Multiplex Immunoassay Protocol

Introduction of Multiplex Immunoassay Protocol

Multiplex immunoassays allow simultaneous detection of various analytes within the same test sample. However, current platforms capable of multiplexing are often complex and expensive. Here, we describe a low-cost planar array on a chip capable of simultaneously detecting up to 80 different analytes using the MagArray MR-110 chip reader. Each chip is composed of 80 giant magnetoresistive sensors on which sandwich ELISA immunoassays are built with magnetic nanotags (MNT), an alternative to fluorescent or enzymatic labels. This technology utilizes a magnetic nanoparticle technology derived from the computer disk drive industry for use in diagnostics and makes the MagArray platform an easy-to-operate (i.e., easy as plugging a USB drive into a computer), small (i.e., 22 × 13.5 × 11 cm), portable system (i.e., less than 1 kg) with no moving parts and/or microfluidics. When compared to conventional ELISA, the MagArray system is capable of detecting analyte(s) (i.e., in this case carcinoembryonic antigen [CEA]) at a much lower concentration and across a wider dynamic range. In addition to superior performance in sensitivity, dynamic range, multiplexibility, and size, the MagArray system also possesses the following unique characteristics:

• Real-time read-out: All 80 sensors coated with the capture antibodies of interest are scanned every 4 s. The results are displayed as they are generated, thus allowing researchers to monitor binding with the MNT labels as it takes place. This special feature is extremely useful for “go/no go“ decisions in a fast paced environment.

Figure 1. Comparison of conventional ELISA to MagArray sensitivities for the biomarker CEA.

• Complex sample: Even optical interference caused by red blood cells is not a factor for the MagArray technology. While sensitivity is slightly less than serum, whole blood can be used as a suitable sample type as the chips are often insensitive to matrix effect. This special feature should not be taken for granted and needs to be checked for every new analyte.
• Sequential addition of antibodies: It has long been recognized that cross-reactivity among the reagents in a multiplex assay is often a bottleneck in assay development. The open well system allows for detection antibodies to be sequentially added at any stage during the process. Researchers can identify and isolate cross-reactivity issues in multiplex assays, as well as design assay procedures to circumvent certain crossreactivity problems.
• No wash: The chip sensors only detect magnetic particles that have bound to the analytes of interest, but do not detect the bulk of unbound particles which float freely in the assay liquid. The net signal (i.e., bound particle fraction) is therefore accurately measured and equilibrates after several minutes. Thus, there is no need to wash before or after the addition of the MNT. This is of particular advantage when analyzing low affinity proteins that tend to get washed away during typical assay procedures.
• Small sample volume: Assays can be run using as little as a 20 μL sample after dilution, thus allowing researchers to run multiplex assays on minute samples from small animals such as mice or rats or on limited human samples that cannot be readily re-obtained. Because of the advantages and characteristics listed above, the MagArray technology is an ideal platform of choice for detecting multiple analytes simultaneously.

Methods of Multiplex Immunoassay Protocol

Assay

Each chip has 8 built-in reference sensors, passivated and chemically isolated from the reaction well, that are used for establishing the level of electrical signal which is independent of assay chemistry during an assay (i.e., baseline or reference signal). The steps of the standard assay are described below, while the shortened steps for the express assay are described in the Notes.

1. Allow cartridges and samples to sit at room temperature for 30 min.
2. Dilute sample to desired concentrations in Reaction Buffer.
3. Add 100 μL of sample to the chip well.
4. Cover chip well with Scotch tape.
5. Place chip cartridge into foam holder on Rocking Shaker and shake for 2 h at 350 +/− 20 rpm.
6. Prepare detection antibody to the desired concentrations in Reaction Buffer and allow it to warm to room temperature for 30 min.
7. Remove chip cartridge from Rocking Shaker and remove Scotch tape from chip well.
8. Wash the chips by aspirating out sample with central vacuum system by using a Transfer Pipette to fill the chip well with Rinsing Buffer and repeat washing step 2 more times for a total of three washes.
9. Add 100 μL of Detection Antibody Solution to the chip well.
10. Incubate for 1 h at room temperature.

Measurements

1. Open “MagArray Chip Reader“ program on computer.
2. Enter assay run information: Operator Initials, Sample Number, and Run Number.
3. Input or load spotting pattern provided by MagArray.
4. Insert Plastic Chip Cartridge into the MagArray MR-110a reader.
5. Click on “Press to Start Measurement“.
6. Wait until calibration is complete.
7. Wash the chip(s) by aspirating out detection antibody with central vacuum system by using a Transfer Pipette to fill the chip well with Rinsing Buffer and repeat washing step 2 more times for a total of three washes.
8. Immediately add 100 μL of MNT solution.
9. Let measurement run for as short as 2 min from addition of MNT solution.
10. Click on “STOP RECORDING“ button in the MagArray Chip Reader program.
11. Data processing software is integrated in the data acquisition and the user can get a result sheet after stopping the assay.

Disposal

1. Remove chip cartridge from the MagArray MR-110a reader.
2. Fill chip well with bleach and allow to soak for at least 30 min.
3. Pour bleach into sink and allow chip to dry.
4. Put the chip cartridge into a designated container for disposal.