Rational recombinant XMRV antigen preparation and bead coupling for multiplex serology in a suspension array

Sheikholvaezin, Ali; Blomberg, Fredrik; Öhrmalm, Christina; Sjösten, Anna; Blomberg, Jonas

doi:10.1016/j.pep.2011.08.007

Cited by 8 publications

(11 citation statements)

References 33 publications

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“…We developed new rapid and rational ways to produce antigens for microarrays, both what we call “megapeptides”, extraordinarily long synthetic peptides, in essence synthetic proteins (described in this paper), and by simple production of recombinant proteins [ 10 ].…”

Section: Discussionmentioning

confidence: 99%

“…Antibodies to a pathogen are traditionally detected to one antigen at a time using agent-specific ELISA (enzyme-linked immunosorbent assays). However, many different antibodies can now be detected simultaneously, e.g., with SMIA (suspension multiplex immunoassay) [ 7 , 8 , 9 , 10 ], a rational and economic technique. SMIA is a cost-effective serological method where antigen and serum consumption can be minimized, its background is generally low, and the dynamic range wider than in ELISA.…”

Section: Introductionmentioning

confidence: 99%

“…SMIA is a cost-effective serological method where antigen and serum consumption can be minimized, its background is generally low, and the dynamic range wider than in ELISA. Unlike the latter, it provides multiple results per analysis [ 7 , 8 , 9 , 10 ].…”

Section: Introductionmentioning

confidence: 99%

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Serology in the Digital Age: Using Long Synthetic Peptides Created from Nucleic Acid Sequences as Antigens in Microarrays

et al. 2016

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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 optimal serological performance, can successfully be used as detection antigens in a suspension multiplex immunoassay (SMIA). Megapeptides can quickly be created just from pathogen sequences. A combination of rational sequencing and bioinformatic routines for definition of diagnostically-relevant antigens can, thus, rapidly yield efficient serological diagnostic tools for an emerging infectious pathogen. Results: We designed megapeptides using bioinformatics and viral genome sequences. These long peptides were tested as antigens for the presence of antibodies in human serum to the filo-, herpes-, and polyoma virus families in a multiplex microarray system. All of these virus families contain recently discovered or emerging infectious viruses. Conclusion: Long synthetic peptides can be useful as serological diagnostic antigens, serving as biomarkers, in suspension microarrays.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Serology in the Digital Age: Using Long Synthetic Peptides Created from Nucleic Acid Sequences as Antigens in Microarrays

et al. 2016

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“…The fact that three PCRs were needed to investigate the presence of APEC makes it not a practical approach and highlights the need to use faster and more accurate methods to screen for the presence of the APEC associated VGs. A number of methods may be suitable for multiplex analysis in one assay, such as Luminex that allows multiplexing of up to 100 analytes in a single well of a microtiter plate (Sheikholvaezin, et al, 2011).…”

Section: Discussionmentioning

confidence: 99%

Studies on avian pathogenic Escherichia coli in commercial broiler chicken in South East Queensland

Awawdeh¹,

Turni²,

Henning³

et al.

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Avian pathogenic Escherichia coli (APEC) is the causative agent of avian colibacillosis, a localised or systemic infection resulting in clinical diseases such as colisepticemia, chronic respiratory disease and swollen-head syndrome. Globally, avian colibacillosis is the leading cause of morbidity and mortality in poultry, and it has been associated with massive economic losses and welfare problems.This organism is of public health significance as APEC is communicable to humans. The diagnosis of avian colibacillosis relies on clinical signs, typical pathological lesions and culture of E. coli from affected tissue(s). Antimicrobial therapy is often used both for treatment and control. Previous overseas studies have characterised APEC and identified virulence genes (VGs) that can be used as molecular markers for the identification of APEC. Little is known about APEC in broiler chickens in Australia.The aim of this thesis was to gain a better understanding of the epidemiology of APEC in Australian broiler flocks and how factors including presence of VGs and phylogenetic group can improve the identification of this pathotype in Australia. Firstly, three faecal DNA extraction methods were evaluated. Faeces were collected from healthy chickens and chickens with colibacillosis from commercial broiler farms in South East Queensland (SEQ). The extracted DNA was screened by a pentaplex-PCR for five APEC-associated VGs (iroN, iutA, iss, hlyF and ompT). DNA extracted from E. coli isolates cultured from the cloaca and organs of the birds were screened using the same PCR.Repeated bead beating plus column elution was the preferred DNA extraction method, as it yielded good PCR quality and adequate quantity DNA. However, identifying APEC by direct detection of the five VGs from the faecal material was not feasible as all of these genes were also detected in all of the birds. However, the VGs were more commonly detected in E. coli isolates cultured from birds with colibacillosis.A cross-sectional study was performed to estimate APEC farm-level prevalence in healthy broiler chickens in SEQ and to identify potential risk factors associated with the carriage of APEC. At the farm-level, all of the 40 farms sampled were positive for APEC, that is at least one bird per farm carried APEC, while the within-farm prevalence was 63% (95% Confidence Interval: 55.8, 70.2).Higher APEC within-farm bird-level prevalence was significantly associated with the usage of well water as a source of drinking water, failure to disinfect the water line after each flock, farm visitors not showering before entering the shed, distances greater than 20 metres between the car park and the poultry shed and the presence of wild birds within 50 metres of the shed. Chlorinating the drinking water combined with automatic water filtration reduced within-farm bird-level APEC prevalence. Page | 3Therefore, based on the results concluded from the multivariable model, improving biosecurity and water treatments might reduce APEC prevalence, decrease the risk of co...

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“…Nine XMRV proteins, covering the gag and env genes, were designed (Table 1). Their design, expression, and purification and the verification of their antigenicity were reported previously (61).…”

Section: Figmentioning

confidence: 99%

No Evidence for Xenotropic Murine Leukemia-Related Virus Infection in Sweden Using Internally Controlled Multiepitope Suspension Array Serology

Blomberg

Sjösten

et al. 2012

Clin Vaccine Immunol

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ABSTRACTMany syndromes have a large number of differential diagnoses, a situation which calls for multiplex diagnostic systems. Myalgic encephalomyelitis (ME), also named chronic fatigue syndrome (CFS), is a common disease of unknown etiology. A mouse retrovirus, xenotropic murine leukemia-related virus (XMRV), was found in ME/CFS patients and blood donors, but this was not corroborated. However, the paucity of serological investigations on XMRV in humans prompted us to develop a serological assay which cover many aspects of XMRV antigenicity. It is a novel suspension array method, using a multiplex IgG assay with nine recombinant proteins from theenvandgaggenes of XMRV and 38 peptides based on known epitopes of vertebrate gammaretroviruses. IgG antibodies were sought in 520 blood donors and 85 ME/CFS patients and in positive- and negative-control sera from animals. We found no differences in seroreactivity between blood donors and ME/CFS patients for any of the antigens. This did not support an association between ME/CFS and XMRV infection. The multiplex serological system had several advantages: (i) biotinylated protein G allowed us to run both human and animal sera, which is essential because of a lack of XMRV-positive humans; (ii) a novel quality control was a pan-peptide positive-control rabbit serum; and (iii) synthetic XMRV Gag peptides with degenerate positions covering most of the variation of murine leukemia-like viruses did not give higher background than nondegenerate analogs. The principle may be used for creation of variant tolerant peptide serologies. Thus, our system allows rational large-scale serological assays with built-in quality control.

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Rational recombinant XMRV antigen preparation and bead coupling for multiplex serology in a suspension array

Cited by 8 publications

References 33 publications

Serology in the Digital Age: Using Long Synthetic Peptides Created from Nucleic Acid Sequences as Antigens in Microarrays

Serology in the Digital Age: Using Long Synthetic Peptides Created from Nucleic Acid Sequences as Antigens in Microarrays

Studies on avian pathogenic Escherichia coli in commercial broiler chicken in South East Queensland

No Evidence for Xenotropic Murine Leukemia-Related Virus Infection in Sweden Using Internally Controlled Multiepitope Suspension Array Serology

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