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Immunanalytik
Thomas Scheper TCITCITCI Institut fürTechnische Chemie
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Thomas Scheper TCITCITCI Institut fürTechnische Chemie
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Thomas Scheper TCITCITCI Institut fürTechnische Chemie
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Thomas Scheper TCITCITCI Institut fürTechnische Chemie
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Thomas Scheper TCITCITCI Institut fürTechnische Chemie
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Thomas Scheper TCITCITCI Institut fürTechnische Chemie
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Thomas Scheper TCITCITCI Institut fürTechnische Chemie
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P bl P l kl l M kl l
Thomas Scheper TCITCITCI Institut fürTechnische Chemie
Problem: Polyklonal zu Monoklonal
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Thomas Scheper TCITCITCI Institut fürTechnische Chemie
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Thomas Scheper TCITCITCI Institut fürTechnische Chemie
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Assay: heterogen – homogeny g g
Thomas Scheper TCITCITCI Institut fürTechnische Chemie
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Thomas Scheper TCITCITCI Institut fürTechnische Chemie
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Thomas Scheper TCITCITCI Institut fürTechnische Chemie
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Thomas Scheper TCITCITCI Institut fürTechnische Chemie
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Thomas Scheper TCITCITCI Institut fürTechnische Chemie
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Thomas Scheper TCITCITCI Institut fürTechnische Chemie
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Development of lateral flow assays with Sartorius and Dr Noack A G
Thomas Scheper TCITCITCI Institut fürTechnische Chemie
Development of lateral flow assays with Sartorius and Dr. Noack A.G.
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What is a Rapid Test?What is a Rapid Test?
Relies on the natural function of antibodies Relies on the natural function of antibodies (Biosensor)(Biosensor)
Simple to useSimple to useSimple to useSimple to use
Test resultTest result within a few minuteswithin a few minutes
No (or simple) instrumentation requiredNo (or simple) instrumentation required
Inexpensive Inexpensive productionproduction
Sensitivities similar to or greater than ELISASensitivities similar to or greater than ELISASensitivities similar to or greater than ELISA Sensitivities similar to or greater than ELISA
Detection level in the ng/mL range (we need pg/ml!!)Detection level in the ng/mL range (we need pg/ml!!)
Thomas Scheper TCITCITCI Institut fürTechnische Chemie
g g ( g )g g ( g )
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Components of a Rapid TestComponents of a Rapid Test
Capture (test) line Control lineCapture (test) line Co t o e
S l P dMembrane Absorbent Pad
Sample PadWith labelled AK
Conjugate PadCo jugate ad
Thomas Scheper TCITCITCI Institut fürTechnische ChemieAdhesive
Plastic backing
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Thomas Scheper TCITCITCI Institut fürTechnische Chemie
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Application for Rapid TestsApplication for Rapid Testspp ppp p
Environmental Food Testing Military VeterinaryEnvironmental Food Testing Military Veterinary
Water Testing GM Foods Germ Warfare Feline Cancer
Pesticides
Dust Mite
E-Coli
Salmonella
Explosives
Chemical
BSE
Canine HeartDust Mite Testing
Salmonella Chemical Warfare
Canine Heart Worm
Disease STD‘s Fertility Drugs of Abuse
Malaria Chlamydia Pregnancy Cocainea a a
Hepatitis B
T b l i
C a yd a
Syphilis
HIV
eg a cy
LH
FSH
Coca e
Cannabis
E tThomas Scheper TCITCITCI Institut für
Technische Chemie
Tuberculosis HIV FSH Ecstasy
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Botulinum Neurotoxin
Thomas Scheper TCITCITCI Institut fürTechnische Chemie
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• Produced by the sporeforming, anaerobic Clostridium BotulinumClostridium Botulinum
• 7 immunologically distinct forms (indicated with letters from A-G) are acknowledged
• Most poisonous substance known (mouse• Most poisonous substance known (mouse intraperitoneal LD50 (BoNT/D) of 0.4 ng/kg)
• Consists of a 50-kDa light (L) chain and a 100-kDa heavy (H) chain linked by a disulfide bond
• The toxin causes flaccid muscle paralysis
Thomas Scheper TCITCITCI Institut fürTechnische Chemie
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Food Monitoring
Fodder MonitoringFodder Monitoring
BiBioweapon
Thomas Scheper TCITCITCI Institut fürTechnische Chemie
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Medical application of Botulinum Neurotoxin
• Strabismus
• Migraine headacheMigraine headache
• Spasticity
• Various dystonias
• Stroke
• Plastic surgeryPlastic surgery
Thomas Scheper TCITCITCI Institut fürTechnische Chemie
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AimsAims
D l t fD l t f
ss
Development of an Development of an immunochromatographic assay for immunochromatographic assay for g p yg p ybotulinum toxin Type Dbotulinum toxin Type D
Sensitivity Sensitivity << 50 pg/mL 50 pg/mL
E l ti f th t t ith i ti t tE l ti f th t t ith i ti t tEvaluation of the test with existing test Evaluation of the test with existing test methods.methods.
Thomas Scheper TCITCITCI Institut fürTechnische Chemie
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Lateral Flow AssayLateral Flow Assay-- Sandwich assay Sandwich assay (multi epitopes)(multi epitopes)
-- Inhibition assay Inhibition assay (single epitope)(single epitope)
-- Competitive assay Competitive assay (single epitope)(single epitope)
-- Serological assay Serological assay (antibody detection)(antibody detection)
Botulinum Toxin: Two differentBotulinum Toxin: Two differentBotulinum Toxin: Two different Botulinum Toxin: Two different antibodies are needed to assure antibodies are needed to assure
specificityspecificityThomas Scheper TCITCITCI Institut für
Technische Chemie
specificityspecificity
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Sandwich assaySandwich assay
P l l l Ab Detector
yy
Polyclonal Ab Detector reagent
Botulinum Toxin Type D Analyte
Monoclonal Ab Capture Monoclonal Ab Reagent
Thomas Scheper TCITCITCI Institut fürTechnische Chemie
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Detector Reagent
Mobile sandwich Analytecomplex
Specification Reagent
CaptureSecondary Ab
n Reagent
Capture Reagent
Secondary Ab
Thomas Scheper TCITCITCI Institut fürTechnische Chemie
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Rapid TestRapid Test0.5 cm
Absorbent Pad
ap d estap d est
• cellulose paper wick
Thomas Scheper TCITCITCI Institut fürTechnische Chemie
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0.5 cm Rapid TestRapid TestMembrane
ap d estap d estMembrane
• Nitrocellulose Membrane (Unisart CN 200, Sartorius AG)
Capture (test) zoneCapture (test) zone• Coating of 1.25 mg/mL goat anti g g g
mouse IgG or streptavidine
• Dispensing volume 0 8 µl/cm• Dispensing volume 0.8 µl/cm.
• The membrane was dried 2h at 30 °C
Thomas Scheper TCITCITCI Institut fürTechnische Chemie
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Rapid TestRapid Test0.5 cm ap d estap d est
Sample Pad
Gl fib t i (R 2040 R• Glass fibre strips (Reemay 2040, Reemay Ltd.)
• Impregnation with 20 mM borate buffer + Sucrose+ Protein + Tenside
Thomas Scheper TCITCITCI Institut fürTechnische Chemie
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BoNT Type D ImmunoassayBoNT Type D ImmunoassayPolyclonal B NT A tib dBoNT Antibody(Chicken)Labelled
BoNT Type Ddifferent EpitopesLabelled
B
different Epitopes
M l l
B
Biotinylated Polyclonal
MonoclonalAnti BoNT Antibody(Mouse)Biotinylated Polyclonal
BoNT Antibody(Goat)
(Mouse)
Thomas Scheper TCITCITCI Institut fürTechnische Chemie
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BoNT Type D ImmunoassayBoNT Type D Immunoassayyp yyp y
Chicken IgY Anti-Chicken
BoNT IgY
Mouse IgG
4 hours 28 Min
Thomas Scheper TCITCITCI Institut fürTechnische Chemie
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ProcedureProcedure
P ti f t d d t ti• Preparation of standard concentration range + polyclonal Ab Detector Reagent+ monoclonal Ab Specification Reagent
• Sample pre treatment (Incubation) (4 h at 37°C)• Sample pre-treatment (Incubation) (4 h at 37 C)
• 150 µL of sample (18 min)
• 100 µL washing buffer (15 min)
• Total test time 4.5 h
• label: latex black carbonThomas Scheper TCITCITCI Institut für
Technische Chemie
• label: latex, black carbon
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SamplesSamplesSa p esSa p es
5 pg/mL50 pg/mL0,5 ng/mL5 ng/mL50 ng/mL Negative5 pg/mL50 pg/mL0 5 ng/mL5 ng/mL50 ng/mL Negative5 pg/mL50 pg/mL0.5 ng/mL5 ng/mL50 ng/mL Negative
Thomas Scheper TCITCITCI Institut fürTechnische Chemie
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ResultsResultsDevelopment of an immunochromatographic Development of an immunochromatographic
esu tsesu tsp g pp g p
assay (latex or black carbon)assay (latex or black carbon)
S i i iS i i i 0 / L b / l!!0 / L b / l!!Sensitivity Sensitivity << 50 ng/mL but not pg/ml!! 50 ng/mL but not pg/ml!!
Improved test time (4 5 h)Improved test time (4 5 h)Improved test time (4.5 h)Improved test time (4.5 h)
Quantitative analysis in principle possibleQuantitative analysis in principle possible
with image analysis (linear behavior) with image analysis (linear behavior)
⇒⇒ Enhancement necessaryEnhancement necessary
Thomas Scheper TCITCITCI Institut fürTechnische Chemie
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Use of gold labelled antibodies(detector antibodies)
Thomas Scheper TCITCITCI Institut fürTechnische Chemie
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SamplesSamplesSamplesSamples
5 pg/mL50 pg/mL0,5 ng/mL5 ng/mL50 ng/mL Negative5 pg/mL50 pg/mL0 5 ng/mL5 ng/mL50 ng/mL Negative5 pg/mL50 pg/mL0.5 ng/mL5 ng/mL50 ng/mL Negative
Thomas Scheper TCITCITCI Institut fürTechnische Chemie
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Polyclonal B NT A tib dBoNT Antibody(Chicken) BoNT Type D
different Epitopes
B
different Epitopes
M l l
B
Biotinylated Polyclonal
MonoclonalAnti BoNT Antibody(Mouse)Biotinylated Polyclonal
BoNT Antibody(Goat)
(Mouse)
Thomas Scheper TCITCITCI Institut fürTechnische Chemie
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BoNT Type D Immunoassay IIBoNT Type D Immunoassay II
Goat IgGB gB
BoNT
Mouse IgG
Thomas Scheper TCITCITCI Institut fürTechnische Chemie
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Outlook: SamplesOutlook: Samples
500 ng/mL 5 ng/mL 50 pg/mL
50 ng/mL
g
0,5 ng/mL negative
Thomas Scheper TCITCITCI Institut fürTechnische Chemie
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Algae toxin microcystineAlgae toxin microcystineCyanobacteria produce highly potent toxins,Cyanobacteria produce highly potent toxins, e.g. microcystins (hepatotoxic) or anatoxins (neurotoxic).
Mi i li ib lMicrocystins are cyclic nonribosomal peptides.
Microcystins consist of several uncommon non-proteinogenic amino acids:
- Dehydroalanine derivatives
- Special b-amino acid ADDA ((all S all E) 3 Amino 9 methoxy 2 6 8((all-S,all-E)-3-Amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-diene acid)
Thomas Scheper TCITCITCI Institut fürTechnische Chemie
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Detection of microcystineDetection of microcystine
P blProblem:
- Accumulation of microcystins in theAccumulation of microcystins in thefood chain
- Toxicity for plants and animalsincluding humansg
- Critical microcystine concentration inliquid samples
Thomas Scheper TCITCITCI Institut fürTechnische Chemie
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Immobilized secondary antibody (testline)
Immobilized antigen forproducing a test signalAnalyte
Gold/Latex-labelled antibody
(testline)
Membrane
Conjugate fleecePlastic component
Sample adsorption fleece
Adsorption fleece
Competitive test formatep
Signal intensity is anti-proportional in relation to the antigen concentration
Thomas Scheper TCITCITCI Institut fürTechnische Chemie
to the antigen concentration
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FACS – Fluorescence Activated Cell Sorting
488 nm laser FSC Detector
SSC
Deflection
Detector
Deflection plate
Sorted scells
Thomas Scheper TCITCITCI Institut fürTechnische Chemie
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FACS – Fluorescence Activated Cell Sorting
Correlation between the amount of surface IgG1and secretion rate.
Thomas Scheper TCITCITCI Institut fürTechnische Chemie
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FACS – Fluorescence Activated Cell Sorting
Cells covered with a surface affinity matrix, which captures the secreted product with antibodies linked to cell surface proteins via biotin-avidin bridges.
Thomas Scheper TCITCITCI Institut fürTechnische Chemie
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Protein analysis via immunoreactionsProtein analysis via immunoreactions
Thomas Scheper TCITCITCI Institut fürTechnische Chemie
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1 immobilization of antibodies 2 sample injection1. immobilization of antibodies 2. sample injection
3 hi 4 l ti3. washing 4. elution
antibody cartridge antigen, (sample) byproducts
Thomas Scheper TCITCITCI Institut fürTechnische Chemie
y g g p yp
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system control
sample
system controldata processing
equilibration buffer
waste
elution buffercartridge fluorescence
detector
waste
pump injection valve selector
Thomas Scheper TCITCITCI Institut fürTechnische Chemie
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8
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AT III concentration [µg/ml]
080 100 120 140 160 180 200 220 240 260 280 300
withoutBSA addition
addition of1mg/ml BSA
addition of10 mg/ml BSA
Thomas Scheper TCITCITCI Institut fürTechnische Chemie
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120
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0 200 400 600 800
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Peakno.0 200 400 600 800
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MV-15%
Peakno.(mean value 99,97; standard deviation 5.97; cv=6.04%) (mean value 99,79; standard deviation 4.11; cv=4.12%)
Thomas Scheper TCITCITCI Institut fürTechnische Chemie
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7070
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00 40 80 120 160 200 240 280
Cultivation time [h]
Thomas Scheper TCITCITCI Institut fürTechnische Chemie
Cultivation time [h]
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Aptamere
Thomas Scheper TCITCITCI Institut fürTechnische Chemie
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Was sind Aptamere ?Was sind Aptamere ?
Aus dem Griechischen „haptein“ : t bi d “ h ft “„an etwas binden“, „haften“
Oligonukleotide (DNA oder RNA) mit der FähigkeitOligonukleotide (DNA oder RNA) mit der Fähigkeit, beinahe jedes Zielmolekül mit hoher
Bindungsstärke und hoher Spezifität erkennen und g pbinden zu können
Thomas Scheper TCITCITCI Institut fürTechnische Chemie
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F kti i i d l k lF kti i i d l k lFunktionsprinzip der molekularen Funktionsprinzip der molekularen ErkennungErkennungErkennungErkennung
nach Schlüsselnach Schlüssel--SchlossSchloss--Prinzip/ Prinzip/ InducedInduced--fitfit--MechanismusMechanismus
Bildung eines AptamerBildung eines Aptamer--ZielmolekülZielmolekül--KomplexesKomplexes
h h Affi ität d S ifität üb d Zi l l külh h Affi ität d S ifität üb d Zi l l külhohe Affinität und Spezifität gegenüber dem Zielmolekülhohe Affinität und Spezifität gegenüber dem Zielmolekül
Inhibierung der biologischen Aktivität des ZielmolekülsInhibierung der biologischen Aktivität des Zielmoleküls
Thomas Scheper TCITCITCI Institut fürTechnische Chemie
(www.ufz.de)
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SELEX SELEX -- KreisprozessKreisprozessI k b ti & Bi dI k b ti & Bi dInkubation & BindungInkubation & Bindung
WaschenWaschenEl tiEl tiElutionElutionAmplifikationAmplifikationR i iR i iReinigungReinigung
Thomas Scheper TCITCITCI Institut fürTechnische Chemie
(Proske et al. 2005)
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Vorteile von Aptameren gegenüberVorteile von Aptameren gegenüberVorteile von Aptameren gegenüber Vorteile von Aptameren gegenüber AntikörpernAntikörpernpp
Identifikation durch automatisierbaren Identifikation durch automatisierbaren in vitroin vitro ProzessProzessbenötigt keine Tiere oder Zelllinienbenötigt keine Tiere oder Zelllinienmit gewünschte Eigenschaften, unter definierten mit gewünschte Eigenschaften, unter definierten Selektionsbedingungen (z B pHSelektionsbedingungen (z B pH Wert) herstellbarWert) herstellbarSelektionsbedingungen (z.B. pHSelektionsbedingungen (z.B. pH--Wert) herstellbarWert) herstellbargegen gering immunogene Zielmolekülegegen gering immunogene Zielmoleküleleicht reproduzierbar, geringe Batchleicht reproduzierbar, geringe Batch--to Batch Variationto Batch Variationleicht reproduzierbar, geringe Batchleicht reproduzierbar, geringe Batch to Batch Variationto Batch Variationpharmakokinetischen Parameter veränderbarpharmakokinetischen Parameter veränderbarKopplung mit Reportermolekülen & Immobilisierung Kopplung mit Reportermolekülen & Immobilisierung dede-- und renaturierbarund renaturierbarkein Beweis für Immunogenitätkein Beweis für Immunogenität & Toxizität & Toxizität
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Aptamer AnwendungsbeispieleAptamer Anwendungsbeispiele
Aptamere sind geeignet für viele Anwendungen in• Aptamere sind geeignet für viele Anwendungen in Bereichen wie• DiagnostikDiagnostik• Therapeutik• Apheresis• Imaging• Umweltanalytik• Proteomics• Proteomics• Biochips• Spezifische Prozesskontrolle (Kontaminationskontrolle)p ( )
Thomas Scheper TCITCITCI Institut fürTechnische Chemie
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Wie funktionieren DNA Chips?
Oligonukleotide als
DNA-Target-Moleküle
Hybridisierungkomplementärer
EinzelsträngeOligonukleotide alsSondenmoleküle
e s gezur Doppelhelix
Chip
Thomas Scheper TCITCITCI Institut fürTechnische Chemie
Chip
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Wie funktionieren Proteinchips?Wie funktionieren Proteinchips?
Thomas Scheper TCITCITCI Institut fürTechnische Chemie
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Vorteile der Aptamer ChipsVorteile der Aptamer ChipsAptamere monoklonale Antikörper
- Neue in vitro Technologie - hohe Kreuzreaktivitäten(Neue Moleküle-Patentierbarkeit),- immer in gleicher Qualität - Intrazelluläre Targetsimmer in gleicher Qualität - Intrazelluläre Targetsherstellbar sind in vivo kaum möglich- mehr Modifikationen möglich,z B für Immobilisierungenz.B. für Immobilisierungen- Alle Zielmoleküle möglich: - Nicht viele Lösungsmittel möglichz.B. „kleine Moleküle“, zelltoxische; wasserunlösliche; intrazelluläre;wasserunlösliche; intrazelluläre;Zielmoleküle, die sichnur in einem Zuckerrest oder - als Proteine in AnwendungPhosphatrest unterscheiden;... empfindlichPhosphatrest unterscheiden;... empfindlich- Hohe Spezifität durch speziellein vitro Methodennicht immunogen
Thomas Scheper TCITCITCI Institut fürTechnische Chemie
- nicht-immunogen,
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Probleme bei Antikörper-ProteinchipsProbleme bei Antikörper Proteinchips
•Proteine verhalten sich nicht so einfach wie Nukleinsäuren
•Funktion der Proteine abhängig von:P i h f lli 3D k L i bili–Präziser, sehr anfälliger 3D- Struktur, Langzeitstabilität
–muss im Array erhalten werdenP i P i I k i•Protein-Protein Interaktionen:
–Bindungsstärken und –Stabilität nicht standardisiert wie bei NukleinsäurenNukleinsäuren
Aptamer-Methodik ist komplementär zu und in einigen FällenAptamer Methodik ist komplementär zu und in einigen Fällen geeigneter als Antikörper-Methodik⇒ Substitution der Antikörper durch Aptamere
Thomas Scheper TCITCITCI Institut fürTechnische Chemie
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Vergleich DNA / Protein / Aptamer Chip
DNA-Chip AK-Chip Aptamer-Chip
Transkriptomanalyse
Glasssubstrat
Proteomanalyse
Membransubstrat
Proteomanalyse
Neue Membran
Thomas Scheper TCITCITCI Institut fürTechnische Chemie