Luminex® 分析

Life Technologies推出Invitrogen™ Luminex®分析,用于细胞因子和信号转导蛋白的定量分析和检测。 这些分析可同时检测每个样本中的多个靶点,且可获得批次间一致的出色性能和可重复的结果。 进行多重分析时,越多…越好!


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About Novex® Multiplex Luminex® Assays

Luminex® Assays
We produce Luminex® assays in formats to suit the individual needs of virtually any lab. Flexibility is one of the many advantages of the Luminex® xMAP® multiplex platform; we’ve taken advantage of this to offer a large selection of premixed Luminex® assay panels for use in cytokine screening, pathway analysis, and simple profiling. In addition, we offer single-target Luminex® bead kits that you can combine to make your own assay panels or can be added to pre-mixed Luminex® assays to create larger panels. Finally, we can build custom Luminex® assay panels for your targets of interest.

Luminex® assays are easy to set up and run, using a procedure that is very similar to an ELISA (Figure 1). Life Technologies has a team of dedicated Field Application Scientists (FASs) ready to help if you are new to Luminex® xMAP® technology or using Luminex® assays for the first time. Our FASs come to your lab to guide you through running a Luminex® assay, and setting up the Luminex® instrument and software. They can also help troubleshoot virtually any problem that may arise. Request your personal Luminex® consultation today.

Tables 1, 2, and 3, demonstrate the superiority of Novex® multiplex Luminex® assays and protein standards (included in the assays). Unlike some other assays, we use the maximum number of beads in each Luminex® assay, so that data are collected from at least 100 events per bead region per target. Counting at least 100 events, compared to the 50 recommended by some manufacturers, leads to lower %CVs and more precise assays. Luminex® assays are subject to rigorous quality control and validation prior to release; we extensively test cross-reactivity to help ensure the specificity of each assay for its intended target. In addition, the protein standards we supply are precisely calibrated to reference materials for maximum consistency.

Luminex protocol
 
图1. Fast and easy Novex® multiplex Luminex® assay protocol.
Beads of defined spectral properties are conjugated to protein-specific capture antibodies and added along with samples (including standards of known protein concentration, control samples, and test samples), into the wells of a filter-bottom microplate. Target protein binds to the capture antibodies over the course of a 2-hr incubation. After washing the beads, protein-specific, biotinylated detector antibodies are added and incubated with the beads for 1 hr. Next, excess biotinylated detector antibodies are removed, and streptavidin-conjugated fluorescent protein, R-Phycoerythrin (SAV-RPE), is added and incubated for 30 min. SAV-RPE binds to the biotinylated detector antibodies, forming a four-member, solid-phase sandwich. After washing to remove unbound SAV-RPE, the beads are analyzed with a Luminex® detection system. By monitoring the spectral properties of the beads and the amount of associated R-Phycoerythrin (RPE) fluorescence, the concentration of one or more proteins can be determined.

仅供研究使用。 不得用于人或动物的治疗或诊断用途。

表1. 100 Counts Strong for More Reliable Data

Novex® multiplex Luminex® assays are configured to enable data to be collected from 100 beads per target. Luminex® assay protocols from some providers may require collecting data from only 50 events per region per target (in other words they may require that only 50 beads for each target is read by the detection system). These data indicate that %CVs and assay precision are improved by collecting data from the full 100 bead events compared to collecting data from only 50 bead events.

Sample Description
%CV (MFI)
50 Bead events
(0.5X Beads/ well)
100 Bead events
(1X Beads/ well)
Average (Serum) 8.7 12.0
Average (Plasma) 14.8  7.3
Average (TCS) 8.5 5.2
Average (All) 10.5 6.9

2表 . Better-Calibrated Standards

Novex® mulitplex Luminex® assays are calibrated to NIBSC standards (a set of international reference standards) when available, or to published intracellular cell models for phosphoprotein activity to help ensure that measurements are relevant. Protein standards are tested before and after lyophilization, in single as well as multiplexed assays, for consistency in standard integrity and performance.

Novex® Multiplex Luminex® Assay Standards
  Expected
(pg/ml)
Calc.
(pg/ml)
%
Differ
GM-CSF 1,964 1,988 1
IFN-γ 1,078 948 -12
IL-2 1,432 1,489 4
IL-4 982 1,089 11
IL-6 1,040 1,102 6
IL-8 1,357 1,266 -7
IL-10 954 910 -5
TNF-α 2,375 2,278 -4
 
 
Competitor Standards
  Expected
(pg/ml)
Calc.
(pg/ml)
%
Differ
GM-CSF 500 271 -46
IFN-γ 500 445 -11
IL-2 500 484 -3
IL-4 500 237 -53
IL-6 500 301 -40
IL-8 500 306 -39
IL-10 500 768 54
TNF-α 500 3,620 624
 

3表 . Better Assay Specificity

We measured assay response from dozens of like-target proteins to help ensure that the antibodies selected for use in Novex® multiplex Luminex® assays are highly specific for their desired targets. The antibodies used in the competitor’s assays are not as rigorously tested and may show nonspecific assay results. In this table we compare results (fluorescence units shown) for the indicated similar target proteins using either Novex® multiplex Luminex® assays or assays from another manufacturer. None of the Luminex® assays showed significant signal from similar, nontarget proteins, whereas a few of the assays from the other supplier showed significant cross-reactivity with nontarget proteins.

Novex® Multiplex Luminex® Assay
Fluorescence Units
  IL-6 IL-10 TNF-
α
IFN-
γ
IL-6 6,827 31 22 12
IL-10 12 8,151 23 12
TNF-
α
7 30 6,642 10
IFN-
γ
8 35 25 3,540
Zero 11 10 13 7
 
 
Competitor Assay
Fluorescence Units
  IL-6 IL-10 TNF-
α
IFN-
γ
IL-6 8,878 15 32 40
IL-10 5 7,090 8 12
TNF-
α
132 15 9,999 330
IFN-
γ
90 20 206 14,248
Zero 3 16 5 9