Cross reactivity testing at Quansys Biosciences
Cross-reactivity testing at Quansy Bioscience
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 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.
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.
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.
- 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.)