Modification of anti-DNA antibodies with carbon quantum dots

Antibodies represent a specialized glycoprotein group and form the main base of vertebrate immune system. There are 5 immunoglobulin classes in mammalian serum: IgG, IgM, IgA, IgD and IgE [1]. In avian species the immunoglobulins G are replaced by highly functionally similar immunoglobulins Y and there are also IgA and IgM present [2]. Antibodies are often used in variety of applications, including ELISA for the detection of various analytes [3], virions [4] or tumor markers [5], cell capture for subsequent analysis [6] and magnetic immunoseparation [7,8]. For bulk production, antibodies extracted from chicken egg yolks immunized with the specific antigen are often used [9].
Anti-DNA antibodies were first discovered in 1957 [10] and they can bind to single stranded or double stranded DNA [11,12]. Anti-dsDNA antibodies can be found in the blood of systemic lupus erythematodes (SLE) patients. This autoimmune disease is manifesting by formation of immunocomplexes [13]. The antibodies usually belong to class IgM in normal patients or IgG in SLE patients [14] and they can be transiently found in the blood of patients with some viral infections (HIV, BK or B19 virus) [15]. Anti-dsDNA antibodies usually bind to sugar-phosphate backbone, base pairs or some double strand conformations. Anti-ssDNA antibodies should be able to bind to bases, nucleotides, oligonucleotides and sugar-phospate backbone [16]. Commercially available are also antibodies against DNA adducts such as those with cisplatin [17]. The combination of suitable antibodies can help identify the secondary structures of DNA [18].
To enhance the antigen detection limit it is possible to label the antibodies with highly fluorescent nanoparticles such as quantum dots (QDs) [3]. QDs have unique fluorescent characteristics, including wide absorbance spectrum with narrow emission spectrum, high quantum yield or excellent photostability [19]. They are usually formed by a semiconductor crystals, such as CdTe, CdS or ZnS, but they can also be prepared from carbon materials with polymer coating [6]. These materials include nanodiamonds [20], graphene [21], graphite [22], single- and multi-wall carbon nanotubes [23], citric acid [24] or sucrose [25]. The prepared carbon quantum dots usually show high fluorescence under UV light [26].
In this work, we studied anti-DNA antibodies produced in chicken egg yolks as a tool for DNA structure detection. We used four different anti-DNA antibodies: anti-dsDNA, afi-dsDNA, anti-ssDNA and afi-ssDNA. Their reactivity towards various sized DNA molecules was evaluated using dot blot method. The successful modification of anti-DNA antibodies with carbon quantum dots was determined by ELISA-like method with fluorescent detection. .

Figure 1: The dot blot assay for evaluation of antibody reactivity with single and double stranded DNA of different length. (A) The sample order on Zeta Probe membrane. (B) Dot blot assay with 15 nM DNA and 22 μg/mL of antibodies. (C) Dot blot assay with 100 μg/mL of DNA and 22 μg/mL of antibodies.

Table 1: The colorimetric intensity quantification of dot blot assay.

Figure 2: The characterization of MWCNT-CQDs. (A) Visualization under ambient and UV (254 and 312 nm) light. (B) Absorbance spectrum of MWCNT-CQDs. (C) The fluorescence spectra of MWCNT-CQDs after various excitation wavelength.

Figure 3: Modification of anti-DNA antibodies with MWCNT-CQDs determined by ELISA-like method with fluorescent detection. (A) Fluorescence spectra (excitation wavelength 330 nm) of microtiter plate wells coated with DNA fragment after incubation with anti-ssDNA antibodies. (B) Fluorescence spectra (excitation wavelength 330 nm) of microtiter plate wells coated with DNA fragment after incubation with anti-ssDNA antibodies modified with MWCNT-CQDs. (C) Fluorescence spectra (excitation wavelength 230 nm) of microtiter plate wells coated with DNA fragment after incubation with anti-ssDNA antibodies. (D) Fluorescence spectra (excitation wavelength 230 nm) of microtiter plate wells coated with DNA fragment after incubation with anti-ssDNA antibodies modified with MWCNT-CQDs.

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