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A detailed characterization of metal-tagged antibodies is the prerequisite for the implementation of quantitative concepts in inductively coupled plasma–mass spectrometry (ICP-MS)-based bioanalysis or future medical diagnosis. In this paper, the common modification with bifunctional ligands containing maleimide residues as a reactive group was investigated in detail via size exclusion chromatography (SEC)-ICP-MS and liquid chromatography–time-of-flight (LC-TOF)-MS to determine the preservation of the antibody structure after tagging. Mouse monoclonal IgG modified with metal-coded tags (MeCATs) was used as a model system. Several antibody fragments were identified carrying different numbers of metal tags. In a second step, a functionality test was performed with isolated fragments where the antigen specificity was tested in a dot blot immunoassay.
Immunoassays are analytical methods used to track both clinical and environmental parameters. Antibodies or other proteins with similar recognizing activity, are employed, often immobilized onto a modified surface. Addressable microarrays are based on single-stranded DNA oligonucleotides which are normally used to detect aptamers or relevant gene sequences. The use of antibody-oligonucleotide conjugates allows non-directed antibody immobilization from an immunoassay to be converted into DNA hybridization events on the array. Consequently, the diagnostics platform is multiplexed and addressable. Figure 1 is a model representation of the whole biosensor construct. In brief, a surface is coated with streptavidin, and decorated with biotin-modified oligonucleotides of a controlled size and known sequence. These oligonucleotides on the surface also contain a furan motif, which upon irradiation and in the presence of a photosensitizer, is oxidized via single oxygen to a reactive intermediate which crosslinks the immediate opposing base when hybridized with its complementary strand [1].
Antibody-oligonucleotides conjugates still display several concerns due to their heterogeneicity, difficulty of characterization and high price [2]. The being the case, we aim at developing stable, robust, reproducible and well characterized quimeras for the application on te above described immunoarray. Mild chemical conditions are crucial for the antibody stability, therefore bivalent crosslinkers have been employed [2]. Even though the use of these bilinkers is standard for other substrate conjugation (i.e., enzymes, drug payloads, etc.), there is no literature available on the conjugation of small oligonucleotides (< 30 mer) to antibodies using this method. Several protein-nucleic acid conjugates have been therefore developed, and successfully characterized using MALDI-ToF and gel electrophoresis techniques.
Deficiencies in research antibody quality lead to economic losses up to several billions of Euros annually. Poorly documented antibody experiments lead to a flood of publications without any chance to be reproduced and without any scientific value.The dominance of antibody specifications in complex analytical procedures is often highly underestimated. Most journals and funding agencies lack the expertise to identify major flaws in antibody experiments and their documentation. Most companies sell insufficiently characterized and documented antibodies. Recombinant antibodies are not the solution for antibody validation.
This author interview is by Dr Michael G. Weller, Head of Division 1.5 Protein Analysis at Federal Institute for Materials Research and Testing (BAM). BAM (www.bam.de/en) is a senior scientific and technical institute with responsibility to the German Federal Ministry for Economic Affairs and Energy. Dr Weller's review paper, Quality issues of research antibodies is available for download in Analytical Chemistry Insights.
Fast and robust purification of antibodies from human serum with a new monolithic protein A column
(2017)
The use of antibodies is becoming more and more important. Therewith the need for fast and robust methods for their purification and columns with a long lifetime is growing. In this application, we describe a fast and robust protein A affinity chromatography method with a new monolithic column. Antibodies were successfully purified in less than 1.5 minutes with a flowrate of 5 mL/min highlighting the advantages of this column and method.
A recent Opinion Article by Jan Voskuil (JV), Aeonian Biotech, discussed the challenges in validation of research antibodies. It was openly peer reviewed by Michael Weller (MW), Federal Institute for Materials Research and Testing (BAM), Germany. In this blog, both discuss these challenges, the reproducibility crisis and open data.
Die neue Crosslinking-Methode ist hilfreich, um Immunglobuline des Isotyps G ortspezifisch an ihrer Bindungsstelle mit Protein A oder G zu konjugieren. Die Kopplungen von Protein A und G konnten erfolgreich an Maus- und Human-IgG durchgeführt, sowie die Bedingungen untersucht und optimiert werden. Die Aktivierung von Protein G mit Glutaraldehyd erfolgt am besten bei pH 8 und die anschließende Kopplung mit Maus-IgG1 bei einem pH-Wert von 6. Jedoch wurden mit SIAB und Sulfo-SIAB als Crosslinker im Vergleich zu Glutaraldehyd noch höhere Signale erhalten. Für die Kopplung von SIAB sind 40% DMSO im Reaktionspuffer günstig, währenddessen Sulfo-SIAB gut wasserlöslich ist daher keine Lösungsvermittler benötigt. Es ergab sich ein optimaler pH-Wert von 7,4 um Protein A mit SIAB zu aktivieren und den gleichen pH-Wert um die Kopplung mit Maus-IgG1 durchzuführen. Für die Kopplung von Protein G mit Maus-IgG1 hingegen zeigten die Experimente, dass ein leicht saurer pH-Wert bei pH 6 für den IgG-Kopplungsschritt am günstigsten ist. Während die Inkubationszeit von Maus-IgG1 mit Protein G bei 16 h liegt, muss mit Protein A bis zu 40 h inkubiert werden, um das Kopplungsmaximum zu erreichen. Des Weiteren wurde das Crosslinking von Human-IgG (Herceptin) mit SIAB untersucht. Dabei zeigte sich, wie in der Abbildung 47 zu sehen ist, dass Protein A und G vergleichbar gut an Human-IgG zu koppeln sind. Für das Crosslinking von Protein G mit Maus-IgG1 ist dagegen ein deutlich besserer Umsatz im Vergleich zu Protein A zu erkennen (Abbildung 48). Heterobifunktionale Linker können mit der reaktiveren Gruppe die erste Bindung eingehen, um erst nach Zugabe eines weiteren Reagenzes die zweite Bindung auszubilden und zudem intramolekulare Reaktionen möglichst zu vermeiden.
Type-I pyrethroids are frequently used for disinfection purposes on airplanes from and to tropical areas as a preventive health measure to control or kill the insect vectors of human diseases including dengue, yellow fever and malaria. The aim of the presented work was the development of such a simple, rapid and effective method for pyrethroid analysis
A novel stationary phase for affinity separations is presented. This material is based on sintered borosilicate glass readily available as semi-finished filter plates with defined porosity and surface area. The material shows fast binding kinetics and excellent long-term stability under real application conditions due to lacking macropores and high mechanical rigidity. The glass surface can be easily modified with standard organosilane chemistry to immobilize selective binders or other molecules used for biointeraction. In this paper, the manufacturing of the columns and their respective column holders by 3D printing is shown in detail. The model system protein A/IgG was chosen as an example to examine the properties of such monolithic columns under realistic application conditions. Several specifications, such as (dynamic) IgG capacity, pressure stability, long-term performance, productivity, non-specific binding, and peak shape, are presented. It could be shown that due to the very high separation speed, 250 mg antibody per hour and column can be collected, which surpasses the productivity of most standard columns of the same size. The total IgG capacity of the shown columns is around 4 mg (5.5 mg/mL), which is sufficient for most tasks in research laboratories. The cycle time of an IgG separation can be less than 1 minute. Due to the glass material's excellent pressure resistance, these columns are compatible with standard HPLC systems. This is usually not the case with standard affinity columns, limited to manual use or application in low-pressure systems. The use of a standard HPLC system also improves the ability for automation, which enables the purification of hundreds of cell supernatants in one day. The sharp peak shape of the elution leads to an enrichment effect, which might increase the concentration of IgG by a factor of 3. The final concentration of IgG can be around 7.5 mg/mL without the need for an additional nanofiltration step. The purity of the IgG was > 95% in one step and nearly 99% with a second polishing run.
The trafficking of illegal drugs by criminal networks at borders, harbors, or airports is an increasing issue in public health as these routes ensure the main supply of illegal drugs. The prevention of drug smuggling, including the installation of scanners and other analytical devices to detect ultra-small traces of drugs within a reasonable time frame, remains a challenge. The presented immunosensor is based on a monolithic affinity column with a large excess of immobilized hapten, which traps fluorescently labeled antibodies as long as the analyte cocaine is absent. In the presence of the drug, some binding sites of the antibody will be blocked, which leads to an immediate breakthrough of the labeled protein, detectable by highly sensitive laser-induced fluorescence with the help of a Peltier-cooled complementary metal-oxide-semiconductor (CMOS) camera. Liquid handling is performed with high-precision syringe pumps and microfluidic chip-based mixing devices and flow cells. The biosensor achieved limits of detection of 23 pM (7 ppt) of cocaine with a response time of 90 seconds and a total assay time below 3 minutes. With surface wipe sampling, the biosensor was able to detect 300 pg of cocaine. This immunosensor belongs to the most sensitive and fastest detectors for cocaine and offers near-continuous analyte measurement.