A luminex assay was used to determine relative IgG titers to the HIV-1 YU2 gp120 antigen, as described previously (Brown et al., 2012 (link)). Briefly, magnetic carboxylated fluorescent beads (Luminex Corporation) were coupled to gp120 in a two-step carbodiimide reaction. Serum samples diluted at 1:100 were then incubated with antigen-coated beads overnight at 4 °C. Following bead washing, gp120-specific IgG1 was then detected with PE-conjugated mouse anti-human IgG1 (Southern Biotech #9052-09), diluted to 1.3 μg/ml in assay buffer, to each well. Median Fluorescence Intensity corresponding to each sample was measured using a BioPlex array reader (BioPlex 3D, Bio-Rad). A minimum of 50 beads per well were collected for analysis.
Bio plex 3d
Manufactured by Bio-Rad
Sourced in United States
The Bio-Plex 3D is a multiplex immunoassay system that enables the simultaneous detection and quantification of multiple analytes in a single sample. It utilizes magnetic beads coated with specific capture antibodies to capture and measure target proteins or other biomolecules.
Automatically generated - may contain errors
Lab products found in correlation
Magnetic carboxylated fluorescent beads, by DiaSorin (1 mentions)
Optilyse solution, by Beckman Coulter (1 mentions)
Ab7356, by Merck Group (1 mentions)
Skyhigh, by MultiSciences Biotech (1 mentions)
Bio plex amine coupling kit, by Bio-Rad (1 mentions)
Bio plex pro magnetic cooh beads, by Bio-Rad (1 mentions)
4 protocols using bio plex 3d
1
Luminex Assay for HIV-1 gp120 IgG Titers
2
Multiplex Immunoassay Analysis of Plasma
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The plasma sample was centrifuged (1000g, 15 min, 4° C) to remove particulates and mixed with wash buffer in a ratio of 1:4. The standards were added 250 μl of standard diluent HB, vortexed 5s and incubated 30 mins. Prepared a fourfold standard dilution series and blank sample. Each well was added 50 μl 1x beads and washed 2 times with 100 μl Bio-Plex Wash Buffer. Then, added 50 μl standards, a plasma sample, blank to well and incubated on shaker at 850 rpm for 30 mins. After washing 3 times, add 25 μl 1x detection antibody and incubate 30 mins. After washing 3 times, added 50 μl 1x streptavidin-PE and incubated for 10 mins. After washing 3 times, resuspended beads in 125 μl assay buffer and generated data with Bio-Plex 3D (Bio-Rad, USA). The data were analyzed using MILLIPLEX Analyst software (V5.1). The fluorescence value of the standard sample was used to obtain the fitting curve, Coefficient of variation (CV), Accuracy, and Sensitivity with the 5-parameter logistic method. Calculated the concentration of immunologic factors by substituting the sample’s fluorescence value into the fitting curve.
3
Comprehensive Immune Profile Assessment
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Venous blood was taken from all participants from the cubital vein, in the morning and strictly on an empty stomach, using a vacuum EDTA tube. Storage and transportation were carried out strictly at 18–20 °C in a vertical position for no more than 12 h. An automatic hematology analyzer, Mindray BC-3200 (Shenzhen Mindray Bio-Medical Electronics Co., Ltd., Shenzhen, China), was used to determine the parameters of the general blood test (hemoglobin, leukocytes, erythrocytes, platelets).
Indicants of immune status, T-cells (CD3+, CD4+, CD8+, immunoregulatory index CD4+8+), B-cells (CD19+), natural killer (NK) cells (CD3+56/16+), TNK cells (CD3-56/16+), and neutrophil phagocytic activity (hereinafter, NPA) were defined by the method of flow cytometry with a cytometer, from Cyflow Space (Partec GmbH, Munster, Germany). For levels of IgA, IgM, and IgG, a multiplex immunological assay with XMap technology on a Bioplex 3D (Bio-Rad Laboratories, Inc., Hercules, CA, USA) was used. Monoclonal antibodies from Beckman Coulter (Beckman Coulter Inc., Brea, CA, USA) were used for combined dying, according to the instructions of the manufacturer, with a no-wash procedure of lysis fixation using Optilyse solution (Beckman Coulter Inc, CA, USA).
Indicants of immune status, T-cells (CD3+, CD4+, CD8+, immunoregulatory index CD4+8+), B-cells (CD19+), natural killer (NK) cells (CD3+56/16+), TNK cells (CD3-56/16+), and neutrophil phagocytic activity (hereinafter, NPA) were defined by the method of flow cytometry with a cytometer, from Cyflow Space (Partec GmbH, Munster, Germany). For levels of IgA, IgM, and IgG, a multiplex immunological assay with XMap technology on a Bioplex 3D (Bio-Rad Laboratories, Inc., Hercules, CA, USA) was used. Monoclonal antibodies from Beckman Coulter (Beckman Coulter Inc., Brea, CA, USA) were used for combined dying, according to the instructions of the manufacturer, with a no-wash procedure of lysis fixation using Optilyse solution (Beckman Coulter Inc, CA, USA).
4
Multiplex ELISA and Immunoprecipitation Assay
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Sandwich-based ELISA kits were used to detect PON1 (RayBiotech), FUT8 (LSBio), and ATF6 (Novus Biologicals) following the manufacturer’s protocol. For ELISA detection of SLC35C1, 96-well microtiter plates were manually pre-coated with SLC35C1 antibody (CSB-PA839285LA01HU, Cusabio) similar to HLE. Absorbance was measured at 450 nm in a microplate reader (Multiskan SkyHigh). For bead-based ELISA, established conjugatable ELISA antibodies for PON3 (ab242568, Abcam), VWF (AB7356, Sigma), and MPO (EPX01A-12038–901, Invitrogen) and Quantikine calibrated standards (R&D Systems) were used. Cross-linking-mediated coimmunoprecipitation was performed as described above, incubated with identifiable capture beads targeting non-PON1 proteins, washed with wash buffer and then incubated with a biotinylated antibody for PON1 for 5 hr at 4 °C. After multiple washes, the beads were incubated with streptavidin-conjugated phycoerythrin for 30 min. The capture antibody was conjugated to unconjugated Bio-Plex Pro Magnetic COOH Beads (Bio-Rad) using Bio-Plex Amine Coupling Kit (Bio-Rad), except for MPO (ProcartaPlex system, Invitrogen) and used in a multiplexed fashion. Linearity of the assay was validated during measurement by dilution series of both lysates and standards. Flow cytometry-based multiplex quantification was performed using Bio-Plex 3D (Bio-Rad).
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