Automated Magnetic Bead Washing of Metabolic Hormone Assays

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Automated Magnetic Bead Washing of Metabolic Hormone Assays
Authored by: Paul Held Ph.D., Principal Scientist, Applications Department, BioTek Instruments, Inc. Winooski, VT

1 Introduction
2 Basis for the Assay
3 Materials and Methods
4 Washing Instructions
5 Results
6 Discussion
7 Result
8 Discussion
9 References
10 Acknowledgements
11 Related articles

Introduction

Metabolic syndrome is a group of risk factors for coronary heart disease, stroke, peripheral vascular disease and type 2 diabetes. Adults with metabolic syndrome exhibit a degree of low-grade inflammation that is likely caused by disregulated production of pro- and anti-inflammatory factors. As a result, research in this area encompasses a myriad of different analytes including cytokines, acute phase proteins, biomarkers for diabetes, obesity related hormones and cardiovascular disease. Many multiplex assays for these analytes use ferrite beads as the solid substrate, which require a bead-retaining magnetic field during the wash steps. Here we describe the use of a magnetic bead washer to automate the wash steps used with a metabolic hormone assay panel.

Hormones produced by cells and organs of the endocrine system, including adipocytes, the pancreas, and the gastrointestinal tract, play a critical role in regulating the body’s metabolic state. Metabolic syndrome represents a group of risk factors in an individual that include: central obesity, atherogenic dyslipidemia, insulin resistance, prothrombotic state, raised blood pressure and a pro-inflammatory state. People with metabolic syndrome are at increased risk of coronary heart disease, stroke, peripheral vascular disease and type 2 diabetes. The syndrome is closely associated with obesity, and as many as 47 million US adults are believed to have metabolic syndrome ^[1].

Metabolic hormones regulate many metabolic syndrome risk factors. As a result of this epidemic’s severity, a large amount of research is being performed on the disorder’s regulatory hormones. There are a number of different hormones that act in concert, so it is advantageous to measure a number of different analytes simultaneously from the same sample (multiplex). The MILLIPLEX™ MAP Metabolic Hormone Assay Panel (Millipore Corporation, Billerica, MA) uses xMAP® bead technology (Luminex Corporation, Austin, TX) to perform a 13-plex assay on samples, meaning that thirteen assays are simultaneously run using the same sample.

Despite using a multiplex reaction, many researchers still need to assay large numbers of samples. Microsphere-based assays were originally designed for use in conjunction with manual or semi-automated vacuum wash systems ^[2]. While these systems worked well for low throughput demands, they did not lend themselves well to high-throughput analysis. To remedy this situation and provide a platform that is readily automatable, new microsphere technologies, partially coated with super-paramagnetic iron oxide, were developed.

Here we describe the important wash parameters of the assay and demonstrate the ability of a magnetic bead washer to automate the wash steps in a biomagnetic multiplex assay.

Basis for the Assay

MILLIPLEX MAP assay beads are comprised of polystyrene microspheres, about 5.6 microns in diameter that have been impregnated with ferrite particles and a mixture of two colored dyes. The relative volumes of the dyes are carefully adjusted to provide 100 distinct colors to differentiate beads and provide a means to simultaneously measure up to 100 analytes in a microplate well. Capture of analyte molecules is provided by conjugating analyte-specific primary antibodies to the surface of the microsphere and using fluorescently-labeled reporter tags that bind to the captured molecule. In the case of metabolic hormone assays, the capture molecule would be a primary antibody against the hormone analyte and the reporter tag would be a secondary antibody to a different epitope on the protein labeled with biotin. Fluorescent readout would be provided using streptavidin labeled with phycoerythrin, which binds directly to the biotin moiety of the secondary antibody. The amount of bound phycoerythrin is proportional to the amount of analyte (Figure 1).

Figure 1. Overview of xMAP assay including (A) conjugation of capture probes; (B) binding of analyte to be quantified; (C) binding of reporter tag to produce analytical signal.

A magnetic bead washer such as BioTek’s ELx405™(Figure 2) offers full plate washing of magnetic microspheres. Two different magnets, each with high-energy neodymium iron boron magnets to rapidly separate and retain micrometer and nanometer beads, can accommodate 96- and 384-well microplate formats. In some cases, the magnet may be removed for easy cleaning or when working with non-magnetic assays.

Figure 2. BioTek’s ELx405 Magnetic Bead Washer.

A two-laser cytometer is used for detection where the microspheres are channeled individually through the detection volume defined by one green and one red laser. The green laser excites the Phycoerythrin reporter tag and generates the analytical signal used to quantify analytes. The red laser excites the dye mixture inside the bead and identifies the analyte being measured by the bead type (Figure 3).

Figure 3. Principle of signal generation. Microspheres enter the flow cytometer (A) where a green laser (B) generates signal for analyte quantification and a red laser (C) identifies analyte via bead type.

Materials and Methods

A BioTek Instruments ELx405 Magnetic Bead Washer was used to automate the wash steps in a Millipore MILLIPLEX MAP Human Metabolic Hormone Magnetic Bead Panel, catalog number HMHMAG-34K. A series of calibration curves were generated and assayed according the assay kit instructions. Briefly, seven working multiplex standards were generated by a 1:3 serial dilution of the reconstituted Human Metabolic Standard. These standards contained 13 different analytes.

After reconstitution, 25 μL each of assay buffer, standards, matrix solution, and beads were dispensed into the assay microplate wells. The reaction was allowed to incubate for 2 hours at room temperature (RT) with agitation on a plate shaker. After incubation, the plate was washed using the ELx405 Magnetic Bead Washer as described in the washing instructions below.

After washing, 50 μL of detection or secondary antibody reagent was added and allowed to incubate for 60 minutes at RT with agitation, followed by the addition of 50 μL of SAPE reagent. After a 30-minute incubation with agitation to allow for reporter tag binding, the plate was again washed as described in the washing instructions, except that 100 μL of sheath fluid residual was left. Samples were then read on a Luminex 200 reader with XPONENT software (Figure 4).

Figure 4. MILLIPLEX MAP Assay Protocol using ELx405 Magnetic Bead Washer Protocol (see Table 1). Following an initial soak time of 60 seconds to capture the beads, the wash protocols consist of series of dispense, soak and aspiration steps under magnetization.

Washing Instructions

Automated plate washing was performed using the ELx405 Magnetic Bead Washer. The programming “link” function of the washer was used to link a soak routine (SOAK60) to a wash procedure (MAGX3) depending on the plate matrix. The soak routine allows for a 60 second bead capture by the magnet, while the wash procedure automates dispense and aspiration. The wash procedure has also utilized soak routines between cycles to allow the recapture of any beads resuspended during the fluid dispense. The specific parameters for each procedure are listed in Table 1. These parameters have been optimized for both bead retention and washing efficiency.

**Table 1. ELx405 Magnetic Bead Washer Protocol for MILLIPLEX MAP.**
Link File	Millipore
Program Name	SOAK60	MAGX3
File Type	Soak	Wash

Method
Wash Buffer		A
Plate Type		96
Number of Cycles		3
Soak/Shake		Yes
Soak Duration	60 sec	60 sec
Shake Before Soak		No
Prime		No
Prime Volume
Prime Flow Rate

Dispense
Dispense Volume		200
Dispense Flow Rate		5
Dispense Height		130
Horizontal Dispense Position		0
Horizontal Y Position		0
Bottom Wash First		No
Bottom Dispense Volume
Bottom Flow Rate
Bottom Dispense Height
Bottom Dispense Position
Prime		No
Prime Volume
Prime Flow Rate

Aspiration
Aspiration Height		35
Horizontal Aspiration Position		0
Aspiration Rate		6
Aspiration Delay		0
Crosswise Aspirate		No
Crosswise Aspirate On Final
Crosswise Height
Crosswise Horizontal Position
Final Aspiration		Yes
Final Aspiration Delay		0 msec

Results

Using known concentrations of analyte, a series of standard curves can be generated for each metabolic hormone biomarker by plotting the fluorescent signal against concentration. These standard curves can then be interpolated to determine the concentrations of unknown samples. As with ELISA reactions, in order to obtain useable results, efficient washing to remove nonspecific antibody binding is critical. Additionally, retention of the bead is important in bead-based assays, unlike ELISA reactions where the solid surface is the microplate itself.

As shown in Figure 5, washing magnetic bead-based multiplex assays in 96-well microplate format with an automated washer such as the ELx405 Magnetic Bead Washer results in very reliable data. These standard curves can be used to calculate unknown sample concentrations with a high degree of confidence.

Figure 5. Standard curves for the MILLIPLEX MAP 13-plex Human Metabolic Hormone Panel.

There are several different means to wash multiplex magnetic bead based assays, including bottom filtration, which provide accurate calibration curves. Equally important is the degree of precision obtained with these different methods. As demonstrated in Figure 6, comparisons of magnetic bead wash precision were made using the ELx405 Magnetic Bead Washer, a commercially available magnetic strip washer and standard filter washing via vacuum filtration. While the standard curves were all essentially the same (data not shown), when the precision of the replicate data for each of the three cases was analyzed in a histogram, it was noted that the ELx405 produced significantly higher precision and was not prone to high variance data indicative to loss of beads by filter plate clogging.

Figure 6. Histogram depicting the frequency of data points in the standard curve with CVs depicted on the x-axis.

Discussion

The ability to multiplex several related, but distinctly different analytes in the same assay well saves considerable amounts of time, reagents, as well as precious sample. However, with increasing numbers of analytes in a multiplexed assay, the potential for cross-reactivity and non-specific interference increases. A vigorous wash routine that effectively retains the assay beads is an important factor in preventing artifactual signal. In this example the metabolic hormones: amylin; ghrelin; GIP; GLP-1; glucagon; IL-6; insulin; leptin; MCP-1; pancreatic polypeptide; PYY; and TNF-α were assayed simultaneously with very good results in part due to the efficient washing provided by the automated washer.

Result

As shown in Figure 5, washing magnetic bead-based multiplex assays in 96-well microplate format with an automated washer such as the ELx405 Magnetic Bead Washer results in reliable data. These standard curves can be used to calculate unknown sample concentrations with a high degree of confidence.

There are several different means to wash multiplex magnetic bead based assays, including bottom filtration, which provide accurate calibration curves. Equally important is the degree of precision obtained with these different methods. As demonstrated in Figure 6, comparisons of magnetic bead wash precision were made using the ELx405 Magnetic Bead Washer, a commercially available magnetic strip washer and standard filter washing via vacuum filtration. While the standard curves were all essentially the same (data not shown), when the precision of the replicate data for each of the three cases was analyzed in a histogram, it was noted that the ELx405 produced higher precision and was not prone to high variance data indicative of loss of beads by filter plate clogging.

Discussion

The ability to multiplex several related, but distinctly different analytes in the same assay well saves time, reagents, as well as sample. However, with increasing numbers of analytes in a multiplexed assay, the potential for cross-reactivity and non-specific interference increases. A vigorous wash routine that effectively retains the assay beads is an important factor in preventing artifactual signal. In this example the metabolic hormones: amylin; ghrelin; GIP; GLP-1; glucagon; IL-6; insulin; leptin; MCP-1; pancreatic polypeptide; PYY; and TNF-α were assayed simultaneously with good results in part due to the efficient washing provided by the automated washer.

References

↑ Ford ES, Giles WH, Dietz WH (2002). Prevalence of metabolic syndrome among US adults: findings from the third National Health and Nutrition Examination Survey. JAMA 287(3):356-359.
↑ Held, P. Using the ELx50 Filter Microplate Washer to Perform the Wash Processing Steps Required for Luminex xMAP Assays. BioTek Application note, www.biotek.com.