Luminex™ Assays Support—Getting Started

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 microplate. The 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 a 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. The Luminex™ technology is compatible with the following Luminex™ analyzers:

• MAGPIX™ System—affordable, efficient, and compact
• Luminex™ 100/200™ System—versatile, efficient, and widely used in multiplexing
• FLEXMAP 3D™ System—high throughput (up to 500 simultaneous assays) and automation compatible

Luminex™ xMAP™ technology is based on polystyrene or paramagnetic microspheres, or beads, that are internally dyed with red and infrared fluorophores of differing intensities. Each dyed bead is given a unique number, known as a “bead region”, allowing the differentiation of beads. For Novex™ multiplex immunoassay kits, individual bead sets are then coated with a capture antibody qualified for one specific analyte. Multiple analyte-specific beads can then be combined in a single well of a 96-well assay to detect and quantify multiple targets simultaneously, using one of the Luminex™ instruments for analysis. We offer multiplex assays using either polystyrene or paramagnetic beads. 

Please watch this video to learn how to measure multiple proteins simultaneously using the Novex™ multiplex bead-based kits on the Luminex™ instrument platform.

ELISA is a simple and powerful way to quantify individual proteins specifically in complex samples. The selectivity of ELISA is achieved through the use of qualified single- or double-antibody sandwich technology, and accurate quantitation is achieved through the use of calibrated standards. ELISAs can detect low-level proteins and can be performed in a 96-well format with only 60 minutes of hands-on time. In addition, the results obtained with ELISAs are generally very reproducible.

While ELISA has been established as a standard method of protein analysis, multiplexing methods that enable the measurement of multiple analytes simultaneously in a single sample address a number of specific limitations:

• ELISA allows for the measurement of only one analyte at a time in a given sample, limiting investigators’ increasing need to measure multiple targets in their research studies.
• The low available volume of many samples being studied may limit the number of times analyses can be conducted. This is especially true in small animal research, in pediatric testing, and in microplate assays providing limited sample volumes. The ability to assay multiple analytes in a single small-volume sample enables more effective use of each sample.
• Difficulties in data interpretation can arise when comparing analyte levels measured by multiple ELISAs, each assay having been performed with different sample aliquots and each susceptible to systematic errors leading to decreased precision and accuracy.
• Many analytes require assays with broad dynamic ranges to avoid repeat testing or out-of-range values. Multiplex assays can be designed to have large dynamic ranges for all of the analytes, or ranges tailored to various expected analyte concentrations.

Novex™ multiplex assays, which are based on Luminex™ xMAP™ technology, provide a versatile platform that gives users more flexibility and a greater array of options for analyte detection. Whether you are testing for single or multiple analytes, Novex™ multiplex assays deliver accurate analytical performance using efficient, easy-to-follow protocols. Each of these assays has undergone the same development, validation, manufacturing, and quality control standardization we conduct for our ELISAs. Each lot of Novex™ multiplex assays as well as ELISA assays is fully qualified with the appropriate sample type (i.e., species-specific serum, plasma, and cell culture supernatants), and each lot is evaluated based on the following performance characteristics:

• Specificity—each analyte is screened to make sure there is no significant cross-reactivity with other analytes in the multiplex test
• Sensitivity—each analyte is evaluated for both functional sensitivity (differentiation from background) and lower limit of detection (LLOD)
• Precision/accuracy—multiplex assays have good intra-assay precision (<10% CV), inter-assay precision (<10% CV), and lot-to-lot consistency (<20% CV); these values are comparable to or better than most ELISA tests

Novex™ multiplex assays are regularly tested against the matching ELISAs. Analytical comparison of the two assay formats assures that multiplex assays can effectively be used to obtain comparable results. As such, studies that use ELISAs to quantify multiple analytes in samples can confidently be transitioned to the use of Novex™ multiplex assays and obtain improved analysis efficiency, economy, and performance. An example is shown below:

Murine GM-CSF in tissue culture supernatant fluid was tested by ELISA (y-axis) and in a Novex multiplex assay (x-axis). Correlation of values over 3 logs of sample dilution was 0.9868

Please see this White Paper for more data.

Here is an overview of Luminex™ instruments we offer for multiplex detection and analysis.

The MAGPIX™ System is a compact benchtop instrument with a multiplex capability of up to 50 analytes. It is a robust and cost-effective multiplexing tool. Streamlined startup and shutdown protocols, and minimal maintenance requirements, make the system easy to use—ideal for both new and experienced users. It features simple out-of-the-box setup and interactive software. The MAGPIX System analyzes magnetic beads immobilized with a magnet, excites the beads using light-emitting diodes (LEDs), and then detects and analyzes the beads using a CCD camera.

Please review the following table for instrument setup:

Note: Before running the assay, set the probe height appropriately, and calibrate and verify the system.

*Volume can be adjusted during acquisition to optimize bead count.

Serum samples should be collected in pyrogen/endotoxin-free tubes. Whole blood should be allowed to sit at room temperature for 15–30 min to clot. Spin at 1,000–2,000 x g for 10 min in a 4°C refrigerated centrifuge to separate the cells. Transfer the supernatant to a chilled clean polypropylene tube with a sterile Pasteur pipette. Maintain the samples at 2–8°C while handling. 

If the serum is to be analyzed at a later date, apportion it into 0.5 mL aliquots and store at –80°C. Avoid multiple freeze-thaw cycles. When possible, avoid the use of hemolyzed or lipemic sera. We recommend that upon thawing the samples be clarified by centrifugation (14,000 rpm for 10 min) and/or filtered, prior to analysis, to prevent clogging of the filter plates and/or probe. Follow the assay procedure provided with the kit for appropriate dilutions.

Separate the cells from the plasma samples by centrifugation at 2,000 x g for 10 min in a refrigerated centrifuge. Centrifugation at this force is necessary to deplete the sample of platelets. Transfer the supernatant to a chilled clean polypropylene tube with a sterile Pasteur pipette. Maintain the samples at 2–8°C while handling. 

If the plasma is to be analyzed at a later date, apportion it into aliquots in polypropylene microcentrifuge tubes and store at –80°C. Avoid multiple freeze-thaw cycles. When you are ready to analyze them, allow the samples to thaw on ice. All plasma samples should be clarified by centrifugation (14,000 rpm for 10 min at 4°C) in a refrigerated microcentrifuge immediately prior to analysis. Follow the assay procedure provided with the kit for appropriate dilutions.

Cells should be in log-phase growth. Stimulate cells as desired in appropriate cell culture flasks. Using sterile technique, remove the desired volume of conditioned cell culture medium with a pipette and transfer the medium to clean polypropylene microcentrifuge tubes. Centrifuge the medium at 14,000 rpm for 10 min at 4°C in a refrigerated microcentrifuge to remove any cells or cellular debris. Aliquot the clarified medium into clean polypropylene microcentrifuge tubes. These samples are ready for the assay. Alternatively, clarified medium samples can be aliquoted and stored at –80°C for future analysis. Avoid multiple freeze-thaw cycles. Frozen samples should be allowed to thaw on ice just prior to running the assay. Thawed samples should be clarified by centrifuging at 14,000 rpm for 10 min at 4°C in a refrigerated microcentrifuge prior to analysis to prevent clogging of the Luminex™ probe and/or filter plate. Follow the assay procedure provided with the kit for appropriate dilutions.

We recommend following the protocol provided below, which was developed using the Tissue Extraction Reagent I (Cat. No. FNN0071), and shows good correlation between ELISA and Luminex™ technology. This procedure has been applied to multiple tissue types. However, we recommend that you optimize for each tissue sample type used. Similar extraction reagents/lysis buffers may be used. 

1. Add protease inhibitors to the Tissue Extraction Reagent just before use.
2. Weigh the tissue sample.
3. Add 10 mL of the Tissue Extraction Reagent per 1 gram of tissue.
4. Homogenize the tissue.
5. Centrifuge the sample at 10,000 rpm for 5 min to pellet the tissue debris.
6. Collect the supernatant. Follow the assay procedure provided with the kit for appropriate dilutions; this is to prevent/minimize potential inhibition of antibody-antigen binding by the detergent present in the extraction or lysis buffer. In general, tissue homogenates or cell lysates (depending on the lysis buffer used) need to be diluted 5- to 10-fold to reduce the detergent concentration to ≤0.01%. However individual kits/samples may require further dilution with assay diluent or standard diluent based on the concentration of the cytokine/protein of interest.

Note: If the samples are to be stored, aliquot them and freeze at –80°C. Avoid multiple freeze-thaw cycles.

Use only freshly collected urine samples. Dilute 2-fold with the Assay Diluent provided in the kit. The final dilution of the sample will be 4-fold, and all results should be multiplied by 4. As needed, clarify samples by centrifugation (14,000 rpm for 10 min) and/or filter them prior to analysis to prevent clogging of the filter plates and/or probe.

Collect synovial fluid into non-heparinized tubes and spin at 1,000 x g for 10 min within 30 min of sample collection. The acellular portion of synovial fluid should be stored at –80°C before subsequent analysis. All samples need to be clarified by centrifugation (14,000 rpm for 10 min) and/or filtered prior to analysis to prevent clogging of the filter plates. Dilute samples 1:1 with Assay Diluent prior to addition to the assay. Reference: Raza K et al. (2005) Arthritis Research &Therapy 7(4): R784–R795.

All samples need to be clarified by centrifugation (14,000 rpm for 10 min) and/or filtered prior to analysis to prevent clogging of the filter plates. However, CSF has relatively low viscosity, and unless there is presence of an infected state (abundance of WBCs), it should not require clarification and can be directly applied to the assay. Dilute 2-fold with Assay Diluent provided in the Neuroscience Buffer Kit (Cat. No. LNB0001). 

The bronchoalveolar lavage (BAL) should be collected in a sterile syringe and kept on ice until you are ready to analyze it. Alternatively, BAL can be aliquoted and frozen in usable sample sizes (such that exposure to freeze-thaw is limited to one time). All samples need to be clarified by centrifugation (14,000 rpm for 10 min) and/or filtered prior to analysis to prevent clogging of the filter plates. Dilute 2-fold with Assay Diluent before applying to the plate.

Cervical sponges should be placed on ice immediately upon collection. Samples should be stored at –20°C for up to one week and then stored at –80°C until ready for assay. After thawing, sponges should be weighed and placed into Eppendorf tubes, using forceps cleaned with ethanol after each transfer. Add 200 μL of ice-cold extraction buffer (recipe below) to each tube and incubate overnight at 4°C. The sponges and extraction buffer can then be transferred to microcentrifuge tubes with 0.2 μm cellulose acetate filters and centrifuged at 13,000 rpm for 10 min at 4°C. The eluate can then be tested for cytokine expression.

Extraction Buffer

50 mM HEPES, pH 7.5 

1 mM Na₃VO₄

150 mM NaCl 

1 mM NaF

1 mM EDTA 

0.1% Tween™ 20

25 mM EGTA

10% glycerol

Isolate the site around the tooth and insert a piece of periodontal filter paper into the gum pocket around the tooth for 30 seconds. Remove the filter paper and extract 4 times with 50 μL PBS for 5 min each at room temperature. The individual extractions can be combined and analyzed. Dilute 2-fold with Assay Diluent before applying to the assay.

We have not specifically tested saliva samples in-house. Saliva contains several proteolytic enzymes. It would be important to centrifuge samples, and be sure not to pipet any cellular material or debris into the assay plate. We would suggest adding some anti-protease in the sample (for example, trasylol or aprotinine, 10 to 50 U/mL) to protect the protein from enzyme degradation. You may treat the sample as you would a supernatant; follow the procedure described for cell culture supernatant. 

The protocols mentioned below have been applied to several human and mouse cell lines. You should optimize the cell extraction procedures for your own applications. 

Cell Lysis Procedure

Non-adherent cells:

Pellet cells by low-speed centrifugation. Remove the medium from the pellet, and wash twice with ice-cold PBS. Remove the PBS, and resuspend the cell pellet in cell lysis buffer (recommended cell lysate concentration is 2–5 mg/mL). Incubate 15 min on ice with occasional vortexing. Transfer the lysate to a microcentrifuge tube and centrifuge at 14,000 rpm for 10 min at 2–8°C. Aliquot the cleared lysate into clean microcentrifuge tubes, and determine total protein concentration.

Adherent cells:

Remove the tissue culture medium from the cells, and wash twice with ice-cold PBS. Remove the PBS, add cell lysis buffer (recommended cell lysate concentration is 2–5 μg/mL), and incubate 15 min on ice. Collect the cell lysate and transfer it to a microcentrifuge tube. Centrifuge at 14,000 rpm for 10 min at 2–8°C. Aliquot the cleared lysate into clean microcentrifuge tubes, and determine total protein concentration. Lysates should be frozen and stored at –80°C or analyzed shortly after collection. Avoid multiple freeze-thaw cycles of frozen samples. Thaw completely, mix well, and clarify by centrifugation (14,000 rpm for 5 min) prior to analysis to prevent clogging of the filter plates.

Recommended Cell Lysis Buffers:

NP40 Cell Lysis Buffer (Cat. No. FNN0021) 

Note: Lysates prepared with NP40 Lysis Buffer must be diluted at least 5-fold prior to running the assay.

Or

50 mM Tris, pH 7.4

50 mM NaF

260 mM NaCl

1 mM Na₃VO₄

5 mM EDTA 

0.02% NaN₃

1% Nonidet P40 

The NP40 Cell Lysis Buffer (without protease inhibitor cocktail and PMSF) is stable for 2–3 weeks at 2–8°C, or for 6 months when stored in aliquots at –20°C. 

 Add fresh to the NP40 Cell Lysis Buffer just before use:

• 1 mM PMSF (stock 0.3 M in DMSO)
• Protease inhibitor cocktail (Sigma, Cat. No. P-2714)

Alternate Cell Extraction Buffer

Cell Extraction Buffer (Cat. No. FNN0011) 

Note: Lysates prepared with Cell Extraction Buffer must be diluted at least 10-fold prior to running the assay.

Or

10 mM Tris, pH 7.4

2 mM Na₃VO₄

100 mM NaCl 

1% Triton X-100

1 mM EDTA 

10% glycerol

1 mM EGTA 

0.1% SDS

1 mM NaF 

 0.5% deoxycholate

20 mM Na₄P₂O₇ 

The Cell Extraction Buffer (without protease inhibitor cocktail and PMSF) is stable for 2–3 weeks at 2–8°C, or for 6 months when stored in aliquots at –20°C. 

Add fresh to the Cell Extraction Buffer just before use:

• 1 mM PMSF (stock 0.3 M in DMSO)
• Protease inhibitor cocktail (Sigma, Cat. No. P-2714)

While most of our Multiplex Luminex™ Assay kits can be combined with other Singleplex Assay kits to permit higher-level multiplexing, there are a few exceptions and limitations. Please carefully read the product manual for kit-specific guidelines. Instructions for combining bead mixtures and Detector Antibody mixtures in the development of multiplexed assays are shown below.

Note: Before preparing multiplexed assays, it is important to verify that each analyte is represented by a unique bead region. This assures the compatibility of each bead in the development of multiplexed assays. Up to 10 bead concentrates (premixed panel multiplexes and/or singleplexes) can be combined to increase the number of proteins being monitored. The buffer systems used for each kit must also be compatible. In general, Luminex™ assay kits that use Buffer Kit Cat. No. LHB0001 should not be multiplexed with Luminex™ Assay kits that use Buffer Kit Cat. No. LHB0002. Also, protein standards may be analyzed alone or combined with other protein standards for higher levels of multiplexing. Do not combine more than 4 vials.

Multiplexing Antibody Beads:

The volume required for a multiplexed assay can be calculated using the formulas presented below:

Total Volume of 1X Antibody Bead Mixture Required

0.0275 mL x (number of wells) = ________________

Volume of Each 10X Antibody Bead Concentrate Required

[total volume of 1X Antibody Bead mixture required] ÷ 11 = ______

Working Wash Solution Required

[total volume of 1X Antibody Bead mixture] – [(volume of 10X Antibody Beads) x (number of plexes)] = ________

To prepare a 1X Antibody Bead mixture for a multiplexed assay, pipette the beads and Working Wash Solution (using the volumes calculated with the formulas presented above) into a foil-wrapped tube. Vortex the tube for 30 sec, then sonicate for 30 sec. The mixture is ready to be used in a multiplexed assay. If desired, this Antibody Bead mixture can be stored overnight at 2–8°C. 

Multiplexing Detector Antibodies:

The volume required for a multiplexed assay can be calculated using the formulas presented below:

Total Volume of 1X Detector Antibody Mixture Required

0.110 mL x (number of wells) = ________________

Volume of Each 10X Detector Antibody Concentrate Required

[total volume of 1X Detector Antibody mixture required] ÷ 11 = ______

Detector Antibody Diluent Solution Required
[total volume of 1X Detector Antibody mixture] – [(volume of 10X Detector Antibody) x (number of plexes)] =________

To prepare a 1X Detector Antibody mixture for a multiplexed assay, pipette the Detector Antibody concentrates and Detector Antibody Diluent (using the volumes calculated with the formulas presented above) into a tube. Mix gently. The mixture is ready to be used in a multiplexed assay. If desired, this Detector Antibody mixture can be stored overnight at 2–8°C. 

Multiplexing Standards:

Protein standards may be analyzed alone, or combined with other protein standards for higher levels of multiplexing. Do not combine more than 4 vials.

If the plate cannot be read on the day of the assay, cover and store the plate in the dark overnight at 2–8°C for reading the following day without significant loss of fluorescence intensity. When you are ready to read the plate, bring the plate to room temperature on an orbital plate shaker (still protected from light). Aspirate the Working Wash Solution from the stored plates and add 100 μL of fresh Working Wash Solution. Place the plate on an orbital shaker for 2–3 min at 500–600 rpm prior to analysis.