Optimization of immunoassay parameters in multiplex in the high throughput protein detection technique Proximity Extension Assay
The ability to detect protein-based biomarkers, which are linked to different diseases like colorectal cancer, is very important as a diagnostic tool. Usually complex biological samples like blood are studied which will contribute to different technical issues when performing an assay. The aim with...
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| Formato: | Second cycle, A2E |
| Lenguaje: | Inglés Inglés |
| Publicado: |
2012
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| Acceso en línea: | https://stud.epsilon.slu.se/5068/ |
| _version_ | 1855570783822151680 |
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| author | Källman, Christine |
| author_browse | Källman, Christine |
| author_facet | Källman, Christine |
| author_sort | Källman, Christine |
| collection | Epsilon Archive for Student Projects |
| description | The ability to detect protein-based biomarkers, which are linked to different diseases like colorectal cancer, is very important as a diagnostic tool. Usually complex biological samples like blood are studied which will contribute to different technical issues when performing an assay. The aim with the project is to optimize and develop the high throughput protein detection technique Proximity Extension Assay, PEA, into a 96-plex panel, in hopes of discovering an expression profile for colorectal cancer. PEA was developed by Olink Bioscience and allows specific proteins in a sample to be quantitatively transformed into nucleic acid sequences that are subsequently detected and quantified with real-time PCR. Two proximity probes containing oligonucleotide sequences bind pairwise to target protein and when brought in proximity, a DNA polymerase will extend a hybridization arm from one probe over to the second forming a double-stranded DNA sequence that can serve as a template in real-time PCR. The results showed that there is no significant difference in sensitivity, specificity, recovery or efficiency between assays performed in single plex, lower or higher degree of multiplex. Higher sensitivity of the assay was achieved by optimization of factors such as, concentrations of proximity probes and hybridization oligo arm. The results also show that the recovery will not be affected by higher concentration of plasma or by using other assay formats. Work proceeds to develop a 96-plex panel with just as high sensitivity and recovery, which would make PEA the most, multiplexed immunoassay with high sensitivity so far. |
| format | Second cycle, A2E |
| id | RepoSLU5068 |
| institution | Swedish University of Agricultural Sciences |
| language | Inglés Inglés |
| publishDate | 2012 |
| publishDateSort | 2012 |
| record_format | eprints |
| spelling | RepoSLU50682012-11-27T14:03:49Z https://stud.epsilon.slu.se/5068/ Optimization of immunoassay parameters in multiplex in the high throughput protein detection technique Proximity Extension Assay Källman, Christine Food science and technology The ability to detect protein-based biomarkers, which are linked to different diseases like colorectal cancer, is very important as a diagnostic tool. Usually complex biological samples like blood are studied which will contribute to different technical issues when performing an assay. The aim with the project is to optimize and develop the high throughput protein detection technique Proximity Extension Assay, PEA, into a 96-plex panel, in hopes of discovering an expression profile for colorectal cancer. PEA was developed by Olink Bioscience and allows specific proteins in a sample to be quantitatively transformed into nucleic acid sequences that are subsequently detected and quantified with real-time PCR. Two proximity probes containing oligonucleotide sequences bind pairwise to target protein and when brought in proximity, a DNA polymerase will extend a hybridization arm from one probe over to the second forming a double-stranded DNA sequence that can serve as a template in real-time PCR. The results showed that there is no significant difference in sensitivity, specificity, recovery or efficiency between assays performed in single plex, lower or higher degree of multiplex. Higher sensitivity of the assay was achieved by optimization of factors such as, concentrations of proximity probes and hybridization oligo arm. The results also show that the recovery will not be affected by higher concentration of plasma or by using other assay formats. Work proceeds to develop a 96-plex panel with just as high sensitivity and recovery, which would make PEA the most, multiplexed immunoassay with high sensitivity so far. Möjligheten att upptäcka proteinbaserade biomarkörer, som är kopplade till olika sjukdomar som kolorektal cancer, är mycket viktig som ett diagnostiskt verktyg. Vid studier av komplexa biologiska prover som blod bidrar matrisen vanligtvis till olika tekniska problem. Syftet med projektet är att optimera och utveckla proteindetektionsmetoden Proximity Extension Assay, PEA, till en 96-plex panel, med förhoppning om att upptäcka en uttrycksprofil för kolorektal cancer. PEA har utvecklats av Olink Bioscience och tillåter specifika proteiner i ett prov att omvandlas till nukleotidsekvenser som senare upptäcks och kvantifieras med realtids-PCR. Två prober, så kallade proximity probes, består av oligonukleotidsekvenser som binder parvis till målproteinet. När de kommer i närhet, extenderar ett DNA-polymeras en hybridiseringsarm från en prob över till den andra och bildar en dubbelsträngad DNA-sekvens som kan fungera som en templat i realtids-PCR . Resultaten visade att det inte finns någon signifikant skillnad i känslighet, specificitet, återhämtning eller effektivitet mellan de assays som genomfördes i single-plex, lägre eller högre grad av multiplex. Högre känslighet uppnåddes genom att optimera faktorer som koncentrationen av prob respektive hybridiseringsarm. Resultaten visar också att återhämtningen inte påverkas av högre koncentration av plasma eller genom att använda andra analysformat. Arbete fortsätter med att utveckla en 96-plex panel med lika hög känslighet och återhämtning vilket skulle göra PEA till den hittills mest multiplexade immunoassay med hög känslighet. 2012-11-16 Second cycle, A2E NonPeerReviewed application/pdf en https://stud.epsilon.slu.se/5068/1/kallman_c_121116.pdf Källman, Christine, 2012. Optimization of immunoassay parameters in multiplex in the high throughput protein detection technique Proximity Extension Assay . Second cycle, A2E. Uppsala: (NL, NJ) > Dept. of Food Science <https://stud.epsilon.slu.se/view/divisions/OID-550.html> urn:nbn:se:slu:epsilon-s-1886 eng |
| spellingShingle | Food science and technology Källman, Christine Optimization of immunoassay parameters in multiplex in the high throughput protein detection technique Proximity Extension Assay |
| title | Optimization of immunoassay parameters in multiplex in the high throughput protein detection technique Proximity Extension Assay |
| title_full | Optimization of immunoassay parameters in multiplex in the high throughput protein detection technique Proximity Extension Assay |
| title_fullStr | Optimization of immunoassay parameters in multiplex in the high throughput protein detection technique Proximity Extension Assay |
| title_full_unstemmed | Optimization of immunoassay parameters in multiplex in the high throughput protein detection technique Proximity Extension Assay |
| title_short | Optimization of immunoassay parameters in multiplex in the high throughput protein detection technique Proximity Extension Assay |
| title_sort | optimization of immunoassay parameters in multiplex in the high throughput protein detection technique proximity extension assay |
| topic | Food science and technology |
| url | https://stud.epsilon.slu.se/5068/ https://stud.epsilon.slu.se/5068/ |