antigen capture
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2022 ◽  
Vol 2022 (1) ◽  
pp. pdb.prot103127
Author(s):  
Edward A. Greenfield

In an antigen capture assay for hybridoma screening, the detection method identifies the presence of the antigen. Often this is achieved by labeling the antigen directly. In this assay, the polyvinyl chloride (PVC) wells of a high-binding-capacity ELISA plate are first coated with an affinity-purified rabbit anti-mouse immunoglobulin and then incubated with hybridoma tissue culture supernatant. Monoclonal antibodies in the supernatant are “captured” on the coated PVC surface and detected by screening with biotin- or histidine (His)–tagged antigen. The antigen can be labeled to a high specific activity and thus very little antigen is required for this procedure.


2022 ◽  
Vol 2022 (1) ◽  
pp. pdb.prot103135
Author(s):  
Edward A. Greenfield

A dot blot is widely used to determine the productivity of a given hybridoma. This assay can also be used to screen a fusion or subclone plate for productive hybridoma clones. First, a nitrocellulose membrane is coated with an affinity-purified goat or rabbit anti-mouse immunoglobulin and then incubated with hybridoma tissue culture supernatant. Monoclonal antibodies in the supernatant are then “captured” on the coated nitrocellulose membrane surface and detected by screening with horseradish peroxidase (HRP).


Author(s):  
Sri Handayani Irianingsih ◽  
Dessie Eri Waluyati ◽  
Desi Puspita Sari ◽  
Hastari Wuryastuty

Abstract Bovine Viral Diarrhea (BVD) is one of the main causes of impaired productivity and reproduction of cows. Antigen capture Elisa (ACE) is one of the serological technique that is sensitive, reliable and used regularly for detecting persistent BVD infection individually which simpler than  multiplex nested PCR. The aim of this study was to determine the agreement between ACE and multiplex nested PCR as a routine laboratory diagnostic technique to detect the presence of BVD infection. A total of 128 cow serum samples consisting of 63 positive and 65 negative samples based on ACE were used in this study. The samples were collected from active and passive surveillance in dairy and beef cattle conducted by Balai Besar Veteriner (BBVet) Wates. The serum samples were then tested molecularly using multiplex nested PCR against BVD. The result showed 48 out of 63  BVDV-1 positive samples were found positive BVD antigen whereas 57 of 65  BVDV-1 negative samples were negative using multiplex nested PCR, . The agreement value between the two different assays based on statistic analysis using Kappa method was 0.64 and classified a good one. The result concluded that the ACE BVD assay was equally suitable as routine diagnosis to determine BVD infected cattle in the farm. Keywords: Antigen capture ELISA; Bovine viral diarrhea; Kappa; Multiplex nested PCR.   Abstrak Bovine Viral Diarrhea (BVD) merupakan salah satu penyebab gangguan produktivitas dan reproduksi sapi. Antigen capture ELISA (ACE) merupakan salah satu teknik serologis yang sensitif, dapat diandalkan dan digunakan secara teratur untuk mendeteksi infeksi BVD persisten secara individual yang lebih sederhana daripada multiplex nested PCR. Tujuan penelitian ini adalah untuk mengetahui kesesuaian antara uji ACE dan multiplex nested PCR sebagai teknik diagnostik laboratorium rutin untuk mendeteksi adanya infeksi BVD. Sebanyak 128 sampel serum sapi yang terdiri dari 63 sampel positif dan 65 negatif berdasarkan ACE BVDV Antigen Test Kit/Serum Plus (Idexx®) digunakan dalam kajian ini. Sampel serum sapi merupakan koleksi dari surveilans aktif dan pasif pada sapi perah dan potong yang dilakukan Balai Besar Veteriner (BBVet) Wates. Sampel serum kemudian diuji secara molekuler menggunakan multiplex nested PCR terhadap BVD. Hasil penelitian menunjukkan bahwa dengan teknik multiplex nested PCR, 48 dari 63 sampel positif BVDV-1 ditemukan positif untuk antigen BVD sedangkan 57 dari 65 sampel negatif BVDV-1 negatif untuk antigen BVD. Analisis statitik berdasarkan perhitungan metoda Kappa menunjukkan nilai kesesuaian antara dua uji sebesar 0,64 dan tergolong bagus. Hasil penelitian menunjukkan kesimpulan bahwa uji ACE BVD sesuai sebagai diagnosis rutin untuk menentukan ternak yang terinfeksi BVD di peternakan. Kata kunci: Antigen capture ELISA; Bovine viral diarrhea; Kappa; Multiplex nested PCR.


2022 ◽  
Vol 2022 (1) ◽  
pp. pdb.prot103143
Author(s):  
Edward A. Greenfield

Immunoprecipitation is rarely used for screening hybridoma fusions because the assays are tedious and time-consuming. However, it can be useful when working with complex antigens because the precipitated antigen is normally detected after sodium dodecyl sulfate (SDS)–polyacrylamide electrophoresis and thus it is simple to discriminate between true and false positives. Furthermore, the assay provides information regarding the molecular weight of the antigen.


2021 ◽  
Author(s):  
Jon Arizti-Sanz ◽  
A'Doriann Bradley ◽  
Yibin B. Zhang ◽  
Chloe K. Boehm ◽  
Catherine A. Freije ◽  
...  

The COVID-19 pandemic, and the recent rise and widespread transmission of SARS-CoV-2 Variants of Concern (VOCs), have demonstrated the need for ubiquitous nucleic acid testing outside of centralized clinical laboratories. Here, we develop SHINEv2, a Cas13-based nucleic acid diagnostic that combines quick and ambient temperature sample processing and lyophilized reagents to greatly simplify the test procedure and assay distribution. We benchmarked a SHINEv2 assay for SARS-CoV-2 detection against state-of-the-art antigen-capture tests using 96 patient samples, demonstrating 50-fold greater sensitivity and 100% specificity. We designed SHINEv2 assays for discriminating the Alpha, Beta, Gamma and Delta VOCs, which can be read out visually using lateral flow technology. We further demonstrate that our assays can be performed without any equipment in less than 90 minutes. SHINEv2 represents an important advance towards rapid nucleic acid tests that can be performed in any location.


2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A218-A218
Author(s):  
Daniel Corey ◽  
Sunil Thomas ◽  
Brandon Cieniewicz ◽  
Linh Nguyen ◽  
Jared Clever ◽  
...  

BackgroundActivated T cells have limited antigen presenting capability due to inefficient capture.1 This process can be enhanced through novel chimeric engulfment receptors (CERs) expressing a human Tim-4 phagocyte receptor that recognizes phosphatidylserine (Ptd-Ser)2 fused to T cell and macrophage/dendritic cell-derived signaling domains. CERs can facilitate antigen capture, processing, and presentation, and impart target-dependent cytotoxic function when expressed in T cells. This combined function is hypothesized to improve tumor clearance and durability of response, making CER T cell products ideal clinical candidates.MethodsWe generated Tim-4 receptors fused to toll-like receptor (TLR)-2 or -8, CD28 or CD3 zeta and tested phagocytic, antigen presentation and cytotoxic function in healthy donor T cells. To assess phagocytosis, target cells treated with a small molecule to induce Ptd-Ser externalization were labeled with pH-Rodo followed by co-culture with CER T cells. Activated CER T cells were evaluated by transmission electron microscopy (TEM) or flow cytometry (FC) for lysosomal uptake of cell fragments. Antigen capture and presentation were characterized by FC for the capacity of human papilloma virus 16 (HPV 16) E7 peptide-pulsed CER T cells to activate and induce proliferation of autologous HPV 16 E7-TCR transduced T cells. Cytotoxic function was evaluated in co-culture assays of CER T cells in the presence of subtherapeutic doses of BTKi (ibrutinib)-treated JeKo-1 lymphoma cells.ResultsTEM imaging demonstrated that CER T cells engulfed target cell fragments, illustrated by multi-vesicular bodies containing tumor fragments (some measuring >0.5 uM) and pseudo-pod like formations around apoptotic target cell blebs. RNA analysis revealed upregulation of TLR, myeloid differentiation, and antigen presentation pathways. In the HPV 16 E7 co-culture model, T-cell surface activation markers CD25 and CD69 were upregulated 41% and 23%, respectively, on E7-TCR-T cells relative to controls. In addition, the percentage of dividing E7-TCR-T cells was increased (44% vs 8%) after 6 days in co-culture. Addition of CER T cells to JeKo- 1 target cells in the presence of BTKi at low effector: target ratios enhanced cytotoxicity by over 99%, demonstrating synergy with a targeted small molecule to fully eliminate lymphoma cells.ConclusionsNovel Tim-4/TLR containing CERs can capture tumor cell fragments and present soluble antigen, a function previously demonstrated to be a barrier to effective antigen presentation in T cells. Enhanced T-cell antigen capture and presentation capability alongside inducible and target-specific cytotoxic function in single T cells represents a significant advancement in the potential for chimeric receptor-based therapies.ReferencesLanzavecchia A, Roosnek E, Gregory T, Berman P, Abrignani S. T cells can present antigens such as HIV gp120 targeted to their own surface molecules. Nature 1988 Aug 11;334(6182):530–2.Caronni N, Piperno GM, Simoncello F, Romano O, Vodret S, Yanagihashi Y, et al. TIM4 expression by dendritic cells mediates uptake of tumor-associated antigens and anti-tumor responses. Nat Commun 2021 Apr 14;12(1):2237.


2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Yixin Xiao ◽  
Fan Yang ◽  
Fumin Liu ◽  
Hangping Yao ◽  
Nanping Wu ◽  
...  

Abstract Background The H9N2 subtype of avian influenza virus (AIV) has become the most widespread subtype of AIV among birds in Asia, which threatens the poultry industry and human health. Therefore, it is important to establish methods for the rapid diagnosis and continuous surveillance of H9N2 subtype AIV. Methods In this study, an antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) and a colloidal gold immunochromatographic test (ICT) strip using monoclonal antibodies (MAbs) 3G4 and 2G7 were established to detect H9N2 subtype AIV. Results The AC-ELISA method and ICT strip can detect H9N2 subtype AIV quickly, and do not cross-react with other subtype AIVs or other viruses. The detection limit of AC-ELISA was a hemagglutinin (HA) titer of 4 for H9N2 subtype AIV per 100 μl sample, and the limit of detection of the HA protein of AIV H9N2 was 31.5 ng/ml. The ICT strip detection limit was an HA titer of 4 for H9N2 subtype AIV per 100 μl sample. Moreover, both detection methods exhibited good reproducibility and repeatability, with coefficients of variation < 5%. For detection in 200 actual poultry samples, the sensitivities and specificities of AC-ELISA were determined as 93.2% and 98.1%, respectively. The sensitivities and specificities of the ICT strips were determined as 90.9% and 97.4%, respectively. Conclusions The developed AC-ELISA and ICT strips displayed high specificity, sensitivity, and stability, making them suitable for rapid diagnosis and field investigation of H9N2 subtype AIV.


Viruses ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 1771
Author(s):  
Brandon J. Beddingfield ◽  
Jessica N. Hartnett ◽  
Russell B. Wilson ◽  
Peter C. Kulakosky ◽  
Kristian G. Andersen ◽  
...  

Infection with Zika virus (ZIKV), a member of the Flavivirus genus of the Flaviviridae family, typically results in mild self-limited illness, but severe neurological disease occurs in a limited subset of patients. In contrast, serious outcomes commonly occur in pregnancy that affect the developing fetus, including microcephaly and other major birth defects. The genetic similarity of ZIKV to other widespread flaviviruses, such as dengue virus (DENV), presents a challenge to the development of specific ZIKV diagnostic assays. Nonstructural protein 1 (NS1) is established for use in immunodiagnostic assays for flaviviruses. To address the cross-reactivity of ZIKV NS1 with proteins from other flaviviruses we used site-directed mutagenesis to modify putative epitopes. Goat polyclonal antibodies to variant ZIKV NS1 were affinity-purified to remove antibodies binding to the closely related NS1 protein of DENV. An antigen-capture ELISA configured with the affinity-purified polyclonal antibody showed a linear dynamic range between approximately 500 and 30 ng/mL, with a limit of detection of between 1.95 and 7.8 ng/mL. NS1 proteins from DENV, yellow fever virus, St. Louis encephalitis virus and West Nile virus showed significantly reduced reactivity in the ZIKV antigen-capture ELISA. Refinement of approaches similar to those employed here could lead to development of ZIKV-specific immunoassays suitable for use in areas where infections with related flaviviruses are common.


2021 ◽  
Vol 8 (9) ◽  
Author(s):  
Georgina C. Girt ◽  
Abirami Lakshminarayanan ◽  
Jiandong Huo ◽  
Joshua Dormon ◽  
Chelsea Norman ◽  
...  

Detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigens in the fluid has important uses in biotechnology, and is integral to many point-of-care SARS-CoV-2 diagnostics. Sandwich enzyme-linked immunosorbent assays (ELISAs) are a sensitive, well-established method of measuring antigens in solutions. They use one ligand to capture and the other ligand to detect the target analyte. Detection is commonly achieved using colorimetric readout obtained upon the reaction of a substrate with HRP-conjugated secondary ligand. Nanobodies, the V H H domain of camelid antibodies, have expanded the repertoire of molecules used in antigen detection. Nanobodies' high affinity for target antigens, their compact structure, their high stability and ease of production has driven research into their use as diagnostic reagents. Guided by a structural understanding of epitopes on the receptor-binding domain of the SARS-CoV-2 Spike protein, we investigated various combinations of engineered nanobodies in a sandwich ELISA to detect the Spike protein of SARS-CoV-2. We have identified an optimal combination of nanobodies. These were selectively functionalized to further improve antigen capture, enabling the measurement of sub-picomolar amounts of SARS-CoV-2 Spike protein in solution. With this combination, the routine detection limit in samples inactivated by heat and detergent corresponded to less than seven focus-forming units of infectious SARS-CoV-2.


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