Cattle

Coordinator: Jayne Hope

Jayne Hope leads the Bovine Immune Mechanisms group at the Institute for Animal Health and is the bovine co-ordinator for the Immunological Toolbox. The contribution to the Toolbox focuses on development of reagents to study cytokine biology and innate immune responses in cattle. Evaluation of reagents for cross-reactivity with other species, notably sheep, is an important aspect of these studies

 

 

Key targets

cattle

  • IL-2 (mAbs, ELISA)
  • TLR2 (mAbs)
  • CCR7 (mAbs)
  • CD34 (mAbs)
  • KIR/Ly49 (mAbs) with Dr Shirley Ellis)

Summary of progress

Other targets

  • IL-17 (mAbs, ELISA) – in collaboration with Dr C Baldwin, US-VIRN
  • Develop FLAG expression and purification systems for bovine cytokines
  • Evaluation of species-cross reactivity of reagents

Summary of progress

 

Progress on key targets for cattle
Updated 22 July 2009

IL-2
Mice were immunised for IL-2 mAb using plasmid DNA and boosted with protein. Four attempts to generate IL-2 mAb by performing mouse immunisation were made in total. Two did not result in seroconversion and in two other fusions, although hybridomas showing IL-2 specificity were established, these did not remain viable during extended culture. Through collaboration with AbD Serotec utilising the Morphosys HuCAL® system anti-bovine IL-2 antibodies were selected and tested against both recombinant and native IL-2. Pairs of antibodies selected as specific for recombinant IL-2 did not recognise native IL-2. The selection of antibodies from the HuCAL® library is currently being repeated. Polyclonal anti-bovine IL-2 antibodies (R&D systems) have been incorporated into newly developed multiparametric intracytoplasmic cytokine assays to demonstrate co-expression of IL-2, IFN-g, and IL-4 within bovine T cells.

TLR2   
Mice were immunised with plasmid encoding bovine TLR-2 and boosted with either monocytes or TLR-2 transfected HEK cells. A total of four attempts were made in mice to generate mAb. Seroconversion was observed but specific hybridomas were not isolated following fusions. We are currently selecting antibodies in the Morphosys HuCAL® system.

CCR7
Mice were immunised with plasmid DNA and boosted with activated T cells. Seroconversion was observed with antibodies detected that recognised a subset of T lymphocytes with similar expression profiles to that observed with human cells/anti-human CCR7 MAb. Hybridomas were selected from fusions and extensively validated. Attempts to confirmation CCR7-specificity using COS cells transfected with CCR7 plasmid showed that the selected mAb were not CCR7 specific despite promising expression profiles on bovine peripheral blood cells.

CD34
Monoclonal antibodies to CD34 have been developed by our collaborators at Texas A & M University. These are being tested at IAH for specificity and use in cellular systems utilising cord blood and neonatal calf blood.

KIR/Ly49
Antibodies to NK receptors were generated through DNA immunisation with protein boost. Methods were developed for the high level expression of KIR and Ly49 proteins and ELISAs set up to facilitate screening of mouse serum samples and hybridoma supernatants. mAb were successfully generated to bovine Ly49 and generic KIR. These mAb recognise the specific proteins to which they were raised and show complex expression patterns on NK cells isolated from bovine blood. mAb expression patterns are mimicked by gene expression as determined by quantitative PCR – assays for these have also been established.

 

Progress on other targets for cattle
Updated 22 July 2009

IL-17
This is a collaborative aspect between the Immunological Toolbox and US-VIRN. We will obtain IL-17 protein from US-VIRN/Kingfisher Biotec and perform mouse immunisations to generate mAb. The first batch of protein has just been received at IAH and immunisations are planned.

Develop FLAG expression and purification systems for bovine cytokines
Recombinant bovine IFNg and IL-2 have been expressed in tagged vectors and purified based on FLAG or His-tags; these were validated for biological activity and utilised as ELISA standards.

Evaluation of species-cross reactivity of reagents
Through significant interactions between IAH and Moredun Research Institute we have demonstrated cross reactivity of bovine IL-4 and IL-12 antibodies with sheep cytokines (see Hope et al, 2005 and Wattegedera et al, 2004 in publications list). We recently demonstrated that of the two commercially available bovine anti-TNF-a Mab (CC327,CC328) only one was able to detect ovine TNF-a despite significant sequence homology between the species. Species cross-reactivity was determined for a large number of reagents in HLDA8. A relatively low number of cross-reactive anti-human reagents were found and these were further validated (see Sopp et al, 2007 in publications list).

 

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