High-throughput screening of T7 phage display and protein microarrays as a methodological approach for the identification of IgE-reactive components

Segundo‐Acosta, Pablo San; Garranzo‐Asensio, María; Oeo-Santos, Carmen; Montero‐Calle, Ana; Quiralte, Joaquín; Cuesta‐Herranz, Javier; Villalba, Mayte; Barderas, Rodrigo

doi:10.1016/j.jim.2018.02.011

Cited by 14 publications

(12 citation statements)

References 38 publications

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“…Talwar et al (2015) developed a T7 phage displayed library to detect sarcoidosis and TB by a panel of novel antigens. San Segundo-Acosta et al (2018) identified allergenic peptides and mimotopes from olive pollen and mustard seeds using T7 phage displayed library. Cytidine-5-triphosphate synthase1-selective inhibitory peptide from random peptide library displayed on T7 phage was discovered by Sakamoto et al (2017).…”

Section: Discussionmentioning

confidence: 99%

Ribokinase screened from T7 phage displayed Mycobacterium tuberculosis genomic DNA library had good potential for the serodiagnosis of tuberculosis

Luo

Wang

Liu

et al. 2019

Appl Microbiol Biotechnol

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Tuberculosis caused by Mycobacterium tuberculosis (M. tuberculosis) is the leading cause of death among infectious diseases in the worldwide. Lack of more sensitive and effective diagnostic reagents has increased the awareness of rapid diagnosis for tuberculosis. In this study, T7 phage displayed genomic DNA library of M. tuberculosis was constructed to screen the antigens that specially bind with TB-positive serum from the whole genome of M. tuberculosis and to improve the sensitivity and specificity of tuberculosis serological diagnosis. After three rounds of biopanning, results of DNA sequencing and BLAST analysis showed that 19 positive phages displayed four different proteins and the occurrence frequency of the phage which displayed ribokinase was the highest. The results of indirect ELISA and dot immunoblotting indicated that representative phages could specifically bind to tuberculosis-positive serum. The prokaryotic expression vector containing the DNA sequence of ribokinase gene was then constructed and the recombinant protein was expressed and purified to evaluate the serodiagnosis value of ribokinase. The reactivity of the recombinant ribokinase with different clinical serum was detected and the sensitivities and specificities in tuberculosis serodiagnosis were 90% and 86%, respectively by screening serum from tuberculosis patients (n = 90) and uninfected individuals (n = 90) based on ELISA. Therefore, this study demonstrated that ribokinase had good potential for the serodiagnosis of tuberculosis.

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Section: Discussionmentioning

confidence: 99%

Ribokinase screened from T7 phage displayed Mycobacterium tuberculosis genomic DNA library had good potential for the serodiagnosis of tuberculosis

Luo

Wang

Liu

et al. 2019

Appl Microbiol Biotechnol

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“…For the 384 PrESTs arrays, normalization and quantification of all microarray images were performed using GenepixPro 7.1 software (Axon Laboratories). After calculation of the median values of the spots and background, GPR files were used to perform inter-array median normalization using Microsoft Office Excel prior to bioinformatics analyses by pomelo II () and MultiExperiment Viewer Analysis (MeV). , The comparison of the AD and control groups was performed as previously described. − To identify seroreactive PrESTs to AD sera by comparing AD patients and healthy individual groups, a t test using pomelo II was performed, in which adjusted p -values of <0.05 (FDR ≤ 0.15) were obtained by permutation testing using 100,000 permutations. Pomelo II generated a heatmap, showing seroreactive PrESTs to AD or controls.…”

Section: Methodsmentioning

confidence: 99%

Multiomics Profiling of Alzheimer’s Disease Serum for the Identification of Autoantibody Biomarkers

et al. 2021

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New biomarkers of Alzheimer's disease (AD) with a diagnostic value in preclinical and prodromal stages are urgently needed. AD-related serum autoantibodies are potential candidate biomarkers. Here, we aimed at identifying AD-related serum autoantibodies using protein microarrays and mass spectrometry-based methods. To this end, an untargeted complementary screening using high-density (42,100 antigens) and low-density (384 antigens) planar protein-epitope signature tag (PrEST) arrays and an immunoprecipitation protocol coupled to mass spectrometry analysis were used for serum autoantibody profiling. From the untargeted screening phase, 377 antigens corresponding to 338 proteins were selected for validation. Out of them, IVD, CYFIP1, and ADD2 seroreactivity was validated using 128 sera from AD patients and controls by PrEST-suspension bead arrays, and ELISA or luminescence Halotag-based bead immunoassay using full-length recombinant proteins. Importantly, IVD, CYFIP1, and ADD2 showed in combination a noticeable AD diagnostic ability. Moreover, IVD protein abundance in the prefrontal cortex was significantly two-fold higher in AD patients than in controls by western blot and immunohistochemistry, whereas CYFIP1 and ADD2 were significantly down-regulated in AD patients. The panel of AD-related autoantigens identified by a comprehensive multiomics approach may provide new insights of the disease and should help in the blood-based diagnosis of Alzheimer's disease. Mass spectrometry raw data are available in the ProteomeXchange database with the access number PXD028392.

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“…After biopanning, 1 536 phages from the third and four rounds of biopanning were amplified onto 96-well plates and transferred to five 384-well plates after 1:2 dilution in PBS 0.1% Tween 20 (PBS-T) . Size diversity of cDNA-derived inserted sequences and the absence of cross-contamination among monoclonal phages (presence of more than one band) were checked by PCR using T7_up2 and T7_down2 primers. , Then, 1 920 phages and controls were printed onto SuperNitro Substrates Nitrocellulose Coating slides (Arrayit) using a MicroGrid II Spotter (GeneMachines, San Carlos, CA) at 20 °C and 50% humidity. Negative controls consisted of empty spots, PBS-Tween 0.1%, or 1 μg/μL bovine serum albumin (BSA, Sigma-Aldrich).…”

Section: Methodsmentioning

confidence: 99%

“…Analysis, normalization, and quantification of all microarray images were performed using GenepixPro 7.1 software (Axon Laboratories). The median values of the spots and background were determined, and interarray median normalization was performed. ,,, To identify seroreactive AD-specific phages by comparing AD patients and healthy individual groups, a t test using pomelo II () was performed, where p -values were obtained by permutation testing using 200 000 permutations. Pomelo II generated a heatmap, showing the phages with a false discovery rate (FDR) value below 0.15 and an unadjusted p -value below 0.05 to minimize for the presence of false positive results (15% of significant tests might be false positives instead of 5% of all positive phages if using only the p -value).…”

Section: Methodsmentioning

confidence: 99%

Identification of Alzheimer’s Disease Autoantibodies and Their Target Biomarkers by Phage Microarrays

et al. 2019

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The characterization of the humoral response in Alzheimer's disease (AD) patients might aid in detecting the disease at early stages. We have combined phage display and protein microarrays to identify AD autoantibodies and their target biomarkers. After enrichment of the T7 phage display libraries from AD and healthy brain tissue mRNA in AD-specific phages, 1536 monoclonal phages were printed on microarrays to probe them with 8 AD and 8 healthy control sera. A total of 57 phages showed higher seroreactivity in AD. In total, 13 out of the 44 unique sequences displayed on the phages were selected for validation using 68 AD and 52 healthy control sera. Peptides from Anthrax toxin receptor 1, Nuclear protein 1, Glycogen phosphorylase, and Olfactory receptor 8J1 expressed in bacteria as HaloTag fusion proteins showed a statistically significant ability to discriminate between AD patients and controls. The identified panel of AD autoantibodies might provide new insights into the blood-based diagnosis of the disease.

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High-throughput screening of T7 phage display and protein microarrays as a methodological approach for the identification of IgE-reactive components

Cited by 14 publications

References 38 publications

Ribokinase screened from T7 phage displayed Mycobacterium tuberculosis genomic DNA library had good potential for the serodiagnosis of tuberculosis

Ribokinase screened from T7 phage displayed Mycobacterium tuberculosis genomic DNA library had good potential for the serodiagnosis of tuberculosis

Multiomics Profiling of Alzheimer’s Disease Serum for the Identification of Autoantibody Biomarkers

Identification of Alzheimer’s Disease Autoantibodies and Their Target Biomarkers by Phage Microarrays

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