title: A statistical approach to determining responses to individual peptides from pooled-peptide ELISpot data.
abstract: To investigate in detail the effect of infection or vaccination on the human immune system, ELISpot assays are used to simultaneously test the immune response to a large number of peptides of interest. Scientists commonly use "peptide pools", where, instead of an individual peptide, a test well contains a group of peptides. Since the response from a well may be due to any or many of the peptides in the pool, pooled assays usually need to be followed by confirmatory assays of a number of individual peptides. We present a statistical method that enables estimation of individual peptide responses from pool responses using the Expectation Maximization (EM) algorithm for "incomplete data". We demonstrate the accuracy and precision of these estimates in simulation studies of ELISpot plates with 90 pools of 6 or 7 peptides arranged in three dimensions and three Mock wells for the estimation of background. In analysis of real pooled data from 6 subjects in a HIV-1 vaccine trial, where 199 peptides were arranged in 80 pools if size 9 or 10, our estimates were in very good agreement with the results from individual-peptide confirmatory assays. Compared to the classical approach, we could identify almost all the same peptides with high or moderate response, with less than half the number of confirmatory tests. Our method facilitates efficient use of the information available in pooled ELISpot data to avoid or reduce the need for confirmatory testing. We provide an easy-to-use free online application for implementing the method, where on uploading two spreadsheets with the pool design and pool responses, the user obtains the estimates of the individual peptide responses.
authors.affiliation: Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, Sweden.
authors.forename: Peter
authors.initials: P
authors.lastname: Ström
authors.affiliation: Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, Sweden.
authors.forename: Nathalie
authors.initials: N
authors.lastname: Støer
authors.affiliation: The Jenner Institute, University of Oxford, UK.
authors.forename: Nicola
authors.initials: N
authors.lastname: Borthwick
authors.affiliation: Weatherall institute of Molecular Medicine, Oxford, UK.
authors.forename: Tao
authors.initials: T
authors.lastname: Dong
authors.affiliation: The Jenner Institute, University of Oxford, UK; Weatherall institute of Molecular Medicine, Oxford, UK.
authors.forename: Tomáš
authors.initials: T
authors.lastname: Hanke
authors.affiliation: Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, Sweden. Electronic address: marie.reilly@ki.se.
authors.forename: Marie
authors.initials: M
authors.lastname: Reilly
mesh_terms: D016915:AIDS Vaccines / Q000276:immunology; D000465:Algorithms; D000068598:Data Accuracy; D058501:Enzyme-Linked Immunospot Assay / Q000379:methods; D015497:HIV-1 / Q000276:immunology; D006801:Humans; D020407:Internet; D007963:Leukocytes, Mononuclear / Q000276:immunology; D016013:Likelihood Functions; D008297:Male; D010455:Peptides / Q000032:analysis / Q000276:immunology*; D016012:Poisson Distribution
chemical_list: D016915:AIDS Vaccines; D010455:Peptides
keywords: Background adjustment; ELISpot assay; EM-algorithm; Peptide pools