Multiplex Bead-based Luminex® Assays

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Multiplex Bead-Based Luminex® Assays—What you need to know

How Multiplex Luminex® Assays Work
Multiplex Luminex® Assay Performance Specifications
Intracellular Protein Assay Kit Contents and Protocol
Soluble Protein (extracellular) Assay Kit Contents and Protocol

How Multiplex Luminex® Assays Work

The multiplex Luminex® assay format differs from conventional ELISA in one significant way— the multiplex capture antibody is attached to a polystyrene bead whereas the ELISA capture antibody is attached to the microplate well.

The use of the suspension bead-based technology enables the multiplexing capabilities of the Luminex® assays. The xMAP® technology uses 5.6 micron polystyrene microspheres, which are internally dyed with red and infrared fluorophores of differing intensities. Each bead is given a unique number, or bead region, allowing differentiation of one bead from another.

Beads covalently bound to different antibodies can be mixed in the same assay, utilizing a 96-well microplate format.

At the completion of the sandwich immunoassay, beads can be read, using the Luminex® 100™ or 200™ detection system, in single-file by dual lasers for classification and quantification of each analyte.

Multiplex Luminex® Assay Performance Specifications

Performance Specifications
Examples of Characterization

Invitrogen takes pride in setting the industry standards when it comes to high-quality assays. With more than 20 years of assay grade antibody and bioactive recombinant protein development experience, Invitrogen has full control over the superior quality each kit. Every individual Antibody Bead Kit and premixed Multiplex Kit comes with a technical performance sheet that outlines the specifications for each marker in the assay (Table 1). These specifications include:

Range of Assay	Broad dynamic range
Recovery	80–120% Serum 80–120% Plasma 80–120% Tissue culture supernatants (TCS) 80–120% Cell lysates
Sensitivity	Physiologically relevant levels, <10 pg/ml (based on detectable signal >2 SD above background)
Specificity	Cross-reactivity tests are performed with other analytes and antibodies (Table 2)
Precision	Inter-assay CV: <10% Intra-assay CV: <10%
Benchmarking to ELISA	Correlates to ELISA data (>90% correlation) (Figure 5)
Linearity of dilution	(R²>0.99) High correlation of coefficient between dilutions of samples vs. expected concentration over the range of the assay
Parallelism to natural samples	(R²>0.99) Recombinant standards are compared to natural samples to ensure equivalency (Figure 6)
Stability	18 months from post QC
Storage	+2-8°C

Calibration

Invitrogen Multiplex Bead-Based Luminex® kits are calibrated to NIBSC standards, an international reference standard, when available, or to published intracellular cell models for phosphoprotein activity ensuring a relevant basis for analysis. Standards are tested before and after lyophilization in single, as well as in multiplex assays to ensure consistency in protein integrity and performance.

Cytokine (Human)	Sensitivity pg/mL	Recovery Serum	Recovery TCS	Recovery Plasma	NIBSC Conv.	Precision
GM-CSF	<15	81%	100%	90%	1 ng = 9 IU	9.1%
IFN-γ	<5	105%	100%	107%	1 ng = 77 IU	8.7%
IL-1β	<15	105%	100%	84%	1 ng = 74 IU	8.8%
IL-2	<6	111%	100%	111%	1 ng = 14 IU	9.5%
IL-4	<5	94%	100%	86%	1 ng = 10 IU	8.6%
IL-5	<3	83%	100%	96%	1 ng = 10 IU	7.4%
IL-6	<3	118%	100%	91%	1 ng = 505 IU	6.8%
IL-8	<3	118%	100%	96%	1 ng = 1.1 IU	8.5%
IL-10	<5	103%	100%	85%	1 ng = 5.3 IU	9.6%
TNF-α	<10	109%	100%	89%	1 ng = 16.4 IU	8.2%

Table 2. Performance characteristic data for Human Cytokine 10-plex 1kit, Cat. no. LHC0001.

Specificity

	IL-1β	IL-2	IL-4	IL-5	IL-6	IL-8	IL-10	TNF-α	IFN-γ
IL-1β	6332	41	37	13	9	37	32	27	18
IL-2	38	11324	47	17	26	40	33	27	15
IL-4	38	41	10503	15	25	40	32	25	12
IL-5	48	54	36	8265	11	34	33	25	16
IL-6	44	72	40	11	6827	42	31	22	12
IL-8	39	39	44	12	27	10002	32	27	19
IL-10	41	51	39	11	12	41	8151	23	12
TNF-α	32	55	36	10	7	60	30	6642	10
IFN-γ	32	41	39	10	8	70	35	25	3540

Table 3. Specificity data for human Cytokine 10-plex panel, showing no measurable cross-reactivity. Numbers represent Fluorescent Units generated when individual recombinant proteins were analyzed independently in a series of 10-plex assays.


Figures 5 & 6. Benchmarking data for Mouse GM-CSF and IL-2, showing strong correlation between the respective Luminex® and ELISA assays. Recombinant protein samples are rigorously (>98% correlation) compared to natural samples. This parallelism ensures that data obtained from the standard curve will accurately reflect natural sample values.

Kit Configurations for Maximum Flexibility

For maximum flexibility in assay design, Invitrogen Luminex® products are sold as either individual analyte-specific Antibody Bead Kits that are multiplex-ready, or as a premixed panel. Invitrogen now offers over 150 kits, encompassing markers from a broad range of species (human, mouse, rat, and monkey), and is expanding its product line activity.

Intracellular Protein Assay Kit Contents and Protocol
Soluble Protein (extracellular) Assay Kit Contents and Protocol

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Intracellular Protein Assays

Invitrogen has developed intracellular protein multiplex bead-based Luminex® assays that quantitate proteins using highly specific antibodies. Additionally, some of the kits available quantitate protein phosphorylation using highly specific phosphorylation site–specific antibodies developed by Invitrogen. Samples are controlled for total protein by multiplexed quantitative assays for each protein, regardless of phosphorylation state.

Sensitivity and specificity of these multiplexed assays are comparable to the results obtained from ELISA and phosphoELISA™ assays and are more sensitive than western blotting. The intracellular protein multiplex bead-based Luminex® assays:

provide accurate intracellular protein kinase quantitation
measure protein phosphorylation relative to total protein,
require very small sample amounts (10 μl of cell extract),
can be run in less than 4 hours
are benchmarked to phosphoELISA™ assay.

A basic outline of the protocol for the intracellular assays is shown below.

View complete intracellular product list

Intracellular Protein Assay Protocol

General Buffer Reagent Kit includes:

Assay Diluent
Wash Solution
Sample Treatment Buffer
Detection Antibody Diluent
10X Anti-Rabbit-RPE Concentrate
10X Anti-Rabbit-RPE Diluent
96-Well Filter Plate
A detailed Protocol

The Individual Antibody Bead Kit includes:

10X Bead Conjugated Capture Antibody Concentrate
10X Detection Antibody Concentrate
Two vials Multiplexed Standards
An Information Sheet

Premixed Catalog Multiplex Kit includes:

General Buffer Reagent kit components
Antibody Bead kit components, with reagents premixed for the number of markers in the premixed panel.

Premixed Custom Multiplex Kit includes:

Premixed 10X Bead Conjugate
Premixed 10X Detection Antibody Concentrate
One vial of each standard required to achieve multiplex
A Certificate of Analysis

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Soluble Protein Assays

Invitrogen offers soluble protein pathway marker assays for accurate quantitation of cytokines, chemokines, and growth factors. Available as singleplex or multiplex kits, the assays:

can detect picogram levels of protein
require a small sample size (50 μl of serum, plasma, cell culture supernatant, or tissue extract)
can be run in 3.5 hours

A basic outline of the protocol for the extracellular assays is shown below.