Enzyme-linked immunosorbent assays (ELISAs) are simple and inexpensive methods used to determine the presence of biomarkers for the diagnosis of multiple diseases and drug development. Output signal depends on the number of enzyme molecules that can be indirectly attached per analyte molecule. Improving the detection sensitivity of the assay would allow a better understanding of cellular mechanisms and decrease the amount of sample required per assay. To increase the assay's sensitivity other groups have tried successive incubations of enzyme labeled avidin and biotinylated anti-avidin antibodies. The main drawbacks of this approach are an increase in assay time and decrease in reproducibility due to the multiple incubations and washes. A better approach would be to deliver a higher number of labels. To this end, we have prepared DNA dendrimers conjugated to anti-biotin antibodies containing up to 300 molecules of horseradish peroxidase (HRP), and compared these reagents to streptavidin-HRP (~2HRP/streptavidin). The assay format consisted of a standard microtiterplate ELISA in which the dendrimer conjugate is a “drop-in” reagent replacing the streptavidin, so that no extra steps are involved. Interleukin 1 alpha (IL-1&alpha) was selected as the model analyte. Upon optimization we were able to detect less than 2 pg/mL of IL-1&alpha, corresponding to greater than 20-fold signal amplification respect to streptavidin-HRP. The conjugate was also used to detect IL-6, TNF alpha, antibodies against beta 2 glycoprotein I (&beta2 GPI) and gastric pariental cell antibody (GPA). For these markers signal amplifications ranged from 5 to 200 fold compared to streptavidin-HRP detection.
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