Standardization of a cytometric p24-capture bead-assay for the detection of main HIV-1 subtypes.

Merbah, Mélanie; Onkar, Sayali; Grivel, Jean‐Charles; Vanpouille, Christophe; Biancotto, Angélique; Bonar, Lydia; Sanders‐Buell, Eric; Kijak, Gustavo H.; Michael, Nelson; Robb, Merlin L.; Kim, Jerome H.; Tovanabutra, Sodsai; Chenine, Agnès‐Laurence

doi:10.1016/j.jviromet.2016.01.009

Cited by 3 publications

(5 citation statements)

References 24 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Many studies highlighting the use of antibody from commercial hybridoma cell (clone 183-H12-5C) in detecting the HIV-1 capsid protein p24, 32 , 33 however until now it has yet to be used for therapeutic purpose of HIV-1/AIDS. With the fact that monoclonal antibodies (mAbs) are highly specific and stable, it becomes an attractive candidate for in vivo treatment.…”

Section: Discussionmentioning

confidence: 99%

Expression of Functional Anti-p24 scFv 183-H12-5C in HEK293T and Jurkat T Cells

Omar¹

2017

Adv Pharm Bull

View full text Add to dashboard Cite

Purpose: More than half of the diagnostic and therapeutic recombinant protein production depends on mammalian-based expression system. However, the generation of recombinant antibodies remains a challenge in mammalian cells due to the disulfide bond formation and reducing cytoplasm. Therefore, the production of functional recombinant antibodies in target cell line is necessary to be evaluated before used in therapeutic application such intrabodies against HIV-1. Methods: The work was to test expression of a single-chain variable fragment (scFv) antibody against HIV-1 Capsid p24 protein in a human mammalian-based expression system using HEK293T and Jurkat T cells as a model. Three expression plasmid vectors expressing scFv 183-H12-5C were generated and introduced into HEK293T. Expression of the scFv was analyzed, while ELISA and immunoblotting analysis verified its binding. The evaluation of the recombinant antibody was confirmed by HIV-1 replication and MAGI infectivity assay in Jurkat T cells. Results: Three plasmid vectors expressing scFv 183-H12-5C was successfully engineered in this study. Recombinant antibodies scFv (~29 kDa) and scFv-Fc (~52 kDa) in the cytoplasm of HEK293T were effectively obtained by transfected the cells with engineered pCDNA3.3-mu-IgGk-scFv 183-H12-5C and pCMX2.5-scFv 183-H12-5C-hIgG1-Fc plasmid vectors respectively. scFv and scFv-Fc are specifically bound recombinant p24, and HIV-1 derived p24 (gag) evaluated by ELISA and Western blot. Jurkat T cells transfected by pCDNA3.3-scFv 183-H12-5C inhibit the replication-competent NL4-3 viral infectivity up to 60%. Conclusion: Anti-p24 scFv 183-H12-5C antibody generated is suitable to be acted as intrabodies and may serve as a valuable tool for the development of antibody-based biotherapeutics against HIV-1.

show abstract

Section: Discussionmentioning

confidence: 99%

Expression of Functional Anti-p24 scFv 183-H12-5C in HEK293T and Jurkat T Cells

Omar¹

2017

Adv Pharm Bull

View full text Add to dashboard Cite

show abstract

Section: Diagnostic Applicationsmentioning

confidence: 99%

“…37,38 The results have shown that the method is 91% more sensitive than the standard ELISA test and also has a lower cost. 37…”

Section: Diagnostic Applicationsmentioning

confidence: 99%

“…36 Tests have also been developed for the detection of the p24 protein in various subtypes of HIV-1. 37,38 The results have shown that the method is 91% more sensitive than the standard ELISA test and also has a lower cost. 37 Fonseca et al 39 developed a new multiplex, bead-based assay for the detection of hepatitis C virus (HCV), using the NS3, NS4, and NS5 recombinant antigens from HCV, individually in the simplex assay and in a combination of them in the multiplex assay.…”

Section: Viral Infectionsmentioning

confidence: 99%

“…37,38 The results have shown that the method is 91% more sensitive than the standard ELISA test and also has a lower cost. 37 Fonseca et al 39 developed a new multiplex, bead-based assay for the detection of hepatitis C virus (HCV), using the NS3, NS4, and NS5 recombinant antigens from HCV, individually in the simplex assay and in a combination of them in the multiplex assay. In this study, 100% sensitivity and specificity were observed, which shows that the method has the potential to become a possible alternative for the diagnosis of HCV, while having a cost that is two to four times lower than the tests usually employed.…”

Section: Viral Infectionsmentioning

confidence: 99%

See 2 more Smart Citations

Diagnostic applications of microsphere-based flow cytometry: A review

Figueiredo

Glória

Chaves

et al. 2022

Exp Biol Med (Maywood)

View full text Add to dashboard Cite

Microsphere-based flow cytometry is a highly sensitive emerging technology for specific detection and clinical analysis of antigens, antibodies, and nucleic acids of interest. In this review, studies that focused on the application of flow cytometry as a viable alternative for the investigation of infectious diseases were analyzed. Many of the studies involve research aimed at epidemiological surveillance, vaccine candidates and early diagnosis, non-infectious diseases, specifically cancer, and emphasize the simultaneous detection of biomarkers for early diagnosis, with accurate results in a non-invasive approach. The possibility of carrying out multiplexed assays affords this technique high versatility and performance, which is evidenced in a series of clinical studies that have verified the ability to detect several molecules in low concentrations and with minimal sample volume. As such, we demonstrate that microsphere-based flow cytometry presents itself as a promising technique that can be adopted as a fundamental element in the development of new diagnostic methods for a number of diseases.

show abstract

In-house ELISA protocols for capsid p24 detection of diverse HIV isolates

Molina,

Vink,

Berkhout

et al. 2023

Virol J

View full text Add to dashboard Cite

Background The capsid p24 (CA-p24) antigen is a component of the viral capsid of human immunodeficiency virus (HIV) that has been commonly used for clinical diagnosis and monitoring of HIV infections in Enzyme-linked Immunosorbent Assays (ELISAs). Commercial CA-p24 ELISAs are widely used in research settings, but these kits are costly and have limited breadth for detecting diverse HIV isolates. Methods Commercial CA-p24 antibodies were used as capture and detection antibodies. Specific CA-p24 ELISAs were established with these antibodies and tested for the detection of HIV-1 isolates with the aim of developing in-house protocols to recognize HIV-1 infections in vitro for research purposes. Results Here we present four protocols for in-house ELISAs to detect HIV CA-p24 using commercial antibodies. The assays were able to detect the CA-p24 antigen of different HIV-1 isolates tested. Comparison between the protocols showed that these in-house ELISAs exhibit high specificity, sensitivity, and reproducibility for CA-p24 quantitation but their reactivity varied per HIV-1 isolate and subtype. Conclusions These optimized ELISA protocols represent valuable tools to investigate HIV-1 infections in research facilities at a lower price than commercial CA-p24 kits.

show abstract

Standardization of a cytometric p24-capture bead-assay for the detection of main HIV-1 subtypes.

Cited by 3 publications

References 24 publications

Expression of Functional Anti-p24 scFv 183-H12-5C in HEK293T and Jurkat T Cells

Expression of Functional Anti-p24 scFv 183-H12-5C in HEK293T and Jurkat T Cells

Diagnostic applications of microsphere-based flow cytometry: A review

In-house ELISA protocols for capsid p24 detection of diverse HIV isolates

Contact Info

Product

Resources

About