scholarly journals Generation of High Affinity Anti-Peptide Polyclonal Antibodies Recognizing Goat αs1-Casein

Molecules ◽  
2020 ◽  
Vol 25 (11) ◽  
pp. 2622
Author(s):  
Aliah Zannierah Mohsin ◽  
Rashidah Sukor ◽  
Jinap Selamat ◽  
Anis Shobirin Meor Hussin ◽  
Intan Hakimah Ismail ◽  
...  
Keyword(s):  
Binding Affinity ◽  
Analytical Methods ◽  
Polyclonal Antibodies ◽  
Sequence Similarity ◽  
High Specificity ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
High Sequence Similarity ◽  
High Affinity

The chemical, technological and allergy properties of goat’s milk are significantly affected by the level of αs1-casein. Detection and quantification of αs1-casein requires high-specificity methods to overcome high-sequence similarity between this protein and others in the casein family. Unavailability of antibodies with high affinity and specificity towards goat αs1-casein hinders the development of immuno-based analytical methods such as enzyme-linked immunosorbent assay (ELISA) and biosensors. Here, we report the generation of polyclonal antibodies (or immunoglobulins, IgGs) raised towards goat αs1-casein N- (Nter) and C-terminal (Cter) peptide sequences. The Nter and Cter peptides of goat αs1-casein were immunized in rabbits for the generation of antisera, which were purified using protein G affinity chromatography. The binding affinity of the antisera and purified IgGs were tested and compared using indirect ELISA, where peptide-BSA conjugates and goat αs1-casein were used as the coating antigens. The Nter antiserum displayed higher titer than Cter antiserum, at 1/64,000 and 1/32,000 dilutions, respectively. The purification step further yielded 0.5 mg/mL of purified IgGs from 3 mL of antisera. The purified Nter IgG showed a significantly (p < 0.05) higher binding affinity towards peptide-BSA and goat αs1-casein, with lower Kd value at 5.063 × 10−3 μM compared to 9.046 × 10−3 μM for the Cter IgG. A cross-reactivity test showed that there was no binding in neither Nter nor Cter IgGs towards protein extracts from the milk of cow, buffalo, horse and camel. High-quality antibodies generated will allow further development of immuno-based analytical methods and future in vitro studies to be conducted on goat αs1-casein.

scholarly journals Development of Murine Monoclonal Antibodies for the Immunohistochemical Diagnosis of Systemic Bovine Aspergillosis

1996 ◽  
Vol 8 (1) ◽  
pp. 68-75 ◽  
Author(s):  
H. E. Jensen ◽  
B. Aalbaek ◽  
P. Lind ◽  
H. V. Krogh ◽  
P. L. Frandsen
Keyword(s):  
Monoclonal Antibodies ◽  
Polyclonal Antibodies ◽  
Fungal Species ◽  
Cross Reactivity ◽  
Water Soluble ◽  
Enzyme Linked Immunosorbent Assay ◽  
Immunohistochemical Techniques ◽  
Immunohistochemical Diagnosis ◽  
Murine Monoclonal Antibodies

Murine monoclonal antibodies (MAbs) against water-soluble somatic antigens (WSSA) and the wall fraction (WF) from Aspergillus fumigatus were produced by fusion of splenocytes from immunized BALB/c mice with mouse myeloma X63-Ag 8.653 cells. The supernatants of in vitro cultured hybridomas were initially screened for reactivity with the WSSA and the WF from A. fumigatus and WSSA of other fungi in an enzyme-linked immunosorbent assay (ELISA). Supernatants reacting only with A. fumigatus antigens were subsequently screened for homologous and heterologous reactivity with immunohistochemical techniques using formalin-fixed, paraffin-embedded tissues from experimentally infected mice. Because of a high immunohistochemical reactivity with homologous fungi, 4 MAbs raised against A. fumigatus WSSA and WF were selected for a further evaluation of cross-reactivity (diagnostic specificity) in immunohistochemical and immunoblotting assays. In immunohistochemical assays, all MAbs raised against WSSA cross-reacted heavily with a number of other fungal species. All 4 MAbs (MAb-WF-AF-1-4) raised against the WF reacted strongly with hyphae of Aspergillus spp.; hyphae of Scedosporium apiospermum were also strongly labeled by MAb-WF-AF-3 and-4. The 2 specifically reacting MAbs (MAb-WF-AF-1 and-2) were of the IgM biotype and were precipitating, and in immunoblotting experiments both bound to a 106-kD antigen of the WF, whereas they did not bind to WSSA of A. fumigatus. One of the 2 aspergillosis-specific MAbs (MAb-WF-AF-1) was used to screen 145 mycotic lesions of cattle. The diagnoses on bovine lesions obtained by MAb-WF-AF-1 were compared with results based on reactivity with heterologously absorbed polyclonal antibodies and, for some lesions, to culture results. In the vast majority of lesions ( n = 133), the MAb-WF-AF-1 and the polyclonal anti-Aspergillus antibodies reacted in a similar pattern, i.e., positively in 41 aspergillosis lesions and negatively in 92 zygomycotic lesions. Hyphae in 3 of 12 lesions that were not stained by the polyclonal antibodies reacted with the specific MAb-WF-AF-1; i.e., aspergillosis was diagnosed. The characteristics of the 2 MAbs (MAb-WF-AF-1 and-2) raised against the WF of A. fumigatus in ELISA and immunoblotting and immunohistochemical assays justify their application for the in situ diagnosis of systemic aspergillosis of cattle.

Immunohistochemical Diagnosis of Systemic Bovine Zygomycosis by Murine Monoclonal Antibodies

1996 ◽  
Vol 33 (2) ◽  
pp. 176-183 ◽  
Author(s):  
H. E. Jensen ◽  
B. Aalbæk ◽  
P. Lind ◽  
H. V. Krogh
Keyword(s):  
Monoclonal Antibodies ◽  
Lymph Nodes ◽  
Rhizopus Oryzae ◽  
Polyclonal Antibodies ◽  
Cross Reactivity ◽  
Water Soluble ◽  
Enzyme Linked Immunosorbent Assay ◽  
The Family ◽  
Murine Monoclonal Antibodies

Murine monoclonal antibodies (Mabs) against water-soluble somatic antigens (WSSA) and the wall fraction (WF) from Rhizopus arrhizus (Rhizopus oryzae) were produced in vitro by fusion of splenocytes from immunized BALB/c mice with mouse myeloma X63-Ag 8.653 cells. Supernatants reacting only with homologous antigens in an enzyme-linked immunosorbent assay were subsequently screened for reactivity with homologous fungi in immunohistochemical techniques. All four Mabs raised against the WF of A. arrhizus failed to react on tissues. However, four of the Mabs raised against the WSSA of R. arrhizus (Mab-WSSA-RA-1 through Mab-WSSA-RA-4) revealed a high homologous reactivity on tissues and the cross-reactivity of these were subsequently evaluated on tissues containing other members of the family Mucoraceae and other unrelated fungi. On tissues and on immunoblots all four Mabs reacted identically and specifically with members of the family Mucoraceae, i.e., Absidia corymbifera, R. arrhizus, and Rhizomucor pusillus. The Mabs were all isotyped as IgM antibodies, were nonprecipitating, and reacted with homologous antigens with molecular masses from 14 to 110 kDa. With WSSA from A. corymbifera and R. pusillus the four Mabs were bound to antigens from 14 to 52 kDa and from 20 to 28 kDa, respectively. The diagnosis of 145 bovine lesions obtained by one of the specific Mabs (Mab-WSSA-RA-1) were compared to results obtained by heterologously absorbed polyclonal antibodies. In most lesions ( n = 140 [∼ 97%]) the Mab and the polyclonal antibodies reacted in a similar pattern, i.e., positively for zygomycosis in 89 lesions, negatively in 41 aspergillosis lesions, and negatively in 10 undiagnosed lesions. Hyphae within two of four lesions in lymph nodes, which were not stained by the polyclonal antibodies, reacted with the specific Mab. However, in another three lesions of lymph nodes stained by the polyclonal antibodies no reactivity was seen with the Mab-WSSA-RA-1. The immunoreactivity of the Mabs (Mab-WSSA-RA-1 through Mab-WSSA-RA-4) raised against WSSA of R. arrhizus justify their application for the in situ diagnosis of systemic bovine zygomycosis.

scholarly journals Efficient CRISPR/Cas9 mediated Pooled-sgRNAs assembly accelerates targeting multiple genes related to male sterility in cotton

Plant Methods ◽  
2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Mohamed Ramadan ◽  
Muna Alariqi ◽  
Yizan Ma ◽  
Yanlong Li ◽  
Zhenping Liu ◽  
...  
Keyword(s):  
Genetic Transformation ◽  
Sequence Similarity ◽  
High Specificity ◽  
Transformation Method ◽  
High Sequence Similarity ◽  
Single Experiment ◽  
Next Generation Sequencing Technology ◽  
Cotton Transformation ◽  
Assembly Method ◽  
Multiple Genes

Abstract Background Upland cotton (Gossypium hirsutum), harboring a complex allotetraploid genome, consists of A and D sub-genomes. Every gene has multiple copies with high sequence similarity that makes genetic, genomic and functional analyses extremely challenging. The recent accessibility of CRISPR/Cas9 tool provides the ability to modify targeted locus efficiently in various complicated plant genomes. However, current cotton transformation method targeting one gene requires a complicated, long and laborious regeneration process. Hence, optimizing strategy that targeting multiple genes is of great value in cotton functional genomics and genetic engineering. Results To target multiple genes in a single experiment, 112 plant development-related genes were knocked out via optimized CRISPR/Cas9 system. We optimized the key steps of pooled sgRNAs assembly method by which 116 sgRNAs pooled together into 4 groups (each group consisted of 29 sgRNAs). Each group of sgRNAs was compiled in one PCR reaction which subsequently went through one round of vector construction, transformation, sgRNAs identification and also one round of genetic transformation. Through the genetic transformation mediated Agrobacterium, we successfully generated more than 800 plants. For mutants identification, Next Generation Sequencing technology has been used and results showed that all generated plants were positive and all targeted genes were covered. Interestingly, among all the transgenic plants, 85% harbored a single sgRNA insertion, 9% two insertions, 3% three different sgRNAs insertions, 2.5% mutated sgRNAs. These plants with different targeted sgRNAs exhibited numerous combinations of phenotypes in plant flowering tissues. Conclusion All targeted genes were successfully edited with high specificity. Our pooled sgRNAs assembly offers a simple, fast and efficient method/strategy to target multiple genes in one time and surely accelerated the study of genes function in cotton.

scholarly journals Examination of the Efficacy and Cross-Reactivity of a Novel Polyclonal Antibody Targeting the Disintegrin Domain in SVMPs to Neutralize Snake Venom

Toxins ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 254
Author(s):  
Shelby S. Szteiter ◽  
Ilse N. Diego ◽  
Jonathan Ortegon ◽  
Eliana Salinas ◽  
Abcde Cirilo ◽  
...  
Keyword(s):  
Snake Venom ◽  
Polyclonal Antibody ◽  
Polyclonal Antibodies ◽  
Cross Reactivity ◽  
Snake Envenomation ◽  
Disintegrin Domain ◽  
The Common ◽  
Neutralization Ability ◽  
New Strategies

Snake envenomation can result in hemorrhage, local necrosis, swelling, and if not treated properly can lead to adverse systemic effects such as coagulopathy, nephrotoxicity, neurotoxicity, and cardiotoxicity, which can result in death. As such, snake venom metalloproteinases (SVMPs) and disintegrins are two toxic components that contribute to hemorrhage and interfere with the hemostatic system. Administration of a commercial antivenom is the common antidote to treat snake envenomation, but the high-cost, lack of efficacy, side effects, and limited availability, necessitates the development of new strategies and approaches for therapeutic treatments. Herein, we describe the neutralization ability of anti-disintegrin polyclonal antibody on the activities of isolated disintegrins, P-II/P-III SVMPs, and crude venoms. Our results show disintegrin activity on platelet aggregation in whole blood and the migration of the SK-Mel-28 cells that can be neutralized with anti-disintegrin polyclonal antibody. We characterized a SVMP and found that anti-disintegrin was also able to inhibit its activity in an in vitro proteolytic assay. Moreover, we found that anti-disintegrin could neutralize the proteolytic and hemorrhagic activities from crude Crotalus atrox venom. Our results suggest that anti-disintegrin polyclonal antibodies have the potential for a targeted approach to neutralize SVMPs in the treatment of snakebite envenomations.

scholarly journals ABD-Derived Protein Blockers of Human IL-17 Receptor A as Non-IgG Alternatives for Modulation of IL-17-Dependent Pro-Inflammatory Axis

2018 ◽  
Vol 19 (10) ◽  
pp. 3089 ◽  
Author(s):  
Marie Hlavničková ◽  
Milan Kuchař ◽  
Radim Osička ◽  
Lucie Vaňková ◽  
Hana Petroková ◽  
...  
Keyword(s):  
Cell Surface ◽  
Clinical Manifestations ◽  
Cell Surface Receptor ◽  
Enzyme Linked Immunosorbent Assay ◽  
Interleukin 17 ◽  
Normal Human Skin ◽  
High Affinity ◽  
Th 17 ◽  
Surface Receptor

Interleukin 17 (IL-17) and its cognate receptor A (IL-17RA) play a crucial role in Th17 cells-mediated pro-inflammatory pathway and pathogenesis of several autoimmune disorders including psoriasis. IL-17 is mainly produced by activated Th-17 helper cells upon stimulation by IL-23 and, via binding to its receptors, mediates IL-17-driven cell signaling in keratinocytes. Hyper-proliferation of keratinocytes belongs to major clinical manifestations in psoriasis. To modulate IL-17-mediated inflammatory cascade, we generated a unique collection of IL-17RA-targeting protein binders that prevent from binding of human IL-17A cytokine to its cell-surface receptor. To this goal, we used a highly complex combinatorial library derived from scaffold of albumin-binding domain (ABD) of streptococcal protein G, and ribosome display selection, to yield a collection of ABD-derived high-affinity ligands of human IL-17RA, called ARS binders. From 67 analyzed ABD variants, 7 different sequence families were identified. Representatives of these groups competed with human IL-17A for binding to recombinant IL-17RA receptor as well as to IL-17RA-Immunoglobulin G chimera, as tested in enzyme-linked immunosorbent assay (ELISA). Five ARS variants bound to IL-17RA-expressing THP-1 cells and blocked binding of human IL-17 cytokine to the cell surface, as tested by flow cytometry. Three variants exhibited high-affinity binding with a nanomolar Kd value to human keratinocyte HaCaT cells, as measured using Ligand Tracer Green Line. Upon IL-17-stimulated activation, ARS variants inhibited secretion of Gro-α (CXCL1) by normal human skin fibroblasts in vitro. Thus, we identified a novel class of inhibitory ligands that might serve as immunosuppressive IL-17RA-targeted non-IgG protein antagonists.

scholarly journals RNA binding to human METTL3-METTL14 restricts N6-deoxyadenosine methylation of DNA in vitro

2022 ◽  
Author(s):  
Shan Qi ◽  
Javier Mota ◽  
Siu-Hong Chan ◽  
Johanna Villarreal ◽  
Nan Dai ◽  
...  
Keyword(s):  
Binding Affinity ◽  
Rna Binding ◽  
Methyl Group ◽  
High Affinity ◽  
Methylation Of Dna ◽  
Dna And Rna ◽  
Close Proximity ◽  
Methylation Activity

Methyltransferase like-3 (METTL3) and METTL14 complex transfers a methyl group from S-adenosyl-L-methionine to N6 amino group of adenosine bases in RNA (m6A) and DNA (m6dA). Emerging evidence highlights a role of METTL3-METTL14 in the chromatin context, especially in processes where DNA and RNA are held in close proximity. However, a mechanistic framework about specificity for substrate RNA/DNA and their interrelationship remain unclear. By systematically studying methylation activity and binding affinity to a number of DNA and RNA oligos with different propensities to form inter- or intra-molecular duplexes or single-stranded molecules in vitro, we uncover an inverse relationship for substrate binding and methylation and show that METTL3-METTL14 preferentially catalyzes the formation of m6dA in single-stranded DNA (ssDNA), despite weaker binding affinity to DNA. In contrast, it binds structured RNAs with high affinity, but methylates the target adenosine in RNA (m6A) much less efficiently than it does in ssDNA. We also show that METTL3-METTL14-mediated methylation of DNA is largely restricted by structured RNA elements prevalent in long noncoding and other cellular RNAs.

scholarly journals Differences in Antibody Responses against Chelonid Alphaherpesvirus 5 (ChHV5) Suggest Differences in Virus Biology in ChHV5-Seropositive Green Turtles from Hawaii and ChHV5-Seropositive Green Turtles from Florida

2019 ◽  
Vol 94 (4) ◽  
Author(s):  
Thierry M. Work ◽  
Julie Dagenais ◽  
Anna Willimann ◽  
George Balazs ◽  
Kate Mansfield ◽  
...  
Keyword(s):  
Life Histories ◽  
Cross Reactivity ◽  
Tumor Formation ◽  
Enzyme Linked Immunosorbent Assay ◽  
Native Form ◽  
Green Turtles ◽  
Tumor Viruses ◽  
The Caribbean ◽  
Lower Antibody Response

ABSTRACT Fibropapillomatosis (FP) is a tumor disease associated with a herpesvirus (chelonid herpesvirus 5 [ChHV5]) that affects mainly green turtles globally. Understanding the epidemiology of FP has been hampered by a lack of robust serological assays to monitor exposure to ChHV5. This is due in part to an inability to efficiently culture the virus in vitro for neutralization assays. Here, we expressed two glycoproteins (FUS4 and FUS8) from ChHV5 using baculovirus. These proteins were immobilized on enzyme-linked immunosorbent assay plates in their native form and assayed for reactivity to two types of antibodies, full-length 7S IgY and 5.7S IgY, which has a truncated Fc region. Turtles from Florida were uniformly seropositive to ChHV5 regardless of tumor status. In contrast, in turtles from Hawaii, we detected strong antibody reactivity mainly in tumored animals, with a lower antibody response being seen in nontumored animals, including those from areas where FP is enzootic. Turtles from Hawaii actively shedding ChHV5 were more seropositive than nonshedders. In trying to account for differences in the serological responses to ChHV5 between green turtles from Hawaii and green turtles from Florida, we rejected the cross-reactivity of antibodies to other herpesviruses, differences in viral epitopes, or differences in procedure as likely explanations. Rather, behavioral or other differences between green turtles from Hawaii and green turtles from Florida might have led to the emergence of biologically different viral strains. While the strains from turtles in Florida apparently spread independently of tumors, the transmission of the Hawaiian subtype relies heavily on tumor formation. IMPORTANCE Fibropapillomatosis (FP) is a tumor disease associated with chelonid herpesvirus 5 (ChHV5) that is an important cause of mortality in threatened green turtles globally. FP is expanding in Florida and the Caribbean but declining in Hawaii. We show that Hawaiian turtles mount antibodies to ChHV5 mainly in response to tumors, which are the only sites of viral replication, whereas tumored and nontumored Floridian turtles are uniformly seropositive. Tumor viruses that depend on tumors for replication and spread are rare, with the only example being the retrovirus causing walleye dermal sarcoma in fish. The Hawaiian strain of ChHV5 may be the first DNA virus with such an unusual life history. Our findings, along with the fundamental differences in the life histories between Floridian turtles and Hawaiian turtles, may partly explain the differential dynamics of FP between the two regions.

scholarly journals Potentiating Antigen-Specific Antibody Production with Peptides Obtained from In Silico Screening for High-Affinity against MHC-II

Molecules ◽  
2019 ◽  
Vol 24 (16) ◽  
pp. 2949
Author(s):  
Yoshiro Hanyu ◽  
Yuto Komeiji ◽  
Mieko Kato
Keyword(s):  
Monoclonal Antibodies ◽  
Binding Affinity ◽  
Antibody Production ◽  
Specific Antibody ◽  
In Silico ◽  
Igg Antibody ◽  
Mhc Ii ◽  
High Affinity ◽  
Specific Antibody Production

Monoclonal antibodies with high affinity and specificity are essential for research and clinical purposes, yet remain difficult to produce. Agretope peptides that can potentiate antigen-specific antibody production have been reported recently. Here, we screened in silico for peptides with higher affinity against the agretope binding pocket in the MHC-II. The screening was based on the 3D crystal structure of a complex between MHC-II and a 14-mer peptide consisting of ovalbumin residues 323–339. Using this 14-mer peptide as template, we constructed a library of candidate peptides and screened for those that bound tightly to MHC-II. Peptide sequences that exhibited a higher binding affinity than the original ovalbumin peptide were identified. The peptide with the highest binding affinity was synthesized and its ability to boost antigen-specific antibody production in vivo and in vitro was assessed. In both cases, antigen-specific IgG antibody production was potentiated. Monoclonal antibodies were established by in vitro immunization using this peptide as immunostimulant, confirming the usefulness of such screened peptides for monoclonal antibody production.

scholarly journals SARS-CoV-2 S1 and N-based serological assays reveal rapid seroconversion and induction of specific antibody response in COVID-19 patients

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Abdullah Algaissi ◽  
Mohamed A. Alfaleh ◽  
Sharif Hala ◽  
Turki S. Abujamel ◽  
Sawsan S. Alamri ◽  
...  
Keyword(s):  
Antibody Response ◽  
Specific Antibody ◽  
Large Scale ◽  
Disease Onset ◽  
High Specificity ◽  
Epidemiological Studies ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Specificity And Sensitivity ◽  
Human Coronaviruses

Abstract As the Coronavirus Disease 2019 (COVID-19), which is caused by the novel SARS-CoV-2, continues to spread rapidly around the world, there is a need for well validated serological assays that allow the detection of viral specific antibody responses in COVID-19 patients or recovered individuals. In this study, we established and used multiple indirect Enzyme Linked Immunosorbent Assay (ELISA)-based serological assays to study the antibody response in COVID-19 patients. In order to validate the assays we determined the cut off values, sensitivity and specificity of the assays using sera collected from pre-pandemic healthy controls, COVID-19 patients at different time points after disease-onset, and seropositive sera to other human coronaviruses (CoVs). The developed SARS-CoV-2 S1 subunit of the spike glycoprotein and nucleocapsid (N)-based ELISAs not only showed high specificity and sensitivity but also did not show any cross-reactivity with other CoVs. We also show that all RT-PCR confirmed COVID-19 patients tested in our study developed both virus specific IgM and IgG antibodies as early as week one after disease onset. Our data also suggest that the inclusion of both S1 and N in serological testing would capture as many potential SARS-CoV-2 positive cases as possible than using any of them alone. This is specifically important for tracing contacts and cases and conducting large-scale epidemiological studies to understand the true extent of virus spread in populations.

scholarly journals Detection and Visualization of an Exopolysaccharide Produced by Xylella fastidiosa In Vitro and In Planta

2007 ◽  
Vol 73 (22) ◽  
pp. 7252-7258 ◽  
Author(s):  
M. Caroline Roper ◽  
L. Carl Greve ◽  
John M. Labavitch ◽  
Bruce C. Kirkpatrick
Keyword(s):  
Xanthan Gum ◽  
Xanthomonas Campestris ◽  
Polyclonal Antibodies ◽  
Xylella Fastidiosa ◽  
Enzyme Linked Immunosorbent Assay ◽  
In Planta ◽  
Mannose Residue ◽  
Pantoea Stewartii ◽  
Vascular Occlusions

ABSTRACT Many phytopathogenic bacteria, such as Ralstonia solanacearum, Pantoea stewartii, and Xanthomonas campestris, produce exopolysaccharides (EPSs) that aid in virulence, colonization, and survival. EPS can also contribute to host xylem vessel blockage. The genome of Xylella fastidiosa, the causal agent of Pierce's disease (PD) of grapevine, contains an operon that is strikingly similar to the X. campestris gum operon, which is responsible for the production of xanthan gum. Based on this information, it has been hypothesized that X. fastidiosa is capable of producing an EPS similar in structure and composition to xanthan gum but lacking the terminal mannose residue. In this study, we raised polyclonal antibodies against a modified xanthan gum polymer similar to the predicted X. fastidiosa EPS polymer. We used enzyme-linked immunosorbent assay to quantify production of EPS from X. fastidiosa cells grown in vitro and immunolocalization microscopy to examine the distribution of X. fastidiosa EPS in biofilms formed in vitro and in planta and assessed the contribution of X. fastidiosa EPS to the vascular occlusions seen in PD-infected grapevines.

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