Reference
Reference TypeLiterature
TitleValidation of peptide epitope microarray experiments and extraction of quality data.
AuthorsTatjana Nahtman; Alexander Jernberg; Shahnaz Mahdavifar; Johannes Zerweck; Mike Schutkowski; Markus Maeurer; Marie Reilly
AffiliationsDepartment of Medical Epidemiology and Biostatistics, Karolinska Institutet, Stockholm, Sweden.
JournalJ Immunol Methods
Year2007
AbstractINTRODUCTION: Within the last decade, the development of antigen microarray slides has enabled the simultaneous measurement of serum reactivity to hundreds of peptides in a single biological sample. Despite this considerable scientific progress, many issues remain regarding the quality, analysis and interpretation of the data these slides produce. There is currently no accepted approach to guide data analysis, and researchers use a wide variety of statistical methods and software tools. We designed and implemented a laboratory experiment to assess the reliability and range of measurement of peptide microarray data, and present graphical and statistical procedures for pre-processing so that quality data can be extracted for addressing biological hypotheses. METHODS: Within the last decade, the development of antigen microarray slides has enabled the simultaneous measurement of serum reactivity to hundreds of peptides in a single biological sample. Despite this considerable scientific progress, many issues remain regarding the quality, analysis and interpretation of the data these slides produce. There is currently no accepted approach to guide data analysis, and researchers use a wide variety of statistical methods and software tools. We designed and implemented a laboratory experiment to assess the reliability and range of measurement of peptide microarray data, and present graphical and statistical procedures for pre-processing so that quality data can be extracted for addressing biological hypotheses.Synthetic peptides spanning the proteins Ag85A, Ag85B, CFP10, MPT51/MPB51, TB10.4 and ESAT-6 were chosen as a paradigm to screen for serum reactivity to Mycobacteria tuberculosis (MTB). We explored various quantitative and graphical methods for presenting the responses from a slide. We replicated assays of samples from five TB-positive individuals to examine reproducibility, and used linear mixed models to investigate the various sources of variability, and to assess the range of measurement. We use our methods to extract data from the five TB-positive individuals and five healthy controls, and analyse the "normalized" responses using the freely available SAM package. RESULTS: Within the last decade, the development of antigen microarray slides has enabled the simultaneous measurement of serum reactivity to hundreds of peptides in a single biological sample. Despite this considerable scientific progress, many issues remain regarding the quality, analysis and interpretation of the data these slides produce. There is currently no accepted approach to guide data analysis, and researchers use a wide variety of statistical methods and software tools. We designed and implemented a laboratory experiment to assess the reliability and range of measurement of peptide microarray data, and present graphical and statistical procedures for pre-processing so that quality data can be extracted for addressing biological hypotheses.Synthetic peptides spanning the proteins Ag85A, Ag85B, CFP10, MPT51/MPB51, TB10.4 and ESAT-6 were chosen as a paradigm to screen for serum reactivity to Mycobacteria tuberculosis (MTB). We explored various quantitative and graphical methods for presenting the responses from a slide. We replicated assays of samples from five TB-positive individuals to examine reproducibility, and used linear mixed models to investigate the various sources of variability, and to assess the range of measurement. We use our methods to extract data from the five TB-positive individuals and five healthy controls, and analyse the "normalized" responses using the freely available SAM package.The ratio of foreground to background signal (on a log scale) provides an appropriate response index. A two-dimensional graphical display clearly illustrates the responses from the control and peptide features on a slide. Mixed model analysis of the repl...
Curation Last Updated2023-08-18 20:30:10
Epitope
Epitope ID49333
Chemical TypeLinear peptide
Linear SequencePSDLGGNNLPAKFLE
Source Molecule NameAntigen 85-A precursor
Source OrganismMycobacterium tuberculosis
Starting Position259
Ending Position273
Epitope Reference Details
Epitope Structure DefinesEpitope containing region/antigenic site
Epitope NameAg85A 259-273
Reference Starting Position259
Reference Ending Position273
Location of Data in ReferenceTable 2
Immunization
Host OrganismHomo sapiens (human)
1st In Vivo Process
In Vivo Process TypeOccurrence of infectious disease
Disease Statetuberculosis
Disease StageChronic;OGMS:0000064
1st Immunogen
Epitope RelationSource Organism
Object TypeOrganism
OrganismMycobacterium tuberculosis
Immunogen Details
Immunogen Reference NameMycobacterium tuberculosis
Immunization Comments
Immunization CommentsPlasma from 5 patients with a clinical history of pulmonary TB were collected. All patients underwent partial pulmonectomy in order to remove scar tissue associated with TB. Material from the lesions of one patient tested positive for viable bacilli, but cultures were negative for the other four patients. Sputum samples from all five patients tested positive for the presence of acid-fast bacilli, and no patient showed serological evidence for HIV or Hepatitis B infection.
B Cell Assay
Qualitative MeasurementPositive
Method/Techniquemicroarray
Measurement ofqualitative binding
Measurement Details
Number of Subjects Tested5
Number of Subjects Responded5
Response Frequency (%)100
Assayed Antibody
Assayed Antibody Source MaterialSerum
Assayed Antibody Purification StatusPolyclonal
Antigen
Epitope RelationEpitope
Chemical TypeLinear peptide
Linear SequencePSDLGGNNLPAKFLE
Source Molecule NameAntigen 85-A precursor
Source OrganismMycobacterium tuberculosis
Starting Position259
Ending Position273
Antigen Details
Antigen Reference NameAg85A 259-273
Assay Reference Details
Assay Comments by IEDB CuratorSynthetic peptides spanning the M. tuberculosis proteins were chosen to screen in a peptide microarray assay. This peptide produced significantly higher responses in TB+ donors than in healthy individuals.
Location of Assay Data in ReferenceFigure 8