Methods Of Determination Immunometric Sandwich Assays

The commercially available assays for BNP and NT-proBNP are classified as immuno-metric assays. Such assays incorporate two antibodies that bind to different regions (epitopes) of the antigen to be detected (i.e., BNP or NT-proBNP) (Fig. 1A). One of these antibodies (capture antibody) is usually bound to a solid phase, and the second antibody (detection antibody) is labeled typically with an enzyme that catalyzes a reaction on which the detection ofthe antigen-antibody complex is based (e.g., fluorescence, luminescence). The immunometric "sandwich" assays typically offer a lower limit of detection, are more precise, and are frequently more specific than other types of assays, such as "competitive immunoassays," thus making them the preferred type of assay (3).

The commercially available, fully automated assays for BNP and NT-proBNP require 15-20 min to measure the analyte but are typically associated with longer turnaround times when sample transport, processing, and reporting of results are included. As an alternative to central laboratory testing, POC whole-blood assays are available that provide results within 20 min (see also Chapter 32). As recommended for cardiac troponin, assays for BNP

Heterophilic Ab

Fig. 1. Principle of immunometric (sandwich) assay with mechanisms of interference. (A) Normal situation. The capture antibody (Ab) is usually bound to a solid phase and binds the antigen to be detected (Ag). The detection antibody binds to a sterically remote region and is labeled with an enzyme that catalyzes the reaction on which the detection ofthe antigen-antibody complex is based. (B,C) Analytic interferences. Heterophilic antibodies may lead either to false positive test results by bridging between the detection and the capture antibody in the absence ofthe antigen or to false negative results by binding to the antigen-binding region of antibodies. Owing to steric hindrance, the antigen cannot bind to the antibodies although it is present in the sample.

Fig. 1. Principle of immunometric (sandwich) assay with mechanisms of interference. (A) Normal situation. The capture antibody (Ab) is usually bound to a solid phase and binds the antigen to be detected (Ag). The detection antibody binds to a sterically remote region and is labeled with an enzyme that catalyzes the reaction on which the detection ofthe antigen-antibody complex is based. (B,C) Analytic interferences. Heterophilic antibodies may lead either to false positive test results by bridging between the detection and the capture antibody in the absence ofthe antigen or to false negative results by binding to the antigen-binding region of antibodies. Owing to steric hindrance, the antigen cannot bind to the antibodies although it is present in the sample.

and NT-proBNP should have a total imprecision of <10% at their medical decision limits, in order to avoid misclassifications owing to poor assay precision. Currently, most, but not all, commercially available BNP and NT-proBNP assays fulfill this criterion.

Was this article helpful?

0 0

Post a comment