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Protein Microarray Capture Molecules and Their Limitations Protein microarray Capture Molekyler og deres begrænsninger

Protein and Antibody Microarray Chips Protein og Antibody microarray Chips

Capture Molecules and Their Limitations - Protein Chips Capture molekylers og deres begrænsninger - Protein Chips

Capture Molecules and their Limitations Capture Molekyler og deres begrænsninger
The most common form of analytical protein arrays are antibody microarrays in which antibodies (or antibody mimics) that bind specific antigens are arrayed on a glass slide at high density. Den mest almindelige form for analytisk protein arrays er antistof microarrays, hvor antistoffer (eller antistof efterligner), der binder specifikke antigener er opstillet på en glas-slide med høj massefylde. A lysate is passed over the array and the bound antigen is detected after washing. En lysate er gået over array og bundet antigen påvises efter vask. The biggest challenge with these methods is producing reagents that identify the protein of interest and with high enough specificity in a high-throughput fashion.  Although antibodies are the traditional reagent of choice for detecting proteins in complex mixtures, polyclonal sera are often not specific and are expensive to produce. Den største udfordring med disse metoder, er der producerer reagenser til at identificere de proteiner af interesse og med høj nok specificitet i en høj overførselshastighed mode. Selv om antistoffer er den traditionelle reagens valg til påvisning af proteiner i komplekse blandinger, polyklonale sera er ofte ikke specifikt og åre dyrere at producere. Also, the conventional hybridoma method of producing highly specific monoclonal antibodies is time-consuming, laborious and costly (8). Også de konventionelle hybridomer metode til at producere meget specifikke monoklonale antistoffer er tidkrævende, besværlige og omkostningskrævende (8).

Several studies using antibodies have recently been conducted despite the obstacles in obtaining specific antibodies. Flere undersøgelser ved hjælp af antistoffer er for nylig blevet gennemført på trods af de forhindringer i at opnå specifikke antistoffer.  In one of the largest studies to date, Sreekumar et al. I en af de største undersøgelser til dato, Sreekumar et al. spotted 146 distinct antibodies on glass to monitor the alternations of protein quantity in LoVo colon carcinoma cells. plettet 146 forskellige antistoffer på glas til at overvåge alternations af protein mængde i LoVo colon carcinom celler. Their results revealed radiation-induced up-regulation of many interesting proteins, including p53, DNA fragmentation factor 40 and 45, tumour necrosis factor-related ligand, as well as down-regulated proteins (58). Deres resultater viste stråling-induceret op-regulering af mange interessante proteiner, herunder p53, DNA fragmentering faktor 40 og 45, tumor nekrose faktor-relaterede ligand, såvel som ned-regulerede proteiner (58).

To date, most antibody microarrays were produced with several dozen or a few hundred commercially available poly- or mono-clonal antibodies.  Although tens of thousands of antibodies are commercially available, this number is insufficient because for most proteins there are no available antibodies.  The fact that many antibodies are glycosylated and contain large protein-based supporting structures means that they often cross-react with more than one target protein.  This can contribute to a large number of false positives (9).  Thus another problem has been obtaining high-specificity antibodies. Til dato har de fleste antistof microarrays blev produceret med flere dusin eller nogle få hundred kommercielt tilgængelige poly-eller mono-klonede antistoffer. Selv om titusinder af antistoffer er kommercielt tilgængelige, dette nummer er utilstrækkelig, fordi de fleste proteiner, der ikke findes antistoffer. kendsgerning, at mange antistoffer er glykosyleret og indeholder store protein-baserede støtte til strukturer betyder, at de ofte på tværs af reagere med mere end ét mål protein. Dette kan bidrage til et stort antal falske positiver (9). således et andet problem har været at opnå høj specificitet antistoffer.

One of the greatest problem with antibody arrays is specificity. En af de største problem med antistoffet arrays er specificitet. Proteins are often present in a very large dynamic range (106); thus, reagents that might have high affinity for one protein, but are low affinity for another will still exhibit detection of the lower affinity protein if it is much more prevalent (9). Proteiner er ofte til stede i et meget stort dynamikområde (106), og således, reagenser, der kan have høj affinitet for et protein, men er lav affinitet til en anden vil stadig udviser afsløring af den lavere affinitet protein, hvis det er langt mere udbredt (9) . One group investigated the ability of 115 well-characterized antibody–antigen pairs to react in high-density microarrays on modified glass slides. En gruppe undersøgte muligheden af 115 vel-karakteriseret antistof-antigen par til at reagere i høj massefylde microarrays på modificeret glasslides. 30% of the pairs showed the expected linear relationships, indicating that a fraction of the antibodies were suitable for quantitative analysis (33). 30% af det par viste den forventede lineære relationer, hvilket indikerer, at en brøkdel af de antistoffer var egnet til kvantitativ analyse (33). Many groups have been using sandwich assays to avoid this problem.  A sandwich assay is performed by spotting the first antibody on the array and then detecting the using a second antibody that recognizes a different part of the proteins. Mange grupper har brugt sandwich assays at undgå dette problem. En sandwich assay udføres ved at afdække de første antistof på array og derefter påvise ved hjælp af et andet antistof, der genkender en anden del af proteiner. This approach dramatically increases the specificity of the antigen detection, but required that a least two high-quality antibodies exist for each antigen to be detected (8). Denne fremgangsmåde dramatisk øger specificiteten af det antigen, afsløring, men krævede, at mindst to af høj kvalitet antistoffer findes for hvert antigen, der skal opdages (8).

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References for Protein and Antibody Microarrays Referencer for protein og Antibody Microarrays

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Introduction and Background to Protein Chips and Antibody Chips. Indledning og baggrund for protein-chips og Antibody Chips.

Types of Antibody and Protein Chips Typer af antistof og Protein Chips