Limits...
Development of a multiplex fluorescence immunological assay for the simultaneous detection of antibodies against Cooperia oncophora, Dictyocaulus viviparus and Fasciola hepatica in cattle.

Karanikola SN, Krücken J, Ramünke S, de Waal T, Höglund J, Charlier J, Weber C, Müller E, Kowalczyk SJ, Kaba J, von Samson-Himmelstjerna G, Demeler J - Parasit Vectors (2015)

Bottom Line: The capability of a multiplex platform for the simultaneous detection of three pasture associated parasite species was evaluated and compared to existing ELISAs.The results between the assays were compared and kappa tests revealed an overall good agreement.This platform provides rapid, high-throughput results and is highly sensitive and specific in comparison to existing serological as well as coproscopical diagnostic techniques.

View Article: PubMed Central - PubMed

Affiliation: Institute for Parasitology and Tropical Veterinary Medicine, Freie Universität Berlin, Berlin, Germany. Sofia.Karanikola@fu-berlin.de.

ABSTRACT

Background: A major constraint for the effective control and management of helminth parasites is the lack of rapid, high-throughput, routine diagnostic tests to assess the health status of individual animals and herds and to identify the parasite species responsible for these helminthoses. The capability of a multiplex platform for the simultaneous detection of three pasture associated parasite species was evaluated and compared to existing ELISAs.

Methods: The recombinant antigens 14.2 kDa ES protein for Cooperia oncophora, major sperm protein for Dictyocaulus viviparus and Cathepsin L1 for Fasciola hepatica were recombinantly expressed either in Escherichia coli or Pichia pastoris. Antigens were covalently coupled onto magnetic beads. Optimal concentrations for coupling were determined following the examination of serum samples collected from experimentally mono-infected animals, before and after their infection with the target species. Absence of cross-reactivity was further determined with sera from calves mono-infected with Haemonchus contortus, Ostertagia ostertagi and Trichostrongylus colubriformis. Examination of negative serum samples was characterised by low median fluorescence intensity (MFI).

Results: Establishment of the optimal serum dilution of 1:200 was achieved for all three bead sets. Receiver Operating Characteristic analyses were performed to obtain cut-off MFI values for each parasite separately. Sensitivity and specificity at the chosen cut-off values were close to, or 100% for all bead sets. Examination of serum samples collected on different days post infection from different animals showed a high reproducibility of the assays. Serum samples were additionally examined with two already established ELISAs, an in-house ELISA using the recombinant MSP as an antigen and a DRG ELISA using Cathepsin L1 for liver fluke. The results between the assays were compared and kappa tests revealed an overall good agreement.

Conclusions: A versatile bead-based assay using fluorescence detection (xMAP technology) was developed to simultaneously detect antibodies against C. oncophora, D. viviparus and F. hepatica in cattle serum samples. This platform provides rapid, high-throughput results and is highly sensitive and specific in comparison to existing serological as well as coproscopical diagnostic techniques.

No MeSH data available.


Related in: MedlinePlus

Results for the triplex assay using serum dilutions of cattle infected with Dictyocaulus, Fasciola and Cooperia. Five parameter logistic regression curves were calculated based on median fluorescence intensity (MFI) values. Beads are coupled with recombinant antigen for the detection of Cooperia oncophora (green), Dictyocaulus viviparus (black) and Fasciola hepatica (red). Artificial mixtures of sera from animals infected with the target species was used. Dilutions are presented as 0.005 ≙ 1:200
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4492007&req=5

Fig2: Results for the triplex assay using serum dilutions of cattle infected with Dictyocaulus, Fasciola and Cooperia. Five parameter logistic regression curves were calculated based on median fluorescence intensity (MFI) values. Beads are coupled with recombinant antigen for the detection of Cooperia oncophora (green), Dictyocaulus viviparus (black) and Fasciola hepatica (red). Artificial mixtures of sera from animals infected with the target species was used. Dilutions are presented as 0.005 ≙ 1:200

Mentions: Determination of the optimal serum dilution was based on the examination of negative and positive control sera in a two-fold dilution series ranging from 1:100 to 1:12,800 for the separate coupled beadsets. The logistic regression curves of the MFI values for all three target species enabled a clear differentiation between the target species and negative control as well as non-target species (Fig. 1) with relatively low background MFI values. Multiplex assays were also performed using an 82-fold dilution series and results were comparable to those obtained in the singleplex assays (Fig. 2).Fig. 1


Development of a multiplex fluorescence immunological assay for the simultaneous detection of antibodies against Cooperia oncophora, Dictyocaulus viviparus and Fasciola hepatica in cattle.

Karanikola SN, Krücken J, Ramünke S, de Waal T, Höglund J, Charlier J, Weber C, Müller E, Kowalczyk SJ, Kaba J, von Samson-Himmelstjerna G, Demeler J - Parasit Vectors (2015)

Results for the triplex assay using serum dilutions of cattle infected with Dictyocaulus, Fasciola and Cooperia. Five parameter logistic regression curves were calculated based on median fluorescence intensity (MFI) values. Beads are coupled with recombinant antigen for the detection of Cooperia oncophora (green), Dictyocaulus viviparus (black) and Fasciola hepatica (red). Artificial mixtures of sera from animals infected with the target species was used. Dilutions are presented as 0.005 ≙ 1:200
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4492007&req=5

Fig2: Results for the triplex assay using serum dilutions of cattle infected with Dictyocaulus, Fasciola and Cooperia. Five parameter logistic regression curves were calculated based on median fluorescence intensity (MFI) values. Beads are coupled with recombinant antigen for the detection of Cooperia oncophora (green), Dictyocaulus viviparus (black) and Fasciola hepatica (red). Artificial mixtures of sera from animals infected with the target species was used. Dilutions are presented as 0.005 ≙ 1:200
Mentions: Determination of the optimal serum dilution was based on the examination of negative and positive control sera in a two-fold dilution series ranging from 1:100 to 1:12,800 for the separate coupled beadsets. The logistic regression curves of the MFI values for all three target species enabled a clear differentiation between the target species and negative control as well as non-target species (Fig. 1) with relatively low background MFI values. Multiplex assays were also performed using an 82-fold dilution series and results were comparable to those obtained in the singleplex assays (Fig. 2).Fig. 1

Bottom Line: The capability of a multiplex platform for the simultaneous detection of three pasture associated parasite species was evaluated and compared to existing ELISAs.The results between the assays were compared and kappa tests revealed an overall good agreement.This platform provides rapid, high-throughput results and is highly sensitive and specific in comparison to existing serological as well as coproscopical diagnostic techniques.

View Article: PubMed Central - PubMed

Affiliation: Institute for Parasitology and Tropical Veterinary Medicine, Freie Universität Berlin, Berlin, Germany. Sofia.Karanikola@fu-berlin.de.

ABSTRACT

Background: A major constraint for the effective control and management of helminth parasites is the lack of rapid, high-throughput, routine diagnostic tests to assess the health status of individual animals and herds and to identify the parasite species responsible for these helminthoses. The capability of a multiplex platform for the simultaneous detection of three pasture associated parasite species was evaluated and compared to existing ELISAs.

Methods: The recombinant antigens 14.2 kDa ES protein for Cooperia oncophora, major sperm protein for Dictyocaulus viviparus and Cathepsin L1 for Fasciola hepatica were recombinantly expressed either in Escherichia coli or Pichia pastoris. Antigens were covalently coupled onto magnetic beads. Optimal concentrations for coupling were determined following the examination of serum samples collected from experimentally mono-infected animals, before and after their infection with the target species. Absence of cross-reactivity was further determined with sera from calves mono-infected with Haemonchus contortus, Ostertagia ostertagi and Trichostrongylus colubriformis. Examination of negative serum samples was characterised by low median fluorescence intensity (MFI).

Results: Establishment of the optimal serum dilution of 1:200 was achieved for all three bead sets. Receiver Operating Characteristic analyses were performed to obtain cut-off MFI values for each parasite separately. Sensitivity and specificity at the chosen cut-off values were close to, or 100% for all bead sets. Examination of serum samples collected on different days post infection from different animals showed a high reproducibility of the assays. Serum samples were additionally examined with two already established ELISAs, an in-house ELISA using the recombinant MSP as an antigen and a DRG ELISA using Cathepsin L1 for liver fluke. The results between the assays were compared and kappa tests revealed an overall good agreement.

Conclusions: A versatile bead-based assay using fluorescence detection (xMAP technology) was developed to simultaneously detect antibodies against C. oncophora, D. viviparus and F. hepatica in cattle serum samples. This platform provides rapid, high-throughput results and is highly sensitive and specific in comparison to existing serological as well as coproscopical diagnostic techniques.

No MeSH data available.


Related in: MedlinePlus