SUPPLEMENTARY METHODS: Protocols for antibody measurement
Anti-nuclear antibodies (ANA)
Anti-nuclear antibody levels were analysed with REAADS ANA Test ELISA Kits (Corgenix UK,
Ltd) using kit reagents and buffers according to manufacturer’s instructions, modified for use in
sheep with a polyclonal rabbit anti-sheep immunoglobulin secondary antibody (Ig/HRP P0163;
Dako UK, Ltd), following [1]. This secondary reagent should detect all Ig isotype subclasses. Kit
antigens included an array of purified mammalian nuclear and cytoplasmic antigens derived from
HEp-2 (Human Epithelial cell line 2) cells: RNP, Sm, SSA, SSB, Scl-70, Jo-1, CENP-B, Ribosomal
P, DNA and histones. Thawed samples were diluted 1:50 on ice, transferred in 100ul aliquots to the
antigencoated plates, and incubated at 37°C for 2 hrs. Wells were emptied, washed 4 times, and
secondary antibodies were added at 0.1625 ug/ml. After incubation at 37°C for 1 hr, wells were
emptied, washed 4 times and 100ul of TMB/H2O2 substrate were added per well. Plates were
incubated at RT in the dark for 15 mins, and then 100ul stop solution (1N sulphuric acid) were
added per well. Absorbance (optical density, or O.D.) was then measured at 450nm on an Emax
Precision Microplate Reader (MDS Analytical Technologies, USA).
Plasma-free blanks were run for both human and ovine-specific secondary antibodies, and two
controls were run on every 96-well plate: a Soay neonate “background control” sample with ovinespecific secondary antibody, and a human ANA-positive control with human-specific secondary
antibody. These controls were run to enable correction for variation in laboratory conditions on the
rates of ELISA reactions (e.g., due to variation in ambient room temperature). For each sample, a
second, independent assay was run using a different kit on a different day, and we took the mean of
the sample duplicates minus the mean of the background control duplicates on the two plates as our
measure of ANA for further analysis (see [1] for further details).
Other antibody assays
For the total assays, we used sheep capture antibodies purchased from AbD Serotec (Catalogue
numbers: IgA: AHP949, IgM: AHP950, IgG: 5184-2104) diluted to 2µg of antibody per ml of
0.06M Carbonate buffer at pH 9.6. For the KLH assays, we used KLH capture antigen purchased
from Calbiochem (provided at 8.33mg/ml, Catalogue number: 374813), diluted to 2µg antigen per
ml of 0.06M Carbonate buffer at pH 9.6. For the T.circumcincta assays, we used T. circumcincta L3
somatic antigen, diluted to 2µg per ml of 0.06M Carbonate buffer at pH 9.6. L3 somatic antigen
was prepared by re-suspending T circumcincta L3 in PBS (~5 x 105 larvae per ml) in Lysing Matrix
D tubes (MP Biomedicals) and homogenising in a Precellys® 24 tissue homogeniser. Debris was
pelleted by centrifugation at 16, 000 × g at 4oC and the somatic antigen containing supernatant
stored at -80oC prior to use. Total protein concentration of the L3 antigen preparation was estimated
using a Pierce™ BCA Protein Assay Kit (Thermo Scientific).
In each assay, 50µl of appropriately diluted antigen solution was added to each well of a Nunc
immuno 96-microwell plate, which was subsequently covered and incubated overnight at 4oC. The
wells were then washed three times in Tris-buffered saline-Tween (TBST) using a plate washer.
Then 50µl of an appropriately diluted Soay sheep plasma sample was added to each well. Sample
dilutions used (following optimisation procedure described above) were as follows: Total IgA:
1:3200; total IgM: 1:25600; total IgG: 1:1638400; anti- T. circumcincta IgA: 1:50; anti- T.
circumcinta IgM: 1:800; anti- T. circumcinta IgG: 1:12800; anti- T. circumcinta IgE: 1:100; antiKLH IgA: 1:50; anti-KLH IgM: 1:400; anti-KLH IgG: 1:400.
The plates were then covered and incubated at 37oC for 1 hour. They were then washed five times
with TBST and 50µl per well of the appropriate rabbit anti-sheep detection antibody conjugated to
horseradish peroxidise (HRP) was added (anti-ovine IgA-HRP, anti-ovine IgM-HRP, anti-ovine
IgG-HRP: all AbD Serotec, cataologue numbers: AHP949P, AHP950P and 5184-2504,
respectively). For the anti-T.circumcincta IgE assay, instead of the detection antibody step
described above, 50µl of anti-ovine IgE (mouse monoclonal IgG1, clone 2F1, [2]) diluted 1:100 in
TBST was added to each well, followed by 1 hour incubation at 37oC, five washes with TBST and
then the addition of 50µl of goat anti-mouse IgG1-HRP detection antibody (AbD Serotec catalogue
number: STAR132P), diluted to1µg in 8000ul of TBST to each well. All plates were then covered
and incubated at 37oC for 1 hour. They were then washed five times with TBST and 100µl of
SureBlue TMB 1-Component microwell peroxidase substrate (KPL) was added per well and then
left to incubate for 5 minutes in the dark at 37oC. Reactions were then stopped by adding 100µl 1M
HCl and optical densities (ODs) were read immediately at 450nm using a Thermo Scientific
Multiskan GO Spectrophotometer.
Each assay on each selected Soay sheep plasma sample was performed twice on separate ELISA
plates. On each plate we also included three sample-free wells as negative controls, and three wells
into which 50µl of dilute plasma (to same level as Soay sheep samples) from the same parasite-free
domestic sheep, from which a large amount of plasma had been obtained, as plate controls. We
excluded 26 duplicates across all assays for which there was obviously poor correspondence across
duplicate OD scores, presumably due to human error. We then checked the correlation of ODs
across duplicate plates and re-ran both plates if r < 0.80. For subsequent analyses, we took the
average OD across the duplicate runs minus the average of the six negative control well ODs across
the two plates as our assay measure.
References:
[1] Graham, A. L., Hayward, A. D., Watt, K. A., Pilkington, J. G., Pemberton, J. M. & Nussey, D.
H. (2010) Fitness correlates of heritable variation in antibody responsiveness in a wild mammal.
Science, 330, 662-665.
[2] Bendixsen, T., Windon, R. G., Huntley, J. F., MacKellar, A., Davey, R. J., McClure, S. J. &
Emery, D. L. (2004) Development of a new monoclonal antibody to ovine chimeric ige and its
detection of systemic and local ige antibody responses to the intestinal nematode trichostrongylus
colubriformis. Veterinary Immunology and Immunopathology, 97, 11-24.