Abstract:Background: Antibodies to microbes, or to autoantigens, are important markers of disease. Antibody detection (serology) can reveal both past and recent infections. There is a great need for development of rational ways of detecting and quantifying antibodies, both for humans and animals. Traditionally, serology using synthetic antigens covers linear epitopes using up to 30 amino acid peptides. Methods: We here report that peptides of 100 amino acids or longer (“megapeptides”), designed and synthesized for opti… Show more
“…A microliter of serum can then give results from many (say, 50) antigens. So far, sensitivity has been similar to that of comparable commercial tests [2][3][4].…”
Section: In My Group We Have Developed a Multiplex Serological Technsupporting
confidence: 54%
“…Immunoassay (SMIA) ( Figure 2) [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16]. We have used it for research on diagnosis and seroprevalence.…”
Section: In My Group We Have Developed a Multiplex Serological Technmentioning
confidence: 99%
“…A comparable collection of singleplex ELISAs, if it exists, will nearly always be more expensive and laborious. SMIA consumes less of expensive antigen, and less sample, than standard singleplex assays [2]. The ability to economize with antigen enables use of purified, more costly, antigens.…”
Section: In My Group We Have Developed a Multiplex Serological Technmentioning
High throughput multiplex serological systems enable the small developer to set up tests at small cost, for microbes for which there are no commercial tests, and for aspects which have not been addressed by them. An example is testing for Zika and Tick Borne Encephalitis virus antibodies, where antigenic cross-reactions make diagnosis problematic. Our technique variant, Suspension Multiplex Immunoassay (SMIA) allows many samples to be tested for antibodies to many antigens in a short time. Computational compensation for cross-reactions is possible if a SMIA panel contains most of the potentially cross-reacting antigens. Using antibody avidity and pattern of reactivity to whole virus and nonstructural protein, antibodies due to vaccination and infection, respectively, as well as probable degree of protection, can be determined with high throughput. These multiplex techniques hold great promise for future diagnostic development. Theoretically, even large scale serological monitoring, like blood donor pathogen testing, could be done inexpensively and rationally with multiplex serology developed in house. However, the quality control demands are steep and in most cases out of scope for a single laboratory. There remain however a number of clinical applications where in house multiplex serology can be performed with adequate quality control under high throughput conditions.
“…A microliter of serum can then give results from many (say, 50) antigens. So far, sensitivity has been similar to that of comparable commercial tests [2][3][4].…”
Section: In My Group We Have Developed a Multiplex Serological Technsupporting
confidence: 54%
“…Immunoassay (SMIA) ( Figure 2) [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16]. We have used it for research on diagnosis and seroprevalence.…”
Section: In My Group We Have Developed a Multiplex Serological Technmentioning
confidence: 99%
“…A comparable collection of singleplex ELISAs, if it exists, will nearly always be more expensive and laborious. SMIA consumes less of expensive antigen, and less sample, than standard singleplex assays [2]. The ability to economize with antigen enables use of purified, more costly, antigens.…”
Section: In My Group We Have Developed a Multiplex Serological Technmentioning
High throughput multiplex serological systems enable the small developer to set up tests at small cost, for microbes for which there are no commercial tests, and for aspects which have not been addressed by them. An example is testing for Zika and Tick Borne Encephalitis virus antibodies, where antigenic cross-reactions make diagnosis problematic. Our technique variant, Suspension Multiplex Immunoassay (SMIA) allows many samples to be tested for antibodies to many antigens in a short time. Computational compensation for cross-reactions is possible if a SMIA panel contains most of the potentially cross-reacting antigens. Using antibody avidity and pattern of reactivity to whole virus and nonstructural protein, antibodies due to vaccination and infection, respectively, as well as probable degree of protection, can be determined with high throughput. These multiplex techniques hold great promise for future diagnostic development. Theoretically, even large scale serological monitoring, like blood donor pathogen testing, could be done inexpensively and rationally with multiplex serology developed in house. However, the quality control demands are steep and in most cases out of scope for a single laboratory. There remain however a number of clinical applications where in house multiplex serology can be performed with adequate quality control under high throughput conditions.
“…The peptides were synthesized under conditions optimized for maximum coupling yield of long peptides, see e.g. [24]. Lyophilized peptides were dissolved in dimethyl sulfoxide (DMSO) (Sigma D2650).Figure 1.Megapeptides from the N terminus of hantavirus N proteins used in the present work.…”
Introduction: Hantaviruses are globally distributed zoonotic pathogens. Great diversity and high antigenic cross-reactivity makes diagnosis by traditional methods cumbersome.Materials and methods: ‘Megapeptides’, 119–120-mers from the amino terminus of the nucleoprotein of 16 hantaviruses, representing the four major branches of the hantavirus phylogenetic tree, were utilized in a novel IgG-based hantavirus suspension multiplex immunoassay (HSMIA) for detection of past hantavirus infections in 155 North European human samples. We compared HSMIA with established EIAs and focus reduction neutralization test (FRNT).Results and discussion: The Puumala hantavirus (PUUV) component in the HSMIA gave concordant results with a PUUV IgG EIA in 142 sera from Northern Sweden (of which 31 were EIA positive, 7 borderline and 104 EIA negative, sensitivity 30/31 = 97%, specificity 104/ 104 = 100%, 134/135 = 99% concordance), with another immunoassay in 40 PUUV IgG positive sera from Finland (36/40 = 90% sensitivity), and was concordant in 8 of 11 cases with PUUV and DOBV neutralization titers, respectively. Two major IgG reactivity patterns were found: (i) a PUUV-specific pattern covering phylogroup IV and its serogroups B and C; and (ii) a Dobrava virus (DOBV)-specific pattern, covering the serogroup A portion of phylogroup III. In addition, we found several minor patterns with reactivity to only one or two megapeptides indicating additional hantaviruses infecting humans in the Swedish and Finnish populations.Conclusion: The broadly reactive and rational HSMIA yielded results highly correlated with the established PUUV EIAs and the NT results. It is a sensitive and specific assay, which will be suited for efficient serosurveillance of hantaviruses in humans. Its use in animals should be further investigated.
“…Peptide microarray-based immunoassays on the other hand have been used less extensively in clinical research and unlike protein microarray-based immunoassays, they utilise single linear epitopes from immuno-dominant regions of a protein as antigens [9][10][11] . Apart from mimicking well-defined epitopes that are highly specific to a particular pathogen 9,12,13 , this approach provides additional advantages over proteinbased arrays, including reduced antigen cost, relative technical simplicity of peptide synthesis and the enhanced shelf-life of peptide array chips. Using a library of previously validated linear epitopes, we developed a synthetic peptide-based microarray for the detection of antibodies against multiple infectious diseases in a single microarray chip.…”
Background: Despite the considerable progress that has been achieved in the development of vaccines, infectious diseases continue to be the major cause of morbidity and mortality in developing countries. The first year of life remains the time of greatest risk with more than half of all deaths caused by infectious diseases in children under the age of five years occurring during this period. For most infections, antibodies are the strongest immune correlate of protective immunity and characterizing the breadth of the antibody repertoire against infectious diseases in early life is key to profiling individual disease risk.
Methods:In order to comprehensively profile the antibody repertoire against infectious diseases in early life, we developed a synthetic peptide-based microarray to simultaneously measure antibodies against forty one common infectious diseases. 82 empirically-validated linear B-cell epitopes were selected from the Immune Epitope Database (IEBD), expressed as synthetic peptides and printed onto microarray glass slides (microarray chip). The chip was used to simultaneously measure antigen-specific IgG and IgA in serum and mucosal samples from thirty eight infants and young children presenting to hospital with different illnesses. We also used the data from the microarray to estimate antibody decay kinetics for the five most frequently detected antigens during the first year of life.
Results:A synthetic peptide microarray to measure antibodies against forty one infectious diseases was successfully developed. Although the combination of antigens that were recognized were different for each child, antigens derived from Epstein-Barr virus, poliovirus, Streptococcus pneumoniae, plasmodium falciparum and varicella zoster were recognized by most study participants. While the combination of antigens recognized in serum was generally similar as those recognized in mucosal samples, antibodies to antigens such as herpesvirus, rubella and echinococcus were more predominant in either serum or mucosal samples. With the exception of the pneumococcus, we observed a progressive decline in serum IgG specific to all the infections above in the first six months of life. Pneumococcal IgG in serum exhibited a continuous rise in the first six months of life.
Conclusions:We successfully developed a synthetic peptide microarray and used it to profile the diversity of systemic and mucosal antibody against multiple infectious diseases in children. The data from the slide provide a snapshot overview of the breadth and kinetics of infectious disease antibodies early in life and opens the opportunity for conducting in-depth multi, multi-target serological studies of infectious diseases in future.
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