Figure 1.

Schematic representation of antibody detection by agglutination-PCR (ADAP). (a) The sample containing the target antibody analyte is incubated with a pair of antigen–DNA conjugates. Each conjugate bears an oligonucleotide sequence comprising either the 5′-(red) or 3′-(green) half of a full amplicon. (b) Next, antibodies within the sample agglutinate the antigen–DNA conjugates and position them for ligation upon the addition of a bridging oligonucleotide (blue) and DNA ligase. (c) The newly generated amplicon (red/green) is exponentially amplified with primers that bind their respective sites (red and green arrows) and quantified by real-time qPCR. The immune complex of antibodies and antigen–DNA conjugates shown here represents the proposed mechanism for detecting polyclonal antibodies with relatively large antigens at high concentrations. For monoclonal and anti-small molecule antibody detection, as well as when antibody concentration is significantly lower than that of antigen–DNA conjugates, the complex likely consists of a single antibody bound to two antigen–DNA conjugates (Figure S5).