Sandwich Antigen Detection Immunoassays Immunoassay formats that require blockers
This is a sensitive and robust method which captures the target antigen between two antibodies (capture and detection antibody). The capture antibody is bound to the solid phase. The antigen-containing sample is applied followed by a wash step to remove unbound antigen. A detection antibody is added that binds directly to the antigen. The capture and detection antibodies must bind to non-overlapping epitopes on the antigen. Either monoclonal or affinity-purified polyclonal antibodies can be used as capture and detection. The antigen can be measured with a conjugated detection antibody (direct detection) or a matched set of unlabeled detection and conjugated secondary antibodies (indirect detection).
Detection Antibody Secondary Antibody
Detector Label
Detector Label
Detection Antibody Capture Antibody Antigen
Capture Antibody Antigen
Direct Detection
Indirect Detection
Antibody detection Assay (IgG, IgM & IgA)
The antigen is immobilized on the solid phase by direct absorption. The antibody-containing sample is applied followed by a wash step. Detection of the antibody can then be performed using an conjugated detection antibody (direct detection) or a matched set of unlabeled detection and conjugated secondary antibodies (indirect detection). Direct detection is shown below.
Detector Label
Detection Antibody
Detector Label
Detection Antibody
Patient’s IgG Ab
Patient’s IgM Ab
Antigen
Antigen
IgG Detection
IgM Detection
Competitive Assay
A competitive binding process between the patient’s target analyte and labeled antigen (inhibitor antigen). Antibody specific for the target analyte is coated onto the solid phase. Patient sample and labeled inhibitor antigen are incubated with the pre-coated antibody and compete for binding sites. Unbound labelled antigen/analyte is removed by washing. The more analyte in the sample, the less the labelled inhibitor antigen will bind to the antibody. Therefore, the weaker the assay signal, the higher the concentration of analyte in the patient sample. This is a common method for small antigens that have only 1-2 epitopes.
Detector Label
Patient Analyte
Inhibitor Antigen
Capture antibody
Direct Detection
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