Development of a competitive enzyme‐linked immunosorbent assay for detecting cytomegalovirus antibody

Wreghitt, T.G.; Hicks, Joanna; Gray, Julia; O'Connor, Candice

doi:10.1002/jmv.1890180204

Cited by 19 publications

(3 citation statements)

References 11 publications

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“…HCMV serology. HCMV antibodies in sera were assayed by a standard competitive enzyme immunosorbent assay (Northumbria Biologicals Ltd, Cramlington, United Kingdom) as described by Wreghitt et al (48). A positive result was recorded when the optical density (OD) of the sample was less than half the OD of the negative control.…”

mentioning

confidence: 99%

Fine specificity of cellular immune responses in humans to human cytomegalovirus immediate-early 1 protein

A.J.

Allport

Zanten

et al. 1991

J Virol

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Cell-mediated immunity is important in maintaining the virus-host equilibrium in persistent human cytomegalovirus (HCMV) infection. The HCMV 72-kDa major immediate early 1 protein (IE1) is a target for CD8+ cytotoxic T cells in humans, as is the equivalent 89-kDa protein in mouse. Less is known about responses against this protein by CD4+ T cells, which may be important as direct effector cells or helper cells for antibody and CD8+ responses. Proliferative-T-cell responses to HCMV IE1 were studied in normal seropositive subjects. Peripheral blood mononuclear cells from 85% of seropositive subjects proliferated in response to HCMV from infected fibroblasts, and of these, 73% responded to recombinant baculovirus IEL. Responding cells were predominantly CD3+ CD4+. IEl antigen preparations, including baculovirus recombinant protein, transfected rat cell nuclei, and synthetic peptides, induced IEl-specific T-cell lines which cross-reacted between the preparations. The fine specificity of these IEl-specific T-cell lines was studied by using overlapping synthetic peptides encompassing the entire sequence of the IE1 protein. The regions of the IEl molecule recognized were identified and these varied between individuals, possibly reflecting differences in major histocompatibility complex (MHC) class II haplotype. In one subject, the peptide specificities of proliferative and MHC class I-restricted cytotoxic determinants on IE1 were spatially distinct. Thus, no single immunodominant T-cell determinant within HCMV IE1 was identified, suggesting that multiple peptides or a region of the 72-kDa TEl protein would be required to induce specific T-cell responses in humans.

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mentioning

confidence: 99%

Fine specificity of cellular immune responses in humans to human cytomegalovirus immediate-early 1 protein

A.J.

Allport

Zanten

et al. 1991

J Virol

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“…Comparison between the result of indirect and competitive ELISA suggested that the IgG titre difference might be deceptively greater in the competitive ELISA. A possible explanation is that the latter assay is more sensitive as has been reported in other studies (Goda et al 2000, Reddington et al 1991, Wreghitt et al 1986). Mice sera used in both assays may contain non-immune IgGs and other serum factors, which can bind nonspecifically to the immobilised antigen and consequently cause background interference.…”

Section: Effect Of Advax Tm -1 On H190 Immunogenicitymentioning

confidence: 62%

“…Similar to the observation on the increase in antibody quality due to the use of Advax TM -1 (Figure 4-14), the increase in antibody quality in Figure 4-15 was marginal and not statistically significant. The increase could be detected in competitive ELISA assay, but not in indirect ELISA, which was possibly because the former assay has a higher sensitivity (Goda et al 2000, Reddington et al 97 1991, Wreghitt et al 1986). The finding that increasing number of H190 tandem repeats from two to three and four copies of H190 did not result in a statistically significant increase in antibody quality indicated that two copies of H190 was sufficient to induce a high quality of antibodies.…”

Section: Effect Of Increasing Number Of H190 Tandem Repeats On Specifmentioning

confidence: 99%

Engineering of virus-like particles for alternative vaccine candidate targeting a hypervariable peptide antigen element

Anggraeni¹

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A promising alternative to replace the current egg-or cell culture-based technology for vaccine production from live viruses is virus-like particle (VLP) technology based on a microbial platform. VLPs are macromolecular assemblies of viral capsid proteins, which have been shown to tolerate insertion of antigen modules via genetic recombinant technology, yielding modular VLPs. Many studies on modular VLPs presume that when a peptide antigen element is taken out from the intact proteins and then modularised on VLPs, it is unable to fold into its native structure. However, until now, presentation of a peptide antigen element on a VLP and the impact of the display strategy to present the antigen element on the quality of the resulting antibodies (i.e. the ability of the antibodies to recognise the intact protein) are not fully understood. This thesis aims to understand the underlying fundamentals regarding modularisation of peptide antigen elements on VLPs for induction of high-quality antibodies. A hypervariable receptor-binding domain, Helix 190 (H190), from the hemagglutinin protein of influenza A virus was used as a model for modularisation on VLPs from murine polyomavirus (MuPyV) VP1 protein. Four major findings are presented. Firstly, two display strategies, i.e. arraying of H190 in tandem repeats and the use of helix promoter elements, were shown to display H190 in its immunogenic form equally. However, modularisation using tandem repeat display induced antibodies of a higher quality than modularisation using helix promoter elements.Secondly, the quality of antibodies induced by the tandem repeat display bearing two copies of H190 was optimum, thus no significant improvement was observed following the use of adjuvant or increasing the copy number of H190. Additionally, the increase in the copy number of H190 was shown to reduce the assembly capability and solubility of modular VP1 in an environment that was optimised for wild-type VP1. Thirdly, this thesis shows the novel finding in the use of flanking ionic elements to stabilise VLP precursors, termed as capsomeres, bearing two copies of H190 containing a hydrophobic stretch, which caused aggregation. Fourthly, the first steps towards obtaining the atomic crystal structure of presented H190 on a modular protein were performed, i.e. a mild and satisfactory laboratory process was developed to achieve high-purity modular VP1 capsomeres, unattainable using previously established expression and purification process of wild-type MuPyV VP1. This thesis shows a step forward towards understanding the presentation of a peptide antigen element on a VLP that enables induction of highquality antibodies, and towards VLP engineering to manipulate the aggregation and solubility of modular VP1. VLP technology based on a microbial platform presented here is iii a potentially safe and effective alternative vaccine candidate that targets a hypervariable peptide antigen element. The speed of the microbial platform allows a rapid response to the hypervariability of the pe...

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A biotin—avidin-amplified inhibition enzyme immunoassay for detection of CMV antibodies in human serum

Nielsen

Hansen

Andersen

et al. 1987

Journal of Virological Methods

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Development of a competitive enzyme‐linked immunosorbent assay for detecting cytomegalovirus antibody

Cited by 19 publications

References 11 publications

Fine specificity of cellular immune responses in humans to human cytomegalovirus immediate-early 1 protein

Fine specificity of cellular immune responses in humans to human cytomegalovirus immediate-early 1 protein

Engineering of virus-like particles for alternative vaccine candidate targeting a hypervariable peptide antigen element

A biotin—avidin-amplified inhibition enzyme immunoassay for detection of CMV antibodies in human serum

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