Cross-Reactivity Testing at Quansys Biosciences

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

Method

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.

Spot Order

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.)

Read more tech tips


About Us

"We are dedicated to the development of protein arrays that aid researchers and clinicians in better understanding, diagnosing, and treating disease. Our products and services provide value to customers by improving the accuracy and efficiency of their testing."

Quality

Quansys Biosciences is an ISO 9001 and ISO 13485 registered company. We pride ourselves in the quality of our product and our commitment to customer satisfaction.


Quansys Bioscience
Fax: 435-750-6869
888-QUANSYS (782-6797)