Higher than 90% of HIV-1 proviruses are considered faulty and incapable of viral replication. Whereas replication competent proviruses are of main concern with respect to illness development or transmission, research have proven that even faulty proviruses will not be silent and may produce viral proteins, which can contribute to irritation and immune responses.
Viral protein expression additionally has implications for immune-based HIV-1 clearance methods, which depend on antigen recognition. Thus, delicate assays geared toward quantifying each replication-competent proviruses and faulty, but translationally competent proviruses are wanted to grasp the contribution of viral protein to HIV-1 pathogenesis and decide the effectiveness of HIV-1 remedy interventions.
Beforehand, we reported a modified HIV-1 gag p24 digital enzyme-linked immunosorbent assay with single molecule array (Simoa) detection of cell-associated viral protein. Right here we report a novel p24 protein enrichment technique coupled with the digital immunoassay to additional lengthen the sensitivity and specificity of viral protein detection.
Immunocapture of HIV gag p24 adopted by elution in a Simoa-compatible format resulted in increased protein restoration and decrease background from varied organic matrices and pattern volumes. Quantification of as little as 1 fg of p24 protein from cell lysates from cells remoted from peripheral blood or tissues from ART-suppressed HIV contributors, in addition to simian-human immunodeficiency virus-infected non-human primates (NHPs), with excessive restoration and reproducibility is demonstrated right here.
The appliance of those enhanced strategies to patient-derived samples has potential to additional the research of the persistent HIV state and study in vitro response to therapies, in addition to ex vivo research of translationally competent cells from a wide range of donors.
Measuring the Inducible, Replication-Competent HIV Reservoir Utilizing an Extremely-Delicate p24 Readout, the Digital ELISA Viral Outgrowth Assay
Quantifying the inducible HIV reservoir supplies an estimate of the frequency of quiescent HIV-infected cells in people in addition to in animal fashions, and can assist verify the efficacy of latency reversing brokers (LRAs). The quantitative viral outgrowth assay (QVOA) is used to measure inducible, replication competent HIV and generate estimations of reservoir dimension.
Nonetheless, conventional QVOA is time and labor intensive and requires giant quantities of lymphocytes. Given the significance of reproducible and correct evaluation of each reservoir dimension and LRA exercise in remedy methods, efforts to streamline the QVOA are of excessive precedence.
We developed a modified QVOA, the Digital ELISA Viral Outgrowth or DEVO assay, with ultra-sensitive p24 readout, able to femtogram detection of HIV p24 protein in distinction to the picogram limitations of conventional ELISA. For every DEVO assay, 8-12 × 106 resting CD4 + T cells from aviremic, ART-treated HIV + contributors are plated in limiting dilution and maximally stimulated with PHA, IL-2 and uninfected allogeneic irradiated PBMC.
CD8-depleted PHA blasts from an uninfected donor or HIV-permissive cells (e.g., Molt4/CCR5) are added to the cultures and virus allowed to amplify for 8-12 days. HIV p24 from tradition supernatant is measured at day Eight by Simoa (single molecule array, ultra-sensitive p24 assay) confirmed at day 12, and infectious models per million CD4 + T cells (IUPM) are calculated utilizing the utmost chance technique.
In all DEVO assays carried out, HIV p24 was detected within the supernatant of cultures as early as Eight days put up stimulation. Importantly, DEVO IUPM values at day Eight had been comparable or increased than conventional QVOA IUPM values obtained at day 15.
Curiously, DEVO IUPM values had been comparable with or with out the addition of allogeneic CD8-depleted goal PHA blasts or HIV permissive cells historically used to increase virus. The DEVO assay makes use of fewer resting CD4 + T cells and supplies an evaluation of reservoir dimension in much less time than commonplace QVOA.
This assay affords a brand new platform to quantify replication competent HIV throughout restricted cell availability. Different potential purposes embrace evaluating LRA exercise, and measuring clearance of contaminated cells throughout latency clearance assays.
Amine-Aldehyde Chemical Conjugation on a Potassium Hydroxide-Handled Polystyrene ELISA Floor for Nanosensing an HIV-p24 Antigen.
The enzyme-linked immunosorbent assay (ELISA) has been broadly used for illness surveillance and drug screening as a result of its comparatively increased accuracy and sensitivity. Fantastic-tuning the ELISA is necessary to raise the precise detection of biomolecules at a decrease abundance.
In direction of this finish, increased molecular seize on the polystyrene (PS) ELISA floor is essential for environment friendly detection, and it might be attained by immobilizing the molecules within the appropriate orientation. It’s extremely difficult to immobilize protein molecules in a well-aligned method on an ELISA floor as a result of cost variations.
We employed a 3-(aminopropyl) triethoxysilane (APTES)- and glutaraldehyde (GLU)-coupled PS floor chemical technique to reveal the excessive efficiency with ELISA. A potassium hydroxide therapy adopted by an equal ratio of 1% APTES and GLU attachment was discovered to be optimum, and an extended incubation with GLU favored most sensitivity.
p24 is an important early secreting antigen for diagnosing human immunodeficiency virus (HIV), and it has been used for environment friendly detection with the above chemistry. Three completely different procedures had been adopted, they usually led to the improved detection of the HIV-p24 antigen at 1 nM, which is a 30-fold increased degree in comparison with a standard ELISA floor.
The floor chemical functionalization proven right here additionally shows the next specificity with human serum and HIV-TAT. The above method with the designed floor chemistry is also beneficial for illness prognosis on different sensing surfaces involving the interplay of the probe and the analyte in heterogeneous check samples.
Growth of a brand new recombinant p24 ELISA system for prognosis of bovine leukemia virus in serum and milk.
Bovine leukemia virus (BLV) is a retrovirus that causes enzootic bovine leucosis. Right here, we designed a p24 enzyme-linked immunosorbent assay (ELISA) to detect antibodies particular for BLV capsid protein p24 (encoded by the gag gene) in bovine serum samples.
The p24 gene was inserted into an Escherichia coli expression system, and recombinant proteins (GST-p24, p24, and His-p24) had been purified. His-p24 was essentially the most appropriate antigen for utilizing within the ELISA. The cut-off level was calculated from a receiver working attribute curve derived from a set of 582 subject samples that beforehand examined constructive or unfavourable by BLV-CoCoMo-qPCR-2, which detects BLV provirus.
The brand new p24 ELISA confirmed nearly the identical specificity and sensitivity as a industrial gp51 ELISA equipment when used to check subject serum samples, and allowed monitoring of p24 antibodies in uncooked milk and whey.
Human HIV1 p24 ELISA Kit |
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| SL3525Hu | Sunlong | 96 Tests | EUR 468 |
Rat HIV-1 p24 core protein (HIV-1 p24) antibody ELISA Kit |
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| MBS2614549-10x96StripWells | MyBiosource | 10x96-Strip-Wells | EUR 3290 |
Rat HIV-1 p24 core protein (HIV-1 p24) antibody ELISA Kit |
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| MBS2614549-48StripWells | MyBiosource | 48-Strip-Wells | EUR 295 |
Rat HIV-1 p24 core protein (HIV-1 p24) antibody ELISA Kit |
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| MBS2614549-5x96StripWells | MyBiosource | 5x96-Strip-Wells | EUR 1800 |
Rat HIV-1 p24 core protein (HIV-1 p24) antibody ELISA Kit |
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| MBS2614549-96StripWells | MyBiosource | 96-Strip-Wells | EUR 450 |
Goat HIV-1 p24 core protein (HIV-1 p24) antibody ELISA Kit |
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| MBS2614545-10x96StripWells | MyBiosource | 10x96-Strip-Wells | EUR 3545 |
Goat HIV-1 p24 core protein (HIV-1 p24) antibody ELISA Kit |
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| MBS2614545-48StripWells | MyBiosource | 48-Strip-Wells | EUR 310 |
Goat HIV-1 p24 core protein (HIV-1 p24) antibody ELISA Kit |
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| MBS2614545-5x96StripWells | MyBiosource | 5x96-Strip-Wells | EUR 1940 |
Goat HIV-1 p24 core protein (HIV-1 p24) antibody ELISA Kit |
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| MBS2614545-96StripWells | MyBiosource | 96-Strip-Wells | EUR 475 |
Human HIV-1 p24 core protein (HIV-1 p24) antibody ELISA Kit |
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| NSL1389Hu | Sunlong | 96 wells | EUR 468 |
Mouse HIV-1 p24 core protein (HIV-1 p24) antibody ELISA Kit |
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| MBS2614547-10x96StripWells | MyBiosource | 10x96-Strip-Wells | EUR 3290 |
Mouse HIV-1 p24 core protein (HIV-1 p24) antibody ELISA Kit |
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| MBS2614547-48StripWells | MyBiosource | 48-Strip-Wells | EUR 295 |
Mouse HIV-1 p24 core protein (HIV-1 p24) antibody ELISA Kit |
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| MBS2614547-5x96StripWells | MyBiosource | 5x96-Strip-Wells | EUR 1800 |
Mouse HIV-1 p24 core protein (HIV-1 p24) antibody ELISA Kit |
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| MBS2614547-96StripWells | MyBiosource | 96-Strip-Wells | EUR 450 |
Human HIV-1 p24 core protein (HIV-1 p24) antibody ELISA Kit |
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| MBS2602892-10x96StripWells | MyBiosource | 10x96-Strip-Wells | EUR 3040 |
Human HIV-1 p24 core protein (HIV-1 p24) antibody ELISA Kit |
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| MBS2602892-48StripWells | MyBiosource | 48-Strip-Wells | EUR 285 |
Human HIV-1 p24 core protein (HIV-1 p24) antibody ELISA Kit |
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| MBS2602892-5x96StripWells | MyBiosource | 5x96-Strip-Wells | EUR 1670 |
Human HIV-1 p24 core protein (HIV-1 p24) antibody ELISA Kit |
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| MBS2602892-96StripWells | MyBiosource | 96-Strip-Wells | EUR 405 |
Human HIV-1 p24 core protein (HIV-1 p24) antibody ELISA Kit |
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| EIA06774h | Enlibio | each | EUR 430 |
Bovine HIV-1 p24 core protein (HIV-1 p24) antibody ELISA Kit |
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| MBS2610168-10x96StripWells | MyBiosource | 10x96-Strip-Wells | EUR 3545 |
Bovine HIV-1 p24 core protein (HIV-1 p24) antibody ELISA Kit |
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| MBS2610168-48StripWells | MyBiosource | 48-Strip-Wells | EUR 310 |
Bovine HIV-1 p24 core protein (HIV-1 p24) antibody ELISA Kit |
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| MBS2610168-5x96StripWells | MyBiosource | 5x96-Strip-Wells | EUR 1940 |
Bovine HIV-1 p24 core protein (HIV-1 p24) antibody ELISA Kit |
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| MBS2610168-96StripWells | MyBiosource | 96-Strip-Wells | EUR 475 |
Canine HIV-1 p24 core protein (HIV-1 p24) antibody ELISA Kit |
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| MBS2614543-10x96StripWells | MyBiosource | 10x96-Strip-Wells | EUR 3545 |
Canine HIV-1 p24 core protein (HIV-1 p24) antibody ELISA Kit |
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| MBS2614543-48StripWells | MyBiosource | 48-Strip-Wells | EUR 310 |
Canine HIV-1 p24 core protein (HIV-1 p24) antibody ELISA Kit |
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| MBS2614543-5x96StripWells | MyBiosource | 5x96-Strip-Wells | EUR 1940 |
Canine HIV-1 p24 core protein (HIV-1 p24) antibody ELISA Kit |
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| MBS2614543-96StripWells | MyBiosource | 96-Strip-Wells | EUR 475 |
Rabbit HIV-1 p24 core protein (HIV-1 p24) antibody ELISA Kit |
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| MBS2614550-10x96StripWells | MyBiosource | 10x96-Strip-Wells | EUR 3545 |
Rabbit HIV-1 p24 core protein (HIV-1 p24) antibody ELISA Kit |
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| MBS2614550-48StripWells | MyBiosource | 48-Strip-Wells | EUR 310 |
Rabbit HIV-1 p24 core protein (HIV-1 p24) antibody ELISA Kit |
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| MBS2614550-5x96StripWells | MyBiosource | 5x96-Strip-Wells | EUR 1940 |
Rabbit HIV-1 p24 core protein (HIV-1 p24) antibody ELISA Kit |
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| MBS2614550-96StripWells | MyBiosource | 96-Strip-Wells | EUR 475 |
Chicken HIV-1 p24 core protein (HIV-1 p24) antibody ELISA Kit |
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| MBS2614544-10x96StripWells | MyBiosource | 10x96-Strip-Wells | EUR 3545 |
Chicken HIV-1 p24 core protein (HIV-1 p24) antibody ELISA Kit |
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| MBS2614544-48StripWells | MyBiosource | 48-Strip-Wells | EUR 310 |
Chicken HIV-1 p24 core protein (HIV-1 p24) antibody ELISA Kit |
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| MBS2614544-5x96StripWells | MyBiosource | 5x96-Strip-Wells | EUR 1940 |
Chicken HIV-1 p24 core protein (HIV-1 p24) antibody ELISA Kit |
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| MBS2614544-96StripWells | MyBiosource | 96-Strip-Wells | EUR 475 |
Porcine HIV-1 p24 core protein (HIV-1 p24) antibody ELISA Kit |
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| MBS2614548-10x96StripWells | MyBiosource | 10x96-Strip-Wells | EUR 3545 |
Porcine HIV-1 p24 core protein (HIV-1 p24) antibody ELISA Kit |
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| MBS2614548-48StripWells | MyBiosource | 48-Strip-Wells | EUR 310 |
Porcine HIV-1 p24 core protein (HIV-1 p24) antibody ELISA Kit |
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| MBS2614548-5x96StripWells | MyBiosource | 5x96-Strip-Wells | EUR 1940 |
Porcine HIV-1 p24 core protein (HIV-1 p24) antibody ELISA Kit |
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| MBS2614548-96StripWells | MyBiosource | 96-Strip-Wells | EUR 475 |
Human Immunodeficiency Virus type 1 (HIV-1) p24/Capsid Protein p24 ELISA Kit |
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| MBS8123946-5x96Tests | MyBiosource | 5x96Tests | EUR 2200 |
Human Immunodeficiency Virus type 1 (HIV-1) p24/Capsid Protein p24 ELISA Kit |
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| MBS8123946-96Tests | MyBiosource | 96Tests | EUR 480 |
Bovine HIV-1 p24 core protein(HIV-1 p24) Anti-body ELISA Kit |
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| NSL1181Bo | Sunlong | 96 T | EUR 528 |
Guinea pig HIV-1 p24 core protein (HIV-1 p24) antibody ELISA Kit |
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| MBS2614546-10x96StripWells | MyBiosource | 10x96-Strip-Wells | EUR 3545 |
Evaluating the outcomes for the p24 ELISA and gp51 ELISA revealed that p24 antibodies had been detected sooner than gp51 antibodies in three out of eight calves experimentally contaminated with BLV, indicating improved detection with out diminishing BLV serodiagnosis. Thus, the p24 ELISA is a strong and dependable assay for detecting BLV antibodies in serum or milk, making it’s a great tool for large-scale BLV screening.