Custom Protein Applications on the Luminex Platform

Using MagPlex® or SeroMAP™ microspheres, developing your own custom protein assay is simple and straightforward on the Luminex platform. In addition, Luminex Technical Support can offer guidance and troubleshooting as well as more information on assay development support and training courses.

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*The xMAP® Antibody Coupling Kit (Cat# 40-50016) includes all of the reagents and consumables necessary for coupling antibodies to MagPlex microspheres, in one convenient package.

Immunoassay Formats

Serology Immunoassay

A serology immunoassay is used to detect an antibody (target) with the use of a capture protein antigen and a detection antibody. The general steps to performing a serology immunoassay with xMAP technology are as follows:

  1. Couple capture protein antigen to microspheres.
  2. Apply the antibody-containing sample to the antigen-coupled microspheres.
  3. Wash the microspheres, so that unbound antibody is removed.
  4. Apply a labeled detection antibody, specific to the target antibody, to the target-bound microspheres.
  5. Wash the microspheres, so that the unbound detection antibody conjugates are removed.
  6. Read the microspheres on an xMAP system.
  7. Measure the fluorescence signal of the microsphere-bound target to determine the presence and quantity of the target antibody.

Protocol Download: Recommended Protocol for Indirect (Antibody Capture)/Serological Immunoassay Using MagPlex Microspheres – Washed Protocol

Capture Sandwich Immunoassay

A sandwich immunoassay is used to detect an antigen (target) with the use of a capture antibody and a detection antibody, forming a “sandwich.” The general steps to performing a sandwich immunoassay with xMAP technology are as follows:

  1. Couple capture antibody to microspheres.
  2. Apply the antigen-containing sample to the antibody-coupled microspheres.
  3. Wash the microspheres, so that unbound antigen is removed.
  4. Apply a labeled detection antibody, specific to the target antigen, to the target-bound microspheres.
  5. Wash the microspheres, so that the unbound detection antibody conjugates are removed.
  6. Read the microspheres on an xMAP system.
  7. Measure the fluorescence signal of the microsphere-bound target to determine the presence and quantity of the target antigen.

Protocol Download: How to convert ELISA format assays to xMAP using MagPlex beads for MAGPIX

Competitive Immunoassay

Another immunoassay format can be performed through competitive binding. The general steps to performing this type of assay on xMAP technology are as follows:

  1. Couple capture protein antigen to microspheres.
  2. Incubate unlabeled antibody in the presence of its antigen (target).
  3. These bound antibody/target complexes are then added to antigen-coupled microspheres.
  4. The microspheres are washed, so that unbound antibody is removed. (The more target in the sample, the less antibody will be able to bind to the antigen on the microspheres, hence "competition.")
  5. A labeled detection antibody, specific to the primary antibody, is added.
  6. Wash the microspheres, so that the unbound detection antibody conjugates are removed.
  7. Measure the fluorescence signal of the microsphere-bound antibody. The presence and quantity of the target antigen can be inferred from the presence and quantity of the captured antibody.

For competitive immunoassays, the higher the sample target concentration, the weaker the eventual signal. The major advantage of a competitive immunoassay is the ability to use crude or impure samples and still selectively bind any target that may be present.

Data Collection and Analysis

xPONENT® Software Solutions offer simple, easy to use experimental setup and acquisition saving valuable walk away time. xPONENT is compatible with MagPlex Microspheres and has options allowing LIS, Automation, 21 CFR part 11 compliant, and Security options to meet each individual lab’s needs.

Reagents Available for Protein Custom Assay Development

 


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