Bispecific Antibody Detection Using Antigen-Conjugated Synthetic Nucleic Acid Strands

We report here the development of two different sensing strategies based on the use of antigen-conjugated nucleic acid strands for the detection of a bispecific antibody against the tumor-related proteins Mucin1 and epidermal growth factor receptor. Both approaches work well in serum samples (nanomolar sensitivity), show high specificity against the two monospecific antibodies, and are rapid. The results presented here demonstrate the versatility of DNA-based platforms for the detection of bispecific antibodies and could represent a versatile alternative to other more reagent-intensive and time-consuming analytical approaches.


Materials Chemicals
Reagent-grade chemicals (Sodium Chloride, Disodium Hydrogen Phosphate Phosphatebuffered saline (PBS tablets)) were purchased from Sigma-Aldrich (St Louis, Missouri) and used without further purifications.Anti-MUC1 monoclonal antibody, Anti-EGFR and Anti-MUC1/EGFR bispecific antibody were kindly provided by Merck (Darmstadt, Germany).The antibodies were aliquoted and stored at 4 °C or at -20°C for immediate use and at -80 °C for long-term storage.

DNA Sequences
HPLC purified oligonucleotides were purchased from Biosearch Technologies (Risskov, Denmark), Metabion International AG (Planegg, Germany) and Biomers GmbH (Ulm/Donau, Germany).PNA/Peptide chimera probes were purchased from Panagene (South Korea).All sequences were designed using Nupack or IDT oligoanalyzer tools.In the MUC1-PNA strand the sequence in parentheses represents the selected peptide epitope portions that is terminally conjugated to a PNA strand.

EGFR-DNA conjugation
EGFR protein is covalently conjugated to a DNA strand using the Amine Coupling Kit 3 according to the manufacturer's instructions (Dynamic Biosensors).All these kit reagents (i.e., crosslinker, spin desalting column, etc.) have been optimize to maximize the conjugation yield.Specifically, EGFR protein tablets were diluted in PBS buffer (10 mM

Data analysis
The experimental values represent averages of three separate measurements and the error bars reflect the standard deviations.Binding curves were fitted with the following four parameter logistic equation: (1) where, F min and F max are the minimum and maximum fluorescence values, K 1/2 is the equilibrium antibody concentration at half-maximum signal, n H is the Hill coefficient, and [Target] is the concentration of the specific antibody added.
For a more ready interpretation of the results, normalization has been obtained on a 0−1 scale using the following formula: (2) where F T is the fluorescence signal obtained in presence of the target antibody, F 0 is the fluorescence signal obtained in the absence of target and F max represents the maximum fluorescence signal of the platform at a saturating concentration of target (Rel.Fluor.= 1).
Signal Gain (%) values are calculated as the relative signal change registered upon the addition of saturating concentration of target antibodies (or non-specific antibodies) using the following formula: (3) Signal gain (%) = where target is the signal in the presence of different concentration of the target; 0 is the   background signal.
Accuracy tests were performed by adding known amounts of Bispecific antibody into either PBS and blank 10% or 50% plasma samples.Bispecific antibody quantification ([Target]) was performed by using the following formula: 1/ F min and F max are the minimum and maximum fluorescence values, K 1/2 is the equilibrium antibody concentration at half-maximum signal, n H is the Hill coefficient extrapolated from the binding curves in Figure S4.The Percent Coefficient of Variation (CV %) and the Percent Relative Error (BIAS %) of the estimated concentration were calculated using the following formulas: (5) CV % = St.Deviation Mean Value × 100 Added Conc.

× 100
The Percent Stability (STAB %) of the method was assessed after keeping the reaction components at room temperature (RT) for 4h or after three cycle of freeze-thaw and was calculated using the following formula: (7) STAB % = Average Measured Conc.

× 100
The limit of detection (LOD) was determined as the concentration that reaches three standard deviations above a blank signal, instead the low limit of quantitation (LLOQ) is the lowest concentration that can be quantified in a reliable way, meeting BIAS% and CV% criteria.
AGA ATA AAA CGC CAC TG T(6Fam) ACG TG ATC TAA TGG TGA GTC CAC GT T(BHQ1) -3' Input Strand 5'-AGA ATA AAA CGC CAC TG T TTT TTT TTT TTT TTT TTT TTT TTT GAC TCA CCA TTA G -3' MUC1-PNA strand 5'-CAG TGG CGT TTT ATT CT -3' N term -(APDTRPAPGSTAPPA)-C term Here in the reporter strand sequence the italic bases denote the stem forming portion, the underlined bases the loop portion and bold bases the anchoring portion for the input strand.

Figure
Figure S2 (A) General scheme of the antigen-conjugated nucleic acid strands platform for Anti-MUC1 antibody detection.(B) Fluorescence kinetic traces in the presence (100 nM) and absence of Anti-MUC1 antibodies.(C) End point values obtained at increasing concentration of Anti-MUC1 antibodies (fit eq.(1)).(D) Signal gain values (fit eq.(3)) observed at saturating concentration (100 nM) of Anti-MUC1 antibody, and with non-specific antibodies.The experiments were performed in a 20 μL buffer solution (10 mM Na 2 HPO 4 , 137 mM NaCl, 2.7 mM KCl, pH 7.4) at 25°C containing the reporter module (10 nM), the input module (30 nM) and antibodies as indicated.The fluorescence signals were measured at 520 nm after 30 min of incubation.

Figure
Figure S3 (A) General scheme of the antigen-conjugated nucleic acid strands platform for Anti-EGFR antibody detection.(B) Fluorescence kinetic traces in the presence (100 nM) and absence of Anti-EGFR antibodies .(C) Dose-response curve at increasing concentration of Anti-EGFR antibodies (fit eq.(1)).(D) Signal gain values (fit eq.(3)) observed at saturating concentration (100 nM) of Anti-EGFR antibody, and with non-specific antibodies.The experiments were performed in a 20 μL buffer solution (10 mM Na 2 HPO 4 , 137 mM NaCl, 2.7 mM KCl, pH 7.4) at 25°C containing the reporter module (10 nM), the input module (30 nM) and antibodies as indicated.The fluorescence signals were measured at 520 nm after 30 min of incubation.

Figure S4
Figure S4Binding curves obtained in 384 well plate at increasing concentrations of the BsAb (fit eq.(1)) in (A) buffer solution, (B) 10% and (C) 50% plasma solution.The experiments were performed in a 20 μL buffer solution (10 mM Na 2 HPO 4 , 137 mM NaCl, 2.7 mM KCl, pH 7.4), 10% and 50% plasma solutions each containing the reporter module (10 nM), the input module (30 nM), and Bispecific antibodies as indicated.The fluorescence signals were measured at 523 nm after 30 min information.

Figure
Figure S5 (A) Antibody-responsive DNA circuit designed to activate a strand displacement reaction upon the binding of a bivalent DNA strand (Ab mimic) to two Split #1 and #2 strands.(B) Binding curve obtained (fit eq.(2)) at increasing concentration of the Ab mimic strands using 60 nM of target duplex and 100 nM of Split #1 and #2.The experiments were performed in a 20 μL phosphate buffer solution (50 mM Na 2 HPO 4 , 150 mM NaCl, pH 7.0) and the fluorescence signals were measured at 680 nm.
Na 2 HPO 4 , 137 mM NaCl, 2.7 mM KCl, pH 7.4) to 1 μg/μL and stored at 20°C.The DNA strand modified with a DBCO group at one end is mixed with a crosslinker solution and the excess of crosslinker is removed with a Spin Desalting Column.EGFR protein is added to the solution for an incubation of 1 h at RT and then overnight at 4 °C.Subsequently, the conjugate is purified from unreacted oligos by ion exchange chromatography using a proFIRE device (Dynamic Biosensors, Planegg, Germany) according to the manufacturer's instructions.The ion-exchange column is pre-equilibrated with buffer A (50 mM Na 2 HPO 4 /NaH 2 PO 4 , 150 mM NaCl, pH 7.2) before the injection of 160μL of EGFR-DNA conjugate.The conjugate is eluted with a salt gradient using buffer B (50 mM Na 2 HPO 4 ,/NaH 2 PO 4 , 1 M NaCl, pH 7.2) with a flow rate of 1 mL/min.After purification, buffer exchange of the conjugate is performed using Centrifugal Filter Units with 3 kDa molecular weight cut-off (MWCO) against 10 mM Na 2 HPO 4 , 137 mM NaCl, 2.7 mM KCl, pH7 7.4.Conjugate concentration is finally determined by measuring the absorbance at 260 nm.