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Identification of stroke-associated-antigens via screening of recombinant proteins from the human expression cDNA library (SEREX).

ABSTRACT: Because circulating antibodies against a variety of antigens have been detected in patients with coronary heart disease, carotid atherosclerosis and those who have suffered a stroke, it is suspected that immune response may be one of the mechanisms of atherogenesis The objective of this study is to identify novel antibodies in ischemic stroke patients by screening the expressed recombinant proteins using a human cDNA library (SEREX).To identify the candidate antigens, cDNA library was screened by SEREX using plasma from ten patients with ischemic stroke. Subsequently, via ELISA using recombinant proteins and synthetic peptides, the serum antibody levels were measured in two independent patient/healthy donor (HD) cohorts (142 and 78 in the 2nd screening and a validation cohort, respectively).The initial screening resulted in the identification of six candidate antigens. Of these antigens, replication protein A2 (RPA2) was determined to be the antigen associated with stroke (P?

SUBMITTER: Machida T

PROVIDER: S-EPMC4344740 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Identification of stroke-associated-antigens via screening of recombinant proteins from the human expression cDNA library (SEREX).

Machida Toshio T Kubota Motoo M Kobayashi Eiichi E Iwadate Yasuo Y Saeki Naokatsu N Yamaura Akira A Nomura Fumio F Takiguchi Masaki M Hiwasa Takaki T

Journal of translational medicine 20150222

<h4>Background</h4>Because circulating antibodies against a variety of antigens have been detected in patients with coronary heart disease, carotid atherosclerosis and those who have suffered a stroke, it is suspected that immune response may be one of the mechanisms of atherogenesis The objective of this study is to identify novel antibodies in ischemic stroke patients by screening the expressed recombinant proteins using a human cDNA library (SEREX).<h4>Methods</h4>To identify the candidate an ...[more]

PMID: 25890248

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Project description:BackgroundThe simplicity and potential of minimal invasive testing using serum from patients make auto-antibody based biomarkers a very promising tool for use in diagnostics of cancer and auto-immune disease. Although several methods exist for elucidating candidate-protein markers, immobilizing these onto membranes and generating so called macroarrays is of limited use for marker validation. Especially when several hundred samples have to be analysed, microarrays could serve as a good alternative since processing macro membranes is cumbersome and reproducibility of results is moderate.MethodsCandidate markers identified by SEREX (serological identification of antigens by recombinant expression cloning) screenings of brain and lung tumour were used for macroarray and microarray production. For microarray production recombinant proteins were expressed in E. coli by autoinduction and purified His-tag (histidine-tagged) proteins were then used for the production of protein microarrays. Protein arrays were hybridized with the serum samples from brain and lung tumour patients.ResultMethods for the generation of microarrays were successfully established when using antigens derived from membrane-based selection. Signal patterns obtained by microarrays analysis of brain and lung tumour patients' sera were highly reproducible (R = 0.92-0.96). This provides the technical foundation for diagnostic applications on the basis of auto-antibody patterns. In this limited test set, the assay provided high reproducibility and a broad dynamic range to classify all brain and lung samples correctly.ConclusionProtein microarray is an efficient means for auto-antibody-based detection when using SEREX-derived clones expressing antigenic proteins. Protein microarrays are preferred to macroarrays due to the easier handling and the high reproducibility of auto-antibody testing. Especially when using only a few microliters of patient samples protein microarrays are ideally suited for validation of auto-antibody signatures for diagnostic purposes.

Project description:BackgroundEimeria maxima is one of the most prevalent Eimeria species causing avian coccidiosis, and results in huge economic loss to the global poultry industry. Current control strategies, such as anti-coccidial medication and live vaccines have been limited because of their drawbacks. The third generation anticoccidial vaccines including the recombinant vaccines as well as DNA vaccines have been suggested as a promising alternative strategy. To date, only a few protective antigens of E. maxima have been reported. Hence, there is an urgent need to identify novel protective antigens of E. maxima for the development of neotype anticoccidial vaccines.MethodsWith the aim of identifying novel protective genes of E. maxima, a cDNA expression library of E. maxima sporozoites was constructed using Gateway technology. Subsequently, the cDNA expression library was divided into 15 sub-libraries for cDNA expression library immunization (cDELI) using parasite challenged model in chickens. Protective sub-libraries were selected for the next round of screening until individual protective clones were obtained, which were further sequenced and analyzed.ResultsAdopting the Gateway technology, a high-quality entry library was constructed, containing 9.2 × 106 clones with an average inserted fragments length of 1.63 kb. The expression library capacity was 2.32 × 107 colony-forming units (cfu) with an average inserted fragments length of 1.64 Kb. The expression library was screened using parasite challenged model in chickens. The screening yielded 6 immune protective genes including four novel protective genes of EmJS-1, EmRP, EmHP-1 and EmHP-2, and two known protective genes of EmSAG and EmCKRS. EmJS-1 is the selR domain-containing protein of E. maxima whose function is unknown. EmHP-1 and EmHP-2 are the hypothetical proteins of E. maxima. EmRP and EmSAG are rhomboid-like protein and surface antigen glycoproteins of E. maxima respectively, and involved in invasion of the parasite.ConclusionsOur results provide a cDNA expression library for further screening of T cell stimulating or inhibiting antigens of E. maxima. Moreover, our results provide six candidate protective antigens for developing new vaccines against E. maxima.

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Identification of stroke-associated-antigens via screening of recombinant proteins from the human expression cDNA library (SEREX).

Publications

Identification of stroke-associated-antigens via screening of recombinant proteins from the human expression cDNA library (SEREX).

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