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Cross reactivity testing at Quansys Biosciences

All assays to be multiplexed in an array must be tested for various types of cross-reactivity to determine if the reagents are specific enough to give a true positive signal. For example, the following types of cross-reactivity can occur:

Antigen-Capture Antibody Cross-Reactivity

A capture antibody binds the wrong antigen, which was then either specifically detected by its own detection antibody or nonspecifically detected by another detection antibody. If antigen-capture antibody (Ag-Cab) cross-reactivity is truly present, then the two systems cannot be multiplexed under the conditions tested and different capture antibodies or assay conditions should be screened.

Detection-Capture Antibody Cross-Reactivity

A capture antibody spot binds a detection antibody directly, lighting up in wells where the appropriate antigen was not present. Detection-capture antibody (Cab-Dab) cross-reactivity can often be minimized via reagent or diluent optimization.

Antigen-Detection Antibody Cross-Reactivity

An antigen is captured by the appropriate capture antibody but then detected by an antibody for another system. Antigen-detection antibody (Ag-Dab) cross-reactivity is not necessarily a problem and can even be used to one’s advantage, as the cross-reacting detection antibody can be used as an additional detection for the targeted antigen.

Capture Antibody-Conjugate Cross-Reactivity

The conjugate, such as SHRP, nonspecifically binds directly to a capture antibody. This type of cross-reactivity is rare and is more often due to biotin contamination of the capture antibody. Although rare, capture-conjugate cross-reactivity must be tested for, as this type of cross-reactivity is unacceptable and must be resolved for the assay to be validated.

Antigen-Conjugate Cross-Reactivity

The conjugate, such as SHRP, nonspecifically binds to an antigen bound by its respective capture antibody. This type of cross-reactivity is rare and is more often due to biotin contamination of the antigen. Although rare, antigen-conjugate crossing must be tested for, as this type of cross-reactivity is unacceptable and must be resolved for the assay to be validated.

Example of a Cross-Reactivity Experiment

Method

Individual antigens, individual detection antibodies, and negative controls for both were run on a Q-Plex™ microplate in duplicate in a grid pattern under normal assay conditions. Percent cross-reactivity was calculated by dividing the calculated concentration of a particular antigen run with a particular matched pair by the calculated concentration of the antigen with its intended matched pair.

Experimental Setup and Results

Discussion of Results

Note: Because the CRP assay is a competitive ELISA, the chemiluminescence in the absence of CRP is expected to be bright, and a decrease in chemiluminescence means that something was detected. All other systems are sandwich ELISA.

No significant cross-reactivity (> 1%) was observed. For example:

  • No non-specific binding was observed across rows or columns or in negative control wells.
  • GMCSF antigen was detected only in the presence of GMCSF detection (wells A1and A2) on the GMCSF capture spot (spot 14)
  • G-CSF antigen was detected only in the presence of GCSF detection (wells B3 and B4) on the GCSF capture spot (spot 15)
  • Unlabeled CRP was detected only in the presence of labeled CRP (wells C5 and C6) on the CRP capture spot (spot 13)
  • Systems in the Quansys Human Chemokine or Human Cytokine arrays were detected only in the presence of the appropriate Dab Mix (wells D7:E10, note that IL-8 (spot 5) is in both products).