All assays to be multi-plexed in an array must be tested for various types of cross-reactivity to determine if the reagents are specific enough to give 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 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. 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. 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
Individual antigens, individual detection antibodies, and negative controls for both were run on a Q-PlexTM 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.
Expirimental 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.
- GM-CSF antigen was detected only in the presence of GM-CSF detection (wells A1and A2) on the GM-CSF capture spot (spot 14)
- G-CSF antigen was detected only in the presence of G-CSF detection (wells B3 and B4) on the G-CSF 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 Hu Chemokine or Hu 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.)