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.
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-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).