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Bead-Based Chemistries to Quantitate Antibody Responses to Multiple Plasmodium Falciparum and Plasmodium Vivax Antigens
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| 자료유형 | 학위논문 |
|---|---|
| 서명/저자사항 | Bead-Based Chemistries to Quantitate Antibody Responses to Multiple Plasmodium Falciparum and Plasmodium Vivax Antigens. |
| 개인저자 | Ghag, Sonam Kaur. |
| 단체저자명 | University of Washington. Chemistry. |
| 발행사항 | [S.l.]: University of Washington., 2019. |
| 발행사항 | Ann Arbor: ProQuest Dissertations & Theses, 2019. |
| 형태사항 | 136 p. |
| 기본자료 저록 | Dissertations Abstracts International 81-05B. Dissertation Abstract International |
| ISBN | 9781088321324 |
| 학위논문주기 | Thesis (Ph.D.)--University of Washington, 2019. |
| 일반주기 |
Source: Dissertations Abstracts International, Volume: 81-05, Section: B.
Advisor: Rathod, Pradipsinh K. |
| 이용제한사항 | This item must not be sold to any third party vendors.This item must not be added to any third party search indexes. |
| 요약 | Each year malaria impacts approximately 219 million people and causes over 400,000 deaths. The worldwide parasite burden remains high, even with the availability of efficacious antimalarial drugs and vector control tools such as insecticides and bed nets. Despite large investments in malaria vaccine development, there is currently no deployable vaccine for malaria. However, individuals who live in malaria endemic regions develop naturally acquired immunity (NAI). Thus, good vaccine candidates may be identified by studying serum antibody responses in malaria patients who live in endemic areas.Protein arrays, which interrogate patient serum antibody binding to many proteins simultaneously, are the primary method of antigen prioritization. However, protein arrays are fabricated by generating unpurified malaria antigens in bacteria and directly spotting lysates onto slides without validation of correct antigen folding or purification. Results from protein arrays can be affected by the sample impurity where positive binding events may be masked by background protein interactions or unrecognizable epitope presentation. Thus, technical improvements are needed to control protein production and protein exposure to patient sera. More recently, multiplexing bead-based assays have been applied to investigate malaria patient immune responses where spectrally distinct bead sets are coated with unique antigens of interest. For such assays, antigens traditionally are individually purified before coupling onto beads. To produce a more high-throughput method of patient immune response dissection using a large panel of antigens, bead-based chemistries were developed to modify multiplexing beads with a small linker molecule or with antigen capturing antibody. These modified beads capture malaria antigens directly from translation lysates, essentially purifying antigens in a single step. Both chemistries utilized the capture of recombinant antigens via a highly specific protein tag, HaloTag or C-tag. Although both systems successfully capture tagged antigens from translation lysates, the C-tag capture system proved to be more reliable and reproducible. Using recombinant malaria antigen merozoite surface protein 1 (MSP1)-coated beads and standard anti-MSP1 detecting antibodies, a 105-fold signal increase was observed in comparison to green fluorescence protein (GFP)-coated beads. Malaria patient sera responses to recombinant MSP1 were also measured. When assessed against MSP1-coated beads, serum from a high immune responsive patient gave an 8.5-fold increase in comparison to GFP-coated beads, while a low immune responsive patient gave a 2-fold increase. These results agree with previously reported protein microarray dissection of the same patient serum samples. The bead chemistries developed here make it possible to interrogate multiple malaria antigens simultaneously without demanding timely antigen purification. Furthermore, the multiplexing assay present antigen epitopes in a more accessible configuration while utilizing small amount of precious patient sera. The platform developed here will provide a high-throughput method of identifying specific patient antibody-antigen binding events and has the potential to customize bead arrays to model populations with high antigenic variation. |
| 일반주제명 | Chemistry. Biochemistry. Biology. |
| 언어 | 영어 |
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