Development of immunoassays for the investigation of peptide antibody interactions in array format
Creators: name: Palermo, Andrea
Publisher: name: Karlsruhe
Year: 2020
Subjects: peptide array
peptide-antibody interaction
microfluidic channel
immunoassay

description: Antibodies perform a variety of important tasks in the immune system to protect us from diseases. They are trained to specifically target pathogens, which might be toxins, bacteria or viruses. However, they can also harm us by erroneously mark endogenous structures for degradation, which contributes to autoimmune diseases. Studying the immune response by determining, which antibodies are amplified during a successful immune reaction, and which part of the pathogen neutralizing antibodies are binding to, would help to develop new vaccine candidates and contribute to the common knowledge of the underlying mechanism. In addition to the information, what antibodies specifically bind to, it is of interest, in which concentration they are available in the blood and with which affinity they bind their target. Peptide arrays provide an ideal platform for the study of antibody-protein interactions, with the peptides mimicking antibody targets. Using a minute sample volume, several thousand peptides can be examined simultaneously. To study identified antibody-peptide interactions in terms of kinetics, on-line measurements in microfluidic channels, so called continuous flow assays, have to be performed. This work is divided into two main parts, covering the screening for epitopes and the development of an experimental setup to characterize the peptide-epitope interaction. In the first part, the immune response of healthy European individuals towards the tetanus, diphtheria and pertussis vaccination and two established epitopes (PALT(A)xET and PEFxGSxP) originating from enterovirus and probably Staphylococcus aureus, was investigated using peptide arrays. Vaccination specific antibodies were identified by determining their linear epitope in amino acid resolution. Therefore, the linear amino acid sequence of tetanus, diphtheria and pertussis toxin was synthesized in fragments (peptides) in array format onto a solid substrate and incubated with serum of 19 healthy European individuals. For the tetanus toxin, an epitope was identified (929ExxEVIVxK937), which was targeted by 8 out of 19 individuals. The amino acids crucial for antibody binding (the `antibody fingerprint'), were determined for this epitope, by substitution analysis. In a substitution, every amino acid is substituted by all of the other 19 amino acids while the rest of the sequence remains conserved. The antibody fingerprint can be determined, since the substitution of crucial amino acids leads to the loss of antibody binding. The `antibody fingerprints' were found to be strongly conserved among individuals for the three investigated linear epitopes (929ExxEVIVxK937, PALT(A)xET and PEFxGSxP). Further, it was further verified by affinity batch chromatography and ELISA, that antibodies binding the identified epitope originating from the sequence of the tetanus toxin, also bind to the native tetanus toxin. This indicates, that the identified epitope is tetanus specific. For the mapping of the diphtheria toxin linear and cyclic peptides of different length were applied. Five prominent regions were identified in the mapping of cyclic peptides using pooled sera. For the pertussis toxin, three regions of interest could be identified, which also were reported in literature for their ability to induce toxin recognizing antibodies in rodents. However, no distinct antibody fingerprints could be determined for a variety of peptides covering the identified regions. Altogether, the homology in between fingerprints of different individuals targeting the same epitope (929ExxEVIVxK937, PALT(A)xET and PEFxGSxP) is striking, given the randomness of antibody formation. In the second part, an experimental setup was realized to incubate peptide arrays in an automated fashion in microfluidic channels to perform continuous flow assays. The interactions of fluorescently labelled antibodies with the peptides can be characterized via the detection of fluorescence using an epi-fluorescence setup design. For this purpose, fluorescence images are taken at short intervals and the increase of the fluorescence signal over time is determined. The fluorescence intensity of the fluorophore DL550 was found to be temperature dependent under assay conditions. However, for a constant temperature (24 +/- 0.1°C), the intensity can be approximated as being stable for 50 min of continuous exposure. For the fast production of peptide arrays needed during preliminary experiments, spotting of pre-synthesized peptides (bearing a C-terminal cysteine) on 3D-Maleimide surfaces was established. To have an additional label free imaging method at hand, VSI was established for the quality control of peptide arrays. Height profiles obtained by VSI did not significantly deviate from AFM measurements. Spots down to approximately 1 nm height can be resolved on a comparable large field of view of (1.74 x 1.31) mm within a minute. Two different approaches for the integration of peptide arrays into a microfluidic channel have been investigated. Molding channel structures in PDMS is a fast technique, but the channel design was determined to be restricted to have a minimum distance between channels to prevent leakage. By adding up to 2% of graphene particles to the PDMS prior to polymerization, the background fluorescence of PDMS was reduced by approximately 70%. The fabricated channel proved to be leak-tight up to a flow of 3 ml/min when screwed to a torque of 10 N/cm. However, the clamping led to a deformation of the PDMS channels. This results in non-homogeneous channel cross sections and is thus not apt for a concentration analysis, which requires a constant channel height. The second type of channel fabrication investigated is adhesive layer bonding using Ordyl SY355. It is the first approach to bond a glass substrate to a functionalized surface, used for micro array fabrication. This method has the potential to make microarrays available to other microsystem technologies. Fluidic in- and outlets are laser drilled into a glass substrate and the adhesive layer is applied and structured lithographically to form the walls of the channel. The parameter window for this lithographic process was determined to be quite broad with exposure doses ranging from (150-220) mJ/cm² and bonding pressures ranging from (220-735) N/cm². Process temperatures over 100°C were found to be critical, since damage of the functionalized surface occurred. The magnitude of fracture strength of the bond was estimated by random pulling tests to be about 305 +/- 50 N/cm². This results in a theoretical pressure resistance of ~ 4.9 bar and proved to be leak proof for a flow rate of up to 750 mL/min. The designed experimental setup, including the integration of peptide arrays into microfluidic channels, was shown to fulfill the requirements for performing continuous flow assays on peptide arrays. By conducting the calibration of fluorescence signal intensity to antibody concentration in a future project, the setup will be ready to further determine antibody titers.