Abstract
We present general means to greatly increase the sensitivity of antibody-based assays. Augmentation relies on a 'tadpole' protein-DNA chimera whose protein moiety binds most classes of mammalian antibodies but not avian immunoglobulin Y (IgY). We used this tadpole in affinity capture assays followed by real-time PCR to quantify numerous molecules, including prostate-specific antigen (PSA) in human serum, with great sensitivity and accuracy.
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Acknowledgements
We thank L. Bjorck for providing the LG protein coding sequence, and E. O'Shea and J. Weissman laboratories for providing the pDEST17-HIS-INFA-TAP expression vector. We thank D. Pincus for purifying the INFA-TAP protein, A. Gordon for helpful discussions on the statistical analysis of data, and K. Benjamin for helpful conversations during preparation of the manuscript. This work was supported by grants P50 HG002370 from the US National Human Genome Research Institute, R33 CA114306 from the National Cancer Institute, and U54 AI057156 from the National Institute of Allergy and Infectious Diseases.
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I.B. synthesized and purified LG tadpoles, conjugated antibodies to magnetic beads, performed binding assays and quantified data. K.Y. made the expression constructs, performed polyacrylamide gel analysis, control ELISAs and PCR measurements. R.Y. suggested the use of the LG protein and had input into project design. O.R. and R.B. had input into project design and interpretation. I.B. and R.B. wrote the manuscript and guarantee its integrity.
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Supplementary Figures 1–7, Supplementary Table 1 (PDF 275 kb)
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Burbulis, I., Yamaguchi, K., Yu, R. et al. Quantifying small numbers of antibodies with a 'near-universal' protein-DNA chimera. Nat Methods 4, 1011–1013 (2007). https://doi.org/10.1038/nmeth1127
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DOI: https://doi.org/10.1038/nmeth1127
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