Filtern
Erscheinungsjahr
- 2016 (8) (entfernen)
Dokumenttyp
- Zeitschriftenartikel (3)
- Vortrag (3)
- Sonstiges (1)
- Posterpräsentation (1)
Schlagworte
- Antibodies (2)
- Reproducibility (2)
- immunoassay (2)
- Airport security (1)
- Amino acid analysis (1)
- Antibody ID (1)
- Blindwert (1)
- Certified reference materials (1)
- Crisis (1)
- ELISA (1)
Eingeladener Vortrag
- nein (3)
Amino acid analysis is considered to be the gold standard for quantitative peptide and protein analysis. Here, we would like to propose a simple HPLC/UV method based on a reversed-phase separation of the aromatic amino acids tyrosine (Tyr), phenylalanine (Phe), and optionally tryptophan (Trp) without any derivatization. The hydrolysis of the proteins and peptides was performed by an accelerated microwave technique, which needs only 30 minutes. Two internal standard compounds, homotyrosine (HTyr) and 4-fluorophenylalanine (FPhe) were used for calibration. The limit of detection (LOD) was estimated to be 0.05 µM (~10 µg/L) for tyrosine and phenylalanine at 215 nm. The LOD for a protein determination was calculated to be below 16 mg/L (~300 ng BSA absolute). Aromatic amino acid analysis (AAAA) offers excellent accuracy and a precision of about 5% relative standard deviation, including the hydrolysis step. The method was validated with certified reference materials (CRM) of amino acids and of a pure protein (bovine serum albumin, BSA). AAAA can be used for the quantification of aromatic amino acids, isolated peptides or proteins, complex peptide or protein samples, such as serum or milk powder, and peptides or proteins immobilized on solid supports.
Zusammenfassung Selektivität
1. Widersprüchliche Definitionen in unterschiedlichen Bereichen
2. Analytische Definition (pragmatisch): Selektivität ist die Fähigkeit einer Methode zur Unterscheidung aller interessierender Stoffe in der Probe.
3. Tests: Blindwerte, Leerwerte, Blank Values; Peakform
4. Wichtige Einflussgrößen: Trennleistung, Auflösung, Peakkapazität, Trennkapazität
5. Verbesserung durch: Kopplungsverfahren, Änderung der chromatographischen „Selektivität“, Orthogonalität
6. Wichtig ist auch die Komplexität der Probe(n)
7. Selektivität in der Immunchemie/Biochemie wird oft als Kreuzreaktion gemessen.
Zusammenfassung Robustheit
1. Sehr individuelle Kriterien – sinnvolle Auswahl notwendig!
2. Wichtige Testphasen:
a)Änderung von Methodenparametern
b)Stabilität über die Zeit
c)Überführung in die Routine
3. Ringversuche bzw. Laborvergleichsmessungen
4. Wichtigstes Vergleichskriterium: Relative Verfahrensstandardabweichung
5. Extratipp: Maximale Diversität der Testproben sicherstellen (besonders bezüglich der Matrix)
Affinity extraction is one of the most powerful separation methods. Regarded to be indispensable in some research fields, such as proteomics, the application in other fields is still scarce or even waning. Many researchers fear the perceived complexity, cost and unreliability of this approach. The presentation gives a broad overview of the most important methods and discusses some important points for their implementation. Having considered these aspects, it should be easier to establish an affinity separation approach to resolve challenging analytical questions.
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.
A robust and sensitive method for the detection of the explosive trinitrotoluene (TNT) was developed. The detection limit was determined to be around 0.5 µg/L. The fast signal response of less than 1 minute shows that this approach is suitable for security and other time-critcal applications. In addition, the very low cross-reactivity highly reduces the number of false-positives in relation to competing techniques, including sniffer dogs. Due to the multianalyte ability of the SAW system, several explosives might be detected in parallel.
According to several recent studies, an unexpectedly high number of landmark papers seem to be not reproducible by Independent laboratories. Nontherapeutic antibodies used for research, diagnostic, food analytical, environmental, and other purposes play a significant role in this matter. Although some papers have been published offering suggestions to improve the situation, they do not seem to be comprehensive enough to cover the full complexity of this issue. In addition, no obvious improvements could be noticed in the field as yet. This article tries to consolidate the remarkable variety of conclusions and suggested activities into a more coherent conception. It is concluded that funding agencies and journal publishers need to take first and immediate measures to resolve these problems and lead the way to a more sustainable way of bioanalytical research, on which all can rely with confidence.
An improved antibody against the explosive pentaerythritol tetranitrate (PETN) was developed. The immunogen was designed by the concept of bioisosteric replacement, which led to an excellent polyclonal antibody with extreme selectivity and immunoassays of very good sensitivity. Compounds such as nitroglycerine, 2,4,6-trinitrotoluene, 1,3,5-trinitrobenzene, hexogen (RDX), 2,4,6-trinitroaniline, 1,3-dinitrobenzene, octogen (HMX), triacetone triperoxide (TATP), ammonium nitrate, 2,4,6-trinitrophenol and nitrobenzene were tested for potential cross-reactivity. The detection limit of a competitive enzyme-linked immunosorbent assay (ELISA) was determined to be around 0.5 µg/L. The dynamic range of the assay was found to be between 1 µg/L and 1000 µg/L, covering a concentration range of three decades. This work shows the successful application of the bioisosteric concept in immunochemistry by exchange of a nitroester to a carbonate diester. The antiserum might be used for the development of quick tests, biosensors, microtitration plate immunoassays, microarrays and other analytical methods for the highly sensitive detection of PETN, an explosive frequently used by terrorists, exploiting the extreme difficulty of its detection.
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.