Development of the covalent antibody-DNA conjugates technology for detection of IgE and IgM antibodies by immuno-PCR

Abstract

Immuno-PCR (iPCR) is one of the methods used for the detection of a wide range of analytes and features the high sensitivity of the polymerase chain reaction (PCR) method. iPCR uses antibodies coupled to DNA, followed by the amplification of the attached DNA using RT-PCR. Two major types of antibody-DNA conjugates are currently used, which are obtained as a result of non-covalent (biotin-streptavidin) or covalent interactions. Using a strain-promoted azide-alkyne cycloaddition (SPAAC), we synthesized covalent DNA-antibody conjugates, optimized the reaction conditions, and developed an efficient protocol for the purification of conjugates, with which all unreacted antibodies and oligonucleotides are separated. Covalent DNA-antibody conjugates were tested with iPCR assays that were previously developed for the detection of IgE and IgM antibodies with the use of the supramolecular complex of 5'- and 3'-biotinylated DNA and streptavidin. The results show that the modification of antibodies with amino groups did not allow us to obtain monolabeled antibodies or antibodies with a strictly defined number of DNA-labels. The degree of labeling determined by the dyes introduced through the azido group reflects the actual labeling degree statistically. If the average labeling degree for azido groups is 1.1, the conjugates contain 25% mono-labeled antibodies, 50% double-labeled antibodies, and 25% unlabeled ones. The specificity of the monoclonal antibody to human IgE (BE5) changed after conjugation with the oligonucleotide. The sensitivity of iPCR in the detection of IgM antibodies produced against the LeC disaccharide using a covalent conjugate was similar to that of a supramolecular complex of 5'- and 3'-biotinylated DNA and streptavidin, but the new procedure is two steps shorter.

Fig 1. Synthesis of covalent DNA-antibody conjugate.

Fig 2. Anion-exchange (a) and gel-filtration (b) chromatograms of antibody (Ab-N3 DOL = 6.4) and oligonucleotide 1:1 reaction products treated with 3-fold excess of sCy5-DBCO.

(a) 9.16 min: unmodified antibody; 11.7 min: conjugates and unreacted ODN. (b) 18.6 min: conjugates; 22–27 min: unreacted ODN.

Fig 3. Results of anion-exchange chromatography obtained by purification of conjugation reaction products involving an antibody with Ab-N3 DOL = 1.1.

(a) Anion-exchange chromatogram of conjugation reaction products involving an antibody with Ab-N3 DOL = 1.1. The numbers of fractions are indicated on the enlarged fragment (fraction 1: unreacted antibody; fractions 2–6: peak 11–12.5 min). Right: SDS-PAGE in reducing (b) and non-reducing (c) conditions. The sample numbers correspond to the fractions, MA2 is the original antibody. L: protein ladder (b: PageRuler Prestained Protein Ladder, 10 to 180 kDa; c: Spectra Multicolor Broad Range Protein Ladder).

Fig 4. Results of anion-exchange chromatography obtained during purification of the conjugation reaction products involving an antibody with Ab-N3 DOL = 2.6.

(a) Anion-exchange chromatography of the conjugation reaction products involving an antibody with Ab-N3 DOL = 2.6. (b) SDS-PAGE under non-reducing conditions. 5–9: fractions collected between 11 and 13 minutes. L: Spectra Multicolor Broad Range Protein Ladder.

Fig 5. Results of the second stage of purification of conjugation reaction involving antibody Ab-N3 DOL = 2.6.

(a) Gel-filtration chromatography of fractions obtained after anion-exchange chromatography (Fig 4) of conjugation reaction with the antibody Ab-N3 DOL = 2.6. (b) Electrophoregram of fractions collected during gel-filtration chromatography. The track numbers correspond to the designated fractions. MA2 is the original antibody. L: Spectra Multicolor Broad Range Protein Ladder.

Fig 6. Results of monolabeled conjugate testing (concentrations: 40 pM, 400 pM, and 4 nM) by iPCR of samples with a known concentration of anti-Le^C IgM.

Fig 7. Results of the different degrees of labeling conjugates tested by iPCR on a panel of samples with a known concentration of anti-Le^C IgM.

The numbers of labels per AB corresponds to conjugates with the same number of ODN molecules per antibody. DNA-Stvd is a variant of iPCR using a streptavidin supramolecular complex [19].

Fig 8. Titration curves of allergic and healthy donor sera obtained from the iPCR analysis of IgE on the recombinant allergen Alt a 1.

(a) Titration curves of allergic sera. (b) Titration curves of healthy donor. Data are shown for the BE5-ODN conjugate with two labels at a concentration of 10 ng/ml concentration. (c) Titration curves of the BE5-ODN conjugate and unconjugated antibody BE5 in ELISA when analyzing the serum of an allergic patient (20-fold dilution). The assay was performed on strips with and without sorbed antigen.