Utilization of commercial plasma administered to the index case
An aliquot of the commercial plasma that had been administered to the
index case described previously, which apparently contributed to a fatal
anaphylactic reaction was available for analysis. This sample was
aliquoted into 10 mL and 2 mL vials and stored at
-80oC.
Indirect ELISA to detect
BSA-specific IgE
This protocol was adapted from Gershwin et al. 2012 with
modifications. ELISA plates were coated with 1 ug/well BSA in
bicarbonate-carbonate buffer pH 9.6 overnight. ELISA wells were blocked
with 0.5% rabbit serum albumin (RSA) in coating buffer. Each well in a
96 well plate was filled with 200 µL of 0.5% RSA for blocking. The
plates were incubated at 4oC overnight. After
incubation concluded, the wells were washed with 300 µL of PBSt (PBS +
0.1% Tween-20) by an electronic plate washer 3 times. Plates were
coated and blocked in bulk and stored at 4˚ C until use within one week.
The commercial plasma that was administered to the index case was used
as a positive control. Unvaccinated horse serum was used as a negative
control. PBS was used as a blank to establish background absorbance. Two
wells were designated as the positive control, two as the negative
control and two as the blank. Undiluted serum samples (100 µL) were
added to each remaining well in duplicate and incubated at room
temperature for 2 hours. A standard curve was not available and optical
density (OD) values were used to analyze the data.
Plates were washed as described above. Anti-horse IgE horse radish
peroxidase (HRP)10 was diluted out to 1:500 with wash
buffer, added to each well (100 µL) and incubated for 1 hour at room
temperature. The plates were washed again. Tetramethylbenzidine (TMB)
(100 µL) was added to each well and left to incubate for 30 minutes at
room temperature protected from the light. After 30 minutes 100 µL of
sulfuric acid (2 M SO2H4) was added to each well to terminate the
reaction. Plates were read with an electronic plate reader at 450 nm and
recorded to obtain the optical density.
All horse samples including 0, 14, 30, 90, 180, 270, and 365 day
post-vaccination, if available, were tested by way of ELISA for the
presence of BSA-specific IgE. All horses classified as a reactor were
tested by ELISA. Plasma from the plasma donor samples from commercial
source were all tested by ELISA. All plates contained the same positive
control, negative control and blanks. The OD value for blank wells was
averaged for each plate and then subtracted from each sample well to
correct for background absorbance. The adjusted duplicate sample OD
values were then averaged together for each horse.
Statistical analysis
Statistical analysis was completed with Prism (v.8) on the BSA-specific
IgE ELISA data. Horses in the healthy vaccine group had their
post-vaccine Ig OD measurements compared with baseline values. Means
were determined for each timepoint for all age groups. Differences
between groups at specific timepoints were determined with t-tests.
Significance was defined as a p -value <0.05. Data was
then compiled in Microsoft Excel as a spreadsheet function.
RESULTS
ELISA BSA-specific IgE
The OD value for the positive control mean across all plates was 0.340.
The OD value for the negative control mean across all plates was 0.062.
The OD value for the blank control mean across all plates was 0.071. The
OD value for the positive control mean was significantly different from
the negative control mean (p value <0.001). The OD
value for the negative control mean was significantly different from the
OD value blank control mean (p value = 0.008).
Healthy Horses
The mean OD value +/- standard deviation for day 0 BSA-specific IgE in
all healthy horses (0.108 +/- 0.089) was not significantly different
when compared to the mean OD value +/- standard deviation (SD) for 14
days post-vaccination BSA-specific IgE in all healthy horses (0.124 +/-
0.118) (p value = 0.43) (Figure 1). The mean OD value +/- SD for
day 0 BSA-specific IgE in all healthy horses (0.108 +/- 0.089) was
significantly higher when compared to the mean OD value +/- SD for 180
days post-vaccination BSA-specific IgE in all healthy horses (0.073 +/-
0.069) (p value = 0.007) (Figure 1).
When comparing the fold change between day 0 BSA-specific IgE OD values
and 14 days post-vaccination BSA-specific IgE OD values, 63% of horses
between the ages of 3 and 9 years had a fold increase greater than 1.5
(Figure 2 and Table 1). The percentage of horses displaying a fold
change greater than 1.5 decreased with age across age groups. Only 29%
displayed a fold change greater than 1.5 in the 10-14 year old age
group, 0% in the 15-19 year old age group and 5.6% in the 20 -32 year
old age group (Table 1).
The mean BSA-specific IgE OD values +/- SD at time points 0, 14, 30, 90,
180, 270 and 365 days after vaccination were compared across age groups
(Table 1). The mean BSA-specific IgE OD value +/- SD for horses 3-9
years of age at day 0 was 0.072 +/- 0.086 and at 14 days
post-vaccination had increased to 0.125 +/- 0.09 (p-value = 0.013)
(Table 1). The mean BSA-specific IgE OD value +/- SD decreased to 0.070
+/- 0.070 at 180 days post-vaccination with days 0 and 180 being
significantly different (p-value = 0.035) (Table 1). All other
timepoints compared to day 0 within this age group were not
significantly different. When comparing all other age groups’ mean
BSA-specific IgE OD values at 14 days after vaccination and day 0, the
differences were not significant. When comparing all other age groups’
mean BSA-specific IgE OD values at day 0 and 180 days after vaccination,
the differences were significant for age groups 10-14 years (p value =
0.001) and 20-32 years (p value = 0.028). In all of these age groups,
the mean BSA-specific IgE OD values decreased at 180 days
post-vaccination compared to time 0 and 14 days post-vaccination (Table
1). No other time points in any age group were significantly different
when compared to day 0.
Anamnestic response of BSA-specific IgE expression in response to annual
vaccination in healthy horses was most common in younger horses, aged
horses rarely demonstrated a > 1.5 fold change in
BSA-specific IgE expression within 14 days of annual booster vaccination
(Table 1).
Among young adult horses that were evaluated over the course of a year,
two-thirds of the horses demonstrated a greater than 1.5 fold increase
in BSA-specific IgE expression without adverse event (Figure 2). Among
horses greater than 10 years of age, it was uncommon to have a marked
increase in BSA-specific IgE expressed following booster vaccination
(Table 1).