion and weight diversity. Although TVP etiology has not been defined, a Birnaviridae family member, named chicken proventricular necrosis virus (CPNV) is considered as a potential factor of a disease. This study was undertaken in order to reproduce TVP and to evaluate its etiology. Broiler chickens of the TVP-infected group were inoculated with TVP positive proventriculi homogenate on the 24th day of life. Samples were collected, on infection day and 14 days post-infection (dpi). The 14 dpi anatomo- and histopathological evaluation, revealed that we have succeeded to reproduce TVP. TVP-infected birds gained 30.38% less body weight. In the TVP-infected group a seroconversion against picornaviruses, fowl adenoviruses (FAdV) and infectious bursal disease viruses (IBDV) was recorded with an ELISA test. Using RT-PCR and PCR, CPNV was detected in proventriculi and FAdV in spleens and livers of infected birds, 14 dpi. Our study supports that CPNV is involved in the development of TVP. We did not record the presence of IBDV in TVP or control birds, despite our recording of a seroconversion against IBDV in TVP infected birds. CPNV and IBDV belong to the same family, which allows us to assume serological cross-reactivity between them. The role of FAdV needs further evaluation.

scholarly journals Evaluation of mobile real-time polymerase chain reaction tests for the detection of severe acute respiratory syndrome coronavirus 2

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Chukwunonso Onyilagha ◽  
Henna Mistry ◽  
Peter Marszal ◽  
Mathieu Pinette ◽  
Darwyn Kobasa ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Real Time ◽  
Point Of Care ◽  
Middle Eastern ◽  
Turnaround Time ◽  
Cross Reactivity ◽  
Clinical Samples ◽  
Diarrhea Virus ◽  
Highly Sensitive ◽  
Transmissible Gastroenteritis

AbstractThe coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), calls for prompt and accurate diagnosis and rapid turnaround time for test results to limit transmission. Here, we evaluated two independent molecular assays, the Biomeme SARS-CoV-2 test, and the Precision Biomonitoring TripleLock SARS-CoV-2 test on a field-deployable point-of-care real-time PCR instrument, Franklin three9, in combination with Biomeme M1 Sample Prep Cartridge Kit for RNA 2.0 (M1) manual extraction system for rapid, specific, and sensitive detection of SARS-COV-2 in cell culture, human, and animal clinical samples. The Biomeme SARS-CoV-2 assay, which simultaneously detects two viral targets, the orf1ab and S genes, and the Precision Biomonitoring TripleLock SARS-CoV-2 assay that targets the 5′ untranslated region (5′ UTR) and the envelope (E) gene of SARS-CoV-2 were highly sensitive and detected as low as 15 SARS-CoV-2 genome copies per reaction. In addition, the two assays were specific and showed no cross-reactivity with Middle Eastern respiratory syndrome coronavirus (MERS-CoV), infectious bronchitis virus (IBV), porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis (TGE) virus, and other common human respiratory viruses and bacterial pathogens. Also, both assays were highly reproducible across different operators and instruments. When used to test animal samples, both assays equally detected SARS-CoV-2 genetic materials in the swabs from SARS-CoV-2-infected hamsters. The M1 lysis buffer completely inactivated SARS-CoV-2 within 10 min at room temperature enabling safe handling of clinical samples. Collectively, these results show that the Biomeme and Precision Biomonitoring TripleLock SARS-CoV-2 mobile testing platforms could reliably and promptly detect SARS-CoV-2 in both human and animal clinical samples in approximately an hour and can be used in remote areas or health care settings not traditionally serviced by a microbiology laboratory.

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