Site-specific DNA-antibody conjugates for specific and sensitive immuno-PCR

Abstract

Antibody conjugates are widely used as diagnostics and imaging reagents. However, many such conjugates suffer losses in sensitivity and specificity due to nonspecific labeling techniques. We have developed methodology to site-specifically conjugate oligonucleotides to antibodies containing a genetically encoded unnatural amino acid with orthogonal chemical reactivity. These oligobody molecules were used in immuno-PCR assays to detect Her2(+) cells with greater sensitivity and specificity than nonspecifically coupled fragments, and can detect extremely rare Her2(+) cells in a complex cellular environment. Such designed antibody-oligonucleotide conjugates should provide sensitive and specific reagents for diagnostics, as well as enable other unique applications based on oligobody building blocks.

Fig. 1.

Oligobody construction for immuno-PCR. (A) Scheme depicting site-specific immuno-PCR. A complementary single-stranded oligonucleotide (68 nt) (blue) is annealed to the oligobody (red). T4 ligase is added to form a circular oligonucleotide, which is now the template for RCA. The antibody-oligonucleotide conjugate is bound to Her2 on cells and phi29 polymerase is added to isothermally amplify the DNA, creating multiple copies of the 20 nt sequence. Small complementary oligonucleotides derivatized with Alexa Fluor 488 (20 nt) are then added to obtain a fluorescent signal. (B) Residues (K169 or S202) in anti-Her2 Fab mutated to pAcF for site-specific conjugation are shown in sphere form in orange, in the light chain (LC) in blue and the heavy chain (HC) in red. The Her2 antigen (green) is distant from all mutations. (C) Oliognucleotide conjugation to Fab. Either anti-Her2 pAcF (K169X, lanes 1, 3) or wild-type Fab (lanes 2, 4) was incubated without (lanes 1, 2) or with (lanes 3, 4) 3 mM aminoxy-modified ssDNA (100 mM methoxy aniline, 37 °C, 16 h). Reactions were analyzed by SDS-PAGE (Top), or transferred to nitrocellulose and incubated with anti-kappa-HRP (Middle) or a biotinylated antisense oligonucleotide, then detected with streptavidin-HRP (Bottom). The anti-kappa-HRP and streptavidin-HRP blots were developed colorimetrically with the metal enhanced DAB kit (Pierce). (D) Purified site-specific oligonucleotide conjugates and nonspecifically labeled oligonucleotide conjugate. Lanes 2 and 3 correspond to oligonucleotide site-specifically coupled to anti-Her2 pAcF mutant Fab. Lane 4 has multiple oligonucleotides (1–6) coupled to various lysines in anti-Her2 wild-type Fab.

Fig. 2.

Oligobody sensitivity and specificity in detecting Her2⁺ cells by immuno-PCR. Immuno-PCR on (A–C) SK-BR-3 (Her2^Hi) and (E–G) MDA-MB-231 (Her2^-). Site-specific oligonucleotide-Fab conjugates (K169pAcF or S202pAcF) or the nonspecifically labeled oligonucleotide-Fab conjugate were added to fixed Her2 cell lines and immuno-PCR was performed as previously described. Hoechst 33342 was used as the nuclear stain (blue) and Alexa Fluor 488 complementary fluorescent probes were used to detect Her2 (green). (D and H) Fluorescence from the immuno-PCR signal was quantified with ImageJ and error bars correspond to the standard deviation from triplicate frames. One-way ANOVA test indicated p value < 0.001. Tukey’s post hoc test, p value *< 0.05, **< 0.01, ***< 0.001. Exposure times: A–C, 40 ms; E–G, 200 ms. Scale bar, 25 μm.

Fig. 3.

Immuno-PCR in a complex mixture derived from human blood. Cells are identified by nuclear staining (blue). The anti-CK antibodies (anti-CK19, 1∶100, Dako and anti-panCK, 1∶100, Sigma) (red) stained both the (A) SK-BR-3 cells and (B) MDA-MB-231 cells, whereas the Her2 immuno-PCR with K169pAcF oligobody (green) only stained (A) SK-BR-3 cells. Anti-CD45 (1∶125, AbD Serotec) (gray) only stain WBCs. (C) MDA-MB-231 cells were stained with CellTracker Red (Invitrogen) (orange) and both SK-BR-3 and MDA-MB-231 cells were spiked into WBCs. SK-BR-3 cells were stained with Her2 immuno-PCR (green) and CK antibodies (red), whereas MDA-MB-231 cells were only stained with CK antibodies (red). (D) Approximately 1,000 cells were spiked into approximately three million WBCs and scanned on a fluorescent microscope (1∶3,000). The Alexa Fluor 488 standard deviation of the mean (SDOM) of a representative amount of cells were graphed. (E) Eleven SK-BR-3 cells were spiked into 1.4 million WBCs, scanned and all cells were identified by immuno-PCR. Scale bar, 25 μm.