Serotype- and serogroup-specific detection of African horsesickness virus using phage displayed chicken scFvs for indirect double antibody sandwich ELISAs

Wyngaardt, Wouter Van; Mashau, Cordelia; Wright, Isabel M.; Fehrsen, Jeanni

doi:10.4142/jvs.2013.14.1.95

Cited by 6 publications

(3 citation statements)

References 12 publications

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“…Since its invention in 1972, the enzyme-linked immunosorbent assay (ELISA) [ 1 ] has been used until today as a reliable tool to detect and quantify virus concentrations in humans [ 2 – 8 ], animals [ 9 – 11 ], and plants [ 12 – 18 ]. To quantify virus concentrations in a sample, double antibody sandwich ELISA (DAS-ELISA) is a common tool where an enzyme-linked antibody binds specifically to the coat protein of the virus, which again is bound to the surface of a microtiter plate by another specific antibody.…”

Section: Introductionmentioning

confidence: 99%

Non-linear transformation of enzyme-linked immunosorbent assay (ELISA) measurements allows usage of linear models for data analysis

Lange

Rotärmel

Müller

et al. 2022

Virol J

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Background In research questions such as in resistance breeding against the Beet necrotic yellow vein virus it is of interest to compare the virus concentrations of samples from different groups. The enzyme-linked immunosorbent assay (ELISA) counts as the standard tool to measure virus concentrations. Simple methods for data analysis such as analysis of variance (ANOVA), however, are impaired due to non-normality of the resulting optical density (OD) values as well as unequal variances in different groups. Methods To understand the relationship between the OD values from an ELISA test and the virus concentration per sample, we used a large serial dilution and modelled its non-linear form using a five parameter logistic regression model. Furthermore, we examined if the quality of the model can be increased if one or several of the model parameters are defined beforehand. Subsequently, we used the inverse of the best model to estimate the virus concentration for every measured OD value. Results We show that the transformed data are essentially normally distributed but provide unequal variances per group. Thus, we propose a generalised least squares model which allows for unequal variances of the groups to analyse the transformed data. Conclusions ANOVA requires normally distributed data as well as equal variances. Both requirements are not met with raw OD values from an ELISA test. A transformation with an inverse logistic function, however, gives the possibility to use linear models for data analysis of virus concentrations. We conclude that this method can be applied in every trial where virus concentrations of samples from different groups are to be compared via OD values from an ELISA test. To encourage researchers to use this method in their studies, we provide an R script for data transformation as well as the data from our trial.

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Section: Introductionmentioning

confidence: 99%

Non-linear transformation of enzyme-linked immunosorbent assay (ELISA) measurements allows usage of linear models for data analysis

Lange

Rotärmel

Müller

et al. 2022

Virol J

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“…Using phage display technology, antibodies have been produced against CR6261 (influenza virus) and KB001 (Pseudomonas aeruginosa) (Nelson et al, 2010). The Nkuku® phage library has been used to determine epitopes of footand-mouth disease virus (Opperman et al, 2012) and to generate antibodies for African horse sickness virus to use in diagnostic tests (Van Wyngaardt et al, 2004;Van Wyngaardt et al, 2013).…”

Section: Applications Of Phage Displaymentioning

confidence: 99%

“…A different approach to using animal-produced antibodies is the use of phage display. The single chain variable fragment (scFv) antibodies have been produced and used in the development of ELISA diagnostic tests for bluetongue virus (Fehrsen et al, 2005;Rakabe et al, 2011) and African horse sickness (Van Wyngaardt et al, 2004;Van Wyngaardt et al, 2013).…”

Section: Selection Of Scfvs Specific To Tvicatlmentioning

confidence: 99%

Recombinant expression and enzymatic characterisation of Trypanosoma vivax cathepsin L-like protease (TviCATL) for single chain variable fragment antibody production.

Ramjeawon

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Humans and animals in sub-Saharan Africa are at risk of African trypanosomiasis (AT), caused by tsetse fly-transmitted protozoan blood parasites of the Trypanosoma genus. Animal African trypanosomiasis (AAT), or nagana, is caused by T. brucei, T. congolense and T. vivax and negatively impacts livestock farming and consequently the economy of the continent. Since AAT occurs in rural areas, affordable rapid diagnostic tests (RDTs) and drugs are required. Diagnostic tests focus on antibody detection; however, antigen detection is more favorable since anti-trypanosome antibodies persist in blood for years following recovery. Due to the parasite’s defense by antigenic variation, development of a vaccine is unlikely. Molecules that are essential for parasite survival, such as peptidases, are currently being targeted for diagnosis and chemotherapy. A cathepsin-L-like cysteine protease from T. vivax, TviCATL, is released by dying parasites in the host bloodstream and was shown to be a diagnostic target for detecting host antibodies. To achieve diagnosis of current infections, detection of TviCATL is being explored. The overall aim of this study was to enzymatically characterise TviCATL; and to study the interaction of antibodies against the TviCATL antigen which could be used as a chemotherapeutic drug for the diagnosis of T. vivax infections. The protease, TviCATL, was recombinantly expressed in E. coli using the pET-28a expression vector and purified using a nickel chelate affinity column. The resulting 47 kDa protein was identified using western blot and was shown to hydrolyse H-D-Ala-Leu-Lys-AMC and was inhibited by bestatin and E-64 and had optimal activity between pH 6.5 and 7.5. The crossreactivity between TviCATL and antibodies produced against other Trypanosoma spp cysteine proteases was evaluated in western blots, and results confirmed cross-reactivity. In addition, chicken anti-TviCATL antibodies were able to detect TviCATL in TviCATL-spiked bovine serum. The production of antibodies using the Nkuku® phage library was employed as an alternative to the animal-based antibody production and single-chain variable fragment (scFvs) antibodies were selected by panning against the TviCATL antigen. After four rounds of panning, TviCATL-scFvs binders were enriched and four clones gave the highest signal when evaluated using a monospecific ELISA. Due to the low values obtained, optimisation of panning is necessary for improved results. Optimisation of recombinant expression and purification of the identified scFvs for use in a sandwich ELISA were explored to this end. This study showed that TviCATL is a promising chemotherapeutic and diagnostic target for African animal trypanosomiasis.

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Construction of Chicken and Ostrich Antibody Libraries

Fehrsen,

Wemmer,

van Wyngaardt

2023

Methods in Molecular Biology

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Serotype- and serogroup-specific detection of African horsesickness virus using phage displayed chicken scFvs for indirect double antibody sandwich ELISAs

Cited by 6 publications

References 12 publications

Non-linear transformation of enzyme-linked immunosorbent assay (ELISA) measurements allows usage of linear models for data analysis

Non-linear transformation of enzyme-linked immunosorbent assay (ELISA) measurements allows usage of linear models for data analysis

Recombinant expression and enzymatic characterisation of Trypanosoma vivax cathepsin L-like protease (TviCATL) for single chain variable fragment antibody production.

Construction of Chicken and Ostrich Antibody Libraries

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