荧光标记抗原法(FLAQ)对HIV-1 p24Gag快速定量

Marianne Gesner; Mekhala Maiti; Robert Grant; Marielle  Cavrois

doi:10.21769/BioProtoc.1366

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Peer-reviewed

Fluorescence-linked Antigen Quantification (FLAQ) Assay for Fast Quantification of HIV-1 p24^Gag

荧光标记抗原法(FLAQ)对HIV-1 p24Gag快速定量

MG Marianne Gesner

MM Mekhala Maiti

RG Robert Grant

Marielle Cavrois email

发布: 2014年12月20日第4卷第24期 DOI: 10.21769/BioProtoc.1366 浏览次数: 12259

评审: Yu ChenAnonymous reviewer(s)

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Cover of Journal of Virology, featuring study using the protocol.

Jul 2014

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Abstract

The fluorescence-linked antigen quantification (FLAQ) assay allows a fast quantification of HIV-1 p24^Gag antigen. Viral supernatant are lysed and incubated with polystyrene microspheres coated with polyclonal antibodies against HIV-1 p24^Gag and detector antibodies conjugated to fluorochromes (Figure 1). After washes, the fluorescence of microspheres is measured by flow cytometry and reflects the abundance of the antigen in the lysate. The speed, simplicity, and wide dynamic range of the FLAQ assay are optimum for many applications performed in HIV-1 research laboratories.

Keywords: Viral Production (病毒生产)

Materials and Reagents

Sphero Protein A Polystyrene Particles (6-8 µm) (Spherotech, catalog number: PAP-60-5 )
Human anti-p24^Gag HIV-1 IIIB polyclonal antibodies (ImmunoDX, catalog number 2503 )
Normal Human IgG (Sigma-Aldrich, catalog number: I2511 )
p24^Gag recombinant protein (Abcam, catalog number: ab43037 )
Anti-p24^Gag KC57 clone conjugated to fluorescein isothiocyanate (FITC) or to phycoerythrin also called RD1 (Beckman Coulter, catalog numbers: 6604665 and 6604667 )
1x phosphate-buffered saline (PBS) without calcium and magnesium (Corning, catalog number: 21-031-CV )
Bovine serum albumin (BSA) (Axenia Biologix, catalog number: S200 )
Triton X-100 (Thermo Fisher Scientific, catalog number: BP151-500 )
16% paraformaldehyde (PFA) solution (Electron Microscopy Sciences, catalog number: 15710 )
Fluorescence-activated cell sorting (FACS) staining buffer (see Recipes)

Equipment

Eppendorf centrifuge
Rotating mixer
96-well V-bottom plate (optional) (Thermo Fisher Scientific, catalog number: 12-565-216 )
Thermo Fisher lids for 96-well microplates (Thermo Fisher Scientific, catalog number: 14-245-53A )
Plate sealers (VWR International, catalog number: 62402-921 )
Eppendorf tubes
Multichannel pipetman (optional)
Pipetman
Tips (1 ml, 200 µl, and 10 µl)
Reagent reservoirs (optional)
Tissue culture centrifuge for Eppendorf tubes or for 96 well plates (optional)
37 °C incubator
Flow cytometer
Note: The FLAQ requires a flow cytometer equipped with a blue laser excitation (488 nm) and one measurement parameter. The photomultiplicator tube (PMT) with a 525/50 nm or a 585/42 band pass filter is used for the detection of FITC or RD1 (phycoerythrin) signals, respectively.

Software

Forecyt (Intellicyt)
FlowJoX software (Tree Star) or other FACS analysis software

Procedure

English

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如何引用

Gesner, M., Maiti, M., Grant, R. and Cavrois, M. (2014). Fluorescence-linked Antigen Quantification (FLAQ) Assay for Fast Quantification of HIV-1 p24^Gag. Bio-protocol 4(24): e1366. DOI: 10.21769/BioProtoc.1366.